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Role of the salivary gland in ion and water regulation during feeding in the female tick, dermacentor… Kaufman, William Reuben 1971

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z • J n ROLE OF THE SALIVARY GLAND IN ION AND WATER REGULATION DURING FEEDING IN THE FEMALE TICK, DERMACENTOR ANDERSONI by W i l l i a m R. Kaufman B.Sc., McGILL UNIVERSITY, MONTREAL, I965 M.Sc., McGILL UNIVERSITY, MONTREAL, I967 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n the Department of ZOOLOGY We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o the r e q u i r e d s t a n d a r d The U n i v e r s i t y o f B r i t i s h C o l u m b i a O c t o b e r , 1971 In p r e s e n t i n g t h i s t h e s i s in p a r t i a l f u l f i l m e n t o f the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r reference and study . I f u r t h e r agree t h a t permiss ion f o r e x t e n s i v e copying o f t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s r e p r e s e n t a t i v e s . It i s understood that copying or p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l ga in s h a l l not be a l lowed without my w r i t t e n p e r m i s s i o n . Depa rtment The U n i v e r s i t y o f B r i t i s h Columbia Vancouver 8, Canada i i ABSTRACT Of t h e t o t a l w ater l o s t d u r i n g t h e f e e d i n g p e r i o d by Dermacentor a n d e r s o n i f e m a l e s , 75$ w » s e x c r e t e d i n t h e s a l i v a , l e s s t h a n 5% was e v a p o r a t e d from t h e integument, and t h e re m a i n d e r was l o s t i n the f e c e s . N i n e t y - f i v e p e r c e n t of t h e t o t a l e x c r e t e d sodium and 18$ o f t h e t o t a l e x c r e t e d p o t a s s i u m was l o s t i n t h e s a l i v a . The complementary p e r c e n t a g e s o f t h e l a t t e r i o n s were e x c r e t e d i n t h e f e c e s . S a l i v a was shown t o be produced by a s e c r e t o r y r a t h e r t h a n a f i l t r a t i o n mechanism. S i n c e s a l i v a r y g l a n d s s e t up i n v i t r o s e c r e t e d f l u i d when o f f e r e d a d r e n a l i n , n o r a d r e n a l i n o r dopamine, b u t n o t when o f f e r e d n a t u r a l hemolymph from s a l i v a t i n g t i c k s , I proposed t h a t s e c r e t i o n i s n o r m a l l y under n e u r a l r a t h e r t h a n e n d o c r i n e c o n t r o l . Moreover, s i n c e I n - v i t r o g l a n d s were i n s e n s i t i v e t o p i l o c a r p i n e and r e l a t i v e l y i n s e n s i -t i v e t o s e r o t o n i n , t h i s s u g g e s t e d t h a t t h e n a t u r a l t r a n s m i t t e r s u b s t a n c e may be a c a t e c h o l a m i n e . The p r e s e n c e o f n e r v e s i n c l o s e a s s o c i a t i o n w i t h s a l i v a r y g l a n d t i s s u e was c o n f i r m e d h i s t o l o g i c a l l y and u l t r a s t r u e t u r a l l y . F l u i d t r a n s p o r t ceased when c h l o r i d e i n t h e e x t e r n a l medium was r e p l a c e d w i t h a c e t a t e , and was i n h i b i t e d by 95$ when r e p l a c e d w i t h n i t r a t e . The r a t e o f f l u i d s e c r e t i o n was p r o -p o r t i o n a l t o t h e c h l o r i d e c o n c e n t r a t i o n I n t h e e x t e r n a l medium. I l l The e l e c t r o p o t e n t i a l d i f f e r e n c e r e c o r d e d a c r o s s t h e s a l i v a r y a c i n i by means of -glass m i c r o e l e c t r o d e s was about 30 mV lumen n e g a t i v e , and t h e s a l i v a t o hemolymph r a t i o f o r c h l o r i d e i n v i t r o was about 1.6. These d a t a i n d i c a t e d t h a t c h l o r i d e I s a c t i v e l y t r a n s p o r t e d by t h e s a l i v a r y e p i t h e l i u m . The r a t e o f f l u i d t r a n s p o r t was I n f l u e n c e d by t h e Na:K r a t i o i n t h e medium. The r a t e o f f l u i d s e c r e t i o n was maximal i n medium c o n t a i n i n g 210 m e q / l l t e r Na and 10 m e q / l i t e r K. R e p l a c i n g t h e 10 m e q / l i t e r K w i t h Na i n h i b i t e d t h e s e c r e t o r y r a t e by 80$. However the p r o g r e s s i v e i n c r e a s i n g o f t h e K l e v e l beyond 10 m e q / l i t e r a t t h e expense of Na a l s o i n h i b i t e d t h e r a t e o f f l u i d s e c r e t i o n , l e a d i n g t o complete c e s s a t i o n when b o t h c a t i o n s were I n e q u i m o l a r c o n c e n t r a t i o n . F l u i d t r a n s -p o r t a l s o ceased when g l a n d s were bath e d i n normal R i n g e r s o l -u t i o n c o n t a i n i n g 10"^ M o u a b a i n . The l a t t e r r e s u l t s s u g g e s t e d t h a t t h e energy r e q u i r e d f o r f l u i d s e c r e t i o n comes from a N a , K - a c t i v a t e d •pump ATPase'. The s a l i v a r y g l a n d i s composed o f t h r e e d i s t i n c t t y p e s of a c i n u s ( t y p e s I , I I , I I I ) . The t y p e I a c i n u s i s composed o f two c e l l t y p e s and t h e t y p e s I I and I I I a c i n i f i v e c e l l t y p e s each. The u l t r a s t r u c t u r a l f e a t u r e s o f t h e c e l l named here t h e " w a t e r c e l l 1 ' i n t h e t y p e I I I a c i n u s a r e v e r y s i m i l a r t o t h o s e d e s c r i b e d f o r o t h e r f l u i d - t r a n s p o r t i n g e p i t h e l i a . The maze o f i n t r a c e l l u l a r c h a n n e l s a s s o c i a t e d c l o s e l y w i t h numerous m i t o c h o n d r i a s u g g e s t e d t h a t f l u i d t r a n s p o r t c o u l d be e x p l a i n e d by t h e s t a n d i n g - g r a d i e n t h y p o t h e s i s of Diamond and B o s s e r t ( I 9 6 7 , 1 9 6 8 ) . But t h e l a t t e r model can o n l y e x p l a i n t h e transport of an lso-osmotic or a hyperosmotic f l u i d . Since the f l u i d secreted by ln-vltro salivary glands was hypo-osmotic to the bathing medium under a l l conditions tested, the primary f l u i d secreted into the acinus must be subsequently rendered hypo-osmotic by means of solute reabsorption. The site of reabsorption was not localized, but reabsorption was suspected to occur in the duct system, either by the duct ce l l s themselves as in mammals, or conceivably by the f i b r i l l a r c ells of the type I acinus. V TABLE OF CONTENTS CHAPTER Page ONE GENERAL INTRODUCTION 1) I n t r o d u c t i o n 2 2) G e n e r a l B i o l o g y of Dennacentor a n d e r s o n l 3 3 ) Water B a l a n c e i n T i c k s - L i t e r a t u r e Review 6 k) Statement o f t h e Problem 12 TWO ROUTES OF WATER AND ION LOSS INTRODUCTION " 15 MATERIALS AND METHODS 1) R e a r i n g Methods 20 2 ) Net Weight I n c r e a s e o f t h e Females 22 3 ) Water Loss Through t h e Integument 22 k) C o l l e c t i o n of Dry Feces 23 5) P r e p a r a t i o n o f R a b b i t Hemoglobin 2k 6 ) D e t e r m i n a t i o n o f Hemoglobin C o n c e n t r a t i o n 25 ?) Method o f S a m p l i n g Hemolymph and S a l i v a o f T i c k s 29 8 ) D e t e r m i n a t i o n o f I o n C o n c e n t r a t i o n 31 9 ) Osmotic P r e s s u r e 36 1 0 ) P r o t e i n D e t e r m i n a t i o n o f Feces 36 11) C l e a r a n c e o f I n u l l n and 3 - 0 - M e t h y l g l u c o s e 36 1 2 ) D e t e r m i n a t i o n o f Hemolymph Volume 38 RESULTS 1) Net Weight I n c r e a s e o f F e e d i n g Females kO 2 ) T o t a l I n g e s t i o n and T o t a l L o s s e s 40 3 ) Routes of Water L o s s : Q u a n t i t a t i v e E v a l u a t i o n 4 5 4 ) Routes of I o n L o s s : Q u a n t i t a t i v e E v a l u a t i o n 52 5) The E x c r e t i o n o f P r o t e i n I n t h e Feces 52 v i CHAPTER Page 6 ) I o n i c and Osmotic Changes i n t h e Hemolymph and S a l i v a D u r i n g a Normal F e e d i n g P e r i o d 57 7 ) The C l e a r a n c e o f Two Non-charged M o l e c u l e s 66 8 ) Hemolymph Volume 67 DISCUSSION 1) Net Weight I n c r e a s e of t h e Females. 71 2 ) T o t a l I n g e s t i o n and T o t a l L o s s e s 72 3 ) I o n C o m p o s i t i o n o f the Feces 73 k) Integumentary Water L o s s 76 5) C o m p o s i t i o n o f Hemolymph and S a l i v a i n F e e d i n g Females 78 6 ) Hemolymph Volume and t h e S t i m u l u s t o S a l i v a t i o n 8 3 7) C l e a r a n c e o f I n u l l n and O - M e t h y l g l u c o s e 8 5 8 ) Summary of C h a p t e r Two 9 3 THREE CONTROL AND MECHANISM OF SALIVARY SECRETION INTRODUCTION 95 MATERIALS AND METHODS 1) E x c i s i o n of the S a l i v a r y Glands 1 0 0 2 ) P o s t - e x c i s i o n Treatment 101 3) E x p e r i m e n t a l Media 103 k) E x p e r i m e n t a l P r o c e d u r e s 108 5) Weight of t h e Glands 110 6 ) The E f f e c t o f Drugs on S e c r e t o r y Rate 110 7) P o t e n t i a l D i f f e r e n c e Measurements 111 RESULTS 1) Development of the I n - V l t r o P r e p a r a t i o n 116 2 ) Time Course of S e c r e t o r y A c t i v i t y U s i n g t h e P u l s e Method 125 3 ) The C o n t r o l o f S e c r e t i o n s Pharmacology 131 4) The E f f e c t o f V a r i o u s A n i o n s on t h e Rate o f S e c r e t i o n and C o m p o s i t i o n o f S a l i v a 136 5) E f f e c t o f E x t e r n a l Sodium and P o t a s s i u m on S a l i v a t i o n 148 v i i CHAPTER Page 6) Magnesium C o n c e n t r a t i o n i n Medium and S a l i v a 152 7) E l e c t r o p o t e n t i a l D i f f e r e n c e A c r o s s t h e S a l i v a r y Gland 153 8) The E f f e c t of Ouabain on S e c r e t o r y Rate 156 9) The E f f e c t o f Osmotic P r e s s u r e on S e c r e t i o n 158 DISCUSSION 1) The I n - V i t r o P r e p a r a t i o n 162 2) The C o n t r o l o f S e c r e t i o n 162 3) Mechanism o f F l u i d S e c r e t i o n and I o n T r a n s f e r 167 4) The E f f e c t of Osmotic P r e s s u r e on S e c r e t i o n 171 5) Summary o f C h a p t e r Three 172 FOUR HISTOLOGY AND ULTRASTRUCTURE OF THE SALIVARY GLAND INTRODUCTION 174 MATERIALS AND METHODS 177 RESULTS 1) The S a l i v a r y D ucts 179 2) The P y r a m i d a l A c i n u s 180 3) The G r a n u l e - S e c r e t i n g A c i n u s (Type I I I ) 182 4) The G r a n u l e - S e c r e t i n g A c i n u s (Type I I ) I89 DISCUSSION , . 1) The Ducts 192 2) The P y r a m i d a l A c i n u s 193 3) The G r a n u l e - S e c r e t i n g A c i n u s (Type I I I ) 1§4 4) The G r a n u l e - S e c r e t i n g A c i n u s (Type I I ) . 206 5) Summary of C h a p t e r Four 208 FIVE SUMMARY REMARKS 243, CITED REFERENCES . 24? vi i i LIST OF FIGURES Figure Page 1 Errors i n determination of chloride concen-t r a t i o n when using f r a c t i o n a l volumes of a 1-jil pipette of uniform "bore 35 2 The net weight increase of a large, hetero-geneous group of t i c k s from several feedings expressed i n terms of Log-, n of the weight r a t i o weight on day "X" x u 42 unfed weight 3 The d a i l y production of feces (dry weight) expressed as a percentage of the t o t a l feces (dry weight) produced during the complete feeding period ( i . e . the sum of a l l the points equals 100$) 42 4 Integumentary water loss 48 5 V i s i b l e spectra of rabbit hemoglobin, equine hemin, t i c k homogenate and t i c k feces 56 6 Ion concentrations i n hemolymph and s a l i v a with time during the feeding period 6 l 7 Osmotic pressure i n hemolymph and s a l i v a with time during the feeding period 62 8 Volume of hemolymph with the progression of feeding 70 9 E f f e c t of saline i n j e c t i o n s into the hemolymph on the subsequent volume of s a l i v a r y secretion 87 10 Diagram of microelectrode arrangement f o r measuring e l e c t r o p o t e n t i a l differences by method A 113 11 The response of an i n - v l t r o gland to adrena- 120 l i n 12 Rate of secretion with time under various protocols 123 i x F i g u r e Page 13 The t i m e c o u r s e o f s e c r e t o r y a c t i v i t y d u r i n g t h e f i r s t p u l s e o f a d r e n a l i n u s i n g t h e P u l s e Method 1 2 7 1 4 I o n c o n c e n t r a t i o n of s a l i v a s e c r e t e d i n v i t r o 130 15 E f f e c t o f drugs on s e c r e t o r y r a t e 134 1 6 S a l i v a r y s e c r e t i o n i n v i v o 138 17 The r e c o v e r y o f g l a n d s b a t h e d i n a c e t a t e and n i t r a t e media when r e t u r n e d t o c h l o r i d e media ( m a i n t a i n e d dosage o f 1 0 - 4 M a d r e n a l i n ) 1 4 2 1 8 R e l a t i o n s h i p between c h l o r i d e c o n c e n t r a t i o n i n t h e b a t h i n g medium and f l u i d s e c r e t o r y r a t e 1 4 4 1 9 C h l o r i d e c o n c e n t r a t i o n of s a l i v a i n v i t r o as a f u n c t i o n o f e x t e r n a l c h l o r i d e c o n c e n t r a t i o n 1 4 4 2 0 The r e l a t i o n s h i p between r a t e o f c h l o r i d e and f l u i d s e c r e t i o n 1 4 6 2 1 E f f e c t of t h e Na«K r a t i o on s e c r e t o r y r a t e 150 2 2 E f f e c t o f NatK r a t i o i n t h e b a t h i n g medium on c o n c e n t r a t i o n of c a t i o n s i n t h e s a l i v a 151 23 E f f e c t o f o s m o t i c p r e s s u r e on s a l i v a t i o n 1 6 0 2 4 E f f e c t o f o s m o t i c p r e s s u r e on s a l i v a t i o n 1 6 0 25 A d i a g r a m a t i c r e c o n s t r u c t i o n o f t h e t y p e I I I a c i n u s showing t h e s p a t i a l r e l a t i o n s h i p s among t h e f i v e c e l l t y p e s 1 8 5 2 6 A h i g h l y s c h e m a t i c r e p r e s e n t a t i o n o f how the s t a n d i n g - g r a d i e n t h y p o t h e s i s c o u l d e x p l a i n w a t e r - t o - s o l u t e c o u p l i n g i n the w a t e r c e l l o f th e t y p e I I I a c i n u s 2 0 1 2 7 C r o s s s e c t i o n t h r o u g h t h e main e x c r e t o r y d u c t and l o n g i t u d i n a l s e c t i o n t h r o u g h some t y p e I a c i n i 2 1 2 2 8 C r o s s s e c t i o n t h r o u g h t h e main e x c r e t o r y d u c t and l o n g i t u d i n a l s e c t i o n t h r o u g h some ty p e I a c i n i 2 1 2 29 A A p o l a r s e c t i o n t h r o u g h a t y p e I I a c i n u s 2 1 4 29 B A l i g h t l y s t a i n e d s e c t i o n o f t h e e n c a p s u l a t e d -g r a n u l e c e l l showing more c l e a r l y t h e s t r u c -t u r e o f t h e g r a n u l e s 2 1 4 F i g u r e Page 30 S e c t i o n t h r o u g h a " d e f l a t e d " t y p e I I I a c i n u s showing th e f i v e c e l l - t y p e s 216 31 A s e c o n d a r y d u c t 216 32 S e v e r a l s e c t i o n s I n w h i c h the w a t e r c e l l a p p e a r s t o make c o n t a c t w i t h the a c i n a r lumen 218 33 S e c t i o n p a r a l l e l t o t h e e q u a t o r o f an " I n f l a t e d " t y p e I I I a c i n u s 220 34 I n n e r v a t i o n o f t h e t y p e I I I a c i n u s 222 35 A p a r t i c u l a r l y l u c i d v i e w o f t h e fundus c e l l s o f a t y p e I I I a c i n u s i n e q u a t o r i a l s e c t i o n 222 36 S e c t i o n a c r o s s t h e " n o r t h p o l e " r e g i o n o f a t y p e I I I a c i n u s j u s t below th e r e g i o n of t h e e n t r y o f t h e e f f e r e n t d u c t 224 37 The v a c u o l a r c e l l , t y p e I I I a c i n u s 224 38 C r o s s s e c t i o n t h r o u g h t h e w a l l of t h e main e x c r e t o r y d u c t 226 39 C r o s s s e c t i o n t h r o u g h t h e w a l l o f a s e c o n -d a r y d u c t 228 40 A . S e c t i o n t h r o u g h t h e p y r a m i n a l a c i n u s showing t h e f i b r i l l a r c e l l and t h e i n n e r c e l l 230 40 B N e u r o s e c r e t o r y axon i n t h e t y p e I a c i n u s 230 41 S e c t i o n t h r o u g h t h e i n n e r c e l l o f t h e t y p e I a c i n u s 232 42 S e c t i o n t h r o u g h a n a k e d - g r a n u l e c e l l and an e n c a p s u l a t e d - g r a n u l e c e l l o f a type I I o r I I I a c i n u s f rom an u n f e d t i c k 234 43 C r y p t r e g i o n of type I I I a c i n u s 236 44 S e c t i o n t h r o u g h t h e b a s a l end o f t h e same w a t e r c e l l shown i n t h e p r e v i o u s f i g u r e 238 45 S e c t i o n t h r o u g h a t y p e I I a c i n u s showing a w a t e r c e l l and v a c u o l a r c e l l s d e v o i d of g r a n u l e s 2^° 46 The cap c e l l , t y p e I I a c i n u s 242 x i LIST OF TABLES T a b l e Page I The r a t i o s o f engorged t o u n f e d w e i g h t f o r a wide v a r i e t y o f b l o o d - s u c k i n g organisms 4 I I C omparison of r a b b i t h e m o g l o b i n p r e p a r e d i n t h i s l a b o r a t o r y w i t h Sigma b o v i n e h e m o g l o b i n 2 5 I I I C o m p o s i t i o n of r a b b i t whole venous b l o o d 43 IV T o t a l b l o o d ( i . e . meal) removed f r o m r a b b i t 43 V T o t a l w a t e r l o s t by t h e t i c k d u r i n g f e e d i n g 44 V I F a t e o f t h e b l o o d meal a t end o f f e e d i n g 44 V I I Weight l o s s by t i c k s w i t h t h e mouth and anus c o v e r e d a t d i f f e r e n t p e r i o d s d u r i n g f e e d i n g 46 V I I I Sodium c o n t e n t o f t h e f e c e s ( d r y w e i g h t ) c o l l e c t e d o v e r the t o t a l f e e d i n g p e r i o d 49 IX C a l c u l a t i o n o f s a l i v a r y w a t e r l o s s 51 X A The r o u t e s o f e x c r e t i o n f o r sodium 53 X B The r o u t e s o f e x c r e t i o n f o r p o t a s s i u m 53 X I P r o t e i n c o n t e n t o f t i c k f e c e s by the method o f Lowry e t a l . ( 1 9 5 D 5 ^ X I I P o t a s s i u m d i s t r i b u t i o n i n t i c k hemolymph 59 X I I I A I o n d i s t r i b u t i o n i n t i c k body f l u i d s and e x c r e t a 64 X I I I B Summary of i o n d i s t r i b u t i o n s i n hemolymph and s a l i v a f r o m t i c k s w h i c h had f e d f o r a t l e a s t t h r e e days 65 XIV The c l e a r a n c e o f O-methylglucose 67 XV A I n g r e d i e n t s common t o a l l media 1 0 3 XV B E x p e r i m e n t a l media 104 x i i T a b l e XVI X V I I X V I I I X I X XX A c o m p a r i s o n of l n - v l t r o s e c r e t o r y r a t e s among p r e p a r a t i o n s of a s i m i l a r n a t u r e t o t h e one used i n t h i s s t u d y The e f f e c t on s a l i v a t i o n o f i n j e c t i o n o f a d r e n a l i n i n v i v o P o t e n t i a l measurements a c r o s s t h e s a l i v a r y g l a n d s E f f e c t o f o u a b a i n on r a t e o f s a l i v a t i o n i n v i t r o C o r r e l a t i o n of t h e c e l l t y p e s f o r t h r e e s p e c i e s o f i x o d i d t i c k s Page 1 3 1 1 3 9 1 5 4 1 5 7 207 ACKNOWLEDGEMENTS I am g r a t e f u l t o t h e f o l l o w i n g p e r s o n s who c o n t r i b u t e d t h e i r s k i l l s i n t h e e x e c u t i o n of s p e c i f i c e x p e r i m e n t s i Mr. S t e v e B orden and Mrs. D o l o r e s L a u r i e n t e c r e a t e d t h e com-p u t e r programs used f o r t h e a n a l y s e s of the d a t a . The e a r l y c o m p l e t i o n of t h i s s t u d y w o u l d n o t have been p o s s i b l e w i t h o u t t h e i r h e l p i M i s s P a t C o l l e n c a r r i e d out t h e d e t e r m i n a t i o n s of f r e e z i n g p o i n t d e p r e s s i o n ; my w i f e Susan a s s a y e d t h e t i c k f e c e s f o r p r o t e i n c o n t e n t ; Mr. J o e K i c e n l u k c a r r i e d out t h e d e t e r m i n a t i o n s o f magnesium c o n c e n t r a t i o n ; M i s s J o a n M e r e d i t h p r e p a r e d t h e e l e c t r o n m i c r o g r a p h s , and Dr. P. J . R. T a y l o r e x e c u t e d t h e d r a w i n g i n F i g u r e 2 5 . I am a l s o g r a t e f u l t o the N a t i o n a l R e s e a r c h C o u n c i l o f Canada f o r a w a r d i n g me a p o s t -g r a d u a t e s c h o l a r s h i p , and t h e Z o o l o g y Department a t UBC f o r p r o v i d i n g t e a c h i n g a s s i s t a n t s h l p s . F i n a l l y , as one cannot work I n vacuo, i t i s a p l e a s u r e t o acknowledge my s u p e r v i s o r D r. J . E. P h i l l i p s who o f f e r e d i n v a l u a b l e a s s i s t a n c e t h r o u g h o u t t h e p r e p a r a t i o n of t h e t h e s i s , my c o l l e a g u e s M i c h a e l B a l s h i n , Soon Goh, J o e K i c e n l u k and Simon L e w i s who p r o v i d e d "atmosphere" and my w i f e Susan, e s p e c i a l l y f o r h e r encouragement. C i t a t i o n f r o m F r a n z F e r d i n a n d o f T r o i l o , who was i n the h o l y l a n d 1666-69. " I n t h e Jud e a n d e s e r t one f i n d s a v e r y s e r i o u s p e s t . The g r e e n , venemous s c o r p i o n s and t h e ' l o u s e o f Pharoah' (which was one o f t h e t e n p l a g u e s ) a r e abundant t h e r e . These b e a s t s a r e r o u n d , as l a r g e as a penny, and have n i n e l e g s ; t h e y a r e e x t r e m e l y venemous and l u r k u nder s t o n e s and s m a l l p l a n t s . As soon as one s i t s t o r e s t , and t h u s warms t h e groun d , t h i s " l o v e l y c r e a t u r e " emerges, b i t e s , and burrows i n t o t h e f l e s h j t h e y c a n be p u l l e d out p i e c e by p i e c e , o n l y w i t h r e d - h o t i n s t r u m e n t s , b u t even s o , cause p a i n and l e a v e a b l a c k and b l u e s p o t w h i c h i s loathsome t o l o o k upon. Because of t h i s , I was v e r y c a r e f u l n o t t o r e s t on t h e ground i n t h i s d e s e r t . " F. S. Bodenheimer Ha'Chal B e ' A r t z o t Hamlkra ( A n i m a l L i f e i n B i b l i c a l Lands) 1 CHAPTER ONE GENERAL INTRODUCTION 2 Terrestrial blood-sucking arthropods are exposed throughout their l i f e - c y c l e to alternating periods of water abundance and water deprivation. Unlike the situation for their omnivorous or herbivorous relations, the attaining of a meal is a hazardous enterprise. They tend, therefore, to feed only intermittently, and during these periods, to imbibe suf-fici e n t nutrient to see them through long periods of abstinence. Often this involves the uptake of a meal several times the weight of the unfed organism i t s e l f . During the intervening periods of weeks or months they go about the business of growth or gamete production, u t i l i z i n g the resources of the huge blood meal. Although blood i s an extremely nutritious substance, i t also contains a great deal of excess water which i s metabolically useless. Even though blood-suckers can expand to accommodate the meal - often to a remarkable extent - obviously there are limi t -ations imposed on this expansion. Since they abstain from food intake for extended periods of! time, most of them have evolved mechanisms for separating and 'eliminating a good deal of water from the nutrient portion of the blood. Once this is done, how-I ever, their strategy switches ;to one of water-conservation u n t i l i i. 3 t h e subsequent meal i s t a k e n . I n t h i s t h e s i s I s h a l l a t t e m p t t o e x p l o r e some of t h e p h y s i o l o g i c a l mechanisms employed by t h e a d u l t f e m a l e o f one s u c h s p e c i e s o f b l o o d - s u c k e r f o r t h e r e g u l a t i o n of t h e w a t e r and i o n c o n t e n t i n t h e body. Dermacentor a n d e r s o n l i s a p a r t i c -u l a r l y a t t r a c t i v e s p e c i e s f o r s t u d y i n g t h i s p r o b lem because of t h e enormous s i z e o f t h e b l o o d meal i n r e l a t i o n t o t h e u n f e d o r " f l a t " w e i g h t ( T a b l e 1 ) . 2) G e n e r a l B i o l o g y of Dermacentor a n d e r s o n l The t i c k s and m i t e s c o n s t i t u t e the l a r g e a r a c h n i d o r d e r , A c a r i (= A c a r l n a ) . The A c a r i ( A r t h u r , 1961) a r e d i v i d e d i n t o s e v e n s u b - o r d e r s o f w h i c h t h e M e t a s t i g m a t a c o m p r i s e a l l t h e t i c k s . T here a r e two major f a m i l i e s o f t i c k s , A r g a s l d a e o r s o f t t i c k s and I x o d i d a e o r h a r d t i c k s ; a t h i r d f a m i l y , t h e N u t t a l l e l l i d a e , a l s o has been e s t a b l i s h e d t o accommodate a s i n g l e r a r e s p e c i e s ( N u t t a l l e l l a namaqua) whi c h p o s s e s s e s c h a r a c t e r i s t i c s i n t e r m e d -i a t e between t h e A r g a s l d a e and I x o d i d a e . Dermacentor a n d e r s o n i i s f o u n d m a i n l y i n t h e w e s t e r n r e g i o n o f N o r t h A m e r i c a and i s commonly c a l l e d t h e Rocky M o u n t a i n wood t i c k . I t e x t e n d s as f a r e a s t as mid-Saskatchewan, as f a r n o r t h a s J a s p e r , A l b e r t a , and M a c A l i s t e r , B r i t i s h C o l u m b i a , and as f a r s o u t h as A r i z o n a . I t s range does not e x t e n d west of t h e c o a s t mountains i n B r i t i s h C o l u m b i a . The f a c t o r s g e n e r a l l y l i m i t i n g t h e d i s t r i b u t i o n o f t h i s s p e c i e s a r e d i s c u s s e d i n d e p t h by W i l k i n s o n ( 1 9 6 7 ) . Dermacentor a n d e r s o n i t r a n s m i t s two a g e n t s o f e p i d e m i -o l o g i c a l i m p o r t a n c e : (1) t h e Rocky Mountain s p o t t e d f e v e r TABLE I THE RATIOS OP ENGORGED TO UNFED WEIGHT FOR A WIDE VARIETY OF BLOOD-SUCKING ORGANISMS S p e c i e s R a t i o R e f e r e n c e ( H i r u d i n e a ) H l r u d o s p . H l r u d o raedlcinalls  Haemadipsa s p . ( B a t s ) Desmodus r o t u n d u s m a r l n u s ( D i p t e r a ) Aedes a e g y p t l A. t a e n i o r h y n c h u s A. t r l s e r i a t u s A r m l g e r e s s u b a l b a t u s C u l e x p l p i e n s f a t i g a n s A n o p h e l e s q u a d r l m a c u l a t u s ( H e m i p t e r a ) R h o d n i u s p r o l i x u s  T r l a t o m a i n f e s t a n s (3rd i n s t a r ) ( A c a r i ) O r n l t h o d o r o s moubata  B o o p h l l u s m l c r o p l u s  Hyalomma a s l a t l c u m  I x o d e s r i c i n u s  Hyalomma a n a t o l l c u m Dermacentor a n d e r s o n i 6 t o 10 2 t o 5 up t o 10 1.7 2 2 2.5 2 2.5 2 3 6.7 6 85 87 100 45 t o 50 75 D a l e s , (1967) Mann, (1962) Mann, (1962) Wimsatt & G u e r r i e r e , (I962) Gwadz, (1969) W i g g l e s w o r t h , (1943) G o o d c h l l d , (1955) Kaufman, ( p e r s o n a l c omra.) S e i f e r t e t a l . , (1968) B a l a s h o v , (1965) B a l a s h o v , (I965) Snow, (I969) P r e s e n t s t u d y 5 R i c k e t t s i a and (2) a p a r a l y t i c t o x i n produced by t h e t i s s u e s o f th e f e m a l e i t s e l f . B o t h a g e n t s f i n d t h e i r way i n t o t h e h o s t by way o f t h e s a l i v a r y g l a n d s . The s e r i o u s n e s s o f Rocky M o u n t a i n s p o t t e d f e v e r v a r i e s t r e m e n d o u s l y w i t h t h e l o c a t i o n o f the t i c k p o p u l a t i o n . On t h e west s i d e o f s l o p e s i n t h e B i t t e r o o t V a l l e y , Montana, t h e R i c k e t t s i a l s t r a i n i s e x t r e m e l y s e v e r e , c a u s i n g d e a t h i n about 6$% o f t h e c a s e s r e p o r t e d (Arthur» 1961). The t i c k s o f t h e e a s t e r n s l o p e s i n t h e same v a l l e y do n o t c a r r y t h i s v i r u l e n t s t r a i n o f t h e R i c k e t t s i a ( P h i l i p , 1959). N e i t h e r i s t h e s t r a i n dangerous I n B r i t i s h C o l u m b i a ( G r e g s o n , p e r s o n a l communi-c a t i o n ) . The p a r a l y s i s , however, can be severe i n v u l n e r a b l e s p e c i e s s u c h as sheep, c a t t l e and man, w i t h a m o r t a l i t y r a t e o f about 10$. The p o t e n c y o f t h e t o x i n a l s o v a r i e s f r o m one i n d i -v i d u a l f e m a l e t i c k t o a n o t h e r . C o n s e q u e n t l y , t h e d i s e a s e i s c o n d i t i o n e d by t h e c o m b i n a t i o n o f h o s t s u s c e p t i b i l i t y and t i c k v i r u l e n c e ( r e v i e w e d by A r t h u r , 1961). The a d u l t t i c k s o v e r - w i n t e r i n l o o s e r o c k and s o i l i n a s t a t e o f q u i e s c e n c e u n t i l e a r l y F e b r u a r y ? t h e i r peak of a c t i v i t y i s i n m i d - A p r i l and l a t e May. They t h e n " q u e s t " on t h e upper l e v e l s o f t a l l g r a s s and bushes u n t i l a s u i t a b l e h o s t b r u s h e s a g a i n s t them (Gregson, 1964). A f t e r a t t a c h m e n t t o the h o s t f e e d i n g p r o c e e d s f o r a number of d a y s . C o p u l a t i o n t a k e s p l a c e on the h o s t . The males r e q u i r e about f i v e days and t h e f e m a l e s one o r two days o f f e e d i n g , b e f o r e c o p u l a t i o n b e g i n s . The males d e t a c h f r o m the h o s t and f i n d t h e f e m a l e s w h i c h , t h r o u g h o u t t h e p r o c e s s o f m a t i n g , c o n t i n u e t o suck b l o o d . A f t e r the mated f e m a l e s a r e r e p l e t e ( s i x t o e i g h t days i n t h e w i l d ) , t h e y f a l l 6 t o t h e ground and w i t h i n s e v e r a l days b e g i n t o l a y 2000 - 4000 eggs ( A r t h u r , i960). The f e m a l e s even r e s o r b many o f t h e i r own t i s s u e s i n f a v o u r o f t h i s p r o c e s s . S e v e r a l weeks l a t e r , t h e sp e n t f e m a l e s w i t h e r and d i e (Gregson, 1966). L a r v a e h a t c h f r o m t h e eggs w i t h i n a few weeks and by e a r l y summer b e g i n q u e s t i n g f o r a h o s t on t h e s h o r t e r g r a s s e s . U s u a l l y t h e l a r v a l h o s t s a r e ground s q u i r r e l s and f i e l d m i c e . The l a r v a e engorge w i t h i n f o u r days o r s o , d r o p t o t h e ground and moult i n t o nymphs w i t h i n 14 d a y s . S i m i l a r l y , t h e nymphs f e e d on s m a l l mam-mals f o r s i x o r seven days and moult i n t o a d u l t s w i t h i n t h r e e weeks. The a d u l t s do n o t f e e d u n t i l t h e f o l l o w i n g s p r i n g ( W i l k i n s o n , 1968). 3) Water B a l a n c e i n T i c k s - L i t e r a t u r e Review The f i r s t m a j or c o n t r i b u t i o n t o o u r u n d e r s t a n d i n g o f w a t e r b a l a n c e i n b o t h a r g a s i d and i x o d i d t i c k s come f r o m t h e work o f Lees (1946a, 1947). He co n c e r n e d h i m s e l f m a i n l y w i t h exchange of w a t e r v i a t h e c u t i c u l a r s u r f a c e a t v a r i o u s e x p e r i m e n t a l tempera-t u r e s and r e l a t i v e h u m i d i t i e s . He n o t e d t h a t t h e p e r m e a b i l i t y o f t h e integument v a r i e d w i t h t h e s t a g e i n t h e l i f e c y c l e , and emphasized t h a t w a t e r exchange o c c u r r e d a l m o s t e x c l u s i v e l y t h r o u g h t h e c u t i c l e i t s e l f as opposed t o t h e s p i r a c l e s . L e e s ' comments (19^6a), on the w a t e r r e l a t i o n s of t h e e n g o r g i n g f e m a l e ( I x o d e s r i c i n u s ) a r e e s p e c i a l l y p e r t i n e n t t o t h i s s t u d y . He showed t h a t $0% of t h e w a t e r i m b i b e d i n t h e b l o o d meal was e l i m i n a t e d by t h e t i c k , and r e p o r t e d a t t h e same t i m e t h a t t h e p e r m e a b i l i t y o f t h e c u t i c l e t o w a t e r i n c r e a s e d 7 c o n s i d e r a b l y a t a c r i t i c a l t e m p e r a t u r e o f 3 2 ° C ( L e e s , 1 9 4 7 ) . The c r i t i c a l t e m p e r a t u r e ( o r t r a n s i t i o n t e m p e r a t u r e ) i s t h a t t e m p e r a t u r e above w h i c h t h e r a t e o f t r a n s p i r a t i o n s u d d e n l y i n c r e a s e s v e r y r a p i d l y ( W i g g l e s w o r t h , 1 9 6 5 ). Ramsay (1935) p o s t u l a t e d t h a t a t t h e c r i t i c a l t e m p e r a t u r e , t h e wax l a y e r of the c u t i c l e undergoes a change o f phase, so t h a t t h e m o l e c u l a r o r i e n t a t i o n o f t h e wax i s d i s t u r b e d ( d i s c u s s e d f u l l y by B a r t o n -Browne, 1 9 6 4 ) ; t h e p e r m e a b i l i t y o f the wax t o w a t e r i s t h e n r e d u c e d . W i g g l e s w o r t h (19^5) proposed t h a t t h e c r i t i c a l temper-a t u r e was r e l a t e d d i r e c t l y t o t h e h a r d n e s s o f the wax l a y e r . A m o b i l e " g r e a s e " such as c o v e r s t h e c u t i c l e o f t h e c o c k r o a c h has a low c r i t i c a l t e m p e r a t u r e , b u t a h a r d wax ( e . g . i n Rhodnlus) p r o t e c t e d f r o m a b r a s i o n by means o f t h e o v e r l y i n g "cement" has a h i g h c r i t i c a l t e m p e r a t u r e ( o f t e n above l e t h a l t e m p e r a t u r e ) . F u r t h e r m o r e , t h e w a t e r p r o o f i n g c h a r a c t e r i s t i c o f the c u t i c l e i s in d e p e n d e n t o f the t h i c k n e s s o r degree of s c l e r o t i z a t l o n o f t h e c u t i c l e , b u t i s r e l a t e d t o t h e e f f i c a c y of t h e wax l a y e r a l o n e . C l e a r l y , t h e c r i t i c a l t e m p e r a t u r e i s an i n d e x o f the organism's c a p a b i l i t y f o r w i t h s t a n d i n g d e s s l c a t i o n . R e a l i z i n g t h a t the c r i t i c a l t e m p e r a t u r e f o r t h e c u t i c l e o f Ixodes r i c i n u s was 3 2 ° C, and c o n s i d e r i n g t h a t t h e t i c k s were f e e d i n g on the warm s k i n of sheep, Lees c o n c l u d e d t h a t t h e l o s s o f w a t e r f r o m t h e t i c k was p r o b a b l y c o n s i d e r a b l y i n c r e a s e d w h i l e the t i c k was on t h e h o s t . The enigma i n h i s h y p o t h e s i s a r o s e f r o m the r e s u l t s of h i s o t h e r e x p e r i m e n t s w h i c h showed t h a t t i c k s w h i c h f e d i n s a t u r a t e d o r n e a r - s a t u r a t e d micro,environments were a b l e t o engorge n o r m a l l y and r e t a i n e d t h e same amount o f w a t e r i n t h e i r t i s s u e s as d i d 8 t i c k s f e d i n l o w e r r e l a t i v e h u m i d i t i e s , even though l o s s by-e v a p o r a t i o n must have been v i r t u a l l y e l i m i n a t e d . Lees c o n c l u d e d t h a t under t h e s e c o n d i t i o n s , t h e M a l p i g h i a n t u b u l e s were p o s s i b l y e x c r e t i n g w a t e r w h i c h would o t h e r w i s e have been l o s t by e v a p o r a -t i o n . A f u r t h e r o b j e c t i o n t o t h i s h y p o t h e s i s r e s u l t s f r o m e x a m i n i n g t h e c r i t i c a l t e m p e r a t u r e o f o t h e r i x o d i d t i c k s . Among the 11 s p e c i e s ( i n c l u d i n g Dermacentor a n d e r s o n i ) t h a t Lees s t u d i e d , I x o d e s r l c l n u s was t h e l e a s t r e s i s t a n t t o w a t e r l o s s by e v a p o r a t i o n . The t e n o t h e r s p e c i e s showed c r i t i c a l t e m p e r a t u r e s f r o m 39° C t o 45° C ( L e e s , 194-7). A t 0% RH, t h e c u t i c l e o f dead engorged t i c k s o f t h e s e s p e c i e s l o s t w a t e r o n l y v e r y s l o w l y below 40 C ( l e s s t h a n 1 mg/cm / h o u r ) . D a v i e s and Hobson (1935) e s t i m a t e d t h e t e m p e r a t u r e and r e l a t i v e h u m i d i t y of a s h e e p 1 s s u r f a c e a t t h e base o f t h e woo l t o be about 37° C and 40-80$ RH. L i v e t i c k s on a sheep would o b v i o u s l y l o s e w a t e r even more s l o w l y t h a n dead ones a t 0% RH, n o t o n l y because t h e g r a d i e n t f o r e v a p o r a t i o n w o u l d be l o w e r , b u t because under any g i v e n c o n d i t i o n s l i v e t i c k s c an a b s o r b m o i s t u r e f r o m t h e atmosphere and t h u s , t o v a r y i n g d e g r e e s , c o u n t e r a c t w a t e r l o s s by e v a p o r a -t i o n . The s u r f a c e a r e a o f an engorged Dermacentor a n d e r s o n l f e m a l e , f o r example, i s a t most 4 cm . A t 37 C and 5 0 $ RH, Ixo d e s r l c l n u s l o s t w a t e r a t o n e - h a l f t o o n e - q u a r t e r t h e r a t e a t 0% RH ( L e e s , 1946). I f we assume t h a t Dermacentor a l s o l o s e s w a t e r more s l o w l y a t 50% RH t h a n a t 0% RH, we can e s t i m a t e r o u g h l y f r o m L e e s ' d a t a (1947) t h a t D. a n d e r s o n l would l o s e 2 w a t e r a t t h e r a t e 0.1 mg/cm / h o u r . A t i c k f e e d i n g f o r t e n days and l o s i n g w a t e r a t t h i s r a t e , c o u l d o n l y e v a p o r a t e a maximum of 9 9 6 mg v i a the Integument. T h i s i s o n l y o n e - t h i r d t h e t o t a l amount o f w a t e r t h a t I x o d e s r i c i n u s was e s t i m a t e d t o l o s e d u r i n g f e e d i n g ( L e e s , 1 9 ^ 6 a ) . S i n c e a t r e p l e t i o n , D. a n d e r s o n i i s about t h r e e t i m e s t h e s i z e o f Ixodes r i c i n u s , i t becomes i n c r e a s -i n g l y d i f f i c u l t t o p o s t u l a t e t h e integument as t h e r o u t e f o r e x c r e t i n g t h e t o t a l amount o f e x c e s s w a t e r f o r Dermacentor and most i x o d i d t i c k s , w i t h t h e p o s s i b l e e x c e p t i o n o f Ixodes r i c i n u s . N e v e r t h e l e s s , t h i s i n t e r p r e t a t i o n r e m a i n e d u n c h a l l e n g e d u n t i l G r e g s o n ( 1 9 6 7 ) 1 d i r e c t l y o b s e r v i n g the f r e q u e n t d i s c h a r g i n g o f s a l i v a by D. a n d e r s o n i f e e d i n g on e v e r t e d hamster cheek pouches, p r o p o s e d t h a t t h e e x c e s s f l u i d of t h e b l o o d meal might be i n j e c t e d back i n t o t h e h o s t v i a t h e s a l i v a r y g l a n d s . A l s o c o n t r a r y t o L e e s ' i n t e r p r e t a t i o n , B e l o z e r o v ( 1 9 6 7 ) n o t e d t h a t d u r i n g t h e f i n a l h o u r s o f f e e d i n g (when t h e g r e a t e s t p o r t i o n o f the b l o o d meal i s imbibed) t h e p e r m e a b i l i t y o f t h e integument i s r e d u c e d i n two i x o d i d s p e c i e s (I,, r i c i n u s , and D. m a r g l n a t u s ) a t a g i v e n t e m p e r a t u r e . I t s h o u l d be p o i n t e d o u t t h a t B e l o z e r o v ' s r e s u l t s do n o t c o n t r a d i c t t h o s e o f L e e s , f o r t h e s e two a u t h o r s were m e a s u r i n g d i f f e r e n t p a r a m e t e r s . Lees o b s e r v e d t h e perme-a b i l i t y changes of t i c k s ( a t t h e same s t a g e o f f e e d i n g ) b r o u g h t on by t e m p e r a t u r e i n c r e a s e , whereas B e l o z e r o v c o n s i d e r e d changes i n t h e c u t i c l e t h a t a r e e v i d e n t a t any g i v e n t e m p e r a t u r e b u t a t d i f f e r e n t s t a g e s o f t h e f e e d i n g c y c l e . B e l o z e r o v a l s o s t a t e s t h a t a t t h e same ti m e as t h e p e r m e a b i l i t y of t h e c u t i c l e i s l o w e r e d , t h e b l o o d meal i s made more c o n c e n t r a t e d i n t h e g u t , presumably by s e l e c t i v e e l i m i n a t i o n of w a t e r . C o n s e q u e n t l y , B e l o z e r o v c o u l d n o t p o s t u l a t e t h a t w a t e r was l e a v i n g v i a t h e 10 i n t e g u m e n t . He a l s o s u g g e s t e d , on i n d i r e c t e v i d e n c e ( t h e a b i l i t y t o c o l l e c t r e l a t i v e l y l a r g e volumes of s a l i v a f r o m a c t i v e l y f e e d i n g t i c k s , and t h e i n c r e a s e of s a l i v a r y g l a n d s i z e d u r i n g the f e e d i n g c y c l e ) , t h a t t h e s a l i v a r y g l a n d s were r e s p o n s i b l e . There was precedence f o r t h i s h y p o t h e s i s f r o m h i s work on b l o o d - s u c k i n g gamasid m i t e s w h i c h e x c r e t e e x c e s s w a t e r f r o m t h e s a l i v a r y g l a n d s a f t e r f e e d i n g ( B e l o z e r o v , 1 9 5 8 ) . B e l o z e r o v c o n c l u d e d t h a t i n the t i c k , a t t h e b e g i n n i n g o f f e e d i n g , w a t e r i s e x c r e t e d by i n t e n s i v e e v a p o r a t i o n t h r o u g h a h i g h l y permeable Integument; g r a d u a l l y t h i s p r o c e s s i s p r o b a b l y r e p l a c e d by a c t i v e s a l i v a t i o n j u s t b e f o r e the c o m p l e t i o n o f engorgement. T a t c h e l l ( 1 9 6 7 b ) , w o r k i n g w i t h B o o p h i l u s m l c r o p l u s . the A u s t r a l i a n c a t t l e t i c k , p r o v i d e d d i r e c t e v i d e n c e t h a t f l u i d f r o m t h e f e e d i n g f e m a l e was e n t e r i n g the h o s t . He i n j e c t e d t r i t i a t e d w a t e r i n t o e i g h t a c t i v e l y f e e d i n g t i c k s and t r i e d t o t r a c e the f a t e o f t h i s w a t e r 17 h o u r s l a t e r . He r e c o v e r e d 75% o f t h e t o t a l r a d i o a c t i v i t y i n j e c t e d . Of t h i s amount, was f o u n d i n the h o s t b l o o d and u r i n e ; t h e r e s t was r e c o v e r e d f r o m the t h r e e engorged t i c k s ( 2 . 9 $ ) 1 t h e f i v e t i c k s w hich had n o t dropped o f f by t h i s t i m e ( 5 5 * 8 $ ) and exudate f r o m i n j e c t i o n p u n c t u r e s (6,9%)* He made no a t t e m p t t o c o l l e c t w a t e r e v a p o r a t e d f r o m t h e t i c k s , h o s t , o r f e c e s o f t h e h o s t . However f r o m o t h e r work, he was a b l e t o c a l c u l a t e t h a t o f t h e 2 0 0 pl o f wat e r e x c r e t e d by a 250 mg t i c k , none c o u l d have pass e d out o f t h e anus o f t h e t i c k ( S e i f e r t e t a l . , 1 9 6 8 ) and a maximum of 1 0 - 1 5 yil c o u l d have passed t h r o u g h t h e c u t i c l e ( T a t c h e l l , 1 9 6 7 b ) . I t s h o u l d be n o t i c e d t h a t i n a d d i t i o n t o t h e c o n s i d e r a b l e r e c o v e r y f r o m t h e h o s t f l u i d s , t h e 11 a v e r a g e a c t i v i t y f r o m each o f t h e unengorged t i c k s (11.2$ of the t o t a l "^2°) w a s 1 1 t l m e s t n e a v e r a g e a c t i v i t y f r o m each o f the engorged t i c k s (0.96$ o f t h e t o t a l % 2 0 ) . Thus one can c o r r e -l a t e t h e l o s s o f w a t e r f r o m t h e r e c e n t l y engorged t i c k s w i t h the appearance o f i t i n t h e body f l u i d s o f t h e h o s t ( T a t c h e l l , 1969). As f a r as I am aware, i x o d i d t i c k s and gamasid m i t e s a r e th e o n l y organisms f o r w h i c h t h e r e i s r e a s o n a b l y s t r o n g e v i d e n c e i m p l i c a t i n g t h e s a l i v a r y g l a n d as t h e major o s m o r e g u l a t o r y o r g a n . A t h i r d p o s s i b l e case i s t h e s a t u r n l i d moth A n t h e r a e a p e r n y l ( K a f a t o s , 1968), whose l a b i a l g l a n d may produce i n v i v o o v e r 200 u l o f f l u i d i n two ho u r s ( o r 20$ o f t h e e s t i m a t e d b l o o d v o l u m e ) . The l a b i a l g l a n d s a r e homologous w i t h i n s e c t s a l i v a r y g l a n d s . K a f a t o s however r e j e c t s , i n t h i s c a s e , t h e h y p o t h e s i s advanced by Edwards (1964) f o r H y a l o p h o r a t h a t t h e a d u l t g l a n d may be i n v o l v e d i n t h e r e g u l a t i o n of w a t e r b a l a n c e ; f o r t h e a c t of s e c r e t i o n i n Antheraea does n o t m o d i f y the o s m o t i c p r e s s u r e of t h e hemolymph, and c a n b r i n g about o n l y minor changes i n i o n i c c o m p o s i t i o n . G i v e n t h e t o t a l volume o f s e c r e t i o n , t h e i o n s w h i c h do n o t pass i n t o t h e l a b i a l g l a n d (Na , Mg , Ca , P0^~) c o u l d be c o n c e n t r a t e d i n t h e hemolymph o n l y by 10$. P o t a s s i u m , w h i c h i s h i g h i n l a b i a l f l u i d , c o u l d be d e p r e s s e d i n t h e hemolymph by 20 t o 30$. However t h e volumes o f f l u i d n e c e s s a r y t o b r i n g about t h e s e changes i n hemolymph c o m p o s i t i o n a r e n e v e r s p o n t a n e o u s l y r e l e a s e d by t h e no r m a l moth, e x c e p t d u r i n g escape f r o m t h e cocoon. K a f a t o s b e l i e v e s t h a t under n a t u r a l c i r c u m s t a n c e s , t h e l a b i a l g l a n d i s c o n c e r n e d o n l y w i t h escape from the cocoon - p r o d u c i n g a f l u i d w h i c h s e r v e s as t h e s o l v e n t and b u f f e r f o r the enzyme, 12 c o c o o n a s e . I t I s of c o n s i d e r a b l e i n t e r e s t t h a t the s o f t t i c k s employ a t o t a l l y d i f f e r e n t mechanism f o r o s m o r e g u l a t i o n . I n t h e a r g a -s i d s , t h e o r i f i c e s o f two c o x a l g l a n d s (between t h e f i r s t and s econd coxae) exude a c l e a r f l u i d d u r i n g and a f t e r f e e d i n g . Kaufman ( 1 9 7 1 ) drew up a b a l a n c e s h e e t of i n t a k e and e x c r e t i o n f o r O r n l t h o d o r o s moubata and d e m o n s t r a t e d t h a t t h e volume and i o n c o n c e n t r a t i o n s o f t h e c o x a l f l u i d w o u l d a c c o u n t f o r t h e t o t a l amounts of w a t e r and s a l t s e x c r e t e d . I t had been shown e a r l i e r t h a t no w a t e r was e v a p o r a t e d f r o m t h e integument and no d e t e c t a b l e c h l o r i d e was e x c r e t e d i n M a l p i g h i a n t u b u l a r f l u i d ( L e e s , 1 9 4 6 b ) . The c o x a l g l a n d i s n o t c a p a b l e o f p r o d u c i n g a f l u i d h y p e r o s m o t i c o r h y p e r l o n i c t o t h e hemolymph. However, i f f l u i d e n t e r i n g t h e hemolymph f r o m th e g u t i s i s o - o s m o t i c o r h y p o - o s n m t i c t o t h e hemolymph, t h e c o x a l g l a n d i s c a p a b l e of r e s t o r i n g t h e hemolymph t o a c o n s t a n t c o n c e n t r a t i o n (Kaufman, 1 9 7 1 ) • The c o x a l g l a n d p r oduces i t s f l u i d by a mechanism a t l e a s t somewhat a n a l o g o u s t o t h a t of t h e c r a y f i s h a n t e n n a l g l a n d ( K l r s c h n e r and Wagner, 1 9 6 5 ) and t h e v e r t e b r a t e g l o m e r u l a r k i d n e y ( P i t t s , 1 9 6 3 ) , t h a t i s , by u l t r a f i l t r a t i o n and s e l e c t i v e r e a b s o r p t i o n . 4 ) S t a t e m e n t of t h e P r o b l e m T a t c h e l l was t h e f i r s t t o p r e s e n t c o n c r e t e e v i d e n c e t h a t t h e s a l i v a r y g l a n d s p l a y a r o l e i n the w a t e r r e g u l a t i o n o f an i x o d i d t i c k . Even though h i s s t u d i e s were prompted by t h e o b s e r -v a t i o n s o f G r e g s o n on Dermacentor, i t was n o t i m m e d i a t e l y evident that the same process was occurring i n D. andersoni. In the f i r s t place, D. andersoni voids a considerable amount of feces (undigested host blood i n the main) during the feeding period, whereas Boophllus does not ( S e i f e r t et a l . , 1968). Gregson suggested (personal communication) that because the drops from the anus s o l i d i f i e d within seconds of emerging, they were l i k e l y to contain l i t t l e water; t h i s however remained to be shown q u a n t i t a t i v e l y . Since Involvement of the s a l i v a r y glands i n f l u i d excre-t i o n has been c l e a r l y implicated i n only one species of t i c k ( T a t c h e l l , 1967b), and since t h i s f i n d i n g was possibly i n d i s -agreement with previous suggestions concerning the route of water loss (Lees, 1946a), examination of another species of t i c k was i n order. Furthermore, although Lees (19^7) showed that D. andersoni i s quite r e s i s t e n t to water loss from the c u t i c l e before feeding, and less so a f t e r engorgement, he d i d not follow the intermediate stages and d i d not examine water loss under conditions l i k e l y to p r e v a i l on the natural host. Consequently I wished to extend h i s observations on integu-mentary water loss to Individuals on the host at a l l stages of feeding. Moreover, given the f a c t that the s a l i v a r y glands are active during feeding, no author to date had examined the phys-i o l o g i c a l mechanisms governing the transport of water and ions by the s a l i v a r y glands of t i c k s . For instance, i t was not known whether f l u i d was transported by a f i l t r a t i o n mechanism as In the vertebrate glomerular kidney, the crustacean antennal gland, and the a r g a s i d - t i c k coxal gland, or by a secretory mechanism as in insect Malpighian tubules and mammalian salivary glands. In light of the above, i t was decided f i r s t to quantify as far as possible the amount of water excreted under normal conditions via the three most l i k e l y routes available, namely the integument (including spiracles), the anus (including Malpighian tubule secretion) and the salivary glands. This is considered in Chapter Two. When the salivary glands were shown to be pre-eminent in water excretion during feeding, the mech-anism of secretion by the salivary glands was considered (Chap-ter Three). Finally, I attempted to relate the physiological findings concerning secretion to the ultrastrueture of the salivary gland (Chapter Four) in order to develop working models of the secretory mechanism. . CHAPTER TWO ROUTES OF WATER AND ION LOSS 15 CHAPTER TWO I n t h i s C h a p t e r I s h a l l a t t e m p t t o demonstrate q u a n t i -t a t i v e l y t h e i m p o r t a n c e of t h e s a l i v a r y g l a n d s i n w a t e r b a l a n c e r e l a t i v e t o t h e o t h e r p o t e n t i a l r o u t e s of e x c r e t i o n . I n o r d e r t o do t h i s , I f i r s t had t o a s s e s s t h e t o t a l q u a n t i t y of f l u i d removed f r o m t h e h o s t . S i n c e i n many o t h e r b l o o d - s u c k e r s ( e . g . R h o dnlus) e x c r e t i o n f rom t h e body does not commence u n t i l a f t e r f e e d i n g , the i n c r e a s e i n body w e i g h t a s a r e s u l t of c o m p l e t i o n o f t h e meal may be t a k e n as t h e t o t a l amount o f f l u i d i m b i b e d . However i n Dermacentor, f l u i d s e c r e t i o n o c c u r s w h i l e t h e t i c k i s s t i l l a t t a c h e d t o t h e h o s t , and s o , t h e n e t w e i g h t i n c r e a s e of t h e t i c k a c c o u n t s f o r o n l y a p o r t i o n o f the meal i m b i b e d . I n t h i s case one had t o measure some parame-t e r o t h e r t h a n w e i g h t i n c r e a s e t o e s t i m a t e t h e t o t a l f l u i d i n t a k e . Moreover, s i n c e i t was a n t i c i p a t e d (see C h a p t e r One) t h a t an u n d e t e r m i n e d q u a n t i t y o f s a l i v a i s s e c r e t e d i n t o t h e h o s t , t h e parameter chosen as t h e i n d e x of f l u i d i n t a k e s h o u l d be i n d e p e n d e n t of t h e a c t o f s a l i v a t i o n . I t was t h e r e f o r e d e c i d e d t h a t t h e amount of hemo g l o b i n i m b i b e d would s e r v e t o t r a c e f l u i d i n t a k e , s i n c e i t i s a s u b s t a n c e o c c u r r i n g e x c l u s i v e l y i n t h e meal, i t i s n o t s e c r e t e d by t h e s a l i v a r y g l a n d s , and i t i s r e l a t i v e l y e asy t o a s s a y . A l l t h e hemo g l o b i n t h a t i s removed from t h e h o s t r e m a i n s i n t h e t i c k , o r pa s s e s out o f t h e anus. Knowing t h e v a l u e s f o r t o t a l amount o f hemo g l o b i n i n t h e t i c k and f e c e s , and the hemoglobin concentration in host blood, one can calculate the volume of host blood removed by the tick. The total amount of f l u i d excreted must also be known before one can appreciate the roles played by the various excretory routes available to the tick. Total water loss can be calcualted from the formulai Wt - M - < G + F d r y ) 1) where W^. is the total amount of excreted f l u i d in mg, during feeding, M i s the total amount of meal imbibed in mg, G is the net weight increase of the tick in mg, during feeding, and ^clry *'"8 ^ e c*r^r w e * S h t °^ feces excreted by the tick in mg during feeding. With some Idea of the total quantity of f l u i d lost by the tick, some method had to be devised to measure the routes of water loss, which of course was the ultimate objective of this study. The excreted saliva cannot be collected directly since i t Is injected back into the host. (Indeed, this is probably the main reason that salivation was not suspected earlier as a means of osmoregulation). It was decided therefore to f i r s t estimate water loss via other routes (integument and anus) so that the amount of f l u i d secreted in the saliva could be calcu-lated from the formula i W s=M- ( G + W 1 + F w e t ) 2) where W i s the f l u i d excreted via the salivary glands i s t h e w a t e r l o s s v i a t h e Integument and F W e t * s t h e w e t w e l S h - t o f t n e f e c e s . A l t h o u g h t h e I n t e g u m e n t a r y w a t e r l o s s c o u l d be measured d i r e c t l y , a p r a c t i c a b l e method f o r d i r e c t l y e s t i m a t i n g t h e wet w e i g h t of t h e f e c e s c o u l d n ot be d e v e l o p e d . However an i n d i r e c t method based on i o n d e t e r m i n a t i o n ( r a t h e r t h a n w a t e r d e t e r m i n a t i o n ) was e v e n t u a l l y chosen f o r e l u c i d a t i n g s a l i v a r y and a n a l w a t e r l o s s e s . T h i s w i l l be d e s c r i b e d i n d e t a i l l a t e r . A l t h o u g h an e s t i m a t e o f t h e r e l a t i v e i m p o r t a n c e o f each r o u t e f o r w a t e r e x c r e t i o n was now a v a i l a b l e , i t d i d n o t f o l l o w t h a t e x c e s s i o n s were b e i n g e x c r e t e d v i a each r o u t e i n t h e same p r o p o r t i o n as was w a t e r . I n a d d i t i o n , I hoped t o g a i n some i n s i g h t as t o how osmo- and i o n i c r e g u l a t i o n a r e a c h i e v e d i n t h i s s p e c i e s . To do t h i s , t h e c o n c e n t r a t i o n s o f o s m o t i c a l l y i m p o r t a n t s u b s t a n c e s i n t h e v a r i o u s f l u i d compartments of t h e t i c k must be known. P o r t h e s e r e a s o n s , the c o n c e n t r a t i o n s o f sodium, p o t a s s i u m , c h l o r i d e , and o s m o t i c p r e s s u r e i n t h e hemo-lymph and s a l i v a o f t i c k s were m o n i t o r e d t h r o u g h o u t t h e f e e d i n g p e r i o d . A few d e t e r m i n a t i o n s o f magnesium, phosphate, and g l u -cose were a l s o made. U n f o r t u n a t e l y g u t c o n t e n t s were n o t exam-i n e d s i n c e t h e y were e x t r e m e l y v i s c o u s and i t was d i f f i c u l t t o o b t a i n samples t h a t were n o t c o n t a m i n a t e d w i t h hemolymph. However, t h e c o n c e n t r a t i o n s of sodium and p o t a s s i u m i n t h e f e c e s were measured on a d r y w e i g h t b a s i s . S i n c e i n g e s t i o n o f an enormous meal i m p l i e d t h a t the t i c k was c h a l l e n g e d t o h a n d l e a l a r g e q u a n t i t y of e x c e s s f l u i d , i t was of i n t e r e s t t o see w h e t h e r , i n s p i t e of t h i s l o a d , r e g u l a t i o n 18 o f hemolymph f l u i d volume o c c u r r e d . I t was c o n c e i v a b l e t h a t t h e i n i t i a t i o n of s a l i v a t i o n might be c o r r e l a t e d w i t h a u n i q u e hemolymph volume t h a t r emained s t a b l e f o r t h e r e m a i n d e r of t h e f e e d i n g p e r i o d . Such a c o r r e l a t i o n w ould i m p l y t h a t hemolymph volume o r p r e s s u r e may be t r i g g e r i n g e v e n t s l e a d i n g t o s a l i v a -t i o n . W i t h t h e s e t h o u g h t s i n mind I measured t h e change i n hemolymph volume w i t h t h e p r o g r e s s i o n of f e e d i n g . F i n a l l y , s i n c e e x c r e t o r y organs u s u a l l y f u n c t i o n on one o f two g e n e r a l p r i n c i p l e s - namely a c t i v e s e c r e t i o n o r f i l t r a -t i o n - r e s o r p t i o n ( K i r s c h n e r , 1967) * hoped t o d e c i d e i n f a v o u r o f one o f t h e s e two mechanisms f o r t h e s a l i v a r y g l a n d . One d i s t i n g u i s h i n g f e a t u r e between t h e s e mechanisms i s t h e d i f f e r e n t manner i n w h i c h t h e y h a n d l e m o l e c u l e s of graded s i z e . Organs w h i c h i n i t i a t e e x c r e t i o n by a f i l t r a t i o n p r o c e s s w i l l a l l o w a l l m o l e c u l e s below a c r i t i c a l s i z e , r e g a r d l e s s o f t h e i r c h e m i c a l p r o p e r t i e s , t o pass i n t o t h e p r i m a r y u l t r a f i l t r a t e . V a l u a b l e s u b s t a n c e s so p a s s e d a r e u s u a l l y a b s o r b e d back i n t o the b l o o d o r hemolymph f u r t h e r a l o n g t h e e x c r e t o r y d u c t s , t h u s r e s u l t i n g o n l y I n t h e e x c r e t i o n o f u n d e s l r e d s u b s t a n c e s . F o r i n s t a n c e , t h e a v e r a g e pore d i a m e t e r i n t h e f i l t r a t i o n membrane of t h e v e r t e b r a t e g l o m e r u l a r k i d n e y i s i n t h e o r d e r o f 75-100 A. I n u l i n , w i t h a d i a m e t e r of about 29 A, passes t h r o u g h t h e f i l t r a t i o n membrane, and because i t i s n e i t h e r s e c r e t e d n o r r e s o r b e d f u r t h e r down the d u c t s y s t e m , i t appears i n t h e u r i n e i n t h e same c o n c e n t r a t i o n as i n t h e b l o o d . L i k e w i s e , e x c r e t i o n of i n u l i n i s n o t r e s t r i c t e d by t h e a n t e n n a l g l a n d o f t h e c r a y -f i s h ( K i r s c h n e r and Wagner, 19^5)» n o r ^y t n e c o x a l g l a n d s o f 19 a r g a s i d t i c k s (Kaufman, 1 9 7 1 )t "both of w h i c h f u n c t i o n by f i l t r a -t i o n . S e c r e t o r y systems however u s u a l l y show much g r e a t e r s p e c i f i c i t y as t o w h i c h m o l e c u l e s w i l l be a l l o w e d t o pass - a s p e c i f i c i t y n o t n e c e s s a r i l y c o r r e l a t e d w i t h m o l e c u l a r s i z e per s e , b u t r a t h e r w i t h s t e r i c c o n f o r m a t i o n o r p r e s e n c e of s p e c i f i c r a d i c a l s . F o r a s e c r e t o r y o r gan t o cause w a t e r f l o w , i t must be a b l e t o f i r s t c r e a t e a f a v o u r a b l e o s m o t i c g r a d i e n t , and s o , r e l a t i v e t o a t y p i c a l f i l t r a t i o n membrane, i t must be l e s s permeable t o t h e m a j o r components of t h e s e c r e t e d f l u i d . S i n c e i n most s e c r e t o r y t i s s u e s , the s o l u t e s r e s p o n s i b l e f o r the o s m o t i c + + — g r a d i e n t a r e s m a l l i o n s ( e . g . K , Na , C l e t c . ) , t h e r e q u i r e d low p e r m e a b i l i t y t o t h e l a t t e r u s u a l l y p r e c l u d e s t h e r a p i d movement o f l a r g e r s o l u t e m o l e c u l e s ( e . g . i n u l l n ) a c r o s s t h e membrane. F o r example, t h e M a l p i g h i a n t u b u l e s of D l x l p p u s . w h i c h have been shown t o o p e r a t e by a s e c r e t o r y mechanism (Bamsay, 195*0 » "by and l a r g e do n o t p e r m i t i n u l l n t o pass I n t o t h e u r i n e (U/P r a t i o o f 0 . 0 5 } Ramsay and R i e g e l , 1 9 6 1 ) . The M a l p i g h i a n t u b u l e s o f C a l l l p h o r a s i m i l a r l y e x c l u d e most a n i o n s w h i c h have a h y d r a t e d r a d i u s g r e a t e r t h a n 3.6 A, w i t h the n o t a b l e e x c e p t i o n o f phosphate ( r a d i u s 4 A ) , w h i c h i s t r a n s p o r t e d by a s p e c i f i c c a r r i e r I n t h e membrane ( B e r r i d g e , 1 9 6 9 ) . I t was w i t h t h i s i n mind t h a t I t e s t e d t h e a b i l i t y of t h e s a l i v a r y g l a n d t o e x c r e t e two r e l a t i v e l y l a r g e , non-charged m o l e c u l e s : i n u l l n (MW = about 5 0 0 0 ) and 3 - 0 - m e t h y l g l u c o s e (MW = 1 9 4 ) . MATERIALS AND METHODS 1) R e a r i n g Methods The t i c k s used i n t h i s s t u d y came f r o m a c u l t u r e e s t a b -l i s h e d i n t h i s l a b o r a t o r y f rom w i l d t i c k s k i n d l y p r o v i d e d by Mr. J . D. G r e g s o n , D i r e c t o r of t h e T i c k R e s e a r c h S t a t i o n , CD.A., Kamloops, B r i t i s h C o l u m b i a . a) L a r v a e A t l e a s t two weeks a f t e r h a t c h i n g of t h e eggs, t h e l a r v a e were c o n f i n e d t o t h e shaven e a r and s c a l p r e g i o n of a r a b b i t by means of a c l o t h sac t a p e d t o t h e head. The j u n c t i o n o f tape and s k i n was s e a l e d w i t h " P l a s t l e a s t " ( K r i p p s , V a n c o u v e r ) , a l a t e x p r e p a r a t i o n w h i c h p o l y m e r i z e s } t h e l a t e x h e l p s p r e v e n t the escape o f l o o s e t i c k s f r o m t h e s a c . Engorged l a r v a e were removed f o u r o r f i v e days l a t e r . These m o u l t e d i n t o nymphs i n two weeks when k e p t a t room t e m p e r a t u r e o v e r s a t u r a t e d KNO^ (RH = 9Q%). b) Nymphs An o v o i d a r e n a o f foam r u b b e r 2 cm h i g h was g l u e d t o t h e shaven back of a r a b b i t by means o f " P l a s t i c a s t " . A p i e c e o f c l o t h was g l u e d o v e r t h e r u b b e r , t h u s f o r m i n g an e n c l o s e d a r e a on t h e back o f t h e r a b b i t i n w h i c h t h e nymphs were c o n f i n e d . S i x o r seven days l a t e r , t h e engorged nymphs were removed and s t o r e d o v e r s a t u r a t e d N a C l (RH = 88$) u n t i l t h e y m o u l t e d i n t o a d u l t s t h r e e t o f o u r weeks l a t e r . 21 c) A d u l t s The newly m o u l t e d a d u l t s were s t o r e d o v e r s a t u r a t e d N a C l f o r one month, and t h e n were t r a n s f e r r e d t o c l e a n v i a l s . They were s u b s e q u e n t l y k e p t o v e r s a t u r a t e d KNO^ a t 5 ° C f o r a t l e a s t t h r e e months, b u t u s u a l l y t h r e e t o s i x months. A few days p r i o r t o t h e a d u l t f e e d , the t i c k s were r e t u r n e d t o room t e m p e r a t u r e and k e p t o v e r s a t u r a t e d N a C l . Only r a b b i t s w h i c h had n e v e r been i n f e s t e d w i t h t i c k s were used f o r f e e d i n g a d u l t s , because each s u c c e s s i v e b a t c h o f t i c k s becomes more r e l u c t a n t t o f e e d on a r a b b i t w h i c h has been used t i m e and a g a i n (Loomis, 1 9 6 l j R o b e r t s , 1 9 6 8 ) . Engorged males were d i s c a r d e d . Those engorged f e m a l e s n o t used i n t h e e x p e r i m e n t s were p l a c e d each i n a s e p a r a t e v i a l where t h e y began d e p o s i t i n g t h e i r eggs s e v e r a l days l a t e r ; egg l a y i n g c o n t i n u e d f o r s e v e r a l weeks. A c c o r d i n g t o t h e s e r e a r i n g p r o c e d u r e s t h e minimum g e n e r a -t i o n t i m e was about 30 weeks. A l l s t a g e s of t i c k s were k e p t i n g l a s s v i a l s s e a l e d w i t h c o r k s o r r u b b e r bungs; h o l e s i n t h e s t o p p e r s a l l o w e d gaseous exchange, and f i n e c l o t h s p r e v e n t e d t h e escape o f t i c k s t h r o u g h t h e h o l e s . A b s o r b a n t f i l t e r paper s t r i p s were p r o v i d e d t o i n c r e a s e t h e s u b s t r a t u m and t o a b s o r b t h e f e c e s . When f e e d i n g a l l s t a g e s o f t i c k s , a c a r d b o a r d c o l l a r was p l a c e d a r o u n d t h e neck o f the r a b b i t t o p r e v e n t i t f r o m d i s t u r b -i n g t h e t i c k e n c l o s u r e . I n a d d i t i o n , t h e r a b b i t cages were f i x e d t o w i r e frames around w h i c h were d r a p e d c l o t h " t e n t s " t o p r e v e n t t h e s c a t t e r i n g i n t o t h e room o f any t i c k s w h i c h by chance might have escaped f r o m the r a b b i t . 22 2) Net Weight I n c r e a s e o f the Females A p p r o x i m a t e l y 100 f e m a l e s , I n a known w e i g h t r a n g e , and 40 males were removed from c o l d s t o r a g e (5° C) and were p l a c e d on t h e back o f a r a b b i t . Each day d u r i n g subsequent f e e d i n g a random sample of t e n f e m a l e s was removed. Each t i c k was r i n s e d c l e a n w i t h t a p w a t e r , d r i e d and t h e n weighed. The t i c k s were t h e n used i n subsequent e x p e r i m e n t s . T h i s p r o c e d u r e was r e p e a t e d d u r i n g s e v e r a l such f e e d i n g s . 3) Water L o s s Through t h e Integument T i c k s were a l l o w e d t o commence f e e d i n g and a t d a i l y i n t e r v a l s a few were removed f r o m t h e r a b b i t . The a n a l p l a t e s and m o u t h p a r t s were p l u g g e d w i t h a m i x t u r e o f beeswax and r e s i n ( " t a c k y wax"). The t i c k s were weighed, r e t u r n e d t o t h e r a b b i t , and r e w i g h e d a t 24-hour i n t e r v a l s , u s u a l l y f o r 48 h o u r s , b u t o c c a s s i o n a l l y f o r up t o 96 h o u r s . These c o n d i t i o n s were v i r t u a l l y i d e n t i c a l i n terms o f r e l a t i v e h u m i d i t y (RH) and t e m p e r a t u r e t o t h o s e e x p e r i e n c e d by f e e d i n g t i c k s , but i n t a k e o f b l o o d and e x c r e t i o n o f f e c e s were p r e v e n t e d by the wax p l u g s . R a t e o f i n t e g u m e n t a r y w a t e r l o s s was t a k e n as t h e r a t e of w e i g h t l o s s o v e r t h e f i r s t two days f o l l o w i n g f o r c e d d e t a c h -ment f r o m t h e h o s t . A method s i m i l a r t o t h a t of Lees was used t o measure s u r f a c e a r e a o f t h e t i c k s . The t i c k s were e v i s c e r -a t e d , t h e c u t i c l e c l e a n e d and s e p a r a t e d i n t o s e v e r a l p i e c e s , and t h e o u t l i n e s o f t h e s e p i e c e s marked on m i l l i m e t e r p a p e r . The number of s q u a r e s c o v e r e d by t h e c u t i c l e was t o t a l l e d . The e r r o r i n d e t e r m i n i n g t h e number of c o v e r e d s q u a r e s was e s t i -mated t o be i n t h e o r d e r of 5%* 23 A p o r t i o n of t h e w e i g h t l o s s o b s e r v e d I n t h i s e x p e r i m e n t may be due t o m e t a b o l i c l o s s e s and n o t s o l e l y t o e v a p o r a t i o n of w a t e r . I t was a l s o n o t i c e d t h a t t h e l o c o m o t o r a c t i v i t y of t h e s e e x p e r i m e n t a l t i c k s was i n c r e a s e d because t h e y were n ot a t t a c h e d t o t h e h o s t ; t h i s w o u l d t e n d t o m a g n i f y the c o n t r i b u t i o n of m e t a b o l i c l o s s . I t s h o u l d be emphasized t h e r e f o r e , t h a t t h i s e x p e r i m e n t p r o b a b l y p r o v i d e s a maximum e s t i m a t e of i n t e g u m e n t a r y w a t e r l o s s . The e s t i m a t e a l s o i n c l u d e s any l o s s f r o m t h e s p i r a c l e s , g e n i t a l o r i f i c e , and Gene'1 s or g a n , none of w h i c h was c o v e r e d w i t h " t a c k y wax". k) C o l l e c t i o n o f D r y F e c e s S i x p o l y e t h y l e n e c y l i n d e r s (3 cm d i a m e t e r and 3 cm h i g h ) were g l u e d t o t h e back o f a r a b b i t w i t h epoxy r e s i n o r 1 P l a s t l c a s t ' . The a r e n a o f s k i n s u r r o u n d e d by t h e c y l i n d e r was s p r a y e d w i t h a p l a s t i c s u r g i c a l d r e s s i n g ( " A e r o p l a s t " , Parke D a v i s & C o . ) . T h i s s p r a y - o n f i l m p r e v e n t e d t h e t i c k s ' f e c e s f rom coming i n d i r e c t c o n t a c t w i t h t h e s a l t s produced by t h e r a b b i t ' s s k i n , y e t i t d i d n o t h i n d e r t h e t i c k s f r o m a t t a c h i n g . S i n g l e f e m a l e s were p l a c e d i n e a c h o f t h e f i v e c a p s u l e s and f i v e males were p l a c e d i n the s i x t h . F e c e s were c o l l e c t e d f r o m each c a p s u l e d a l l y and a l l o w e d t o d r y i n s e p a r a t e p o l y e t h y l e n e v i a l s ; t h u s , a d a l l y r e c o r d of f e c e s p r o d u c t i o n was t a l l i e d f o r each fema l e and f o r the group of f i v e m a l e s . As me n t i o n e d i n C h a p t e r One, i t i s n e c e s s a r y f o r t h e f e m a l e s t o c o p u l a t e f o r f u l l engorgement t o be a t t a i n e d . However, t h e males r e q u i r e f i v e o r s i x days of f e e d i n g b e f o r e , t h e y a r e r e a d y t o c o p u l a t e (Gregson, 19**7). There was no p r e v i o u s 2k knowledge on t h e amount of f e c e s produced by m a l e s . F o r t h i s r e a s o n , I k e p t them s e p a r a t e d f r o m t h e f e m a l e s f o r t h e f i r s t s i x d a y s . On t h e s i x t h day of a t t a c h m e n t , t h e males were removed f r o m t h e i r c a p s u l e , and each was put i n one o f t h e o t h e r c a p s u l e s t o mate w i t h a f e m a l e . T h e i r a n a l p l a t e s a l s o were p l u g g e d w i t h epoxy g l u e t o p r e v e n t any o f t h e i r f e c e s b e i n g t a l l i e d w i t h t h o s e o f t h e f e m a l e s . 5) P r e p a r a t i o n o f R a b b i t Hemoglobin P u r i f i e d r a b b i t h e m o g l o b i n was n o t a v a i l a b l e c o m m e r c i a l l y } t h e r e f o r e i t was e x t r a c t e d f r o m r a b b i t b l o o d as f o l l o w s : About 100 ml o f b l o o d was o b t a i n e d by s l i t t i n g the d o r s a l a o r t a o f an a n a e s t h e t i z e d r a b b i t w h i c h was i n j e c t e d w i t h h e p a r i n . The b l o o d was a s p i r a t e d i n t o a Buchner f l a s k w h i c h a l s o c o n t a i n e d h e p a r i n s o l u t i o n . The r e s t o f t h e p r o c e d u r e was c a r r i e d out on i c e o r i n a 5° C c o l d room. The e r y t h r o c y t e s were spun down and washed s i x t i m e s w i t h i s o t o n i c s a l i n e p r o g r e s s i n g f r o m 750 g t o 7700 g ( 1 0 m i n u t e s f o r each wash). W i t h each wash the b u f f y c o a t l a y e r was a l s o d i s c a r d e d . The e r y t h r o c y t e s were r u p t u r e d by f r e e z e -t h a w l n g . The v i s c o u s s l u r r y was t h e n f r o z e n f o r a few days b e f o r e p r o c e e d i n g . The h e m o l y s a t e was a l l o w e d t o thaw, and an e q u a l volume of d i s t i l l e d w a t e r was added t o l o w e r the v i s c o s i t y . The g h o s t s and o t h e r p a r t i c u l a t e m a t t e r were spun down a t 1 5t0 0 0 g f o r one hour and t h e s u p e r n a t a n t was f r e e z e - d r i e d . P u r i t y of t h i s powdered h e m o g l o b i n was d e t e r m i n e d by comparing the a b s o r -bance of t h e s o r e t band (about 4 1 5 niu) o f a s e r i e s o f known c o n c e n t r a t i o n s of t h e powder i n d i s t i l l e d w a t e r w i t h the a b s o r -bance of t h e s o r e t band of c o m m e r c i a l l y p u r i f i e d b o v i n e 2 5 h e m o g l o b i n (Sigma t y p e I I ) d i l u t e d t o t h e same c o n c e n t r a t i o n s . T a b l e I I shows t h a t t h e a b s o r b a n c e s o f b o t h s p e c i e s o f hemo-g l o b i n a t 4 1 5 nyu a r e i n good agreement w i t h each o t h e r . TABLE I I COMPARISON OF RABBIT HEMOGLOBIN PREPARED IN THIS LABORATORY WITH SIGMA BOVINE HEMOGLOBIN C o n c e n t r a t i o n Absorbance a t S o r e t Band* (mg/ml) Oxygenated R a b b i t Hemoglobin Oxygenated B o v i n e Hemoglobin 0 . 0 3 1 2 0 . 1 5 5 0 . 0 6 2 5 0.268 0.273 0 . 0 8 7 5 0 . 4 1 9 0 . 3 8 8 0 . 1 2 5 0 . 5 6 9 0 . 5 6 5 0 . 1 7 5 0 . 8 6 0 0 . 8 1 0 0 . 2 5 1 . 1 6 1 . 1 3 0 . 3 5 1 . 8 5 1 . 7 5 0 . 5 0 > 2 . > 2 . * The e x a c t w a v e l e n g t h of t h e s o r e t peak v a r i e s somewhat w i t h t h e s p e c i e s o f h e m o g l o b i n . C o n s e q u e n t l y , t h e peak a b s o r b a n c e s were r e a d o f f a s c a n n i n g double-beam s p e c t r o p h o t o m e t e r (Bausch & Lomb, SP 6 0 0 ) . I n a l l c a s e s , the peak o c c u r r e d i n t h e v i c i n i t y of 415 myu, t h e range b e i n g 410 t o 420 mu. 6) D e t e r m i n a t i o n of Hemoglobin C o n c e n t r a t i o n The i r o n c o n t e n t of each sample was d e t e r m i n e d a c c o r d i n g t o t h e method of B r e u e r and M i l l t z e r ( 1 9 3 8 ) w i t h o n l y minor m o d i f i c a t i o n s , and t h i s v a l u e was c o n v e r t e d t o h e m o g l o b i n c o n t e n t assuming t h a t 0 . 3 3 7 5 $ o f h e m o g l o b i n i s i r o n (Lemberg and Legge, 1 9 4 9 ) . T h i s method was f o u n d t o be s u p e r i o r t o e i t h e r d i r e c t 26 c o m p a r i s o n o f d i s s o l v e d samples t o pure h e m o g l o b i n s t a n d a r d s by-spec t r o p h o t o m e t r y , o r t o the b e n z i d i n e method ( C r o s b y and F u r t h , 1956) u s i n g c o m m e r c i a l cyanraethemoglobln r e a g e n t as a s t a n d a r d . U s i n g t h e l a t t e r two methods I e x p e r i e n c e d i n t e r f e r e n c e w i t h the r e a c t i o n s f r o m o t h e r components i n t h e f e c e s and homogenates w h i c h r e s u l t e d i n a b e r r a n t s p e c t r a . S i n c e i t was assumed t h a t by f a r t h e g r e a t e s t p o r t i o n of t h e i r o n i n r a b b i t b l o o d , t i c k homogenates, and t i c k f e c e s would come f r o m h e m o g l o b i n and r e l a t e d p o r p h y r i n s , I f e l t t h a t m e a s u r i n g i r o n d i r e c t l y would a c c u r a t e l y r e f l e c t t h e p o r p h y r i n c o n t e n t of t h e samples, and a t the same ti m e would e l i m i n a t e i n t e r f e r e n c e f r o m o t h e r o r g a n i c m a t t e r w h i c h i s ashed away i n t h e method. When f e r r i c i r o n r e a c t s w i t h t h i o c y a n a t e , a s e r i e s of r e d -c o l o u r e d compounds a r e formed. When t h i o c y a n a t e i s i n e x c e s s (as w h i c h i s i n t e n s e l y r e d and r e l a t i v e l y s t a b l e . The c o n c e n t r a t i o n i s d e t e r m i n e d s p e c t r o p h o t o m e t r l c a l l y a t 480 mu ( V o g e l , 1962). S t a n d a r d s o l u t i o n s o f known i r o n c o n t e n t produce a c u r v e w h i c h obeys t h e Beer-Lambert l a w . The i r o n c o n t e n t of each sample c a n be r e a d f r o m t h e s t a n d a r d c u r v e . a) A s h i n g p r o c e d u r e As t h e method used ( B r e u e r and M i l l t z e r , 1938) was o r i g i n a l l y d e v e l o p e d f o r s m a l l q u a n t i t i e s of pure b l o o d , the i n i t i a l s t a g e o f t h e pr o c e d u r e c o n s i s t s of w e t - a s h i n g the b l o o d t o r e l e a s e the i r o n f r o m the h e m o g l o b i n and t o c o n v e r t t h i s i r o n t o t h e f e r r i c s t a t e . I n a d o p t i n g t h i s method f o r use on t i c k i n t h e p r e s e n t method) t h e predominant f e c e s and homogenates of engorged t i c k s , I f o u n d t h i s wet-a s h i n g s t e p was n o t s u f f i c i e n t t o e l i m i n a t e t u r b i d i t y f r o m the f i n a l s o l u t i o n s , so t h e samples were d r y - a s h e d f i r s t and t h e n s u b j e c t e d t o t h e complete method. S m a l l a s h i n g b o a t s , made f r o m heavy p l a t i n u m f o i l , were t h o r o u g h l y c l e a n e d , d r i e d and weighed. A known q u a n t i t y of sample was p l a c e d i n t h e b o a t . A l l t h e b o a t s were h a n d l e d by f o r c e p s and c o n t a i n e d i n a P y r e x p e t r i e d i s h . Samples were d r i e d f o r s e v e r a l h o u r s i n a 6 0 ° C oven, and t h e n d r i e d o v e r -n i g h t a t 100° C i n a m u f f l e f u r n a c e . The samples were t h e n a s h e d f o r f i v e h o u r s a t 460° C. The b o a t s and a s h were t r a n s -f e r r e d t o 2 5-ml E r l e n m y e r f l a s k s f o r i r o n d e t e r m i n a t i o n . R e c o v e r y o f i r o n f r o m t h e a s h i n g p r o c e d u r e was d e t e r m i n e d f r o m s e v e r a l s t a n d a r d s ! (1) a s h i n g 6 . 3 mg PeSO^^HgOf (2) a s h i n g 0.2 ml a l i q u o t s o f s t a n d a r d i r o n s o l u t i o n s (0.1 mg/ml Pe ) used i n t h e method; ( 3 ) a s h i n g 0.2 ml o f a 20 mg/ml s o l u t i o n of p u r i f i e d r a b b i t h e m o g l o b i n i n d i s t i l l e d w a t e r ; and ( 4 ) a s h i n g 5 . 4 mg o f powdered r a b b i t h e m o g l o b i n . The p o s s i b i l i t y of c o n -t a m i n a t i o n was n e g a t e d by a s h i n g an empty boat and t r e a t i n g i t i n a manner i d e n t i c a l t o t h e o t h e r samples. No i r o n c o u l d be d e t e c t e d f r o m t h i s b l a n k . R e c o v e r y o f i r o n f r o m t h e i n o r g a n i c s t a n d a r d s was 98.5 $ 1 b u t t h a t f r o m t h e f o u r samples o f hemoglobin was 77.8$, 7^$» 82,3$» and 79*5%* C o n s e q u e n t l y , t h e d e t e r m i n e d v a l u e s f r o m a l l s u b sequent o r g a n i c samples s u b j e c t e d t o a s h i n g were c o r r e c t e d a s s u ming 80$ r e c o v e r y of i r o n . 28 b) Method f o r d e t e r m i n i n g I r o n F o r t o t a l body c o n t e n t of I r o n , engorged t i c k s were r i n s e d I n d i s t i l l e d w a t e r , weighed , and l a c e r a t e d w i t h a s c a l p e l . The t o t a l c o n t e n t s o f a s i n g l e engorged t i c k were added t o a 1 0-ml P o t t e r E l v e h j e m homogenizer f i t t e d w i t h a t e f l o n p l u n g e r . One o r two ml of d i s t i l l e d w a t e r were added t o l o w e r t h e v i s c o s i t y d u r i n g h o m o g e n l z a t l o n . The homogenate was t r a n s f e r r e d t o a 2 5-ml v o l u m e t r i c f l a s k and s u c c e s s i v e washings of t h e homgenizer were c a r r i e d out w i t h a l i q u o t s of d i s t i l l e d w a t e r u n t i l a l l t h e r e d c o l o u r had been t r a n s f e r r e d f r o m t h e homogenizer. The f r a g m e n t s o f c u t i c l e were t h e n put a s i d e f o r d e t e r m i n a t i o n of sodium c o n t e n t . The volume o f t h i s t u r b i d homogenate was b r o u g h t t o e x a c t l y 2 5 ml w i t h d i s t i l l e d w a t e r . A l i q u o t s of t h i s s u s p e n s i o n ( u s u a l l y 1 ml) were added t o weighed p l a t i n u m b o a t s and ashed as p r e v i o u s l y o u t l i n e d . Each b o a t and i t s ashed c o n t e n t s were t r a n s f e r r e d t o a 2 5-ml E r l e n m e y e r f l a s k c o n t a i n i n g 1 ml d i s t i l l e d w a t e r . One ml c o n c e n t r a t e d s u l p h u r i c a c i d was c a r e f u l l y added t o t h e f l a s k t o d i s s o l v e t h e a s h . T h i s f l u i d was t i t r a t e d w i t h 2% p o t a s s i u m permanganate u n t i l o x i d a t i o n was c o m p l e t e d . T h i s was t e s t e d by s i t t i n g t he f l a s k s i n a 6 0 ° C w a t e r b a t h and o b s e r v i n g t h e d i s a p p e a r a n c e of th e p i n k c o l o u r . Vlhen the p i n k c o l o u r p e r s i s t e d f o r f i v e m i n u t e s , t h e e n d - p o i n t o f o x i d a t i o n was c o n s i d e r e d a t t a i n e d ( B r e u e r and M i l i t z e r , 1 9 3 8 ) . A d r o p of HgOg was added t o each f l a s k t o d e s t r o y t h e p i n k t r a c e . A f t e r c o o l i n g , t h e c o n t e n t s of th e f l a s k s were t r a n s f e r r e d t o 1 0-ml v o l u m e t r i c f l a s k s \ two o r 29 t h r e e washings w i t h d i s t i l l e d w a t e r o f t h e E r l e n m e y e r f l a s k s were a l s o added t o the v o l u m e t r i c f l a s k s . Then 0.5 ml of s a t u r a t e d p o t a s s i u m p e r s u l p h a t e was added, and t h e t o t a l volume brou g h t up t o 10 ml w i t h d i s t i l l e d w a t e r . The c h a r a c t e r i s t i c r e d c o l o u r a p p e a r e d on the a d d i t i o n o f 1.0 ml p o t a s s i u m t h i o c y a n a t e s o l u t i o n . The c o n t e n t s o f t h e f l a s k s were t h o r o u g h l y mixed, and t h e a b s o r -bances r e a d a t 480 mp. on t h e Unlearn SP 500 s p e c t r o p h o t o m e t e r . S i n c e t h e i r o n c o n t e n t of u n f e d t i c k s was t o o low t o be d e t e c t e d by t h i s method, t h e i r o n c o n t e n t o f engorged t i c k s was t a k e n t o r e p r e s e n t t h e n e t uptake of i r o n . 7) Method of S a m p l i n g Hemolymph and S a l i v a of T i c k s The c o l l e c t i o n of a l l f l u i d s was a c c o m p l i s h e d under a s t e r e o m i c r o s c o p e w h i c h was s u r r o u n d e d by a chamber l i n e d w i t h p l a s t i c s h e e t i n g on t h e o u t s i d e and chromatography paper w i t h i n . The m i c r o s c o p e r e s t e d on an I s l a n d i n a t r a y of w a t e r w i t h the o c u l a r s - p r o j e c t i n g t h r o u g h t h e p l a s t i c s h e e t i n g t o t h e o u t s i d e of t h e chamber. The chromatography paper was m o i s t e n e d and the r e l a t i v e h u m i d i t y (EH) was k e p t h i g h by I n t e r m i t t e n t use of a steam v a p o r i z e r . A s i m p l e c o i l h y g r o m e t e r , and t h e p r e v a l a n c e of c o n d e n s a t i o n w i t h i n t h e chamber i n d i c a t e d t h a t t h e RH a p p r o a c h e d 100$. As an a d d i t i o n a l p r e c a u t i o n , the g l a s s s l i d e t o w h i c h t h e t i c k was s e c u r e d d u r i n g t h e c o l l e c t i o n of hemo-lymph, was p l a c e d on m o i s t e n e d f i l t e r paper on the s t a g e o f the m i c r o s c o p e . The arms of the e x p e r i m e n t e r c o u l d g a i n a c c e s s t o t h e m i c r o s c o p e s t a g e t h r o u g h o p e n i n g s i n the f r o n t f l a p of t h e chamber. T h i s chamber was s e t up t o l i m i t e v a p o r a t i o n of the hemolymph d u r i n g t h e t i m e r e q u i r e d f o r c o l l e c t i o n . By u s i n g the v a p o r i z e r i n t e r m i t t e n t l y , c o n d e n s a t i o n of w a t e r on the t i c k was a v o i d e d . The v a p o r i z e r a l s o r a i s e d the t e m p e r a t u r e i n the chamber t o 30° C; t h i s i n c r e a s e d t h e s t r u g g l i n g m o t i o n s of t h e t i c k and t h u s enhanced the r a t e of e x u d a t i o n o f t h e hemolymph. S a l i v a was c o l l e c t e d i n a g l a s s c a p i l l a r y p l a c e d o v e r t h e m o u t h p a r t s o f the t i c k (Gregson, 1957; H o w e l l , 1966} T a t c h e l l , 1967a). No p h a r m a c o l o g i c a l s t i m u l a n t was i n j e c t e d t o enhance s e c r e t i o n . A l t h o u g h t h e a u t h o r s mentioned above o c c a s i o n a l l y have done so i n o r d e r t o i n c r e a s e t h e y i e l d o f s a l i v a , t h e y do c a u t i o n t h a t t h e c o m p o s i t i o n o f t h e s a l i v a f r o m s t i m u l a t e d t i c k s may d i f f e r somewhat f r o m t h a t o f "normal" s a l i v a . B e f o r e p l a c i n g t h e g l a s s c a p i l l a r y o v e r t h e c h e l l c e r a e and hypostome of the t i c k , t h e o r a l end o f t h e tube was d i p p e d i n p a r a f f i n o i l . I n t h i s way, t h e column o f s a l i v a r e l e a s e d i n t o the tube was p r o t e c t e d f r o m e v a p o r a t i o n . The t o t a l t i m e f o r c o l l e c t i o n of s a l i v a v a r i e d w i t h t h e I n d i v i d u a l but was a p p r o x i m a t e l y f i v e t o t e n m i n u t e s . As an added p r e c a u t i o n a g a i n s t e v a p o r a t i o n of s a l i v a , t h e t i c k s were k e p t i n t h e m o i s t chamber d u r i n g t h i s t i m e . The f i l l e d c a p i l l a r i e s were i m m e d i a t e l y submerged under p a r a f f i n o i l . Hemolymph was t a k e n i n t h e f o l l o w i n g wayi A t i c k was s e c u r e d dorsum down t o a g l a s s s l i d e l i n e d w i t h ' P a r a f i l m 1 (American Can Co.) i n o r d e r t o f a c i l i t a t e c o l l e c t i o n . The t e r m i n a l segment o f a l e g was c u t , and s l i g h t p r e s s u r e was a p p l i e d t o t h e body when n e c e s s a r y i n o r d e r t o f a c i l i t a t e f l o w of hemolymph. Hemolymph was c o l l e c t e d i n 1-lambda Druramond " m i c r o c a p s " d i s p o s a b l e p i p e t t e s , and t h e time r e q u i r e d f o r c o l -l e c t i o n was u s u a l l y l e s s t h a n one m i n u t e . A l l samples were i m m e d i a t e l y e j e c t e d f r o m t h e l a m b d a - p i p e t t e s under a dro p of o i l . When more t h a n one lambda was a v a i l a b l e f r o m a s i n g l e t i c k , t h e c o n t e n t s of each p i p e t t e were p o o l e d t o g e t h e r under the p a r a f f i n o i l . As soon as t h e c o l l e c t i o n p e r i o d was c o m p l e t e d , samples of f l u i d f o r t h e d e t e r m i n a t i o n of sodium and p o t a s s i u m were t a k e n f r o m t h e d r o p l e t s p o o l e d under the o i l , and d i l u t e d i n known volumes of d i s t i l l e d w a t e r and sodium swamp r e s p e c -t i v e l y (see b e l o w ) . Samples f o r c h l o r i d e d e t e r m i n a t i o n however were r e a d i m m e d i a t e l y f o l l o w i n g t h e c o l l e c t i o n p e r i o d as de s -c r i b e d below. 8) D e t e r m i n a t i o n of I o n C o n c e n t r a t i o n a) Sodium Sodium was d e t e r m i n e d by f l a m e e m i s s i o n s p e c t r o p h o t o m e t r y u s i n g e i t h e r a Unlearn SP 900 o r a T e c h t r o n AA 120 f l a m e s p e c t r o -photometer. A l i q u o t s o f body f l u i d s (1 pl) were d i l u t e d i n 5 o r 10 ml of d i s t i l l e d w a t e r c o n t a i n e d i n capped p o l y e t h y l e n e v i a l s ( Nalge C o . ) . D i l u t e d samples were f r o z e n f o r up t o s e v e r a l months b e f o r e c a r r y i n g out t h e d e t e r m i n a t i o n s . S t a n d a r d sodium c h l o r i d e s o l u t i o n s ( H a r e l c o ) were s u i t a b l y d i l u t e d t o c o v e r t h e range of 0-50 ^ ieq N a + / l i t e r . Samples of r a b b i t b l o o d , t i c k hemolymph and s a l i v a were u n t r e a t e d b e f o r e d i l u t i n g I n t h e d i s t i l l e d w a t e r , b u t homogenates of whole t i c k s , and t h e d r y f e c e s were ashed f i r s t as p r e v i o u s l y d e s c r i b e d . b) P o t a s s i u m P o t a s s i u m was d e t e r m i n e d I n a manner s i m i l a r t o t h a t f o r sodium by d i l u t i n g 1 /il a l i q u o t s i n 3 o r 5 nil o f 1260 ppm sodium c h l o r i d e s o l u t i o n (500 ppm sodium) t o s t a n d a r d i z e sodium i n t e r -f e r e n c e e f f e c t s ( W i l l a r d e t a l . , 1965)• S t a n d a r d p o t a s s i u m c h l o r i d e s o l u t i o n s ( H a r e l c o ) were a l s o d i l u t e d i n t h i s swamp so as t o c o v e r a range of 0-25 / i e q K + / l i t e r . S o l u t i o n s of t i c k f e c e s were n o t ashed p r i o r t o d i l u t i o n i n swamp. c) Magnesium W i t h o n l y s l i g h t m o d i f i c a t i o n , t h e method used was t h a t of W i l l i s (i960). A l i q u o t s o f one o r two m i c r o l i t e r s were d i s -s o l v e d i n 2 ml o f a 3$ d i s o d i u m EDTA swamp. The EDTA swamp was used t o overcome t h e s u p p r e s s i o n by phosphate o f t h e magnesium a b s o r p t i o n . S t a n d a r d magnesium c h l o r i d e s o l u t i o n s ( H a r e l c o ) were a l s o d i l u t e d i n t h i s swamp so as t o c o v e r a range o f 0-60 jueq Mg / l i t e r . d) C h l o r i d e T h i s a n i o n was d e t e r m i n e d by t h e f i r s t e l e c t r o m e t r i c t i t r a t i o n method o f Ramsay e t a l . (1955) w i t h o n l y s l i g h t modi-f i c a t i o n . P o t e n t i a l s were r e a d o f f a m i l l i v o l t m e t e r (Model 25 pH M e t e r , R a d i o m e t e r Copenhagen). The m i c r o b u r e t t e , p l a t f o r m , and m a g n e t i c s t i r r i n g a p p a r a t u s used were s u p p l i e d by M i s c o , C a l i f o r n i a . Sample s i z e was u s u a l l y 1 pi. Most samples were t i t r a t e d w i t h a 0.2$ s i l v e r n i t r a t e s o l u t i o n ; samples e x p e c t e d t o c o n t a i n more t h a n 200 m e q / l i t e r c h l o r i d e were t i t r a t e d w i t h a 0.4$ s i l v e r n i t r a t e s o l u t i o n . S t a n d a r d s were p r e p a r e d f r o m r e a g e n t grade sodium c h l o r i d e so as t o c o v e r t h e f u l l range of th e e x p e c t e d sample c o n c e n t r a t i o n s ( u s u a l l y 0 - 2 0 0 m e q / l i t e r ) . e) I n o r g a n i c phosphate I n o r g a n i c phosphate was measured a c c o r d i n g t o t h e method of Gomori ( 1 9 4 2 ) w i t h m i n o r m o d i f i c a t i o n s as d e s c r i b e d by S p e i g h t ( 1 9 6 7 ) . T h i s method r e q u i r e d 5 t o 60 pl a l i q u o t s o f hemolymph o r s a l i v a . The r e a d i n g s were t a k e n a t 675 mu on a Unicam SP 500 s p e c t r o p h o t o m e t e r . f ) Measurement o f f r a c t i o n a l volumes The s m a l l e s t volume Drummond m i c r o p i p e t t e a v a i l a b l e was of 1 pl c a p a c i t y . However, a f u l l m i c r o l i t e r of hemolymph c o u l d o f t e n n o t be o b t a i n e d f r o m s m a l l t i c k s . I n t h e s e c a s e s , t h e f r a c t i o n a l volume was d e t e r m i n e d by me a s u r i n g t h e l e n g t h of t h e f l u i d column i n t h e p i p e t t e u s i n g a m i l l i m e t e r r u l e under a low-power d i s s e c t i n g m i c r o s c o p e . S i n c e t h e bore of t h e p i p e t t e was c o n s t a n t , t h e r a t i o of f l u i d l e n g t h t o t o t a l p i p e t t e l e n g t h was d i r e c t l y p r o p o r t i o n a l t o t h e volume o f the sample. T h i s was v e r i f i e d by t r e a t i n g s t a n d a r d s o l u t i o n s i n a manner s i m i l a r t o unknowns. C o n c e n t r a t i o n o f c h l o r i d e ( 0 . 5 - 1 . 0 ^ul) d e t e r m i n e d i n t h i s manner d e v i a t e d f r o m t h e known v a l u e by a maximum o f 6%\ i n t h e case of f u l l p i p e t t e s , t h e maximum d e v i a t i o n was 3$ ( F i g . 1 ) . The d e v i a t i o n s f r o m t h e known v a l u e i n c r e a s e d t o 1 1 $ w i t h volumes f r o m 0 . 2 - 0 . 5 / J l . A l l samples s m a l l e r t h a n 0 . 2 pl were d i s c a r d e d . F i g u r e 1. E r r o r s i n determination of c h l o r i d e c o n c e n t r a t i o n when usi n g f r a c t i o n a l volumes of a 1-ul p i p e t t e of uniform bore. The spread of values increases as the l e n g t h of the f l u i d column decreases, but i t does so s y m m e t r i c a l l y . In other words, there i s no g r e a t e r tendency to e i t h e r underestimate or over-estimate the c h l o r i d e c o n c e n t r a t i o n . The dotted l i n e i n d i c a t e s the range of values f o r a f u l l p i p e t t e . 160-i cr a> E «3 C o 140 H ^ 120 A •G 100 9>. o o o o g oo o o o °o 0 ooo E o T3 CD 80 3 ro 60 40 i— 10 20 30 40 Length of fluid column in ^-X pipette (mm) 36 9) Osmotic P r e s s u r e The o s m o t i c p r e s s u r e s of hemolymph and s a l i v a were d e t e r -mined by t h e c r y o s c o p i c method of Ramsay ( 1 9 ^ 9 ) w i t h one modi-f i c a t i o n ; s m a l l f l u i d samples were c o l l e c t e d i n a Drummond 1 - u l p i p e t t e s e p a r a t e d one f r o m t h e o t h e r w i t h p a r a f f i n o i l ( F i s h e r , S a y b o l t v i s c o s i t y 1 2 5 / 1 3 5 ) • The Beckman thermometer was c a l i b r a t e d u s i n g s t a n d a r d s o l u t i o n s of known o s m o l a r i t y ( F i s k e A s s o c i a t e s I n c . , USA). 1 0 ) P r o t e i n D e t e r m i n a t i o n o f F e c e s P r o t e i n c o n t e n t o f f e c e s was d e t e r m i n e d by t h e method of Lowry e t a l . ( 1 9 5 D u s i n g b o v i n e serum a l b u m i n as t h e s t a n d a r d . O p t i c a l d e n s i t i e s were r e a d a t 700 mu on a Bausch & Lomb ' S p e c t r o n i c 2 0 ' . 11) C l e a r a n c e o f I n u l l n and 3 - 0 - M e t h y l g l u c o s e I n u l i n - c a r b o x y l - C 1 1 1 ' ( 5 0 pC i n 42 mg) and 3 - 0 - m e t h y l - C 1 / 4 , D - g l u c o s e ( 5 0 pC i n 0 . 2 5 ml 7 5 $ e t h a n o l ) were s u p p l i e d by New E n g l a n d N u c l e a r C o r p . To t h e i n u l l n was added 0 . 5 ml of 0 . 9 $ s a l i n e and t h e l n u l i n b r o u g h t i n t o s o l u t i o n by immersing the v i a l b r i e f l y i n h o t w a t e r . To t h e 0 - m e t h y l g l u c o s e was added 0 . 7 5 ml o f 0 . 9 $ s a l i n e . A p p r o x i m a t e l y 1 pi of t h e i n u l l n s o l u t i o n was i n j e c t e d f o r each 10 mg t i c k w e i g h t . I n t h e 0-methyglucose e x p e r i m e n t s , t h e dose was 1 pi f o r each 50 mg t i c k w e i g h t . The p r o c e d u r e f o r i n j e c t i n g t h e above s o l u t i o n s was as f o l l o w s t T i c k s were removed f r o m t h e r a b b i t a f t e r f i v e days of f e e d i n g . (Weight range of t i c k s was 6 0 - 9 0 mg). The s o l u t i o n was I n j e c t e d i n t o a s e v e r e d h i n d - l e g segment f r o m an ' A g l a ' m i c r o m e t e r s y r i n g e (Burroughs Wellcome & Co.) f i t t e d w i t h a t a p e r e d g l a s s p i p e t t e . The p i p e t t e was s e a l e d t o t h e l e g stump w i t h " t a c k y wax" b e f o r e the i n j e c t i o n was p e r f o r m e d , and was l e f t i n p l a c e f o r s e v e r a l m i n u tes a f t e r the i n j e c t i o n of f l u i d . A f t e r r e m o v a l of t h e p i p e t t e , t h e wound was s e a l e d up w i t h the t a c k y wax. Leakage f r o m th e l e g was p r e v e n t e d d u r i n g t h e whole p r o c e d u r e by t h e use a t a p p r o p r i a t e t i m e s of a m o d i f i e d s e r -r e f l n e . The t i m e r e q u i r e d f o r e q u i l i b r a t i o n of t h e t r a c e r s t h r o u g h o u t t h e t i c k was e x p e c t e d t o t a k e s e v e r a l h o u r s . T h e r e -f o r e , t h e s e c r e t i o n o f s a l i v a I n t o g l a s s c a p i l l a r i e s a t t h a t t i m e was a l s o e x p e c t e d t o be s m a l l because th e y i e l d o f s a l i v a d e c r e a s e s w i t h t h e t i m e a f t e r r e m o v a l f r o m th e h o s t (Gregson, 1956). C o n s e q u e n t l y , I f e l t i t n e c e s s a r y t o a l l o w t h e t i c k s t o recommence f e e d i n g and s a l i v a t i o n f o l l o w i n g t h e i n j e c t i o n s . W i t h t h i s i n mind, th e t i c k s were r e t u r n e d t o t h e h o s t o v e r n i g h t . The f o l l o w i n g day t h e y were once a g a i n removed f r o m the r a b b i t , and 1-pl samples of t h e i r hemolymph and s a l i v a were o b t a i n e d as d e s c r i b e d above. Hemolymph and s a l i v a samples were d i s s o l v e d i n 50 pl d i s t i l l e d w a t e r b e f o r e a d d i n g t o 10 ml l i q u i d s c i n t i l -l a t i o n f l u i d ( B r a y , i960). The samples were c o u n t e d by u s i n g a ' N u c l e a r C h i c a g o Mark I ' l i q u i d s c i n t i l l a t i o n c o u n t e r , and t h e v a l u e s were c o r r e c t e d f o r e f f i c i e n c y by t h e c h a n n e l s ' r a t i o method. I n o r d e r t o a s s e s s whether i n u l i n and O-methylglucose were b e i n g l o s t by t h e e x c r e t o r y system, samples of M a l p i g h i a n 38 t u b u l e homogenate and d r i e d f e c e s were a l s o added t o 50 pi d i s t i l -l e d w a t e r and p l a c e d i n B r a y ' s s o l u t i o n . M a l p i g h i a n t u b u l e s and f e c e s f r o m n o n - i n j e c t e d t i c k s , h a n d l e d i n t h e same way, c o n f i r m e d t h a t a u t o f l u o r e s c e n c e was n e g l i g i b l e . 12) D e t e r m i n a t i o n o f Hemolymph Volume I n u l i n - c a r b o x y l - C 1 ^ (50 jpC/25 mg) was d i s s o l v e d i n 5 ml of s i m p l i f i e d t i c k medium ( m o d i f i e d f r o m Rehacek and B r z o s t o w s k i , 1 9 6 9 ) • The medium used h e r e c o n s i s t e d of the s a l t s and s u g a r s enumerated by t h e s e a u t h o r s , b u t l a c k e d t h e amino a c i d s , v i t a m i n s , and a n t i b i o t i c s , n o r was t h e o s m o t i c p r e s s u r e a d j u s t e d i n the manner t h e y d e s c r i b e . The i n j e c t i o n p r o c e d u r e was i d e n t i c a l t o t h e one d e s c r i b e d above e x c e p t f o r dosage. I n t h i s e x p e r i m e n t , 1 pi was i n j e c t e d i n t o t h e u n f e d t i c k s ( a p p r o x i m a t e l y 10 mg i n w e i g h t ) . H e a v i e r t i c k s r e c e i v e d p r o p o r t i o n a t e l y l e s s t r a c e r (1-2 pi i n t i c k s w e i g h i n g up t o 100 mg and 2-3 pi i n t i c k s o v e r 100 mg). The dose g i v e n t o u n f e d t i c k s was h i g h e r because i t was f e l t t h a t 1 ^ i l was t h e minimum volume w h i c h c o u l d be d e l i v e r e d w i t h r e a s o n -a b l e a c c u r a c y . E q u i l i b r a t i o n of t h e t r a c e r was complete w i t h i n an h o u r of i n j e c t i o n s i n c e t h e r a d i o a c t i v i t y p e r u n i t volume of hemolymph rem a i n e d c o n s t a n t f o r t h e n e x t two h o u r s . Hemolymph samples were t h e r e a f t e r t a k e n around two hours a f t e r i n j e c t i o n . The r e s u l t a n t a c t i v i t y r a nged f r o m 200 t o 1200 cpm^ul b e f o r e quench c o r r e c t i o n ; b a ckground a c t i v i t y was l e s s t h a n 20 cpm. A l l hemolymph samples ( u s u a l l y 1 ^ul) were added d i r e c t l y 14 t o B r a y ' s s o l u t i o n f o r d e t e r m i n a t i o n o f C - a c t i v i t y as d e s c r i b e d above. A f t e r c o r r e c t i n g f o r c o u n t i n g e f f i c i e n c y and background a c t i v i t y , t he d i l u t i o n of t h e t r a c e r by the e x t r a c e l l u l a r f l u i d was c a l c u l a t e d f r om the r a t i o : cpm o f 1 pl i n j e c t e d f l u i d cpm of 1 ^Jil sampled hemolymph The s u b t r a c t i o n f r om t h i s r a t i o o f t h e volume i n m i c r o l i t e r s t h a t was i n j e c t e d i n t o t h e t i c k y i e l d s t h e t r u e volume of t h e hemolymph ( o r more a c c u r a t e l y t he " i n u l i n space") a t t h e time of s a m p l i n g . The a s s u m p t i o n i s made t h a t t h e i n j e c t e d i n u l i n has a l s o remained i n t h e e x t r a c e l l u l a r f l u i d as r e p o r t e d by v a r i o u s a u t h o r s (Levenbook, 1958. Ramsay and R i e g e l , 1961). 40 RESULTS 1) Net Weight I n c r e a s e of F e e d i n g Females The n e t w e i g h t i n c r e a s e o f a l a r g e p o p u l a t i o n o f e x p e r i m e n t a l t i c k s i s p r e s e n t e d i n F i g u r e 2. S i n c e t h e f e m a l e s c o n t r i b u t i n g t o t h i s g r a p h came f r o m s e v e r a l b a t c h e s o f t i c k s , were f e d a t d i f f e r e n t t i m e s , and s t a r t e d f r om a wide range o f i n i t i a l u n f e d w e i g h t s (5-20 mg), I chose t o p l o t t h e r a t i o ( w e i g h t on day X / u n f e d w e i g h t ) a g a i n s t time i n d a y s , r a t h e r t h a n t h e a b s o l u t e w e i g h t a g a i n s t t i m e . A f t e r an i n i t i a l l a g on the f i r s t day, t h e n e t w e i g h t i n c r e a s e became l o g a r i t h m i c f r om day two t o r e p l e t i o n , and t h u s r e a c h e d 75 t i m e s t h e u n f e d w e i g h t . S i n c e the m a j o r i t y o f u n f e d f e m a l e s i n t h i s s t u d y weighed 5-15 mg» t h e engor-ged w e i g h t s r a n g e d f r o m 400 t o 1200 mg (708 + 20, mean and S B ) . 2) T o t a l I n g e s t i o n and T o t a l L o s s e s As o u t l i n e d i n t h e I n t r o d u c t i o n and Methods s e c t i o n s , i t was p o s s i b l e t o use t h e amount of i r o n r e c o v e r e d f r o m t h e t i c k and i t s f e c e s as a measure o f t h e t o t a l amount of b l o o d removed f r o m t h e h o s t . F u r t h e r m o r e , t h e t o t a l w a t e r l o s s f r om a l l r o u t e s c o u l d be c a l c u l a t e d ( e q u a t i o n 1) knowing t h e hemo g l o b i n c o n c e n t r a -t i o n of t h e h o s t ' s b l o o d , t h e i n c r e a s e i n body, w e i g h t , and t h e 41 Figure 2. The net weight increase of a large, heterogeneous group of ticks from several feedings, expressed in terms of Log 1 0 of the weight ratio, Weug^°;e^nV^" plotted against time in days. o » females which had not yet copulated, A= females mated and having attained repletion. The curve ( f i t t e d by inspec-tion) represents the population whloh attained normal repletion in seven days. Vertical bars denote the standard error of the mean (SE) wherever the SE i s not included In the points them-selves. Figure 3» The daily production of feces (dry weight) expres-sed as a percentage of the total feces (dry weight) produced during the complete feeding period (i.e. the sum of a l l the points equals 1 0 0 $ ) • Vertical bars denote the SE of the mean wherever the SE i s not included in the points themselves. Days after start of feeding d r y w e i g h t o f the f e c e s d u r i n g t h e whole f e e d i n g p e r i o d . E x p e r i -m e n t a l l y d e t e r m i n e d v a l u e s f o r t h e l a t t e r f a c t o r s a r e shown i n T a b l e s I I I , I V , and V t o g e t h e r w i t h e s t i m a t e s of t o t a l f l u i d i n g e s t e d and l o s t d u r i n g f e e d i n g . TABLE I I I COMPOSITION OF BABBIT WHOLE VENOUS BLOOD Hemoglobin (mg/ml) Sodium ( m e q / l i t e r ) P o t a s s i u m C h l o r i d e ( m e q / l i t e r ) ( m e q / l i t e r ) Mean SE N* 146.5 10.6 4 100.6 2 . 3 11 42.7 1.2 16 8 8 . 5 3 . 6 8 * N i s t h e number of r a b b i t s i n sample. From each r a b b i t , two t o f i v e b l o o d samples were t a k e n , and t h e mean v a l u e of t h e s e samples was used as t h e v a l u e f o r t h e r a b b i t . TABLE IV TOTAL BLOOD ( i . e . MEAL) REMOVED FROM RABBIT T o t a l B l o o d Removed T o t a l Hemoglobin f r o m R a b b i t S e r i a l I n Homogenate of Whole T i c k I n F e c e s Removed f r o m R a b b i t (mg) (mg) (mg) (mg) (Ml ) * 1 1 7 5 1 9 5 370 2 5 3 0 2 3 9 8 2 3 0 1 522 8 2 3 5 6 3 0 5 3 3 6 3 361 247 608 4 1 6 5 39*1-8 4 1 9 3 2 7 7 4 7 0 3 2 2 0 3052 Assuming s p e c i f i c g r a v i t y of whole b l o o d = 1 . 0 5 5 ( L e e s , 1 9 46). TABLE V TOTAL WATER LOST BY THE TICK DURING FEEDING S e r i a l 1 2 3 4 Net Weight Increase (mg) 6 0 0 1 0 3 6 8 7 5 7 6 7 T o t a l Weight D r i e d Feces (mg) 288 6 5 1 3 8 0 3 1 1 Meal Intake T o t a l Water from Table IV Loss (mg) (mg) 2 5 3 0 1 6 4 2 5 6 3 0 3 9 4 3 4 1 6 5 2 9 1 0 3 2 2 0 2142 The r e s u l t s i n Tables IV and V I n d i c a t e that t i c k s removed c o n s i d e r a b l y more f l u i d from the host than they u l t i -mately r e t a i n e d at the t e r m i n a t i o n of f e e d i n g . On the average (Table V I ) , almost 80$ of the t o t a l meal int a k e was excreted by the time r e p l e t i o n was a t t a i n e d . Compared w i t h Rhodnius ( M a d d r e l l , 1964b), which normally excretes only 4 0 - 5 0 $ of the imbibed meal, the t o t a l e x c r e t i o n by Dermacentor i s co n s i d e r -a b l e . The f r a c t i o n s of the t o t a l meal which were voided as s o l i d s and water are shown i n Table V I . S e r i a l 1 2 3 4 Means SE TABLE VI FATE OF THE BLOOD MEAL AT END OF FEEDING P e r c e n t a g e of Meal R e t a i n e d by T i c k 2 3 . 7 18.4 21.0 23.8 21.5 1.2 P e r c e n t a g e o f Meal E x c r e t e d as S o l i d s i n Dry F e c e s 11.4 11.6 9.1 9.7 10.4 0.6 Percentage of Meal Excreted as Water 64.9 7 0 . 0 7 0 . 0 6 6 . 5 6 7 . 8 1.3 *5 3) Routes of Water Losst Quantitative Evaluation It Is clear that there are only three plausible routes for water loss to consider, namely, the anus, the general integumentary surface (including spiracles), and as suggested by Gregson (19^7) and Tatchell (1967b), the salivary glands. a) The integument Table VII and Figure 4A show water loss through the integument during engorgement. In Table VII,the results for 36 ticks were a r b i t r a r i l y divided into weight groups, and a mean weight loss per tick per day was tabulated for each group? from Figure 2, the approximate time that a tick spends in each weight range during feeding can be estimated. The sum of the products of the latter two values yields the total weight loss per tick over the entire feeding period. Over a seven-day normal feeding period, only ko pi of water are lost by the average tick via the integument. Even i f the tick remained attached for a further three days, losing water at the maximum rate measured (about 10 pi per tick per day), the integumentary loss over ten days would be only 70 ^ul, or less than 5% of the total water loss. It i s also worth re-emphasizing at this point that since the method does not distinguish metabolic from evaporation losses, this estimate is probably a maximum value for water loss via the integument.* *Mellanby (1935) points out that weight loss in such experiments do reflect the actual water loss, since In Clmex lectularlus the weight of carbon lost in CO2 equals the weight of metabolic water gained. It is possible that the same holds true for Dermacentor, although this has not yet been demonstrated. 46 TABLE V I I WEIGHT LOSS BY TICKS WITH MOUTH AND ANUS COVERED AT DIFFERENT PERIODS (BY WEIGHT) DURING FEEDING Weight Range of F e e d i n g T i c k s (mg) Number of O b s e r v a t i o n s Mean Weight L o s s P e r Day (mg) + SE Number of Days i n Range T o t a l Los; i n P e r i o d (mg) 0-20 7 0.45+0.27 H 0 . 6 8 20-40 3 1 . 8 4 + 0 . 0 9 1 1 . 8 4 40-100 12 5.91+0.83 H 8.87 100-200 9 8.03+1.23 1 8.03 2 0 0-repletion 5 1 0 .45+ 2 .64 2 20.91 T o t a l f o r whole f e e d i n g p e r i o d 40.33 A l t h o u g h i t a p p e a r e d t h a t h e a v i e r t i c k s l o s t w a t e r more r a p i d l y v i a t h e integument t h a n d i d s m a l l e r ones, i t was q u i t e p o s s i b l e t h a t t h i s s i m p l y r e f l e c t e d t h a t t h e t o t a l s u r f a c e a r e a o f t h e integument was g r e a t e r , r a t h e r t h a n t h a t t h e p e r m e a b i l i t y t o e f f l u x o f w a t e r had i n c r e a s e d . I t was of i n t e r e s t , t h e r e f o r e , t o c a l c u l a t e t h e w a t e r l o s s p e r u n i t s u r f a c e a r e a of integument t h r o u g h o u t the phases o f engorgement. F i g u r e 4 B shows t h a t the r a t e o f w a t e r l o s s p e r u n i t s u r f a c e a r e a (mg HgO/cm /day) a l m o s t d o u b l e d d u r i n g f e e d i n g compared t o t h e v a l u e f o r u n f e d t i c k s (.02 >P >.01). However, t i c k s w e i g h i n g o v e r 150 mg l o s t w a t e r a t a r a t e s i m i l a r t o t h a t f o r u n f e d t i c k s (P>0.5). b) Water L o s s f r o m t h e Anus and S a l i v a r y G l a n d s D i f f e r e n t i a t i n g between t h e q u a n t i t i e s o f f l u i d l o s t v i a th e anus and s a l i v a r y g l a n d s p r o v e d t o be d i f f i c u l t ; one cannot F i g u r e 4. Integumentary water l o s s . A) Water l o s s (mg HgO/tick/day) at d i f f e r e n t stages of fee d i n g i p l o t t e d a g a i n s t mean weight of each stage throughout the fee d i n g p e r i o d . V e r t i c a l and h o r i z o n t a l l i n e s denote the SE of the means. The temperature and r e l a t i v e humidity was tha t prevalent i n the fe e d i n g chamber on the back of the r a b b i t (approximately 33° C, 50$ RH). B) Water l o s s (mg H^O/cm t i c k surface/day) p l o t t e d a g a i n s t mean weight of the t i c k . A curve r e l a t i n g weight of t i c k t o corresponding surface area was drawn from a separate batch of t i c k s (see M a t e r i a l s and Methods f o r d e t a i l s ) and the f i g u r e s appearing i n 4 A were adjusted to c o r r e c t f o r surface area. V e r t i c a l l i n e s denote SE of mean water l o s s , and the width of each bar represents SE of the mean weight f o r each group, (o) = engorged t i c k s . 20 A Weight of engorging tick (mg) 49 c o l l e c t , by d i r e c t means, a l l the f l u i d p a s s i n g t h r o u g h the s a l i v a r y g l a n d s . I was a l s o u n a b l e t o c o l l e c t t h e f e c e s d i r e c t l y f r o m t h e anus w i t h o u t l o s i n g w a t e r by e v a p o r a t i o n . I t was f i n a l l y d e c i d e d t o t a k e advantage of i n f o r m a t i o n on s a l t c o n -c e n t r a t i o n s i n t h e f l u i d s o f the t i c k and h o s t w h i c h p e r m i t one t o c a l c u l a t e f l u i d l o s t v i a t h e s a l i v a r y g l a n d s . These c a l c u -l a t i o n s become p o s s i b l e due t o t h e f o r t u n a t e c i r c u m s t a n c e t h a t once s a l i v a t i o n has commenced, t h e sodium c o n c e n t r a t i o n o f s a l i v a c o l l e c t e d i n g l a s s c a p i l l a r i e s does n o t appear t o change w i t h s i z e o f t h e t i c k (see F i g u r e 6 ) ; t h e r e f o r e , t h e a s s u m p t i o n was made t h a t s a l i v a , e x c r e t e d n a t u r a l l y i n t o t h e h o s t , a l s o showed l i t t l e v a r i a t i o n i n sodium c o n c e n t r a t i o n w i t h t h e phase of engorgement. These c a l c u l a t i o n s a l s o r e q u i r e d v a l u e s f o r the t o t a l l o s s of sodium i n t h e f e c e s of the t i c k ; v a l u e s a r e p r e s e n t e d f o r f o u r i n d i v i d u a l f e m a l e s and f i v e males ( p o o l e d ) i n T a b l e V I I I . TABLE V I I I SODIUM CONTENT OF THE FECES (DRY WEIGHT) COLLECTED OVER THE TOTAL FEEDING PERIOD T o t a l D r y Weight of F e c e s Sodium C o n t e n t Sodium Concen-t r a t i o n of Dry F e c e s (meq/kg Dry Weight) S e r i a l (mg) i n T o t a l D r y F e c e s (ueq) 1 288 651 380 311 10.81 1 1 . 0 5 1 5 . 2 5 1 7 . 8 . 0 8 3 7 . 5 1 7 . 0 4 0 . 2 5 6 . 0 2 1 7 2 3 4 5 males 3 7 . 2 I t I s I n t e r e s t i n g t o n o t e i n p a s s i n g t h a t , based on d r y -w e i g h t measurements, th e a v e r a g e sodium c o n c e n t r a t i o n i n t h e f e c e s o f t h e f e m a l e i s about o n e - s i x t h t h a t i n the f e c e s of the male (38+8 meq Na /kg d r y f e c e s and 217 meq Na /kg d r y f e c e s , r e s p e c t i v e l y ) . A p o s s i b l e e x p l a n a t i o n f o r t h i s w i l l be o f f e r e d l a t e r . To p e r m i t t h e c a l c u l a t i o n of s a l i v a r y and f e c a l w a t e r l o s s , t h e f o l l o w i n g a d d i t i o n a l d a t a ( w i t h t h e i r s t a n d a r d e r r o r s ) were o b t a i n e d ! t h e c o n c e n t r a t i o n of sodium i n t i c k s a l i v a ( l 6 l + 3 m e q / l l t e r ) and the sodium c o n t e n t per mg u n f e d t i c k (0.085 + 0.007 u e q ) . U s i n g these d a t a , one can c a l c u l a t e the l o s s o f w a t e r by t h e s a l i v a r y g l a n d s a c c o r d i n g t o t h e f o l l o w i n g l i n e of r e a s o n i n g : 1) The t o t a l sodium i n g e s t e d equals t h e p r o d u c t of the i n g e s t e d volume of the meal ( T a b l e IV) and the c o n c e n t r a t i o n o f sodium i n t h e meal ( T a b l e I I I ) . 2) The sodium f r o m t h e meal n o t l o s t v i a t h e s a l i v a r y g l a n d s e q u a l s t h e n e t g a i n of sodium by t h e engorged t i c k ( T a b l e IX) p l u s t h e t o t a l sodium c o n t e n t of the f e c e s ( T a b l e V I I I ) . 3) T h e r e f o r e , t h e t o t a l sodium e x c r e t e d by t h e s a l i v a r y g l a n d s e q u a l s t h e d i f f e r e n c e between (1) and (2) above ( T a b l e I X ) . 4) The t o t a l volume of w a t e r l o s t I n the s a l i v a ( T a b l e IX) e q u a l s the t o t a l sodium e x c r e t e d by t h e g l a n d s d i v i d e d by t h e c o n c e n t r a t i o n o f sodium i n t h e s a l i v a . 5) S i n c e t h e t o t a l volume o f w a t e r l o s t v i a t h e integument i s l e s s t h a n 5% of t h e t o t a l w a t e r l o s s ( T a b l e V and T a b l e V I I ) , t h e n t h e f e c a l w a t e r l o s s e q u a l s t h e t o t a l w a t e r l o s s l e s s 51 t h e sum of t h e l o s s v i a the s a l i v a r y g l a n d s and integument, T a b l e IX p r e s e n t s t h e d a t a o b t a i n e d f o r each of t h e f o u r t i c k s i n t h e manner j u s t d e s c r i b e d above. TABLE IX (A) CALCULATION OF SALIVARY WATER LOSS S e r i a l T o t a l Na i n Homogenate of Engorged T i c k (ueq) Net Na R e t a i n e d by Engorged T i c k and I t s C u t i c l e (;ieq) Na' C o n t e n t of the Feces (p.eq; from T a b l e V I I ) 1 28.3 27.28 10.81 2 52.70 51*64 11.05 3 50.00 49.26 15.25 4 36.70 35.90 TABLE IX (E) 17.40 T o t a l N a + I n g e s t e d S e r i a l (/aeq) ,4. Na' L o s t V i a Volume of the S a l i v a r y S a l i v a G l a n d (pi) ipe<l) P e r c e n t a g e of T o t a l Water Loss as S a l i v a 1 239.8 201.71 1261 76.8 2 533-6 470.91 2943 74,6 i. ~> 394.8 330.29 2064 70.9 i. 305.2 .251.90 1574 73.5 Mean % 74.0 SE 1.2 I t i s now p o s s i b l e t o a p p r e c i a t e b o t h the a b i l i t y of the t i c k t o e x t r a c t w a t e r f r o m the meal, and the r o u t e s by whi c h t h i s w a t e r i s e l i m i n a t ed. Whereas Ixodes r i c i n u s can e x t r a c t 70$ of the wat e r i n the meal ( L e e s f 1946) and Rhodnius p r p l i x u s 52 75$ (Wigglesworth, 1 9 3 1 ) i Dermacentor andersonl can eliminate 8 0 - 9 0 $ of the imbibed water. About 5% of t h i s i s l o s t v i a the integument, about 75% v i a the s a l i v a r y glands, and 2 0 - 2 5 $ v i a the anus. I t i s s t i l l not known what proportion of f e c a l water comes through from the mid-gut, and what proportion ( i f any) i s secreted by the Malpighian tubules. 4) Routes of Ion Losst Quantitative Evaluation Since the concentration of sodium i n rabbit whole blood, the s a l i v a of the t i c k , and the sodium content of the body have been determined, i t i s possible to assess the r e l a t i v e importance of the s a l i v a and feces as routes of sodium excretion. I t Is possible to provide a s i m i l a r estimate f o r potassium, since the volume of s a l i v a , the potassium concentration of the s a l i v a , and the potassium content of the feces were determined (Tables X A and X B). Of the t o t a l ingested sodium, 89 + 0 . 6 $ (mean and SE) i s excreted, and of the t o t a l ingested potassium, 60 + 3$ i s excreted. However, whereas 96 + 0 . 6 $ of the excreted sodium i s l o s t i n the s a l i v a , only 18 + 0 . 7 $ of the excreted potassium i s l o s t i n the s a l i v a . Conversely, 82$ of the excreted potassium but only 4$ of the excreted sodium appears i n the feces. 5) The Excretion of Protein In the Feces A considerable portion of the ingested meal i s egested i n the feces i n the form of v i r t u a l l y unchanged hemoglobin (Balashov, 1 9 5 8 , 1 9 6 4 ) . Calculations from the f e c a l iron con-tent revealed that hemoglobin comprises 74 + 6$ (mean and SE) TABLE X A THE ROUTES OF EXCRETION FOR SODIUM s r l a l T o t a l Na T o t a l N a + % of Meal N a + Na Content o f S a l i v a Na C o n t e n t of F e c e s I n g e s t e d (;ueq) E x c r e t e d (,ueq) E x c r e t e d (^ueq) (as % of T o t a l E x c r e t e d ) (fieq) (as % of T o t a l E x c r e t e d ) 1 241 . 2 2 1 3 . 8 8 8 . 6 2 0 3 . 0 95 1 0 . 8 5 . 1 2 5 3 6 . 8 4 8 5 . 0 9 0 . 4 4 7 4 . 0 . 9 8 1 1 . 0 5 2 . 3 3 3 9 7 . 2 3 ^ 7 . 6 8 7 . 5 3 3 2 . 3 9 6 1 5 . 2 5 4 . 4 4 3 0 7 . 0 2 7 0 . 8 8 8 . 2 2 5 3 . ^ 94 17.*+ 6 . 4 TABLE X B THE ROUTES OF EXCRETION FOR POTASSIUM i r i a l T o t a l K + T o t a l K + % of Meal K + K + Content of S a l i v a + K C o n t e n t of F e c e s I n g e s t e d (jueq) E x c r e t e d (/ieq) . E x c r e t e d (/ieq) (as % of T o t a l E x c r e t e d ) tyieq) (as % of T o t a l E x c r e t e d ) 1 1 0 2 . 4 6 5 . 7 64 1 1 . 0 1 6 . 7 5k.7 8 3 . 3 2 2 2 7 . 8 1 ^ 9 . 3 66 2 5 . 6 17 . 2 1 2 3 . 7 8 2 . 9 3 1 6 8 . 6 9 0 . 2 5k 1 8 . 0 2 0 . 0 72 . 2 8 0 . 0 4 1 3 0 . 3 7 2 . 8 56 1 3 . 7 1 8 . 8 5 9 . 1 81 . 2 54 of t h e d r y w e i g h t of t h e f e c e s . That t h i s I r o n t r u l y r e p r e s e n t s h e m o g l o b i n and n o t t h e p o r p h y r i n m o e i t y a l o n e was shown I n s e v e r a l ways. ( 1 ) A l l b u t a s m a l l f r a c t i o n o f t h e f e c e s i s r e a d i l y s o l u b l e i n d i s t i l l e d w a t e r and i m p o r t s a c h a r a c t e r i s t i c r e d d i s h t i n g e t o t h e s o l u t i o n (see a l s o B a l a s h o v , 1 9 5 8 ) . Hemln, on t h e o t h e r hand, does n o t r e a d i l y d i s s o l v e i n w a t e r , and when i n a l k a l i n e s o l u t i o n , i s g r e e n i s h - b r o w n o r g r e e n i s h - b l a c k . ( 2 ) A s o l u t i o n o f f e c e s foams r e a d i l y when v i g o r o u s l y a g i t a t e d ; t h i s a l s o i n d i c a t e s a c o n s i d e r a b l e p r o t e i n c o n t e n t . Hemin s o l u t i o n s ( w h i c h l a c k p r o t e i n ) , and s o l u t i o n s of f e c e s f r o m f r e s h l y m o u l t e d a d u l t s do n o t foam. (3) The v i s i b l e s p e c t r u m of a f e c e s s o l u t i o n r e s e m b l e s v e r y c l o s e l y t h a t of pure hemo-g l o b i n , and i s d i s t i n c t l y d i f f e r e n t f r o m t h a t o f hemin, o r t h a t o f f e c e s f r o m f r e s h l y m o u l t e d a d u l t s ( F i g u r e 5 ) • W F i n a l l y , t h e method of Lowry e t a l . ( 1 9 5 1 ) r e v e a l e d an even h i g h e r p r o t e i n c o n t e n t i n t i c k f e c e s t h a n i n p u r i f i e d b o v i n e h e m o g l o b i n ( T a b l e X I ) . L i k e l y , t h e non-hemoglobin p o r t i o n of d r i e d f e c e s i s a l s o p r o t e i n a c e o u s i n n a t u r e - and p o s s i b l y c o n s i s t s o f plasma p r o t e i n s . TABLE X I PROTEIN CONTENT OF TICK FECES BY THE METHOD OF LOWRY ET AL. ( 1 9 5 D Sample C o n c e n t r a t i o n o f Sample (mg/ml) C o n c e n t r a t i o n of P r o t e i n (as mg/ml Bo v i n e Serum Albumin) T i c k F e c e s 1 2 . 1 5 24.10 1 5 . 5 8 13.0 2 6 . 7 14.0 T i c k F e c e s B o v i n e Hemoglobin (Sigma, t y p e I ) Figure 5« Visible spectra of rabbit hemoglobin, equine hemin, tick homogenate and tick feces. A l l spectra were traced from original records obtained from a chart recorder (Varicord Model kj, Photovolt Corp.) connected to a Bausch & Lomb *Spectronic 6 0 0 1 double-beam spectropho-tometer. a) A solution of hemoglobin in d i s t i l l e d water prepared from the erythrocytes of a rabbit (see Methods). b) A solution of equine hemin (Sigma, type III). A saturated solution i n d i s t i l l e d water at room temperature was diluted l t 3 in d i s t i l l e d water. c) Spectrum of a tick homogenate. The tick was homogenized in d i s t i l l e d water (see Methods), f i l t e r e d , and the resultant solution diluted in d i s t i l l e d water I1I8OO. d) Feces deposited by a feeding tick (2 mg dry feces per ml d i s t i l l e d water). e) An aqueous solution (unknown concentration) of tick feces deposited by a freshly moulted adult. Note the similarity of spectra 'c* and ,d» to that of hemo-globin (spectrum ,a ,)» In •d', the concentration was too high to show the Soret band (415 mu), but the peaks charac-t e r i s t i c of oxyhemoglobin ( 5 ^ 0 , 575 m>0 are quite clear. Also note the similarity between spectrum ,e» and that of hemin (spectrum , b t ) . Absorbance 5 ? 6) I o n i c and Osmotic Changes I n t h e Hemolymph  and S a l i v a D u r i n g a Normal F e e d i n g P e r i o d The p r e v i o u s o b s e r v a t i o n s d e m o n s t r a t e d t h a t the s a l i v a r y g l a n d s p r o v i d e t h e major r o u t e f o r e x c r e t i n g e x c e s s f l u i d f r o m the b l o o d m e a l . I t was t h e n d e c i d e d t o f o l l o w t h e i o n i c com-p o s i t i o n o f t h e hemolymph and s a l i v a d u r i n g t h e f e e d i n g p e r i o d w i t h t h e hope t h a t i t might p r o v i d e c l u e s as t o t h e mechanism of s a l i v a r y s e c r e t i o n . F o r example, Ramsay o b s e r v e d ( 1 9 5 3 ) t h a t t h e p r i m a r y f l u i d s e c r e t e d by t h e M a l p i g h i a n t u b u l e s of D i x l p p u s  morosus was c h a r a c t e r i s t i c a l l y h i g h i n p o t a s s i u m . T h i s s u g g e s t e d t h a t f l u i d s e c r e t i o n i t s e l f may be g e n e r a t e d by the a c t i v e t r a n s p o r t o f p o t a s s i u m . T h i s f e a t u r e I s common t o s e v e r a l i n s e c t s (Ramsay, 1 9 5 2 ; B e r r i d g e , 1 9 6 8 ; I r v i n e , 1 9 6 9 ) and i s p r o b a b l y due t o t h e e x i s t e n c e o f a s p e c i f i c pump f o r p o t a s s i u m . I n some M a l p i g h i a n t u b u l e s ( e . g . Rhoflnlus) t h e r e i s good e v i d e n c e f o r t h e a c t i v e t r a n s p o r t of p o t a s s i u m , sodium and c h l o r i d e ( M a d d r e l l , 1 9 6 9 ) . The heraacyte c o u n t of t h e hemolymph of most i n s e c t s i s u s u a l l y much l e s s t h a n 1 0 0 , 0 0 0 c e l l s / u l ( J o n e s , 1 9 6 4 ) . T h e r e -f o r e many i n v e s t i g a t o r s t e n d t o assume t h a t i o n c o n c e n t r a t i o n of whole hemolymph i s n e g l i g i b l y d i f f e r e n t from t h a t o f c e n t r i f u g e d hemolymph. T h i s a s s u m p t i o n i s based on the f o l l o w i n g r e a s o n i n g 1 A l t h o u g h d i f f e r e n c e s i n i o n c o n c e n t r a t i o n a c r o s s t h e hemacyte membrane may be c o n s i d e r a b l e , t h e c o n t r i b u t i o n of t h e hemacytes t o t h e t o t a l i o n i c p o o l of t h e hemolymph Is s m a l l due t o t h e e x t r e m e l y low h e m a t o c r i t s . T h e r e f o r e , o n l y minor d i f f e r e n c e s a t most s h o u l d o c c u r between the i o n c o n c e n t r a t i o n of whole hemo-58 lymph and t h a t of c e l l - f r e e "plasma". Brady (1967) however, showed a p o s i t i v e c o r r e l a t i o n between c e l l c o u n t and p o t a s s i u m c o n c e n t r a t i o n of whole b l o o d , w h i c h s u g g e s t e d t h a t i n P e r l p l a n e t a . t h e p o t a s s i u m c o n c e n t r a t i o n o f the hemacytes i s c o n s i d e r a b l y h i g h e r t h a n t h a t o f t h e p lasma. He a l s o showed a n e g a t i v e c o r -r e l a t i o n w i t h sodium c o n c e n t r a t i o n . The s i g n i f i c a n c e of t h i s f i n d i n g i s t h a t i n t h e c a s e of P e r l p l a n e t a up t o 50% of t h e hemolymph p o t a s s i u m i s bound up i n the hemacytes, and so does n o t c o n t r i b u t e t o t h e o s m o t i c p r e s s u r e o f p lasma. I n l i g h t o f t h e l a t t e r o b s e r v a t i o n s , some p r e l i m i n a r y e x p e r i m e n t s were r u n t o compare p o t a s s i u m c o n c e n t r a t i o n i n whole and c e n t r i f u g e d hemolymph. No c l e a r d i f f e r e n c e s c o u l d be d e t e c t e d between the two t r e a t m e n t s ( T a b l e X I I ) • M i c r o s c o p i c o b s e r v a t i o n c o n f i r m e d t h a t c e n t r i f u g a t i o n removed a l l t h e p a r t i c u l a t e m a t t e r f r o m t h e hemolymph. A p p a r e n t l y , o n l y a s m a l l p r o p o r t i o n of the t o t a l hemolymph p o t a s s i u m i s s e q u e s t e r e d by t h e hemacytes. I t was t h e r e f o r e d e c i d e d , t h a t any advantage t o be g a i n e d i n c e n t r i -f u g i n g t h e hemolymph p r i o r t o i o n d e t e r m i n a t i o n would l i k e l y be o f f s e t by an i n c r e a s e d e r r o r due t o e x c e s s i v e h a n d l i n g . A l l i o n d e t e r m i n a t i o n s o f hemolymph and s a l i v a were t h u s performed on whole s a m p l e s . The i o n c o n c e n t r a t i o n s of s a l i v a and hemolymph d u r i n g a n o r m a l f e e d i n g p e r i o d a r e shown i n F i g u r e 6. The hemolymph of t h e u n f e d t i c k has a f a i r l y h i g h i o n i c c o n t e n t (280 m e q / l i t e r Na), b u t t h i s f a l l s w i t h the p r o g r e s s i o n of f e e d i n g t o about 160 meq/ l i t e r on t h e t h i r d day ( F i g u r e 6A). T h i s l e v e l i s m a i n t a i n e d u n t i l r e p l e t i o n . L i k e w i s e , c h l o r i d e f a l l s f r o m 1?0 m e q / l i t e r i n i t i a l l y t o 1 2 5 m e q / l i t e r a f t e r f i v e days and t h e n remains r e l a t i v e l y c o n s t a n t ( F i g u r e 6 B ) . S i m i l a r l y , p o t a s s i u m f a l l s f r o m about 20 m e q / l i t e r t o 7 . 5 m e q / l i t e r ( F i g u r e 6C). Compari-sons of sodium and p o t a s s i u m c o n c e n t r a t i o n s i n s a l i v a and hemolymph o f i n d i v i d u a l t i c k s showed no s i g n i f i c a n t d i f f e r e n c e s ; t h e c h l o r i d e c o n c e n t r a t i o n of t h e s a l i v a , however, was about 10% h i g h e r t h a n t h a t of the hemolymph (P = 0 . 0 1 ; p a i r s t - t e s t , Simpson e_t a l . , i 9 6 0 ) o v e r t h e whole time c o u r s e of f e e d i n g . TABLE X I I POTASSIUM DISTRIBUTION IN TICK HEMOLYMPH a) U n p a i r e d samples (Whole and c e n t r i f u g e d samples t a k e n f r o m d i f f e r e n t t i c k s ) P o t a s s i u m C o n c e n t r a t i o n ( m e q / l i t e r ) Whole Hemolymph 1.4.3 5 . 1 1 3 . 8 1 7 . 2 C e n t r i f u g e d Hemolymph 1 1 . 5 1 2 . 4 1 5 . 2 2 2 . 9 Means 1 2 . 6 1 5 . 5 b) P a i r e d samples (Hemolymph from s e v e r a l t i c k s were p o o l e d under o i l . H a l f the p o o l was c e n t r i f u g e d , t h e o t h e r h a l f n o t . ) 2 . 4 4 0 . 0 1 9 . 6 2 . 2 5 0 . 2 6 8 . 0 Figure 6. Ion concentrations In hemolymph (| |) and saliva with time during the feeding period. The v e r t i c a l bars indicate the SE of the means. The concen-trations of sodium, chloride and potassium in the blood meal were 101, 89, and 43 meq/liter, respectively. Figure 7. Osmotic pressure in hemolymph and saliva with time during the feeding period. Paired readings of hemolymph (o ) and saliva ( ) on the same tick are Joined by straight lines. With one exception, the saliva was hypo-osmotic to the hemolymph in a l l cases. Although the osmotic pressure of whole rabbit blood was not measured cryoscopically, the osmotic pressure of human plasma is approx-imately 306 mOsm/liter (Ruch and Patton, 1965)• Tick weight range (mg) 600 i E in O E 500 A o o in in cu k_ Q. O O E w O 400 k 6 2 g o o o I 0 0 ° 300 4- — I 1 1 — 200 300 500 unfed i i 40 50 100 1000 Tick weight (mg) 63 The o s m o t i c p r e s s u r e ( F i g u r e 7) o f t h e hemolymph f e l l f r o m 527 + 21 m O s m / l i t e r (mean and SE) on t h e f i r s t day o f f e e d i n g and remained a t 375 + 6 m O s m / l i t e r t h r o u g h t h e r e m a i n d e r o f t h e f e e d i n g p e r i o d . The s a l i v a i s s l i g h t l y , but c o n s i s t e n t l y h y p o - o s m o t i c (P = 0.02) t o the hemolymph by about 5$ ( s a l i v a = 356 + k m O s m / l i t e r ) . I t i s i m m e d i a t e l y a p p a r e n t t h a t t h e m o l a r c o n c e n t r a t i o n s of t h e two c a t i o n s a r e n o t b a l a n c e d by t h e c o n c e n t r a t i o n o f c h l o r i d e ( T a b l e X I I I ) , so t h a t t h e r e i s a t l e a s t one o t h e r m a j o r a n i o n needed t o a c c o u n t f o r e l e c t r o - n e u t r a l i t y . The l i k e l y c a n d i d a t e s are» amino a c i d s ( h i g h i n some i n s e c t s ) , o r g a n i c a c i d s , i n o r g a n i c phosphate, and b i c a r b o n a t e ( t h e l a s t , h i g h i n h o s t t i s s u e f l u i d ) . D e t e r m i n a t i o n o f i n o r g a n i c phosphate i n t h e s a l i v a of f i v e f e m a l e s showed o n l y a t r a c e of t h i s i o n ( l e s s t h a n 1 m M / l i t e r ) . No I n o r g a n i c phosphate c o u l d be d e t e c t e d i n t h e hemolymph of t h r e e t i c k s . I t would be r e a s o n a b l e t o s u g g e s t t h a t some c o m b i n a t i o n of the r e m a i n i n g a n i o n s l i s t e d complement c h l o r i d e t o b a l a n c e the c o n c e n t r a t i o n o f sodium - t h e m a j o r c a t i o n i n t h e hemolymph and s a l i v a of Dermacentor. A summary o f t h e i o n i c and o s m o t i c p r e s s u r e d a t a a p p e a r s i n T a b l e X I I I . These d a t a r e v e a l t h a t once s a l i v a t i o n has commenced (day t h r e e o r f o u r ) 86$ o f t h e o s m o t i c p r e s s u r e of t h e hemolymph, and 88$ o f t h a t i n t h e s a l i v a can be a c c o u n t e d f o r by sodium, p o t a s s i u m , c h l o r i d e and t h e b a l a n c i n g a n i o n . The i o n i c c o m p o s i t i o n o f t h e d r y f e c e s i s q u i t e d i f f e r e n t f r o m t h a t o f the s a l i v a . T h i s r e f l e c t s t h e d i f f e r i n g i m p o r t a n c e of t h e s e r o u t e s i n t h e e x c r e t i o n o f sodium and p o t a s s i u m ( T a b l e X ) . The p o s s i b l e TABLE X I I I A ION DISTRIBUTION IN TICK BODY FLUIDS AND EXCRETA A l l samples were c o l l e c t e d f r o m t i c k s w h i c h had f e d f o r a t l e a s t t h r e e days ( i . e . a f t e r t h e e q u i l i b r a t i o n of t h e hemolymph t o i t s f i n a l c o m p o s i t i o n ) . Sample Sodium ( m e q / l i t e r ) P o t a s s i u m ( m e q / l i t e r ) C h l o r i d e ( m e q / l i t e r ) Osmotic P r e s s u r e ( m O s m / l i t e r ) Hemolymph Mean SE 162 2 7.5 0 . 4 1 2 4 2 376 6 S a l i v a Mean l 6 l 8 . 7 1 3 6 3 5 6 SE 3 0 . 8 2 4 t - P r o b a b l l i t y * P y0.5 0 . 5 > P > 0 . 1 P < 0 . 0 1 P = 0 . 0 2 F e c e s (meq/kg d r y w e i g h t ) Mean 38 190 SE 8 * The p r o b a b i l i t y t a k e n from t h e t a b l e of t - d l s t r l b u t i o n ( A r k i n and C o l t o n , 1 9 6 3 ) TABLE X I I I B SUMMARY OF ION DISTRIBUTIONS IN HEMOLYMPH AND SALIVA FROM TICKS WHICH HAD FED FOR AT LEAST THREE DAYS Only Mean V a l u e s A r e P r e s e n t e d Hemolymph S a l i v a 1 7 2 . 5 168.7 2 ) T o t a l measured a n i o n s ( C l ~ , PO^-, as m e q / l i t e r ) 124 1 3 6 3) Unknown a n i o n s t o a c c o u n t f o r e l e c t r o n e u t r a l l t y ( m e q / l i t e r ) 48.5 3 2 . 7 4) T o t a l o s m o t i c c o n c e n t r a t i o n as m e q / l i t e r N a C l * 2 0 2 191 5) P e r c e n t a g e o f t o t a l o s m o t i c c o n c e n t r a t i o n w h i c h i s a c c o u n t e d f o r by d e t e r m i n e d i n o r g a n i c c a t i o n s 8 5 . 5 8 8 . 3 6) P e r c e n t a g e o f t o t a l o s m o t i c c o n c e n t r a t i o n w h i c h i s c h l o r i d e s a l t 61.4 7 1 . 2 * T h i s i s t h e e q u i v a l e n t c o n c e n t r a t i o n of pure NaCl w h i c h l o w e r s t h e f r e e z i n g p o i n t o f w a t e r t o t h e same v a l u e as do the s o l u t e s i n t h e hemolymph and s a l i v a . A l t h o u g h the o s m o t i c c o e f f i c i e n t of each s o l u t e i n mixed s o l u t i o n i s not e x a c t l y t h e same as the c o e f f i c i e n t i n pure s o l u t i o n , t h i s f a c t o r i s n e g l e c t e d i n making the c a l c u l a t i o n . 1) T o t a l measured c a t i o n s (Na , K , Mg , as m e q / l i t e r ) 66 mechanisms g o v e r n i n g t h e s e d i f f e r e n c e s w i l l be examined i n t h e d i s c u s s i o n . 7) The C l e a r a n c e o f Two Non-charged M o l e c u l e s a) I n u l l n 14 There was no d e t e c t a b l e C a c t i v i t y i n any of t h e s a l i v a samples s e c r e t e d by t h e s i x t i c k s i n t h i s e x p e r i m e n t , a l t h o u g h t h e hemolymph samples o b t a i n e d f r o m t h e s e t i c k s a t t h e same time e l i c i t e d c o u n t s o f 5 0 0 0 t o 4 , 0 0 0 cpm/yul. C l e a r l y , i n u l l n i s n o t s e c r e t e d by t h e s a l i v a r y g l a n d . The a c t i v i t y i n t h e f e c e s ( c o l l e c t e d j u s t p r i o r t o t a k i n g f l u i d samples) was 310 cpm/mg d r y w e i g h t . A p p a r e n t l y i n u l l n I s o n l y s l o w l y e l i m i n a t e d f r o m t h e hemolymph - e i t h e r v i a t h e M a l p i g h i a n t u b u l e - r e c t a l sac s y s t e m , o r perhaps s i m p l y by p a s s i v e t r a n s p o r t i n t o t h e midgut lumen. b) 3-Q- 1* tCH 3-D-Glucose T h i s m o l e c u l e was c l e a r e d f r o m t h e hemolymph so t h a t o n l y l o w c o u n t s were r e c o v e r e d f r o m t h e t i c k s w h i c h were r e t u r n e d t o t h e r a b b i t f o r a day a f t e r i n j e c t i o n . The S/H ( s a l i v a t o hemo-lymph) r a t i o a t t h i s t i m e was 0 . 1 8 3 ± 0 . 0 3 3 (mean + SE; T a b l e X I V ) . The f e c e s c o l l e c t e d J u s t p r i o r t o t a k i n g t h e f l u i d samples f r o m t h e t i c k s l i s t e d i n T a b l e X I V , e l i c i t e d an a v e r a g e count o f 851 + 1 6 0 cpm/mg d r y w e i g h t (mean + S E ) . From the c a l c u l a t e d w a t e r c o n t e n t of f e c e s ( 5 7 $ )> t h i s v a l u e can be c o n v e r t e d t o an e s t i m a t e d 402 cpm/ul wet f e c e s . The average a c t i v i t y o f hemo-lymph and s a l i v a a t t h e same time were 2 5 3 ± 95 cpm/^1 and 38 + 18 cpm/yul, r e s p e c t i v e l y . TABLE XIV THE CLEARANCE OF O-METHYLGLUCOSE 1 4 C - A c t i v i t y (cpm/ul) S e r i a l Hemolymph S a l i v a S/H R a t i o 1 2 9 3 37 0 . 1 2 6 2 48 17 0 . 3 5 ^ 3 82 12 0.146 4 92 2 5 0 . 2 7 2 5 5 5 3 2 1 0 . 0 3 8 6 110 11 0 . 1 0 0 7 7 6 5 1 6 5 0 . 2 1 6 8 81 17 0 . 2 1 0 Mean 2 5 3 38 0 . 1 8 3 SE 9 5 18 0 . 0 3 3 8) Hemolymph Volume I n u l i n was c o n s i d e r e d t o be a s u i t a b l e s u b s t a n c e f o r me a s u r i n g t h e hemolymph volumes o f t i c k s a t v a r i o u s s t a g e s of engorgement, s i n c e i t was n o t s e c r e t e d i n the s a l i v a , and i t i s n e i t h e r m e t a b o l i z e d n o r known t o be m a l e f l c i e n t when i n j e c t e d i n t o t h e b l o o d o r hemolymph o f many o r g a n i s m s . I n u l i n remains i n t h e e x t r a c e l l u l a r f l u i d space o f s e v e r a l i n s e c t s (Levenbook, 1 9 5 8 ; Loughton and Tobe, 1 9 6 9 ) . One Important p o s s i b i l i t y t o c o n s i d e r was t h a t i n u l i n might have d i f f u s e d i n t o a l a r g e compartment ( s u c h as t h e g u t ) , r e s u l t i n g i n an o v e r e s t i m a t e of hemolymph volume. However, t h e r e l a t i v e l y low a c t i v i t y i n the f e c e s of t i c k s w h i c h were i n j e c t e d w i t h t r a c e r t h e day b e f o r e (page 66), suggested that the gut epithelium was relatively-impermeable to Inulin. The hemolymph volume increased linearly with increasing weight of the tick, and always comprised about 23$ of the body weight (Figure 8 ). Apparently, the tick does not regulate the hemolymph volume to a constant value. 69 Figure 8. Volume of hemolymph with the progression of feeding. Hemolymph volumes were determined from the dilution of injected 14-Inulin - C (see text). The regression curve shows the best straight line through the points. The regression line was determined by the method of least squares and i t s equation i s Y = - 0 . 5 5 + 0 . 2 3 X 150 O 700 Tick weight (mg) 71 DISCUSSION 1) Net Weight I n c r e a s e of t h e Females The p a t t e r n of n e t w e i g h t i n c r e a s e o b s e r v e d i n t h i s study-was i n es s e n c e t h e same as t h a t r e p o r t e d e a r l i e r by Gregson ( 1 9 5 7 ) f o r Dermacentor and by Lees ( 1 9 5 1 ) and Snow ( 1 9 7 0 ) f o r o t h e r i x o d i d t i c k s . The n e t w e i g h t i n c r e a s e o f Dermacentor f e m a l e s a l s o o c c u r s i n two s t a g e s . An i n i t i a l p e r i o d w h i c h l a s t s f r o m f o u r t o s i x days r e p r e s e n t s t h e s l o w phase of g r o w t h , d u r i n g w h i c h t i m e t h e t i c k s I n c r e a s e t h e i r w e i g h t 2 0 - t o 3 0 - f o l d . T h i s i s f o l l o w e d , subsequent t o c o p u l a t i o n , by a r a p i d phase of n e t w e i g h t i n c r e a s e w h i c h n o r m a l l y l a s t s one day. D u r i n g t h i s p e r i o d , t h e t i c k s may undergo a f u r t h e r t r i p l i n g i n w e i g h t . A t r e p l e t i o n , t h e a v e r a g e t i c k weighs 75 t i m e s as much as u n f e d a n i m a l s . C o r -r e l a t e d w i t h t h i s r a p i d i n c r e a s e i s a marked d e c l i n e i n d e f e c a t i o n and s a l i v a t i o n . W i t h t h i s i n mind, i t i s s t i l l p r o b l e m a t i c a l w h e t h e r o r n o t t h e r e m a r k a b l e n e t w e i g h t i n c r e a s e o f t h e femal e i n t h e f i n a l s t a g e o f f e e d i n g i s a c c o u n t e d f o r s o l e l y by an i n c r e a s e d r a t e of i m b i b i t i o n . To answer t h i s , one wou l d have t o e s t i m a t e t h e d a l l y s a l i v a r y l o s s and t h e d a l l y f e c a l w a t e r l o s s . To a t t a i n t h e s e f i g u r e s would r e q u i r e r e p e t i t i o n of t h e e x p e r i -ments o u t l i n e d i n T a b l e s I V , V, V I I I and IX f o r s e v e r a l w e i g h t r a n g e s o f t h e f e e d i n g p e r i o d . 2) T o t a l I n g e s t i o n and T o t a l L o s s e s S i n c e s e v e r a l a s s u m p t i o n s were made i n t h e Methods, e s t i m a t e s of t o t a l i n g e s t i o n and t o t a l l o s s of w a t e r a r e s u b j e c t t o c e r t a i n e r r o r s . Some of t h e s e e r r o r s ( e . g . r e c o v -e r y o f s o l u t e i n an a s h i n g p r o c e d u r e ) can be a c c o u n t e d f o r . H o w e v e r , i t was assumed i n t h e c a l c u l a t i o n s t h a t the meal i s b l o o d f r o m f a i r l y l a r g e b l o o d v e s s e l s ; t h i s i n f a c t i s n o t s o . There i s r e a s o n a b l y good e v i d e n c e t h a t t h e meal comes f r o m c a p i l l a r y beds i n t h e s k i n ( Gregson, I960, 1967). The h e m a t o c r i t , and hence hem o g l o b i n c o n c e n t r a t i o n , i n f i n e c a p i l l a r i e s i s about 96$ t h a t o f venous b l o o d (Guyton, 1966). A l s o , t h i s b l o o d i s r e l e a s e d i n t o a s u b - e p i d e r m a l p o o l t h r o u g h m e c h a n i c a l and c h e m i c a l i n j u r y t o th e c a p i l l a r y w a l l s b e f o r e I m b i b i t i o n ( G r e g s o n , i960, 1967). C o n s e q u e n t l y , a t l e a s t d u r i n g t h e e a r l y s t a g e s of f e e d i n g , t h e b l o o d i s mixed w i t h an unknown amount of t i s s u e f l u i d b e f o r e b e i n g t a k e n by th e t i c k ( S u t t o n and A r t h u r , 19625 S e i f e r t e t a l . , 1 9 6 8 ; Snow, 1 9 7 0 ) ; as a r e s u l t of t h i s , t h e he m o g l o b i n c o n -c e n t r a t i o n o f th e a c t u a l meal i s l i k e l y t o be l o w e r t h a n t h a t o f whole venous b l o o d . A l t h o u g h plasma skimming by t h e t i c k d u r i n g i m b i b i t i o n was n o t d e t e r m i n e d , i t can be shown t h a t as th e plasma p o r t i o n o f th e meal i n c r e a s e s r e l a t i v e t o th e e r y t h r o c y t e p o r t i o n , t h e c a l c u l a t e d s a l i v a volume a l s o i n c r e a s e s . Thus the p e r c e n t a g e s o f s a l i v a r y w a t e r l o s s p r e s e n t e d i n T a b l e IX B 73 a r e p r o b a b l y minimum v a l u e s . N e v e r t h e l e s s , i t w o u l d be d e s i r a b l e t o have an i n d e -pendent measure of s a l i v a r y w a t e r l o s s and a n a l w a t e r l o s s . A t t h e moment I cannot s u g g e s t a p r a c t i c a l method f o r m e a s u r i n g s a l i v a r y w a t e r l o s s d i r e c t l y . Ramsay (1964) d e v i s e d a g r a v i m e t r i c method f o r m e a s u r i n g w a t e r c o n t e n t o f T e n e b r l o f e c e s . S i n c e , however, Dermacentor f e c e s a r e l i q u i d , and t e n d t o s t i c k t o t h e p e r i a n a l a r e a u n t i l f o r c e d away by oncoming f r e s h f e c e s o r by o t h e r m e c h a n i c a l means, i t i s u n l i k e l y t h a t Ramsay's method c o u l d be a d o p t e d f o r Dermacentor. I n a d d i t i o n , Dermacentor c e a s e s t o v o i d f e c e s as soon as i t i s removed f r o m the h o s t . One would have t o c o l l e c t f e c e s f r o m a t i c k w h i c h i s a t t a c h e d , and p r o t e c t t h e f e c e s f r o m e v a p o r a t i o n . 3) I o n C o m p o s i t i o n o f the F e c e s a) Comparison between males and f e m a l e s On a d r y w e i g h t b a s i s , t h e sodium c o n c e n t r a t i o n i n male f e c e s was s i x t i m e s t h a t i n f e m a l e f e c e s . T h i s f i n d i n g i n d i c a t e s t h a t t h e males were e l i m i n a t i n g s a l t and w a t e r somewhat d i f f e r -e n t l y f r o m t h e f e m a l e s . A l t h o u g h t h e r e can be l i t t l e doubt now t h a t t h e s a l i v a i s i m p o r t a n t f o r w a t e r r e g u l a t i o n i n t h e f e m a l e of s e v e r a l i x o d i d t i c k s , f o r a v a r i e t y o f r e a s o n s , i t has n o t y e t been n e c e s s a r y t o p o s t u l a t e t h e same f o r t h e male. F i r s t , t h e growth o f t h e s a l i v a r y g l a n d s d u r i n g t h e f e e d i n g p e r i o d i s n o t as marked i n t h e male as i n t h e f e m a l e ( T i l l , 1961; C h i n e r y , 74 1965). S e c o n d l y , t h e male Imbibes o n l y a modest amount of b l o o d , and so i t i s n o t f a c e d w i t h t h e t a s k o f e x c r e t i n g l a r g e volumes of e x c e s s f l u i d . F i n a l l y , t h e p a r a l y t i c f a c t o r (most p r o b a b l y c a r r i e d i n t h e s a l i v a o f t h e f e m a l e ) i s n o t known t o be t r a n s -m i t t e d by t h e male. One would s u s p e c t t h e n , t h a t a n a l and i n t e g u m e n t a r y w a t e r l o s s m ight s u f f i c e t o a c c o u n t f o r osmoregu-l a t i o n i n t h e male. A l t h o u g h t h e sodium c o n c e n t r a t i o n i n male f e c e s i s so much h i g h e r t h a n t h a t i n f e m a l e f e c e s on a d r y w e i g h t b a s i s , t h i s need n o t be as e x a g g e r a t e d f o r wet f e c e s . I f t h e w a t e r c o n c e n t r a t i o n o f male f e c e s was a l s o h i g h e r t h a n t h a t i n f e m a l e f e c e s , t h i s f a c t a l o n e w o u l d a c c o u n t f o r a h i g h e r sodium c o n c e n t r a t i o n i n d r y male f e c e s . Moreover, th e h i g h e r w a t e r c o n c e n t r a t i o n of male f e c e s w o u l d l e n d s u p p o r t t o t h e h y p o t h e s i s t h a t a n a l w a t e r l o s s i s r e l a t i v e l y more i m p o r t a n t i n t h e male t h a n i n t h e f e m a l e . I n t h e f i n a l a n a l y s i s , r e g a r d l e s s how t h e h i g h sodium c o n c e n t r a t i o n i n male d r y f e c e s i s e x p l a i n e d , t h e r e s u l t s do s u g g e s t t h a t t h e male and f e m a l e a r e h a n d l i n g t h e i r w a t e r and Ions by d i f f e r e n t mechanisms. b) The f e c e s as a r o u t e o f i o n e x c r e t i o n i n t h e f e m a l e The f e m a l e t i c k e x c r e t e s Ions v i a b o t h t h e anus and t h e s a l i v a r y g l a n d s ; however, i t i s c l e a r f r o m T a b l e X, t h a t about 95$ of t h e e x c r e t e d sodium i s l o s t I n t h e s a l i v a , whereas 80$ of t h e e x c r e t e d p o t a s s i u m i s l o s t i n t h e f e c e s . From the meager e v i d e n c e a v a i l a b l e f o r c h l o r i d e , I t would appear t h a t i t i s e x c r e t e d i n a manner s i m i l a r t o sodium. How c o u l d one e x p l a i n t h e d i f f e r e n c e s between sodium and p o tassium? The s i m p l e s t 75 e x p l a n a t i o n t a k e s i n t o a c c o u n t t h a t a major p o r t i o n of t h e f e c e s i s d e r i v e d f r o m h o s t b l o o d p a s s i n g i n t o t h e r e c t a l s a c f r o m t h e midgut. I f t h e midgut e p i t h e l i u m can t r a n s p o r t sodium, c h l o r i d e , and w a t e r i n t o t h e hemolymph, b u t i s r e l a t i v e l y impermeable t o p o t a s s i u m , t h i s w o u l d r e s u l t i n th e o b s e r v e d h i g h p o t a s s i u m c o n t e n t and low sodium c o n t e n t i n t h e rem a i n s of t h e meal e n t e r i n g the r e c t a l s a c . There i s some e v i d e n c e f o r t h i s h y p o t h e s i s . The e q u i l i b r a t e d c o n c e n t r a t i o n o f sodium i n t h e hemolymph (160 m e q / l i t e r ) i s somewhat h i g h e r t h a n t h e c o n c e n t r a t i o n of sodium i n t h e meal (100 m e q / l i t e r ) . The e q u i l i b r a t e d c o n c e n t r a t i o n of c h l o r i d e i n t h e hemolymph (125 m e q / l i t e r ) i s a l s o h i g h e r t h a n t h e c h l o r i d e c o n c e n t r a t i o n i n t h e meal (90 m e q / l i t e r ) . However, t h e e q u i l i b r a t e d c o n c e n t r a t i o n o f p o t a s s i u m i n the hemolymph (7.5 m e q / l i t e r ) i s c o n s i d e r a b l y l e s s t h a n t h a t i n t h e meal (42 m e q / l i t e r ) . These f i g u r e s i n d i c a t e t h a t t h e g u t e p i t h e -l i u m i s t r a n s p o r t i n g sodium and c h l o r i d e , b u t r e l a t i v e l y l i t t l e p o t a s s i u m i n t o t h e hemolymph. The i m p e r m e a b i l i t y of th e g u t e p i t h e l i u m t o p o t a s s i u m has a l s o been s u g g e s t e d f o r O r n l t h o d o r o s moubata (Kaufman, 1971)• A l t e r n a t i v e l y , one cannot r u l e out a n o t h e r p l a u s i b l e e x p l a n a t i o n f o r i o n s e c r e t i o n . S i n c e p o t a s s i u m i s t h e major c a t i o n i n t h e M a l p i g h i a n t u b u l e s e c r e t i o n of s e v e r a l i n s e c t s (Ramsay, 1953; B e r r l d g e , 1968; I r v i n e , 1969)• p o t a s s i u m a b s o r b e d f r o m the midgut might a l s o be r a p i d l y s e c r e t e d by the M a l p i g h i a n 76 t u b u l e s o f the t i c k . R e a b s o r p t i o n o f p o t a s s i u m i n t h e r e c t a l sac w o u l d have t o be l o w e r t h a n i n i n s e c t s s t u d i e d t o d a t e . The ne t r e s u l t w o u l d be a low p o t a s s i u m c o n c e n t r a t i o n I n t h e hemo-lymph and a h i g h c o n c e n t r a t i o n i n t h e f e c e s . A l t h o u g h t h e r e i s no c l e a r - c u t e v i d e n c e w h i c h opposes t h e l a t t e r mechanism, t h e r e a r e some f a c t s a v a i l a b l e w h i c h make I t l e s s a t t r a c t i v e t h a n t h e f i r s t e x p l a n a t i o n . B o t h B a l a s h o v ( 1 9 5 8 ) and T i l l ( 1 9 6 1 ) , on t h e b a s i s of h i s t o l o g i c a l i n v e s t i g a t i o n s , r e p o r t t h a t t h e M a l p i g h i a n t u b u l e s do n o t become v e r y a c t i v e u n t i l a f t e r t h e t i c k has d e t a c h e d f r o m the h o s t . A c c u m u l a t i o n of most o f t h e g u a n i n e i n t h e M a l p i g h i a n t u b u l e s and r e c t a l sac o c c u r s a t t h a t t i m e , and i s p r o b a b l y c o r r e l a t e d w i t h egg matura-t i o n . I have o b s e r v e d t h e l a t t e r i n Dermaoentor as w e l l . I t w o u l d , however, be d e s i r a b l e t o d e t e r m i n e I n d e p e n d e n t l y the q u a n t i t y o f f l u i d and p o t a s s i u m s e c r e t e d by t h e M a l p i g h i a n t u b u l e s d u r i n g t h e f e e d i n g p e r i o d . 4) I n t e g u m e n t a r y Water L o s s Lees ( 1 9 ^ 7 ) r e p o r t e d t h a t t h e w a t e r l o s s p e r u n i t s u r f a c e a r e a f r o m t h e c u t i c l e o f engorged Dermacentor a n d e r s o n i f e m a l e s was about s i x and o n e - h a l f t i m e s t h a t o f u n f e d i n d i v i d u a l s . I n my e x p e r i m e n t s , t h e w a t e r l o s s p e r u n i t s u r f a c e a r e a i n c r e a s e d o n l y about t w o - f o l d i n f e e d i n g f e m a l e s (50 t o 150 mg), b u t f e l l a g a i n i n engorged f e m a l e s t o a l e v e l s i m i l a r t o t h a t f o r u n f e d f e m a l e s ( F i g u r e 4B). The o t h e r a p p a r e n t d i f f e r e n c e f r o m the r e s u l t s of Lees i s t h e magnitude of t h e a c t u a l f i g u r e s f o r w a t e r l o s s . Lees r e p o r t e d a l o s s o f 0 . 2 6 mg RgO/cm /day f o r u n f e d f e m a l e s a t 2 5 ° C i n d r y a i r . I n my e x p e r i m e n t s , u n f e d f e m a l e s l o s t about 4 mg HgO/cm /day a t about 3 3 ° C and about 50$ RH. F o r engorged t i c k s , Lees r e p o r t e d w a t e r l o s s t o be 1.7 mg HgO/ 2 2 cm /day and I fo u n d i t t o be 4 - 5 mg HgO/cm /day. These d i f -f e r e n c e s a r e t o o g r e a t t o be a c c o u n t e d f o r s o l e l y by t h e d i f -f e r i n g e x p e r i m e n t a l c o n d i t i o n s of t e m p e r a t u r e and r e l a t i v e h u m i d i t y . I t i s most l i k e l y t h a t t h e y stem f r o m t h e f a c t t h a t Lees c o v e r e d t h e s p i r a c l e s w i t h a c e l l u l o s e p a i n t , whereas I d i d n o t . A l s o , Lees measured w e i g h t l o s s o v e r 3 0-minute i n t e r v a l s whereas I l e f t t h e t i c k s u n d i s t u r b e d f o r 24 h o u r s a t a t i m e . M e t a b o l i c l o s s of w e i g h t a t 3 3 ° C o v e r 24 hours would g r e a t l y e x c e e d t h a t a t 2 5 ° C o v e r 30 m i n u t e s . I n s p i t e of t h e s e p o t e n -t i a l f a c t o r s , t h e p r e s e n t e x p e r i m e n t s c l e a r l y e s t a b l i s h e d t h a t w a t e r l o s s v i a t h e c u t i c l e and s p i r a c l e s r e p r e s e n t s a n e g l i g i b l e p r o p o r t i o n o f t h e t o t a l w a t e r l o s s . B e l o z e r o v (1967) r e p o r t e d r e s u l t s f o r I x o d e s r i c l n u s w h i c h a p p e a r t o be a t l e a s t q u a l i t a t i v e l y s i m i l a r t o t h o s e f o r D e r m a c e n t o r . Water l o s s f r o m t h e Integument i n c r e a s e d d u r i n g t h e e a r l y s t a g e s of f e e d i n g , but became m i n i m a l a g a i n p r i o r t o detachment. B e l o z e r o v i n t e r p r e t e d t h e s e r e s u l t s as i n d i c a t i n g p e r m e a b i l i t y changes i n t h e c u t i c l e i t s e l f } however, he does n o t m e n t i o n e i t h e r c o v e r i n g t h e s p i r a c l e s o r c o r r e c t i n g h i s f i g u r e s f o r u n i t s u r f a c e a r e a as Lees d i d . Lees does n o t ment i o n an a c t u a l r e d u c t i o n i n c u t i c u l a r p e r m e a b i l i t y j u s t p r i o r t o d e t a c h -ment o f t h e t i c k , b ut t h i s i s p r o b a b l y because he d i d n o t examine i n d i v i d u a l s between t h e u n f e d and engorged s t a g e s . I n c o n c l u s i o n , a l t h o u g h B e l o z e r o v ' s and my f i g u r e s may be u s e f u l i n c l a r i f y i n g t h e c o n t r i b u t i o n o f the Integument i n w a t e r r e g u l a t i o n , c a u t i o n must be e x e r t e d i n d e c i d i n g whether p h y s i c a l 78 o r p h y s i o l o g i c a l changes a r e p r i m a r i l y r e s p o n s i b l e f o r t h e o b s e r v a t i o n s . Lees (19^7) and Beament (1958) worked p r i m a r i l y w i t h dead i n d i v i d u a l s , and o c c l u d e d t h e s p i r a c l e s b e f o r e d e t e r -m i n i n g w a t e r l o s s j c o n s e q u e n t l y , t h e y were a b l e t o c h a r a c t e r i z e t h e p u r e l y p h y s i c a l changes i n t h e c u t i c l e ( t h e wax l a y e r i n p a r t i c u l a r ) as d i s t i n c t f r o m any p h y s i o l o g i c a l r e s p o n s e s of the e x p e r i m e n t a l a n i m a l s . S i n c e B e l o z e r o v and I worked w i t h l i v i n g t i c k s , and I a t l e a s t , w i t h n o n - o c c l u d e d s p i r a c l e s , c l o s e a g r e e -ment between o ur c o n c l u s i o n s and th o s e o f Lees and Beament i s no t w a r r a n t e d . 5) C o m p o s i t i o n o f Hemolymph and  S a l i v a i n F e e d i n g Females There i s c o n s i d e r a b l e i n f o r m a t i o n a v a i l a b l e on the composi-t i o n o f i n s e c t hemolymph w i t h w h i c h t o compare the p r e s e n t r e s u l t s . I n a d d i t i o n , t h e r e have been s e v e r a l a u t h o r s who have examined i o n i c r e g u l a t i o n i n t i c k s . A s i g n i f i c a n t o b s e r v a t i o n i n th e p r e s e n t s t u d y was t h a t d e s p i t e t h e heavy t r a f f i c of i o n s and w a t e r t h r o u g h t h e hemolymph, t h e i o n c o n c e n t r a t i o n s of hemolymph and s a l i v a r e m a i n e d c o n s t a n t a f t e r t h e f i r s t t h r e e days of f e e d i n g . The c o m p o s i t i o n o f t h i s " e q u i l i b r a t e d " hemolymph (and s a l i v a ) i n Dermacentor i s t y p i c a l of t h a t i n most a r t h r o p o d s and p r i m i t i v e i n s e c t s , i n t h a t t h e predominant i o n s a r e sodium and c h l o r i d e ( F l o r k l n and J e u n i a u x , 1964). T h i s i s n ot s u r p r i s i n g c o n s i d e r i n g t h a t t h e d i e t of t h e t i c k a l s o c o n t a i n s an abundance of t h e s e i o n s . L i k e i n s e c t s , the o s m o t i c p r e s s u r e of Dermacentor hemo-lymph i s s l i g h t l y h i g h e r t h a n t h a t of mammals. I n t h e hemolymph of Dermacentor, the c o n c e n t r a t i o n of c h l o r i d e i s n o t h i g h enough t o b a l a n c e t h e c o n c e n t r a t i o n of sodium and p o t a s s i u m . F l o r k i n and J e u n i a u x (1964) p o i n t out t h a t t h e major a n i o n i c components i n a l l i n s e c t s a r e r e p r e s e n t e d by v a r y i n g c o m b i n a t i o n s o f c h l o r i d e , amino a c i d s , i n o r g a n i c p h o s p h a t e , and b i c a r b o n a t e . However, i n o r g a n i c phosphate c o u l d n o t be d e t e c t e d i n t h e hemolymph of Dermacentor. I n o t h e r t i c k s , the t o t a l amino a c i d c o n c e n t r a t i o n i n the hemolymph i s g e n e r a l l y t h e o r d e r o f a few m i l l i m o l a r (6.3 m M / l i t e r i n B o o p h l l u s m i c r o -p l u s - T a t c h e l l , 1969; 1.74 m M / l i t e r i n A r g a s l a g e n o p l a s t l s -Rehacek and B r z o s t o w s k i , 1969? about 2 m M / l i t e r i n O r n l t h o d o r o s  moubata - Kaufman, 1971)• I f t h e same h o l d s t r u e f o r Dermacentor. and even i f a l l the amino a c i d s were i n a n i o n i c f o r m , t h i s o r d e r o f c o n c e n t r a t i o n w o u l d n o t be n e a r l y h i g h enough t o a c c o u n t f o r e l e c t r o n e u t r a l l t y (see T a b l e X I I I B ) . Of c o u r s e , t h e p o s s i b i l i t y t h a t Dermacentor i s a t y p i c a l i n terms of i t s hemolymph amino a c i d c o n c e n t r a t i o n , o r t h a t i t may c o n t a i n i n i t s hemolymph a c o n s i d -e r a b l e c o n c e n t r a t i o n o f o t h e r u n u s u a l a n i o n s ( e . g . s u l p h a t e , o r g a n i c a c i d s , e t c . ) i s one t h a t must n o t be r u l e d o u t . N ever-t h e l e s s , i t a p p e a r s r e a s o n a b l e t o s u g g e s t t h a t b i c a r b o n a t e may e x i s t i n s i g n i f i c a n t c o n c e n t r a t i o n i n t h e hemolymph, s i n c e t h i s a n i o n i s abundant i n t h e meal. On the b a s i s of a s i m i l a r a r g u -ment, Burgen (1967) s u g g e s t e d t h a t b i c a r b o n a t e may a c c o u n t f o r t h e 73 m e q / l i t e r o f non-determined a n i o n s i n t h e s u b m a x i l l a r y g l a n d o f immature r a t s . Kaufman (1971) drew up a b a l a n c e s h e e t of i n g e s t i o n and e x c r e t i o n of c h l o r i d e f o r O r n l t h o d o r o s moubata, and was a b l e t o t r a c e t h e f a t e of t h i s i o n a f t e r i n g e s t i o n . She f o u n d t h a t 80 sodium and c h l o r i d e were t h e major i o n s i n hemolymph and c o x a l f l u i d . The c h l o r i d e c o n c e n t r a t i o n i n t h e hemolymph f e l l d u r i n g f e e d i n g i n a manner s i m i l a r t o t h a t i n Dermacentor f r o m 2 0 0 t o 2 5 0 m e q / l i t e r t o about 160 m e q / l i t e r . A t t h i s c r i t i c a l hemo-lymph c o n c e n t r a t i o n of c h l o r i d e , t h e p r o d u c t i o n of h y p o t o n i c c o x a l f l u i d was i n i t i a t e d , and as a r e s u l t of t h i s p r o c e s s , the hemolymph c o n c e n t r a t i o n r o s e a g a i n s l i g h t l y t o 175 m e q / l i t e r . T h e r e a f t e r , t h e hemolymph c o n c e n t r a t i o n f l u c t u a t e d about t h i s e q u i l i b r a t e d v a l u e t h r o u g h o u t t h e f e e d i n g p e r i o d . S i n c e t h e gu t e p i t h e l i u m o f 0 . moubata c o n t i n u e s t o a b s o r b f l u i d f r o m the meal a f t e r t h e t i c k has s t o p p e d f e e d i n g , c o x a l f l u i d c o n t i n u e d t o appear a f t e r t h e t i c k s d e t a c h e d . F o r a t l e a s t t h r e e h o u r s t h e r e a f t e r , t h e hemolymph c h l o r i d e remained c o n s t a n t a t 175 meq/ l i t e r . S i n c e i t i s d i f f i c u l t t o d e t e r m i n e t h e e x a c t moment a t w h i c h s a l i v a t i o n b e g i n s i n i x o d i d t i c k s , i t was n o t p o s s i b l e t o c o r r e l a t e t h e o n s e t of s a l i v a t i o n w i t h a unique i o n c o n c e n t r a -t i o n ; n o r was i t p o s s i b l e t o d i r e c t l y o b s e r v e t h e momentary e f f e c t o f s a l i v a t i o n on m o d i f y i n g t h e hemolymph c o n c e n t r a t i o n . N o n t h e l e s s i t I s c l e a r t h a t a t t h e ti m e t h a t s a l i v a can f i r s t be c o l l e c t e d , t h e hemolymph c o n c e n t r a t i o n of a l l t h e d e t e r m i n e d i o n s and t h e o s m o t i c p r e s s u r e have r e a c h e d t h e i r e q u i l i b r a t e d l e v e l s , and r e m a i n a t t h e s e l e v e l s u n d o u b t e d l y as a d i r e c t consequence o f the a c t o f s a l i v a t i o n . One may b e t t e r a p p r e c i a t e t h e r e g u l a t o r y r o l e of t h e s a l i v a r y g l a n d s i n Dermacentor from t h e o b s e r v a t i o n t h a t t h e hemolymph o s m o t i c p r e s s u r e i s m a i n t a i n e d a t a f a i r l y c o n s t a n t and h i g h e r o s m o t i c p r e s s u r e ( 3 7 5 m O s m / l i t e r ) t h a n t h a t o f t h e meal ( 3 0 6 m O s m / l i t e r f o r human plasma; Ruch and 81 Patton, 1 9 6 5 ) . This occurs despite the f a c t that the s a l i v a r y glands are faced with secreting a volume of f l u i d which exceeds the ultimate hemolymph volume by nine- to twelve-fold. The only notable difference i n hemolymph composition observed between Dermacentor and Ornlthodoros i s that i n the l a t t e r chloride almost completely balances the molarity of sodium and potassium, whereas i n Dermacentor, chloride c o n t r i b -utes only 72$ of the anions necessary to account f o r e l e c t r o -n e u t r a l i t y . In Ornlthodoros. chloride w i l l s i m i l a r l y balance the monovalent cations i n the coxal f l u i d . In Dermacentor. although chloride makes up a greater proportion of the anions (81$) i n the s a l i v a than i n the hemolymph, s t i l l 19$ of the anions i s unaccounted f o r by c h l o r i d e . Also, the concentration of sodium and potassium s a l t i n the hemolymph and coxal f l u i d of Ornlthodoros accounts f o r 70$ of the observed osmotic pres-sure. In the case of Dermacentor however (Table XIII B), 85$ of the hemolymph osmotic pressure, and 88$ of the s a l i v a osmotic pressure i s accounted f o r by s a l t s of sodium and potassium. The ion concentrations of Dermacentor body f l u i d s may be compared with those of another ixodid t i c k , Boophllus microplus ( T a t c h e l l , 1969b). The hemolymph of Dermacentor contains 74 + 3 $ (mean + SE) of the sodium content of the whole t i c k a f t e r the t h i r d day of feeding. Since the gut contents probably make up the greatest portion of the body, i t follows that the sodium concentration i n the gut at t h i s time must be low i n r e l a t i o n to the hemolymph. Also, the concentration of potassium i n the gut i s probably higher (this i s r e f l e c t e d i n the high concentration o f p o t a s s i u m i n the f e c e s - T a b l e X I I I A ) . One can c o n c l u d e t h e same f o r B o o p h l l u s . f o r t h e c o n c e n t r a t i o n o f sodium and c h l o r i d e i n the hemolymph i s much h i g h e r t h a n t h e i r r e s p e c t i v e c o n c e n t r a t i o n s i n t h e whole t i c k , and the c o n c e n t r a t i o n s o f p o t a s s i u m , magnesium, and c a l c i u m a r e l o w e r i n t h e hemolymph t h a n i n t h e whole t i c k . The f a c t t h a t t h e c o n c e n t r a t i o n s of t h e s e Ions I n whole homogenates of B o o p h l l u s f a l l s as f e e d i n g p r o g r e s s e s ( w i t h t h e e x c e p t i o n of sodium) and t h e n l e v e l s o f f a t a s t a b l e v a l u e , i s i n t e r p r e t e d by T a t c h e l l t o mean t h a t r e g u l a t i o n of hemolymph i o n c o n c e n t r a t i o n , s i m i l a r t o t h a t i n Dermacentor. must be o c c u r r i n g i n B o o p h l l u s . T h i s r e g u l a t i o n i s a l s o b e l i e v e d t o be a r e s u l t of s a l i v a t i o n ( T a t c h e l l , 1967b, 1969b). A s t r i k i n g d i f f e r e n c e between Dermacentor and B o o p h l l u s i s a p p a r e n t when comparing S/H r a t i o s f o r c e r t a i n i o n s . I n t h e f o r m e r , t h e S/H r a t i o s f o r sodium and p o t a s s i u m a r e i n s i g n i f i -c a n t l y d i f f e r e n t f r o m one? i n the l a t t e r , i t a p p e a r s t h a t t he S/H r a t i o f o r sodium i s g r e a t e r t h a n one, and t h e S/H r a t i o f o r p o t a s s i u m i s l e s s t h a n one, a l t h o u g h T a t c h e l l does n o t a f f i r m t h a t t h e o b s e r v e d d i f f e r e n c e s a r e s t a t i s t i c a l l y s i g n i f i c a n t . I n b o t h s p e c i e s , however, t h e S/H r a t i o f o r c h l o r i d e I s 1.1. I n Dermacentor, t h e S/H r a t i o f o r magnesium i n v i t r o i s about 0.1 (see C h a p t e r T h r e e ) . I n B o o p h l l u s . t h e S/H r a t i o i n v i v o I s about 0.7 ( T a t c h e l l , 1969b). The o s m o t i c p r e s s u r e of B o o p h l l u s hemolymph (188 mOsm/ l i t e r ) i s o n l y s l i g h t l y l o w e r t h a n t h a t f o r Dermacentor (202 m O s m / l i t e r ) . The s a l i v a of B o o p h l l u s i s h y p e r o s m o t i c t o the hemolymph (S/H = 1.23)* whereas i n Dermacentor. t h e s a l i v a i s s l i g h t l y ( but s i g n i f i c a n t l y ) h y p o - o s m o t i c t o t h e hemolymph (S/H = 0.94). I f we assume t h a t t h e p r i m a r y s e c r e t i o n i n Derma-c e n t o r i s i s o - o s m o t i c o r h y p e r o s m o t i c t o t h e hemolymph ( t h e u s u a l a s s u m p t i o n t h a t f l o w o f f l u i d i s d r i v e n by a l o c a l o s m o t i c g r a d i e n t ; see C h a p t e r F o u r ) , t h e n r e a b s o r p t i o n o f s o l u t e r e l a t i v e t o w a t e r may o c c u r somewhere between t h e s a l i v a r y a c i n i and t h e o r a l c a v i t y . However, t h i s need n o t be the case i n B o o p h l l u s . The h y p o t h e s i s t h a t t h e s a l i v a r i u m o r b u c c a l c a v i t y may m o d i f y t h e p r i m a r y s e c r e t i o n of t h e s a l i v a r y a c i n i i s r e m i n i s c e n t of th e M a l p i g h i a n t u b u l e - r e c t u m s y s t e m i n i n s e c t s . The rectu m i n i n s e c t s i s o f t e n r e s p o n s i b l e f o r m o d i f y i n g t h e f l u i d p r e s e n t e d t o i t b e f o r e t h a t f l u i d i s e x c r e t e d ( W i g g l e s w o r t h , 1932$ Ramsay, 1952, 1958; P h i l l i p s , 1964 a, b, c, and I969). However, as w i l l be shown i n C h a p t e r T h r e e , g l a n d s s e c r e t i n g i n v i t r o , and th u s i s o l a t e d f r o m t h e b u c c a l c a v i t y , produce a s a l i v a a t l e a s t as hypo - o s m o t i c as t h e y do i n v i v o . One i s o b l i g a t e d , i n l i g h t o f t h i s e v i d e n c e , t o f o c u s more a t t e n t i o n on the r o l e of t h e s a l i -v a r y d u c t s i n t h e r e a b s o r p t i v e p r o c e s s . S i n c e i n B o o p h l l u s t h e s a l i v a i s h y p e r o s m o t i c (as e x p e c t e d f o r a s e c r e t o r y s y s t e m ) , t h i s w ould s u g g e s t t h a t i n t h e l a t t e r s p e c i e s e i t h e r t h e d u c t s s e r v e m e r e l y as a d e l i v e r y s ystem f o r t h e s a l i v a , o r p o s s i b l y t h e y engage i n s e c r e t i o n of s o l u t e t h e m s e l v e s . W i t h t h i s i n mind, i t would be i n t e r e s t i n g t o compare the u l t r a s t r u c t u r e of the d u c t s i n t h e s e two s p e c i e s . 6) Hemolymph Volume and t h e S t i m u l u s t o S a l i v a t i o n The j u s t i f i c a t i o n f o r use of i n u l i n t o measure hemolymph volume by t h e t r a c e r - d i l u t i o n method has a l r e a d y been c o n s i d e r e d (page 6 7 ) . The t i c k does n o t m a i n t a i n i t s hemolymph volume a t a c o n s t a n t v a l u e 5 r a t h e r , the volume i n m i c r o l i t e r s a l w a y s r e p r e -s e n t s about 2 3 $ o f the t i c k ' s w e i g h t i n m i l l i g r a m s . The f a c t t h a t t h e hemolymph rem a i n s a c o n s t a n t p r o p o r t i o n of t h e body w e i g h t as t h e t i c k engorges t o 75 t i m e s i t s u n f e d w e i g h t , s u g g e s t s t h a t t h e volume of t h i s compartment i s under some c o n t r o l . F l u i d i s c o n t i n u a l l y e n t e r i n g the hemolymph f r o m th e g u t . I t i s t h i s f l u i d ( i n a l l , 9 t o 12 t i m e s t h e hemolymph volume a t r e p l e t i o n ) w h i c h the t i c k i s f a c e d w i t h e x c r e t i n g by one means o r a n o t h e r . I t has a l r e a d y been shown t h a t t h e s a l i v a r y g l a n d i s r e s p o n s i b l e f o r t h e b u l k o f t h i s s e c r e t i o n , b u t t h e t r i g g e r t h a t s e t s o f f s a l i v a t i o n i s not known. I n t h e case of O r n l t h o d o r o s moubata, i n i t i a t i o n of c o x a l f l u i d p r o d u c -t i o n was c o r r e l a t e d w i t h a c r i t i c a l low c h l o r i d e c o n c e n t r a t i o n i n hemolymph (Kaufman, 1 9 7 1 ) . The hemolymph c o n c e n t r a t i o n t h e n r a p i d l y a t t a i n e d i t s f i n a l v a l u e w i t h t h e o n s e t of f l u i d e x c r e -t i o n . I t was n o t p r o v e n however, t h a t i t was s p e c i f i c a l l y c h l o r i d e i o n w h i c h p r o v i d e d the s t i m u l u s t o c o x a l f l u i d p roduc-t i o n . M a l p i g h i a n t u b u l e s e c r e t i o n i n Rhodnlus i s known t o be t r i g g e r e d i n i t i a l l y by s t r e t c h r e c e p t o r s i n t h e t e r g o - s t e r n a l m u s c l e s . The s t r e t c h i n g of t h e s e m u s c l e s l e a d s t o t h e r e l e a s e of a d i u r e t i c hormone i n t o the hemolymph. T h i s hormone a c t i v a t e s t h e M a l p i g h i a n t u b u l e s t o s e c r e t e t h e e x c e s s f l u i d w h i c h e n t e r s t h e hemolymph f r o m th e b l o o d meal ( M a d d r e l l , 1962 t o 1 9 6 6 ) . When the hemolymph volume of Dermacentor was i n c r e a s e d 25% o r 85 50$ by I n j e c t i o n s of s a l i n e iso-osmotlc t o t h e hemolymph of f e e d i n g t i c k s (1.2$ NaCl) o r by I n j e c t i o n s o f a medium h y p o - o s m o t i c t o hemolymph, t h e s e d i d n o t i n t h e m s e l v e s p r o v i d e s u f f i c i e n t s t i m u -l u s t o i n i t i a t e o r enhance s a l i v a t i o n i n t o g l a s s c a p i l l a r y t u b e s . I n d e e d , s a l i v a t i o n was i n h i b i t e d as compared t o n o n - i n j e c t e d c o n t r o l s ( F i g u r e 9). W l g g l e s w o r t h (1965) a l s o r e p o r t s (as unpub-l i s h e d o b s e r v a t i o n s ) t h a t i n j e c t i o n s o f " l a r g e amounts" of R i n g e r I n t o t h e body c a v i t y of Rhodnius does n o t cause a r a p i d f l o w of u r i n e . One c a n coax a r e c e n t l y removed t i c k t o s e c r e t e i n t o a c a p i l l a r y tube and t h e n r e s t o r e i t s hemolymph volume by i n j e c t i n g i s o - o s m o t i c o r h y p o - o s m o t i c s a l i n e . T h i s a l s o f a i l e d t o r e i n i t -i a t e s a l i v a t i o n ( F i g u r e 9). The p o s s i b i l i t y s t i l l r e m ains t h a t t h e r e a r e s t r e t c h r e c e p t o r s • l y i n g i n t h e g u t e p i t h e l i u m i t s e l f , o r t h a t a s p e c i f i c c h e m i c a l a g e n t , perhaps f r o m t h e m e a l, i s r e q u i r e d t o t r i g g e r s e c r e t i o n . I do n o t w i s h t o l e a v e t h e I m p r e s s i o n t h a t t h e s e e x p e r i -ments p r e c l u d e t h e i n t e r p r e t a t i o n t h a t a b d o m i n a l s t r e t c h r e c e p -t o r s a r e i n v o l v e d i n s t i m u l a t i n g s a l i v a t i o n . I n l i g h t of the f a c t t h a t d i u r e s i s i n a v a r i e t y of i n s e c t s i s a t l e a s t c o r r e l a t e d w i t h e v e n t s t h a t c o u l d s t i m u l a t e p r o p r i o c e p t o r s (enumerated i n M a d d r e l l , 1964c), i t would be premature t o c o n c l u d e t h a t i n D ermacentor. s t r e t c h r e c e p t o r s a r e n o t t r i g g e r i n g s a l i v a t i o n . B u t t h e above e v i d e n c e , as i t s t a n d s , does n o t d emonstrate the e x i s t e n c e of a b d o m i n a l p r o p r i o c e p t o r s which c o n t r o l s a l i v a t i o n . 7) C l e a r a n c e of I n u l i n and O - M e t h y l g l u c o s e The purpose of t h e s e c l e a r a n c e s t u d i e s was t o demon-s t r a t e w hether m o l e c u l e s l a r g e r t h a n a c e r t a i n d i a m e t e r would 8 6 Figure 9. Effect of saline injections into the hemolymph on the subsequent volume of salivary secretion. Volume was determined by collecting the saliva In calibrated capillary tubes. The percentage of the hemolymph that this represented was calculated, assuming that 23$ of the body weight plus the injected f l u i d equalled hemolymph volume. o = no injections (control) A S injection of 1 . 2 $ NaCl (1 u l per 20 mg tick weight) before collecting saliva. • = injections of 1 . 2 $ NaCl (1 ;ul per 10 mg tick weight before salivation) after collecting f l u i d from some individ-uals i n the control group. Total volume secretion (as % hemolymph volume) o co" rr 8^ o o o o cP t o o o 3 CQ rO O O * o o be u n a b l e t o pass t h r o u g h the s a l i v a r y e p i t h e l i u m . The r e s u l t s of t h e s e e x p e r i m e n t s a l s o p r o v i d e a c l u e as t o the t y p e of mechanism employed i n the p r o d u c t i o n of s a l i v a ( i . e . f i l t r a t i o n -r e s o r p t l o n o r a c t i v e i o n s e c r e t i o n ) . I t w i l l be r e c a l l e d t h a t t h e v e r t e b r a t e g l o m e r u l a r k i d n e y i s a b l e t o h o l d back m o l e c u l e s t h e s i z e of serum a l b u m i n and l a r g e r ; the s l i g h t degree of s i e v i n g o b s e r v e d w i t h s m a l l e r p r o t e i n s , I s r o u g h l y i n a c c o r d w i t h t h e e x i s t e n c e o f p o r e s h a v i n g an average d i a m e t e r o f 75 A ( P i t t s , 1 9 6 3 ) . However W a l l e n i u s ( 1 9 5 4 - quoted by P i t t s ) has shown t h a t i f t h e d i a m e t e r i s c a l c u l a t e d f r om d a t a on t h e s i e v i n g o f d e x t r a n s of g r a d e d m o l e c u l a r s i z e , t h e average d i a m e t e r approaches 100 A. The l o w e r e s t i m a t e o f 75 A, d e r i v e d f r o m t h e s i e v i n g of h e m o g l o b i n , m y o g l o b i n , and egg albumen, may be due t o e l e c t r i c a l h i n d r a n c e t o f i l t r a t i o n ; t h e s e p r o t e i n s a r e known t o p o s s e s s e l e c t r i c a l c h a r g e s a t t h e pH of plasma and g l o m e r u l a r f i l t r a t e , whereas d e x t r a n s a r e uncharged m o l e c u l e s ( P i t t s , 1 9 6 3 ) . A l t h o u g h m o l e c u l a r s i e v i n g may a l s o o c c u r i n s e c r e t o r y systems ( P h i l l i p s and D o c k r i l l , 1 9 6 8 ; P h i l l i p s and Beaumont, 1971)» i t o n l y becomes a p p a r e n t f o r p a r t i c l e s o f much s m a l l e r o d i a m e t e r , ( e . g . 3 t o 8 A; B e r r l d g e , 1 9 6 9 ) . C o n s e q u e n t l y , t h e p e r m e a b i l i t y c h a r a c t e r i s t i c s o f an e x c r e t o r y organ a r e u s u a l l y a c c e p t e d as one c r i t e r i o n f o r d i s t i n g u i s h i n g between th e two mechanisms of e x c r e t i o n ( K i r s c h n e r , 1 9 & 7 ) • However, one s h o u l d c o n s i d e r p r o p e r t i e s o t h e r t h a n m o l e c u l a r s i z e a l o n e when c o n d u c t i n g c l e a r a n c e s t u d i e s . As s u g g e s t e d above, i f a f i x e d charge s h o u l d e x i s t a l o n g t h e pore c h a n n e l I n t h e membrane, a n o m o l l e s may o c c u r I f t h e p a r t i c l e employed i n t h e e x p e r i m e n t i s a l s o c h a r g e d . An i o n p o s s e s s i n g a cha r g e s i m i l a r t o t h a t i n the p o r e , even i f o t h e r w i s e s m a l l enough t o pass t h r o u g h , would tend t o be r e p e l l e d f r o m t h e p o r e . T h i s w o u l d l e a d t o u n d e r e s t i m a t i o n o f the pore d i a m e t e r . I t i s u s u a l l y a d v i s a b l e , t h e r e f o r e , t o s e l e c t n o n - i o n i c s u b s t a n c e s f o r c l e a r a n c e e x p e r i m e n t s i f t h e e x i s t e n c e of a f i x e d charge i n t h e membrane has n o t been d l s p r o v e n . A second p r e c a u t i o n one s h o u l d t a k e i s t o s e l e c t s u b s t a n c e s t h a t a r e n o t l i k e l y t o be m e t a b o l i z e d by t h e a n i m a l . I n u l i n i s a p o l y s a c c h a r i d e t h a t i s n o t m e t a b o l i z e d when i n j e c t e d i n t h e b l o o d of most v e r t e b r a t e s ( P i t t s , 1963) and of s e v e r a l i n s e c t s (Levenbook, 1958? Loughton and Tobe, 1969). The mo n o s a c c h a r i d e , 3-0-methylglucose, i s an anal o g u e of g l u c o s e w h i c h i s t r a n s p o r t e d I n a manner s i m i l a r t o t h e l a t t e r , b u t , a t l e a s t i n t h e r a t , i t i s n o t m e t a b o l i z e d i n t h e stomach, b l o o d , k i d n e y , b l a d d e r o r u r e t h r a (Csaky and W i l s o n , 1956; Csaky and G l e n n , 1957)• C o n s e q u e n t l y i t has been s u g g e s t e d t h a t 0-methyl-g l u c o s e i s a l s o a s u i t a b l e s u b s t a n c e t o use as a t r a c e r f o r t h e s t u d y of s u g a r t r a n s p o r t (Csaky and T h a l e , i960). B e a r i n g i n mind c e r t a i n r e s e r v a t i o n s ( t o be d i s c u s s e d b e l o w ) , I d e c i d e d t h a t I n u l i n and 3-0-methylglucose were a l s o s u i t a b l e f o r t h e p r e s e n t s t u d y . a) I n u l l n The E i n s t e i n - S t o k e s d i a m e t e r of i n u l l n , namely, t h e d i a m e t e r of a sphere w h i c h would d i f f u s e a t t h e same r a t e as does i n u l i n , has been e s t i m a t e d t o be 29 A ( P i t t s , 1963). The s a l i v a r y g l a n d i s c o m p l e t e l y impermeable t o i n u l i n , s i n c e v e r y h i g h a c t i v i t i e s p r e v a i l e d i n t h e hemolymph one day a f t e r i n j e c -t i o n , y e t no a c t i v i t y c o u l d be d e t e c t e d i n t h e s a l i v a . One may c o n c l u d e t h e n , t h a t any u n i f o r m l y - c y l i n d r i c a l a q u e o u s - f i l l e d p o r e s p o s t u l a t e d f o r the b a s a l membrane of t h e s a l i v a r y g l a n d e p i t h e l i u m must have a d i a m e t e r of l e s s t h a n 29 A. There e x i s t s t h e u n l i k e l y p o s s i b i l i t y t h a t i n u l i n i s f i l t e r e d o r s e c r e t e d I n t o t h e a c i n i and t h e n c o m p l e t e l y r e a b s o r b e d ; i t i s n o t f e a s i b l e t o t e s t t h i s by I n j e c t i n g I n u l i n up t h e s a l i v a r y g l a n d i n l i v i n g t i c k s and s u b s e q u e n t l y m e asuring t h e r a d i o a c t i v i t y a p p e a r i n g i n t h e hemolymph. N e v e r t h e l e s s , i t i s u n l i k e l y a p r i o r i t h a t e x t e n s i v e r e a b s o r p t i o n o f i n u l i n o c c u r s . S e c r e t i o n o r f i l t r a t i o n of i n u l i n f o l l o w e d by complete r e s o r p t i o n has n o t been o b s e r v e d i n o t h e r o r g a n i s m s . Indeed, i t would be s u r p r i s i n g f o r a c a r r i e r s i t e , d e s i g n e d t o c o n s e r v e a f o r e i g n s u b s t a n c e ( s u c h as i n u l i n ) , t o have e v o l v e d i n an o r g a n i s m w h i c h n e i t h e r has any use f o r t h a t f o r e i g n s u b s t a n c e , n o r i s n o r m a l l y c o n f r o n t e d w i t h i t . Ramsay and R i e g e l (1961) show t h a t t h e M a l p i g h i a n t u b u l e s of t h e s t i c k i n s e c t cannot r a p i d l y r i d t h e body of i n u l i n ; t h e y r e p o r t a U/P r a t i o o f 0.046 f o r i n u l i n compared t o 0.75 f o r g l u c o s e , O.58 f o r f r u c t o s e , and 0.58 f o r s u c r o s e . N e i t h e r c o u l d t h e y show t h a t m e t a b o l i s m o f i n u l i n o c c u r r e d . My r e s u l t s c o n f i r m t h a t i n Dermacentor. i n u l i n i s e x c r e t e d from the body o n l y v e r y s l o w l y . I n u l i n i s not, r e s o r b e d by t h e c o x a l t u b u l e s of the t i c k , O r n l t h o d o r o s moubata (Kaufman, 1971). Levenbook (1958), c o n d u c t i n g an e x t e n s i v e s t u d y , a l s o c o n f i r m s t h a t i n u l i n i s 91 n e i t h e r e x c r e t e d , n o r m e t a b o l i z e d i n t h e l a r v a l hemolymph of P r o d e n l a e r l d a n i a , and does n o t p e n e t r a t e t h e g u t c e l l s f r o m the hemolymph s i d e . F i n a l l y , i n u l i n i s not s e c r e t e d n o r r e s o r b e d by v e r t e b r a t e t i s s u e s ( P i t t s , 1963). That i n u l i n cannot p e n e t r a t e t h e s a l i v a r y e p i t h e l i u m , can be t a k e n as s u p p o r t i n g e v i d e n c e t h a t t h e s a l i v a r y g l a n d does n o t g e n e r a t e f l u i d f l o w by means of f i l t r a t i o n . b) 3 - 0 - M e t h y l g l u c o s e F o r r e a s o n s d i s c u s s e d i n Methods, t h e hemolymph and s a l i v a were sampled t h e day f o l l o w i n g i n j e c t i o n . That t h e r a d i o a c t i v i t y r e m a i n i n g i n t h e hemolymph a t t h i s t i m e was l o w , i n d i c a t e s t h a t t h e t r a c e r was c l e a r e d f r o m the hemolymph f a i r l y r a p i d l y . P r e s u m a b l y I t was t r a n s p o r t e d v i a r o u t e s t h a t g l u c o s e I t s e l f w o u l d have t a k e n (Csaky and T h a l e , i960). L i k e l y i t was t a k e n up by m e t a b o l l c a l l y a c t i v e t i s s u e s ( m u s c l e s , o v a r y , e t c . ) , though samples o f t h e l a t t e r were n o t t e s t e d f o r t h e p r e s e n c e o f r a d i o -a c t i v i t y . Comparison o f t h e a c t i v i t i e s i n t h e s a l i v a , f e c e s , and M a l p i g h i a n t u b u l e s , however, s u g g e s t s t h a t some 0-methyl-g l u c o s e , o r a t l e a s t a d e r i v a t i v e of i t , p a ssed out of t h e body i n t h e f e c e s , r a t h e r t h a n i n the s a l i v a . The r a d i o a c t i v i t y c o u l d have become i n c o r p o r a t e d i n t o t h e f e c e s by d i f f u s i o n f r o m t h e hemolymph t o the lumen of t h e a l i m e n t a r y c a n a l , o r p o s s i b l y by s e c r e t i o n v i a t h e M a l p i g h i a n t u b u l e s . Perhaps some r a d i o a c t i v i t y 14 was l o s t as ^°2' "k u t * n * s w a s n o ^ measured. The S/H r a t i o f o r O-methylglucose i s a p p r o x i m a t e l y 0.2, showing t h a t a l t h o u g h t h e g l a n d i s permeable t o the m o l e c u l e , O - m e t h y l g l u c o s e i s e i t h e r f i l t e r e d and i s e x t e n s i v e l y r e s o r b e d , 92 o r t h e f l u i d - s e c r e t i n g membranes r e s t r i c t somewhat i t s f r e e p a s s a g e . I f the l a t t e r o p i n i o n i s c o r r e c t , i t would i n d i c a t e t h a t any p o s t u l a t e d a q u e o u s - f i l l e d pores i n t h e membranes a r e s m a l l enough t o show the e f f e c t s o f s t e r l c h i n d r a n c e and v i s c o u s d r a g (Pappenhelmer ejb a l . , 1951s Pappenheimer, 1 9 5 3 ) w i t h O - m e t h y l g l u c o s e . However, i f O - m e t h y l g l u c o s e i s f i l t e r e d f r e e l y t h r o u g h t h e s a l i v a r y e p i t h e l i u m , i t i s r e a s o n a b l e t o s u s p e c t t h a t t h e g l a n d s , s e c r e t i n g s e v e r a l t i m e s the t o t a l body c o n t e n t of i o n s and w a t e r d u r i n g t h e f e e d i n g p e r i o d , m i g h t p o s s e s s a means f o r r e t a i n i n g e s s e n t i a l m e t a b o l i t e s such as g l u c o s e . I n d e e d i t i s s u r p r i s i n g t h a t under normal f e e d i n g c o n d i t i o n s , t h e g l a n d s e x c r e t e even a l i m i t e d q u a n t i t y o f g l u c o s e . N o r m a l l y one cannot d e t e c t s u g a r s i n mammalian u r i n e , f o r i t i s c o m p l e t e l y r e a b s o r b e d i n t h e p r o x i m a l t u b u l e s o f t h e k i d n e y ( P i t t s , 1 9 6 3 ) . A n a l o g o u s t o t h e v e r t e b r a t e k i d n e y , t h e s a l i v a r y g l a n d may pos-s e s s r e s o r p t i v e s i t e s f o r g l u c o s e and o t h e r s m a l l m e t a b o l i t e s . One s h o u l d , however, i n t e r p r e t t h e r e s u l t s of t h e O-methyl-g l u c o s e e x p e r i m e n t w i t h c a u t i o n , s i n c e i t has n o t been demonstra-t e d t h a t t h i s m o l e c u l e i s n o t m e t a b o l i z e d i n t h e hemolymph of t h e t i c k . A l t h o u g h i t i s n o t m e t a b o l i z e d by t h e t i s s u e s of t h e r a t , Csaky and G l e n n ( 1 9 5 7 ) showed t h a t c o n t a m i n a t i n g b a c t e r i a ( s u c h as E. c o l i . ) c a n u t i l i z e O - m e t h y l g l u c o s e as a s o l e s o u r c e of c a r b o n . C o n s i d e r i n g t h a t t h e medium used t o c a r r y t h e t r a c e r i n t o the t i c k was n o t s t e r i l e , and t h a t s a m p l i n g was a c c o m p l i s h e d t h e day f o l l o w i n g i n j e c t i o n , i t cannot be r u l e d out t h a t a s i g n i f i c a n t f r a c t i o n of t h e i n j e c t e d t r a c e r was n o t m e t a b o l i z e d by b a c t e r i a I n t h e hemolymph. I n any c a s e , t h e h i g h e s t S/H 93 r a t i o f o r O - m e t h y l g l u c o s e , under t h e p r e s e n t e x p e r i m e n t a l c o n d i -t i o n s , c a n be a c c e p t e d as 0.2. I n summary t h e i n a b i l i t y of t h e s a l i v a r y g l a n d s t o c l e a r I n u l l n , and t h e p o s s i b i l i t y t h a t t h e g l a n d s were a l s o r e s t r i c t i n g t h e passage o f m o l e c u l e s the s i z e of g l u c o s e , were t a k e n as c i r c u m s t a n t i a l e v i d e n c e t h a t t h e s a l i v a r y g l a n d s were o p e r a t i n g by a s e c r e t o r y mechanism. The q u e s t i o n t h e n a r o s e as t o what s o l u t e s were l i k e l y t o be d r i v i n g f l u i d s e c r e t i o n . U s u a l l y the c o n c e n t r a t i o n o f t h e s o l u t e r e s p o n s i b l e i s h i g h e r i n t h e s e c r e t e d f l u i d t h a n i n t h e b a t h i n g f l u i d ( e . g . p o t a s s i u m i n I n s e c t M a l p i g -h i a n t u b u l e s ) . The o b s e r v e d s l i g h t l y h i g h e r c o n c e n t r a t i o n of c h l o r i d e i n t h e s a l i v a t h a n i n t h e hemolymph s u g g e s t e d t h a t t h i s a n i o n may p l a y a f u n d a m e n t a l r o l e i n t h e mechanism of s a l i v a t i o n . W i t h t h i s h y p o t h e s i s i n mind an i n v i t r o p r e p a r a t i o n of t h e g l a n d was d e v e l o p e d ( C h a p t e r T h r e e ) , f i r s t t o c o n f i r m t h e e x i s t e n c e of a s e c r e t o r y mechanism, and s e c o n d l y , t o c r i t i c a l l y examine the e f f e c t s o f i o n i c c o n c e n t r a t i o n and o s m o t i c p r e s s u r e o f the medium on s a l i v a t i o n . 8) Summary of C h a p t e r Two A) Of t h e t o t a l meal i m b i b e d , o n l y about 20% i s r e t a i n e d by t h e f e m a l e a t r e p l e t i o n . The r e m a i n i n g 80% i s e x c r e t e d - 10% b e i n g d r y f e c e s , and ?0% b e i n g w a t e r . T h i s w a t e r r e p r e s e n t s 80 t o 90% o f t h e t o t a l i m b i b e d w a t e r . Of the t o t a l w a t e r e x c r e t e d by t h e t i c k , 75% i s removed by s a l i v a t i o n , 20 t o 25% i s l o s t i n t h e f e c e s , and t h e r e m a i n d e r ( <5%) i s e v a p o r a t e d f r o m t h e integument and s p i r a c l e s . B) About 57% of the f e c e s i s water? the r e m a i n d e r i s 94 p r o t e i n , 65 t o 9 0 $ being non-digested hemoglobin. Of the t o t a l sodium i n g e s t e d , almost 9 0 $ i s excreted. Of the l a t t e r f i g u r e , over 9 5 $ i s l o s t i n the s a l i v a , and only 5 $ or l e s s i s l o s t by d e f e c a t i o n and Malpighian tubule s e c r e t i o n combined. Of the t o t a l potassium Ingested, about 6 0 $ i s excre t e d . Of the l a t t e r f i g u r e , 16 t o 2 0 $ i s l o s t i n the s a l i v a , and 80 t o 84$ i s ex c r e t e d i n the f e c e s . C) The i o n i c and osmotic concentrations of the hemolymph and s a l i v a s t a b i l i z e a t constant values by the t h i r d or f o u r t h day of f e e d i n g . The volume of e x t r a c e l l u l a r f l u i d i s maintained at 2 3 $ of the body wieght? the l a t t e r i s accomplished even though the t o t a l body weight i n c r e a s e s 75 times over the unfed weight, and the volume of excreted f l u i d passing through t h i s compartment i s about ten times the f i n a l volume of the hemolymph. D) P r e l i m i n a r y support f a v o u r i n g the hypothesis that the s a l i v a r y gland f u n c t i o n s by means of a s e c r e t o r y mechanism was a l s o obtained. The evidence i n c l u d e s i m p e r m e a b i l i t y of the gland to i n u l i n and the r e s t r i c t e d clearance of O-methylglucose. The S/H r a t i o f o r c h l o r i d e was 1 . 1 , but those f o r sodium and potas-sium were i n s i g n i f i c a n t l y d i f f e r e n t from 1 . 0 . This suggests th a t c h l o r i d e may be the t r a n s p o r t e d i o n which d r i v e s f l u i d movement. CHAPTER THREE CONTROL AND MECHANISM OF SALIVARY SECRETION CHAPTER THREE E x p e r i m e n t s d e s c r i b e d i n t h e p r e v i o u s c h a p t e r i n d i c a t e t h a t t h e s a l i v a r y g l a n d i s r e s p o n s i b l e f o r t h e e x c r e t i o n of e x c e s s f l u i d f r o m t h e t i c k d u r i n g f e e d i n g . The p r e s e n t c h a p t e r d e a l s w i t h how t h e g l a n d c a r r i e s out t h i s f u n c t i o n . T h i s q u e s t i o n i s c o n s i d e r e d a t two l e v e l s . F i r s t , does f l u i d s e c r e -t i o n by t h e g l a n d o c c u r by b u l k f i l t r a t i o n under a h y d r o s t a t i c p r e s s u r e g r a d i e n t , o r by t h e a c t i v e s e c r e t i o n of i o n s ? S e c o n d l y , how do su c h f a c t o r s as i o n c o n c e n t r a t i o n s and r a t i o s i n t h e hemolymph, r a t e o f i o n s e c r e t i o n , and o s m o t i c p r e s s u r e o f t h e hemolymph a f f e c t t h e r a t e o f f l u i d s e c r e t i o n ? A p o s s i b l e mechanism f o r t h e c o n t r o l of s e c r e t i o n i s a l s o c o n s i d e r e d . K i r s c h n e r (19^7) has r e v i e w e d t h e f e a t u r e s t h a t d i s t i n -g u i s h f i l t r a t i o n and s e c r e t o r y mechanisms of f l u i d t r a n s f e r . The f o r m e r can be c h a r a c t e r i z e d by f i v e p r o p e r t i e s ! (a) e x c r e -t i o n of h i g h m o l e c u l a r w e i g h t p o l y m e r s , (b) e x c r e t i o n o f g l u c o s e , ( c ) m o r p h o l o g i c a l i d e n t i f i c a t i o n o f a f i l t r a t i o n s i t e , (d) s i m i -l a r i t y i n c o m p o s i t i o n between t h e "plasma" and p r i m a r y u l t r a -f i l t r a t e , and (e) s e n s i t i v i t y of f l u i d f o r m a t i o n t o h y d r o s t a t i c p r e s s u r e . I t i s now u n i v e r s a l l y a c c e p t e d t h a t t h e v e r t e b r a t e g l o m e r u l a r k i d n e y f u n c t i o n s by f i l t r a t i o n ( H e n d r i x e_t a l . , 1936? P i t t s , 1963); one can de m o n s t r a t e t h a t t h e g l o m e r u l a r c a p i l l a r y p r e s s u r e i s s u f f i c i e n t l y h i g h t o d r i v e an u l t r a f i l t r a t e of plasma t h r o u g h the p o r e s of t h e c a p i l l a r y e p i t h e l i u m . T h i s same 96 h y d r o s t a t i c p r e s s u r e p r o p e l s the f l u i d a l o n g the n e p h r i c d u c t s y s t e m . K i r s c h n e r and Wagner (1965) p r e s e n t e d good e x p e r i m e n t a l e v i d e n c e t h a t t h e c r a y f i s h a n t e n n a l g l a n d o p e r a t e s by means of a f i l t r a t i o n mechanism. A l t h o u g h s i m i l a r mechanisms have been s u g g e s t e d f o r e x c r e t o r y organs of o t h e r i n v e r t e b r a t e s (see K i r s c h n e r , 1967), i n many c a s e s c o n c l u s i v e e v i d e n c e a w a i t s the r e f i n e m e n t of e x p e r i m e n t a l t e c h n i q u e s . Organs w h i c h e l a b o r a t e f l u i d by means of a s e c r e t o r y p r o c e s s have f e a t u r e s w h i c h d i f f e r f r o m t h o s e w h i c h o p e r a t e by f i l t r a t i o n : (a) t h e u r i n e c o n c e n t r a t i o n of a s e c r e t e d s u b s t a n c e may be q u i t e d i f f e r e n t f r o m t h e b l o o d c o n c e n t r a t i o n of t h a t s u b s t a n c e , (b) t h e r a t e o f s o l u t e t r a n s p o r t and r a t e of f l u i d s e c r e t i o n e x h i b i t M i c h a e l l s - M e n t o n t y p e k i n e t i c s , ( c ) f l u i d t r a n s p o r t d e r i v e s e n e r g y f r o m c e l l u l a r m e t a b o l i s m of t h e g l a n d -u l a r e p i t h e l i u m , (d) t h e p r i m a r y s e c r e t e d f l u i d i s h y p e r t o n i c o r I s o t o n i c w i t h t h e b a t h i n g medium, and (e) t h e r a t e of f l u i d s e c r e t i o n i s dependent on the p r e s e n c e of c e r t a i n s o l u t e s ( u s u a l l y i o n s ) b u t i s l a r g e l y i n d e p e n d e n t of h y d r o s t a t i c p r e s -s u r e . The l a t t e r c r i t e r i o n r e f l e c t s t h e f u n d a m e n t a l d i f f e r e n c e between s e c r e t i o n and u l t r a f i l t r a t i o n . I n a s e c r e t o r y system, t h e a c t i v e t r a n s p o r t of a s o l u t e e s t a b l i s h e s a c h e m i c a l and o s m o t i c g r a d i e n t a c r o s s o r w i t h i n t h e e p i t h e l i u m ; i t i s t h i s g r a d i e n t w h i c h c o m p r i s e s the p o t e n t i a l energy f o r w a t e r f l o w a c r o s s t h e membrane. How the f l o w of w a t e r c a n be c o u p l e d t o s o l u t e t r a n s p o r t i s d i s c u s s e d by Diamond (1962, 1966). The e s s e n t i a l p o i n t h e r e i s t h a t i f f l u i d has been t r a n s p o r t e d i n t o a r e l a t i v e l y c o n f i n e d space (such as t h e a c i n a r lumen of a s a l i v a r y g l a n d ) , a s u f f i c i e n t l y h i g h h y d r o s t a t i c p r e s s u r e may be generated in the lumen to propel the f l u i d along the duct system. If an external hydrostatic pressure Is applied to the gland over and above this secretory pressure, i t Is capable of hastening only the flow of the luminal f l u i d , not the flow across the secretory epithelium. Por example, i t i s generally accepted that the Malpighian tubules of insects produce urine by means of a secretory mechanism. However in Rhodnlus. the tergo-sternal muscles, and probably the int r i n s i c r e t a l muscles help to expel the drops, of. urine from the rectal sac (Maddrell, 1964c). Simil-arly, Gregson (i960) suggested that the expulsion of saliva from Dermacentor may be aided by a negative pressure in the host tissue as well as by a positive pressure within the s a l i -vary glands, although he did not comment on liow f l u i d flow across the epithelium was generated. One invariably observes contractions of the abdominal musculature in ticks that are salivating into glass capillaries (unpublished observations). It was not known whether these contractions provided a hydro-static pressure gradient to drive f l u i d across a f i l t r a t i o n membrane, or whether the contractions simply augmented the flow of saliva along the excretory ducts. In the previous chapter, preliminary evidence was pre-sented that f l u i d flow in the tick salivary gland i s generated by a secretory mechanism. The salivary gland did not excrete inulin and the passage of O-methylglucose was restricted. If one accepts the theory that water soluble molecules traverse epit h e l i a l membranes through aqueous-filled channels, the impermeability of the salivary gland to inulin suggests that tHe effective diameter of these channels i s considerably smaller than that consistent with a process of f i l t r a t i o n . If one could show in addition, that salivary secretion can occur in the absence of an externally-applied hydrostatic pressure, this would be clear evidence in favour of the secretory hypothesis. Since the tick possesses, i n common with most arthropods, an open circulatory system, removal of the glands from the body should eliminate the hydrostatic pressure which otherwise could conceivably develope across the salivary epithelium. If such glands can then be made to secrete saliva in v i t r o , f i l t r a t i o n i s excluded as a mechanism of salivation. In just such a manner, Ramsay (195*0 f i r s t demonstrated conclusively that the Malpighian tubules of the stick insect Dlxippus morosus operate by means of a secretory mechanism. When bathed in hemolymph, the excised tubules remained alive for over 24 hours; the normal rate of secretion that Ramsay observed was 1.4 nl/minute. However, when the Malpighian tubules were bathed in a r t i f i c i a l media rather than natural hemolymph, they would secrete urine for less than five hours and at about one-third of the rate observed in natural hemolymph (Ramsay, 1955)* The Malpighian tubules of other species fare much better i n a r t i f i c i a l media (Berridge, 1966; Maddrell, 1969? Irvine, 1 9 6 9 )t so that in these cases, tubule function can be examined i n considerable d e t a i l . The salivary glands of Calllphora w i l l also secrete f l u i d in vitro (Berridge and Patel, 196"8) and suffer only minor deterioration in secretory rate over six hours (Berridge, 1970), It was in view of the encouraging successes of the above-mentioned authors working with insects, that I attempted to develope an ln-vitro preparation of the salivary glands of Dermacentor. 100 MATERIALS AND METHODS 1) Excision of the Salivary Glands Each tick was Immobilized dorsum-up with modelling clay in a small dissecting dish. The dorsal alloscutum and part of the scutum was removed by means of a razor-blade scalpel. The tick was covered with disseoting medium (see Table XV B), and most of the viscera (except for the salivary glands) were removed. The rest of the scutum and the dorsal portion of the palps were then pared away to reveal the f u l l length of the chelioerae and part of their retractor muscles* The chelicerae and attached muscles were pulled away to reveal the floor of the hypostome and the origin of the main salivary ducts. An excellent view of the capitular and scutal area thus exposed i s figured in the paper of Gregson (i960). After the f u l l length of the salivary duct (from the salivarium to the f i r s t acini) was exposed and freed of extraneous tissue, the sclero-tlzed portion of the pharyngeal roof was released from the basis oapltull region by means of a transverse-sagittal s l i c e . With the f u l l length of the salivary ducts thus freed, the restraining tracheae and other connective tissue were cut away with fine scissors (Weiss, London); the salivary glands were then transferred In a medicine dropper with fresh medium to a petri dish. The two glands were then completely separated one from the other by s l i c i n g the cuticle joining the two ducts at 101 their origin. A strand of s i l k (about 10 p. in diameter) was tied to the sclerotized cuticle associated with each duct. By means of this fine strand (teased from a piece of thread), the gland could be manipulated. Another petri dish was lined f i r s t with »Saran Wrap' (Dow Chemicals) and then with •Parafilm* (American Can). This petri dish was then f i l l e d with paraffin o i l (•Fisher 1, Saybolt viscosity 1 2 5 / 1 3 5 ) , and the two dissected glands were transferred to i t i n separate drops of medium. 2) Post-excision Treatment The petri dish containing the glands was kept on a cooling-plate (Arthur H. Thomas Co., Philadelphia) through which warm water was circulated from a constant-temperature bath (Colora "Ultra-thermostat"). The temperature of the paraffin o i l was thus maintained at 2 5 - 2 9 ° G« During any single experiment however, the temperature did not vary by more than 0 . 5 ° C. A short length of glass capillary was used to anchor each drop of bathing medium to the Parafllm by surface tensionj the free end of the s i l k thread was draped over the rim of the petri dish and manipulated to draw the proximal portion of the s a l i -vary duct out of the droplet of medium into the l i q u i d paraffin. A l l extraneous connective tissue which s t i l l clung to the duct was teased away to render the duct relatively hydrophobic. At this point, the glands were washed several times with fresh medium. When the appropriate stimulus was applied, the gland secreted a spherical droplet which formed in the o i l at the origin of the duct. Although the droplet of bathing medium was not continuously oxygenated, medical grade oxygen (Canada Liquid 102 Air) was bubbled through the stocks of media from which fresh droplets were obtained. Periodically, as the secreted droplet grew so large that i t s borders approached that of the medium bathing the gland, the secreted droplet was removed with a glass rod and pooled with similar droplets under the o i l , u n t i l their combined volume was sufficient for Ion determinations. When samples for osmotic pressure determinations were required, the procedure was somewhat different from the above. Since these determinations required that several small droplets (about 50 nl) be separated one from the other in the pipette by columns of o i l , these droplets were allowed to enter the pipette directly from the o r i f i c e of the salivary gland. The cumulative volume and rate of secretion for each gland was calculated as follows 1 Since the o r i f i c e of a properly dissected salivary duct was hydrophobic, the secreted droplet of saliva was almost perfectly spherical. Thus the volume of the secreted droplet at any time could be calculated from i t s radius, the l a t t e r , being a linear function of the divisions on the ocular micrometer. At chosen intervals (varying from one to several minutes depending on the rate of secretion) the divisions of the micrometer spanning the dia-meter of the droplet, and the time, were recorded on computer coding forms. The computer (IBM 1130) was programmed to calcu-late the volumes from the r a d i i , and to plot cumulative volume of saliva against time. Mathematically, rate of secretion i s the f i r s t derivative of the lat t e r function. However, since the computer was provided with points separated by f i n i t e 103 intervals, i t would have been relatively d i f f i c u l t (and Indeed unnecessary) to have the computer calculate on equation describ-ing the cumulative secretion with time and then to calculate f i r s t derivatives. Instead, i t was programmed to calculate the slopes of the lines Joining the successive points, and to plot these slopes against the times midway between those succes-sive points. half-unit, the percentage error in estimating the volume of the secreted droplet decreased as the volume of the droplet increased? the maximum error was +5$. 3) Experimental Media Table XV A indicates the components which were common to a l l the experimental and control media. Table XV B l i s t s the additional compounds present i n the diverse experimental media. Since the micrometer scale could be read to the nearest TABLE XV A INGREDIENTS COMMON TO ALL MEDIA Compound Concentration (mg/llter) Succinic acid C i t r i c acid Malic acid Glucose Trehalose Maltose (monohydrate) Sucrose Glycine Proline Glutamlne Glutamic acid Penecillin G Streptomycin sulphate 2000 550 2000 2000 2000 2000 4000 200 530 400 2350 30 100 TABLE XV B EXPERIMENTAL MEDIA C o n c e n t r a t i o n ( m g / l i t e r ) Compound 1* 2 * 3 4 5 6 7 8 G l u c o s e 5 0 0 0 N a C l 6 3 0 0 9 8 0 0 1 4 3 0 5 9 0 0 6 5 0 0 5 3 0 0 NaHCO^ 2 0 0 8 8 6 8 8 6 8 8 6 8 8 6 8 8 6 KC1 1 0 0 0 6 3 5 7530 6 3 5 6 3 5 NaOH 1 1 2 5 1 1 2 5 1 1 2 5 1 1 2 5 1 . 1 2 5 C a C L 2 ' 2 H 2 0 376 662 2 9 0 290 2 9 0 2 9 0 M g C l 2 ' 6 H 2 0 2 6 4 0 2 6 4 0 2 6 4 0 2 6 4 0 NaHgPOj^/HgO 44-90 1 3 8 KHCO^ 1 6 3 0 1 0 5 5 MgSO^ 2 9 0 KOH 1 5 7 8 NaBr 11,420 KB r 1 , 0 1 5 C a ( a c e t a t e ) 2 *H 2 0 3 4 7 3 ^ 7 M g ( a c e t a t e ) 2 * 4 H 2 0 2 7 9 0 2 , 7 9 0 N a ( a c e t a t e ) K ( a c e t a t e ) 10 8 8 6 8 8 6 3^7 2 7 9 0 7 4 3 0 8 3 6 TABLE XV B — C o n t i n u e d Compound 1* 2* 3 ^ 5 6 7 8 9 10 NaNO-j 2080 KNO^ 863 Ca(N0 3) 2»4H 20 ^ 5 Mg(N0 3) 2»6H 20 3 3 3 5 i - i n o s i t o l 400 MOPS A grade / 2093 2093 2093 PIPES A grade / 3^30 3^30 3 , ^ 3 0 3^30 3^30 * The d i s s e c t i n g media do NOT con t a i n the i n g r e d i e n t s of Table I . / "MOPS" i s the a b b r e v i a t i o n f o r Morphollnopropane s u l f o n i c a c i d , and "PIPES" i s the abbre-v i a t i o n f o r plperazine-N, N'-bis (2-ethane s u l f o n i c a c i d ) monosodium monohydrate. 1 D i s s e c t i n g medium ( a f t e r Rehacek and Brzosto w s k i , 1 9 6 9 ) . D i s s e c t i n g medium (from B e r r i d g e , 1 9 6 6 ) . The pH was ad j u s t e d t o 7 . 2 w i t h NaOH. J Reference medium f o r osmotic pressure experiments, and c h l o r i d e medium f o r n i t r a t e -replacement experiment. The pH was adjusted t o 6 . 8 or 7 . 2 w i t h NaOH. L Sodium Ringer. The pH was adjusted t o 7 . 2 w i t h NaOH. ^ P o t a s s i u m Ringer. The pH was adjusted t o 7*2 w i t h KOH. ^ C h l o r i d e Ringer f o r h a l l d e - s u b s t i t u t i o n experiments. The pH was ad j u s t e d to 6 . 8 w i t h NaOH. TABLE XV B — C o n t i n u e d C h l o r i d e medium f o r a c e t a t e - s u b s t i t u t i o n e x p e r i m e n t s . pH a d j u s t e d t o 6 . 8 w i t h NaOH. Bromide R i n g e r . pH a d j u s t e d t o 6 . 8 w i t h NaOH. N i t r a t e R i n g e r . pH a d j u s t e d t o 6 . 8 w i t h NaOH. 'Acetate R i n g e r . pH a d j u s t e d t o 6 . 8 w i t h NaOH. 10? In the experiments which were designed to test the effect of various monovalent anions on f l u i d secretion, the "control" medium was always a chloride Ringer solution; i.e., the major anion was chloride. The precise composition of the chloride medium depended on which anion was being tested, and these com-positions are indicated in Table XV B. The experimental medium in each case possessed the same osmotic pressure and catlonic composition as the chloride Ringer solution, but diverse anionic ratios according to the following schemei Experimental Control Chloride Experimental Range of Anion Tested Medium (Number Anionic Medium Chloride From Table XV B) (Number from Concentration Table XV B) (meq/liter) Nitrate 3 9 0 - 6 5 Acetate 7 10 0-115 Bromide 6 8 0-120 Intermediate anionic ratios were achieved by various mixtures of the appropriate chloride medium and the anionic medium being tested. Similarly, to test the effect of Na*K ratio on f l u i d secretion, experimental media were prepared by mixing the sodium and potassium Ringer solutions in various proportions. The control medium in this case was "normal Ringer solution", which was a mixture of the sodium and potassium media in the propor-tion 9 5 « 5 . Usually, a l l the media were prepared in concentrated form, stored frozen, then thawed and diluted to normal strength prior to use. In experiments designed to test the effect of albumin on long-term a c t i v i t y of the glands, albumin was added to normal 108 Ringer solution so that i t s f i n a l concentration was 1% (W/V). To test the effect of inorganic phosphate on f l u i d secretion, sufficient Mcllvalne's phosphate buffer (pH 7.2)* was added to concentrated normal Ringer solution such that the total phosphate concentration was 4 mM/liter after dilution. To test the effect of osmotic pressure on f l u i d secre-tion, the concentration of medium 3 (Table XV B), the reference medium for these experiments ( A f p s 0.56° C), was increased b y addition of sucrose, NaCl, KC1, or NaNO^. In this way, media of identical a f p but varying solute composition were prepared. The quantities of each solute necessary to raise the Afp by a given amount were calculated from the tables of Wolf and Brown (1968). In order to affirm that the A f p of the media corres-ponded reasonably well to the theoretical values, melting point determinations were made of a NaCl medium and a sucrose medium - both calculated to possess the same Afp. The d i f -ference observed between the two media was just over 1% of their mean value. The difference between this mean value and the theoretical value was within 5$ of the theoretical value. 4) Experimental Procedures Experiments were conducted according to one of two general protocols. a) Maintained dosage method Glands were set up In a "control" Ringer solution Composition in C.R.C. Handbook of Chemistry and Physics. 19^3. 109 - 4 containing 10 M adrenalin; the bathing medium was changed frequently (e.g. every 10 or 15 minutes) from an oxygenated stock solution. After an hour or so, the rate of secretion was recorded and the Ringer solution was replaced by an experi-mental medium. When the secretory rate had equilibrated to a new leve l , the gland was returned to the control medium and the rate of secretion again determined. The secretory rate of the gland, while in the experimental medium, was compared to the mean of the two control rates. b) Pulse method Glands were set up in a control Ringer containing 10"^M adrenalin, and the medium was frequently changed (every 2 to 5 minutes) from an oxygenated stock. The rate of secretion was continually monitored. With each change of the medium, the rate increased u n t i l a maximum was reached. At that point, the Ringer solution was changed to an experimental medium which lacked adrenalin. This medium was also changed frequently u n t i l the secretory rate returned to zero. The gland remained In the experimental medium (with only occasional changes from the oxy-genated stock) for approximately one hour, after which time, the glands were re-exposed to adrenalin; after frequent changes of medium, the new maximum rate was recorded. The gland was then returned to control medium (lacking adrenalin) u n t i l the rate was reduced to zero once again. An hour later, the gland was subjected to adrenalin for a third time, and the maximum rate recorded again. Thus, every gland was subjected to three pulses of adrenalin with "rest" periods between. In the pulse method, the times (after addition of adrena-li n ) at which the maximum rates of the three pulses occurred were noted, and the slope of the line Joining the two control rates was calculated; since the rate of secretion almost invari-ably decreased with time, the calculation of the slope was necessary to estimate what the rate of secretion was l i k e l y to have been in the control medium, at the time that the gland had reached i t s maximum rate in the experimental medium. The assump-tion was made that the f a l l in secretory rate was a linear function with time. That this was so (on the average) was demonstrated experimentally (see "Results", Figure 1 3 ) . A f u l l explanation as to why these two methods were used w i l l also be presented i n the Results section of this chapter. 5) Weight of the Glands In order to allow comparison between secretory rates In this study and those of other secretory epithelia, the wet and dry weights of the salivary glands were determined. After experimenting with a gland, I removed i t from the petri dish, and touched i t to f i l t e r paper according to a standard procedure in order to remove the bulk of extracellular f l u i d adhering to the surface of the gland. This usually took about 30 seconds. The wet weight was determined on a *Sartorlus Selecta" micro-balance to within 10 micrograms. Following this, the gland was dried overnight in an oven at 60° C, and the dry weight recorded. 6) The Effect of Drugs on Secretory Rate Having established that adrenalin stimulated secretion, I l l I wished to determine the s p e c i f i c i t y of the receptor site in the gland. The more discriminatory the site of stimulation to specific drugs, the easier i t becomes to identify the normal agent operating in vivo. Ticks were dissected in medium 2 . DL-DOPA, dopamine, noradrenalin isoproterenol, secrotonin, cyclic AMP ( a l l from Sigma Chemicals), pilocarpine (Nutritional Bio-chemicals) and adrenalin (Eastman Organic) were tested for their a b i l i t i e s to stimulate salivation in v i t r o . Various concentrations of these drugs were prepared in normal Ringer solution. 7) Potential Difference Measurements The electropotential difference across the salivary gland was measured by three methods. A) Mlcroelectrodes were made from Pyrex glass tubing ( 1 . 5 mm O.D.) which was drawn out on a Palmer microelectrode puller (Cambridge) and f i l l e d by boiling in 1.5 M KC1. Only electrodes having a resistance of 2 . 5 to ^0 (usual range, 10 to 20) megohms were subsequently used. The s i l v e r - s i l v e r chloride recording electrode was attached to a 'Medlstor* cathode follower held in a micromanipulator. The glass microelectrode made contact with the s i l v e r - s i l v e r chloride electrode via a KC1 bridge as shown in Figure 10. The indifferent electrode ( s i l v e r - s i l v e r chloride) was fixed with epoxy resin to the rim of the small watch glass that served as the dissecting dish. Thus, when the eviscerated tick was flooded with medium, the indifferent electrode was also covered. The c i r c u i t was not completed u n t i l immediately prior to insertion of the electrode into the acinus. 112 Figure 10. Diagram of microelectrode arrangement for measuring electropotential differences by Method A. shaft to micromanipulator 'Medistor' cathode follower alligator clip adhesive tape rubber bung Ag-AgCI electrode 1.5 M KCI glass cylinder rubber bung glass microelectrode epoxy resin indifferent electrode medium bathing preparation 1 Each tick was dissected in a manner similar to that des-cribed for the in-vltro preparation, up to and including eviscer-ation. Dissecting medium 1 was used. The salivary acini did not offer enough resistance on their own to the advancing glass electrode to permit piercing of the basal plasma membrane; they were deflected aside rather than pierced. Consequently, a "cradle" was constructed from an e.m. copper grid coated with collodion and glued with epoxy resin to a bent dissecting pin. The head of the pin was secured with sealing wax to a glass rod which in turn was held in a micromanipulator. It was thus possible to manoeuvre the cradle under a group of acini floating in the medium, in such a way that a given acinus could not r o l l freely when confronted with the glass electrode. The whole procedure was observed through a dissecting microscope, and the trans-epithelial potentials were visualized on a •Tectronix 1 cathode ray oscilloscope, after the asymmetry potential had been backed off. The eviscerated shell of the dissected tick with the salivary glands intact were bathed in a medium similar to that of Rehacek and Brzostowski ( I 9 6 9 ) . but lacking the vitamins, phenol red and heat-treated hemolymph l i s t e d in their table of composition. B) The second method involved recording the potential difference across the whole gland while the lat t e r was secret-ing In vitro in normal Ringer solution. Saturated KCl - 1% agar gel bridges were made up in PE 90 tubing f i t t e d at one end with a glass capillary. One bridge made contact with the secreted droplet, and the other with the bathing medium. The free ends of the PE tubing were placed in separate vessels containing saturated KCI and calomel electrodes (Radiometer Copenhagen, G 2 0 2 C calomel electrode). The c i r c u i t was comple-ted by connecting the calomel electrodes to a mllllvoltmeter (•Keithly 6021 solid state electrometer). The two agar bridges (held by 1 P r i o r 1 micromanipulators) were manoeuvred into the medium bathing the gland, and the asymmetry potential was recorded. One bridge was then placed in the droplet of saliva that had formed at the o r i f i c e of the duct. The potential difference was recorded after correcting for the asymmetry potential. C) The third method was in most respects identical to the second, except that a glass microelectrode described earlier, rather than a salt bridge, was used to measure the potentials. However, since some groups of acini always rested at the o i l -Ringer interface on the Parafilm covering the petri dish, the cradle described in Method "A" was not required. Electrodes would f i r s t press an acinus to the Parafilm, and then enter the lumen of the acinus when advanced by means of the micromanipu-lator. Occasional recordings across the whole gland (as dis-tinct from across the acinus Itself) were also taken with the glass electrodes to confirm the results obtained with Method "B". RESULTS 1) Development of the In-Vltro Preparation A l l the media used In this study were based on (a) the tick tissue-culture medium of Rehacek and Brzostowski (1969) or (b) complex medium of Berridge ( 1 9 6 6 ) . The latter was the superior of the two; nevertheless, when used without the appro-priate stimulus, only about 10$ of the excised glands secreted at a l l . Of the la t t e r , no gland secreted a f u l l microliter of saliva, and the maximum rate of secretion ( a t room temperature) was 300 nl/hour. In order to Improve the performance of the glands %n v i t r o , a number of potential stimuli were tested on glands set up in Berridge*s medium. The following were without effect* a) To test for possible hormal stimulation of secretion, the bathing medium was enriched with fresh tick hemolymph (up to 30$ V/V) and several glands were set up in pure hemo-lymph from ticks which had been feeding for six or seven days (13 preparations). Such ticks almost invariably secrete saliva when glass capillaries are placed over the mouth parts (see Chapter Two). b) To provide an osmotic shock, the medium was diluted by half with d i s t i l l e d water (3 preparations). c) Vasopressin Is a powerful diuretic hormone In vertebrates. There existed the slim chance that i t could be effective in 117 the tick as well, since the latter could encounter this mole-cule in i t s meal and might have evolved a way of u t i l i z i n g i t for i t s own diuresis. Vasopressin was assayed in doses rang-ing from one-eighth of a mllliunit per ml to 1 3 mllliunits per ml (4 preparations), d) In light of the failure of pure tick hemolymph to stimulate secretion, i t seemed unlikely that a hormonal stimulus t r i g -gered salivation. It was thus natural to suspect that salivation might have been under neural control. Since other authors reported the use of the cholinomimetic drug pilocar-pine to stimulate salivation In vivo (Howell, 1 9 6 6 ? Tatchell, 1 9 6 7 a ; Purnell et a l . , 1 9 6 9 ) , i t seemed reasonable to begin - 7 — 2 by testing pilocarpine ( 1 0 ' to 10 M) and acetylcholine ( 1 0 " ^ M) on the excised glands. Since neither of these were effectual, I turned to serotonin ( 1 0 M to 10 M), since this drug i s known to be a powerful stimulant of secretion when applied to Insect Malpighian tubules (Maddrell, Pllcher, and Gardiner, 1 9 6 9 , 1 9 7 1 ) and to the salivary glands of Calllphora (Berridge and Patel, 1 9 6 8 ) . Certain acidic amino acids (glutamic, aspartic, and cysteic) are known to be powerful excitants of neurones (Eccles, 1964 ) . More recently Usherwood ( 1 9 6 9 ) has suggested that at least in insect skel-etal muscle, a dicarboxyllc acid (such as L-glutamate) is strongly favoured as the natural transmitter at the excita-tory nerve-muscle synapse. Since glutamate and aspartate were both present in Rehacek and Brzostowskl's medium at • -4 -4 4 .3 x 10 M and 1 , 7 x 10 M respectively, and glutamate 118 in Berridge's medium at 1 3 mM/liter, and since these media ( i f unmodified) did not support secretion, i t appeared unlikely that a member of this series of potential stimuli would stimulate salivation in Dermacentor. Finally, a marked stimulation of secretory rate was produced by adrenalin. The threshold dosage lay between 10""^ M and 10"*^ M, and the secretory rate could be progressively accel-erated by increased dosages up to 10"-^ M - the highest concentra-tion tested (Figure 1 1 ) . The usual rate of secretion at 1 0 " ^ M was 5 0 0 0 to 8 0 0 0 nl/hour compared to a maximum of 3 0 0 nl/hour in the absence of adrenalin. Glands continued to perform satisfac-t o r i l y when the protein and T.C. yeastolate were removed from the medium. This modification to Berridge's complex medium resulted in medium 3 of Table XV. Notwithstanding these i n i t i a l successes, the rate of secretion decayed steadily with time. Numerous modifications of the protocol were tested in order to abolish or minimize this decay of a c t i v i t y and to establish the optimal conditions for secretion. The secretory rate was inhibited at pH's below six and above eight, although no consistent pattern emerged In between. Subsequently, a l l media were adjusted to a pH close to neutrality. It was Impracticable to bubble oxygen through the f l u i d bathing the glandj instead, stock media were vigor-ously aerated with medical grade 0 2 (Canada Liquid Air) through-out the duration of the experiment, and the f l u i d bathing the gland was renewed frequently (every 10 or 15 minutes). Since oxygenation tended to drive the pH up, one of the buffers in 119 F i g u r e 11. The r e s p o n s e o f an i n - v i t r o g l a n d t o a d r e n a l i n . G l a n d s were b a t h e d i n B e r r i d g e ' s (1966) complex medium ( i . e . medium 3 c o n t a i n i n g 0.8$ (W/V) l a c t a l b u m i n h y d r o l y s a t e and 0.4$ (W/V) T.C. y e a s t o l a t e ) . Arrows denote t h e t i m e s a t w h i c h s u c c e s s i v e l y h i g h e r c o n c e n t r a t i o n s of a d r e n a l i n were added. T h i s g l a n d (as w e l l as a l l o t h e r s t r e a t e d s i m i l a r l y ) was n o t s t i m u l a t e d t o s e c r e t e when t h e c o n c e n t r a t i o n of a d r e n a l i n was below 10"^ M. Time (min) 121 "Good's s e r i e s " (Good e t a l . , 1966), p r o v i d e d by C a l b i o c h e m C o r p . , was added t o t h e medium. B o t h MOPS (pK =7.2) and PIPES (pK = 6.8) p r o v e d s a t i s f a c t o r y a t a s t r e n g t h o f 10 m M / l i t e r , p r o v i d e d t h e r a t e o f oxygen f l o w i n t o t h e s t o c k s o f media was r e d u c e d t o a minimum. E v e n t u a l l y MOPS was used as t h e r o u t i n e b u f f e r , p r i m a r i l y because i t was n o t a s s o c i a t e d w i t h an i n o r g a n i c c a t i o n . The maintenance o f s e c r e t o r y a c t i v i t y improved m a r k e d l y when d i s s e c t i n g medium 1 was abandoned i n f a v o u r o f medium 2. F u r t h e r improvements i n s e c r e t o r y r a t e r e s u l t e d when t h e N a C l c o n c e n t r a -t i o n i n medium 3 was t r i p l e d . T h i s a d d i t i o n t o medium 3 r e s u l t e d i n t h e no r m a l R i n g e r s o l u t i o n (see " E x p e r i m e n t a l Media" i n Methods s e c t i o n ) w h i c h became t h e e v e n t u a l c o n t r o l medium f o r many o f t h e s e e x p e r i m e n t s . A l t h o u g h t h e p r e p a r a t i o n g r a d u a l l y improved w i t h t h e s e m o d i f i c a t i o n s , s t i l l , t h e r a t e o f s e c r e t i o n d e c l i n e d m a r k e d l y d u r i n g t h e f i r s t two h o u r s a f t e r d i s s e c t i o n . The q u e s t i o n a r o s e as t o whether t h i s d ecay i n r a t e was due t o a " f a t i g u e " c a used by t h e c o n t i n u a l e x p o s u r e o f t h e g l a n d s t o a d r e n a l i n . To t e s t t h i s h y p o t h e s i s , t h e p u l s e method ( d e s c r i b e d i n t h e Methods s e c t i o n ) was i n t r o d u c e d . The r e s u l t s a r e shown i n F i g u r e 12. There was a n o t i c e a b l e i n c r e a s e i n t h e d u r a t i o n o f t h e s e c r e t o r y a c t i v i t y o f t h e p u l s e d g l a n d s compared t o t h a t o f g l a n d s w h i c h were c o n t i n u a l l y b a t h e d i n a d r e n a l i n . Second, t h e decay i n r a t e (which was s t i l l s i g n i f i c a n t ) , became a l i n e a r f u n c t i o n o f t i m e . F i n a l l y , a number o f i n d i v i d u a l p r e p a r a t i o n s o c c a s i o n a l l y showed no a p p r e c i a b l e decay o v e r t h e e x p e r i m e n t a l p e r i o d . The l a t t e r was n e v e r o b s e r v e d when g l a n d s were s u b j e c -t e d t o a m a i n t a i n e d dosage o f a d r e n a l i n . 122 F i g u r e 12. Rate of s e c r e t i o n w i t h time under v a r i o u s p r o t o c o l s . • g l a n d s e x c i s e d i n medium 1 and g i v e n a m a i n t a i n e d dosage of 10 M a d r e n a l i n i n medium 3* O g l a n d s e x c i s e d i n medium 2 and p u l s e d w i t h 10~^ M a d r e n a l i n i n n o r m a l R i n g e r s o l u t i o n . A g l a n d s e x c i s e d i n medium 2 and p u l s e d w i t h 10"^ M a d r e n a l i n i n n ormal R i n g e r s o l u t i o n t o w h i c h was added 1% (W/V) b o v i n e serum a l b u m i n . A i d e n t i c a l t r e a t m e n t t o 11 O 11 e x c e p t t h a t 4 m M / l i t e r i n o r g a n i c phosphate was added t o normal R i n g e r s o l u t i o n . V e r t i c a l b a r s denote t h e SE of the mean. 150-, 0 -I 1 1 1 1 1 0 100 200 300 400 500 Time (min) 12k It i s well established that tissue oulture media must be supplemented with protein i f c e l l s are to survive In vit r o for extended periods (Waymouth, 1965)• The role of proteins in keeping the ce l l s alive i s s t i l l not known with certainty, but they are believed to "detoxify" the medium. They may do this either by protecting the c e l l surface, or by carrying molecules such as fatty acids away from the c e l l surface (Prederickson and Gordon, 19581 Hudman et a l . , 1 9 7 1 ) . The need for albumin is not limited to long-term survival, but may even manifest i t s e l f over a period of hours - especially i f the ce l l s are very active. Consequently, i t seemed reasonable to test the effect of including albumin in the medium on prolonging secre-tory a c t i v i t y of the preparation. Figure 12 shows that although ac t i v i t y of the glands (on the average) did not decay beyond the second pulse, the overall decay was undiminished. A similar experiment was repeated using medium without albumin, but to which was added k mM/liter inorganic phosphate. Phosphate i s an ubiquitous anion in biological f l u i d s ; i t i s especially needed for the synthesis of ATP. Since phosphate was absent from the normal Ringer solution, i t seemed possible that this ion was gradually being leached from the cel l s as a result of f l u i d secretion leading to cessation of ATP synthesis. Furthermore, phosphate i s actively transported by the Malpighian tubules of Calllphora (Berridge, 1 9 6 9 ) . so the possibility existed that phosphate could similarly be used by the tick salivary gland to drive f l u i d secretion. It i s obvious from Figure 12, however, that the rate s t i l l decayed with time, and 125 t h a t t h e i n i t i a l s e c r e t o r y r a t e was s i g n i f i c a n t l y r e d u c e d i n t h e p r e s e n c e o f 4 m M / l i t e r p h o s p h a t e . The development o f t h e l n - v l t r o p r e p a r a t i o n has been t r a c e d i n some d e t a i l because some e x p e r i m e n t s were performed b e f o r e a l l t h e Improvements i n t h e p r o t o c o l were r e a l i z e d . T h i s a c c o u n t s f o r t h e d i v e r s i t y o f p r o c e d u r e s e v i d e n t among t h e v a r i o u s e x p e r i m e n t s t o be d e s c r i b e d below. I t seemed u n n e c e s s a r y t o r e p e a t many o f t h e e a r l i e r e x p e r i m e n t s ( w h i c h were p e r f o r m e d under i n f e r i o r p r o t o c o l ) s i n c e many o f t h e s e s i m p l y d e m o n s t r a t e d q u a l i t a t i v e e f f e c t s o f a g e n t s so t h a t each g l a n d s e r v e d as i t s own c o n t r o l ; c o n s e q u e n t l y , r e p e t i t i o n was c o n s i d e r e d u n l i k e l y t o have r a d i c a l l y a l t e r e d t h e g e n e r a l c o n c l u s i o n o b t a i n e d f r o m e a r l y e x p e r i m e n t s . 2) Time Course o f S e c r e t o r y A c t i v i t y  U s i n g t h e P u l s e Method The maximum r e s p o n s e t o a g i v e n dose o f a d r e n a l i n d i d n o t o c c u r q u i c k l y ( F i g u r e 13). A l t h o u g h s e c r e t o r y a c t i v i t y u s u a l l y commenced w i t h i n f i v e m i n u t e s o f a d d i n g a d r e n a l i n , the r a t e o f s e c r e t i o n r o s e s t e a d i l y , so t h a t t h e maximum r a t e e v e n t u a l l y a c h i e v e d , o c c u r r e d a p p r o x i m a t e l y 25 minutes a f t e r t h e f i r s t a p p l i c a t i o n o f a d r e n a l i n . The medium b a t h i n g t h e g l a n d was f r e q u e n t l y changed t h r o u g h o u t t h i s p e r i o d . A f t e r s e v e r a l changes o f medium however, t h e r e was no s i g n i f i c a n t I n c r e a s e i n t h e r a t e o f s e c r e t i o n . T h i s s u g g e s t s t h a t i t took some t i m e f o r t h e co n -c e n t r a t i o n o f a d r e n a l i n a t i t s s i t e o f a c t i o n t o come t o e q u i l i b -r i u m w i t h t h e c o n c e n t r a t i o n I n t h e medium. On r e m o v a l of a d r e n a l i n , t h e r e s p o n s e o f t h e g l a n d was more r a p i d . Whereas 126 F i g u r e 13. The t i m e c o u r s e o f s e c r e t o r y a c t i v i t y d u r i n g t h e f i r s t p u l s e o f a d r e n a l i n u s i n g t h e p u l s e method. G l a n d s were d i s s e c t e d i n medium 2 and s e t up i n n o r m a l R i n g e r s o l u t i o n . The s o l i d a r r o w s denote a d d i t i o n (1) and r e m o v a l (2) o f 10 J M a d r e n a l i n , r e s p e c t i v e l y . The open arrows i n d i c a t e t h e r e n e w a l o f t h e medium f r o m an oxyg e n a t e d s t o c k . V e r t i c a l b a r s denote SE o f t h e mean. Minutes after adding 10 M adrenalin 128 the time for attainment of the half-maximal rate after adding adrenalin was 11 to 12 minutes, the half time for a c t i v i t y decay after removal of adrenalin was only 2 minutes. The lag in response to the addition of adrenalin suggests that there i s a diffusion barrier interposed between the site of application of adrenalin (i. e . , basal surface of secretory c e l l - see Chapter Four) and the receptor site for this transmitter substance. The reduced half time for decay after removal of adrenalin was possibly a reflection of the presence at the receptor site of an enzyme which can destroy adrenalin. The salivary gland w i l l function in v i t r o , and the volume of the secreted f l u i d may exceed the wet weight of the gland f i v e - or even tenfold over five hours. Moreover, the ion con-centration of the saliva* over this period of time did not fluctuate significantly (Figure lk) ; this suggests that despite a f i v e f o l d decay in the rate of f l u i d secretion, the same active processes generating f l u i d secretion were operative throughout this time. According to the c r i t e r i a proposed by Kirschner (1967), one must conclude that the excretion of excess f l u i d by the salivary gland involves a secretory process rather than f i l t r a t i o n . The usual secretory rate of this ln-vltro preparation was higher than that observed before in preparations of a similar nature (Table XVI). * In the remainder of this chapter, the secreted f l u i d under In vi t r o conditions w i l l be called "saliva"; when necessary, the distinction between natural saliva and in vitro saliva w i l l be made clear. 129 F i g u r e 14. I o n c o n c e n t r a t i o n o f s a l i v a s e c r e t e d i n v i t r o . G l a n d s were d i s s e c t e d i n medium 1 and s u b j e c t e d t o a m a i n t a i n e d -4 dosage of 10 M a d r e n a l i n i n medium 3» V e r t i c a l and h o r i z o n t a l b a r s denote S E of mean c o n c e n t r a t i o n and mean time r e s p e c t i v e l y . The c o n c e n t r a t i o n s o f sodium, c h l o r i d e , and p o t a s s i u m i n t h e medium ifere r e s p e c t i v e l y 105 + 5t 66 + 3» and 8.4 + 0.2 meq/ l i t e r (mean + S E ) . A c c o r d i n g t o a p p r o p r i a t e t - t e s t s , t h e c o n c e n t r a t i o n s o f t h e t h r e e i o n s d i d not change s i g n i f i c a n t l y o v e r s i x h o u r s . A l l f u t u r e e x p e r i m e n t s were c o n d u c t e d w i t h i n t h i s p e r i o d o f t i m e . Time (min) 131 TABLE XVI A COMPARISON OF IN-VITRO SECRETORY RATES AMONG PREPARATIONS OF A SIMILAR NATURE TO THE ONE USED IN THIS STUDY Genus Organ Temper-a t u r e D y s d e r o u s  C a l l l p h o r a  R h o d n l u s  D l x l p p u s C a l p o d e s CalUPhora " S a l i v a r y G l a n d D ermacentor M a l p i g h i a n Room Tubu l e Room 24°C 1 4 - 1 7 C 2 5 - 2 7°C Rate of S e c r e t i o n p e r P r e p a r a t i o n ( n l / m i n ) 1 - 4 10 70 4 - 5 30 40 1 0 0 (may exceed 2 5 0 ) R e f e r e n c e B e r r i d g e , 1966 B e r r i d g e , 1969 M a d d r e l l , 1969 Ramsay, 1 9 5 5 P l l c h e r , 1970 I r v i n e , 1969 B e r r i d g e & P a t e l 1968 T h i s s t u d y 3 ) The C o n t r o l o f S e c r e t i o n i Pharmacology The s a l i v a r y g l a n d w i l l n o t s e c r e t e s p o n t a n e o u s l y when s e t up i n a r t i f i c i a l medium, o r even when s e t up i n hemolymph c o l l e c t e d f r o m t i c k s t h a t have been a c t i v e l y f e e d i n g and (pr e s u m a b l y ) s a l i v a t i n g ; i n b o t h c a s e s , the a p p r o p r i a t e s t i m u l u s f o r s e c r e t i o n i s l a c k i n g . I t i s u n l i k e l y t h e n , t h a t a b l o o d -b o r n e f a c t o r i s r e s p o n s i b l e f o r s e t t i n g the s e c r e t o r y machine i n m o t i o n i n t h e l i v e t i c k , o r i f s o , such a hormone must be 132 e x t r e m e l y l a b i l e . However, t h e f a c t t h a t a d r e n a l i n i n r e a s o n a b l y low c o n c e n t r a t i o n s ( 1 0 " ^ M) was a b l e t o s t i m u l a t e t h e g l a n d . i n v i t r o , b u t a c e t y l c h o l i n e and p i l o c a r p i n e a t much h i g h e r c o n c e n -t r a t i o n s were n o t , s u g g e s t e d t h a t t h e g l a n d s might n o r m a l l y be u n d e r a d r e n e r g i c n e r v o u s c o n t r o l . To t e s t t h e s p e c i f i c i t y o f t h e r e c e p t o r s i t e s , a number o f a l p h a - a d r e n e r g i c d r ugs ( n o r a d r e n -a l i n , dopamine) one b e t a - a d r e n e r g i c d r u g ( i s o p r o t e r e n o l ) and s e v e r a l o t h e r a g e n t s ( s e r o t o n i n , c y c l i c AMP), known f o r t h e i r a b i l i t y t o s t i m u l a t e i n s e c t s e c r e t o r y and n e u r o - m u s c u l a r systems ( B e r r i d g e and P a t e l , 1968} B e r r i d g e , I97O5 M a d d r e l l e t a l . . 1 9 6 9 , 1 9 7 1 ) were a p p l i e d I n v i t r o . I n a d d i t i o n , two i n t e r m e d i a t e s t o t h e b i o s y n t h e s i s o f a d r e n a l i n (Malmejac, 1 9 6 4 ) , n o t n o r m a l l y p o s s e s s i n g a d r e n e r g i c a c t i v i t y t h e m s e l v e s ( p h e n y l a l a n i n e , DL-DOPA), were a s s a y e d . I t i s c l e a r f r o m F i g u r e 15 t h a t t h e s a l i v a r y g l a n d s do n o t r e s p o n d t o p i l o c a r p i n e (10 J M) o r c y c l i c AMP ( 1 0 M). P h e n y l a l a n i n e was n o t t e s t e d s e p a r a t e l y , b u t was p r e s e n t i n a - 4 c o n c e n t r a t i o n o f 2 . 6 x 10 M i n t h e t i c k t i s s u e - c u l t u r e medium o f Rehacek and B r z o s t o w s k l ( 1 9 6 9 ) . As a l r e a d y m e n t i o n e d , t h i s medium does n o t on i t s own s u p p o r t s e c r e t i o n . DL-DOPA was n o t e f f e c t i v e a t 10*"-' M. G l a n d s c a n be s t i m u l a t e d by s e r o t o n i n , b u t o n l y a t h i g h c o n c e n t r a t i o n s ( 1 0 ~ ^ M). The g l a n d s a r e most s e n s i -t i v e t o a l p h a - a d r e n e r g i c d r u g s ( a d r e n a l i n , dopamine, and n o r a d -r e n a l i n ) b u t c a n be s t i m u l a t e d by b e t a - a d r e n e r g i c drugs ( i s o -p r o t e r e n o l ) a t h i g h e r c o n c e n t r a t i o n s . O t h e r a u t h o r s have r e p o r t e d u s i n g o n l y c h o l i n e r g i c drugs ( e . g . p i l o c a r p i n e ) t o s t i m u l a t e s a l i v a t i o n i n v i v o I n t o g l a s s 1 3 3 F i g u r e 1 5 . E f f e c t o f d r u g s on s e c r e t o r y r a t e . The performance o f t y p i c a l I n d i v i d u a l g l a n d s b a t h e d i n v a r i o u s d r u g s a t t h e c o n c e n t r a t i o n s i n d i c a t e d . The t i m e o f a p p l i c a t i o n and r e m o v a l o f each d r u g i s i n d i c a t e d by a r r o w s . A l l g l a n d s were d i s s e c t e d i n medium 2 and s e t up i n n o r m a l R i n g e r s o l u t i o n . A l t h o u g h o n l y s i n g l e p r e p a r a t i o n s a r e shown h e r e , t h e r e s u l t f o r p i l o c a r p i n e was seen i n 12 p r e p a r a t i o n s 1 f o r s e r o t o n i n 10 M and below, 12 p r e p a r a t i o n s ; s e r o t o n i n 10 J M, 2 p r e p a r -a t i o n s ; c y c l i c AMP, 2 p r e p a r a t i o n s ; DL-DOPA, 2 p r e p a r a t i o n s ; N o r a d r e n a l i n , 3 p r e p a r a t i o n s ; i s o p r o t e r e n o l , 3 p r e p a r a t i o n s ; and dopamine, 5 p r e p a r a t i o n s . A d r e n a l i n was used as t h e r o u t i n e s t i m u l a n t i n s e v e r a l hundred p r e p a r a t i o n s . T 1 " 1 1 1 -0 50 200 250 300 Time (min) Time (min) 1 3 5 capillaries (Howell, 1 9 6 6 j Tatchell, 1 9 6 7 * Purnell ejb a l . , 1 9 6 9 ) . Since pilocarpine also Induces salivation when injected Into D. andersoni (Gregson, personal communication) this situation might appear to conflict with the present results for excised glands. However, i t i s possible that pilocarpine injected into ticks acts at the level of the central nervous system to stimu-late afferent adrenergic nerves leading to the salivary glands or that i t may have other non-specific effects. Therefore i t was Important to determine whether adrenalin could also induce salivation when injected into the hemocoele of feeding ticks, A solution of adrenalin ( 1 0 " ^ M), usually in 1 . 2 $ NaCl (approximately iso-osmotic with tick hemolymph) was injected into ticks as described in "Methods" of Chapter Two, In some experi-ments a hypo-osmotic Ringer (medium 3 , Table XV B) was used instead of saline. Three experimental procedures were followed» A) Even without prior injections, ticks can be coaxed to secrete saliva into glass c a p i l l a r i e s . To see whether they would resume salivation after so-called 'spontaneous* s a l i -vation had ceased, 1 pi of adrenalin-solution was injected for each 1 0 mg tick weight. Control ticks were injected with saline alone. B) Immediately after removing and weighing the tick I Injected 1 pi per 2 0 mg tick weight, in order to see whether the rate and volume of spontaneous secretion could be lnoreased above the values characteristic of non-injected ticks. Control ticks were injected with saline alone. C) After some of the experimental ticks in "B" above had 136 terminated salivation, they were re-injected with a volume of adrenalin-solution equal to the volume of secreted saliva. If salivation did not resume within 10 minutes, the injec-tion was considered to be ineffectual. The effect of injected adrenalin in vivo i s shown in Table XVII and Figure 16. Adrenalin was able to Increase the yiel d of saliva sixfold above that from non-injected ticks, provided that i t was injected before the ticks had salivated spontaneously. Although i t caused some resumption of secretion after ticks salivated spontaneously or after re-injection of a volume of adrenalin-solution equal to that secreted by the glands subsequent to the f i r s t injection, this was only a small effect. The non-injected ticks secreted more saliva (in terms of percentage of total hemolymph volume) than those injected with adrenalin (Table XVII). Since adrenalin e l i c i t s a pro-found response from the salivary gland both in vivo and in v i t r o , but pilocarpine appears, from the work of others, to exert an effect only in vivo, this suggests that the former drug probably bears a closer resemblance to the natural transmitter substance. This conclusion i s further substantiated when one considers that the f i n a l concentration of adrenalin i n the hemocoele of Derma-centor was estimated to be between l/100th and l/1000th the concentration of pilocarpine injected into other ticks by the above mentioned authors (see Discussion). 4) The Effect of Various Anions on the Rate  of Secretion and Composition of Saliva In vivo, chloride was the only ion distributed across the 137 Figure 1 6 . Salivary secretion In vivo. The typical secretory pattern of two ticks showing the cumula-tive volume of saliva collected In glass capillaries with time. O tick injected with 1.2$ NaCl (1 pi per 20 mg tick weight) -4 A tick injected with 10 M adrenalin in 1.2$ NaCl. Estimated f i n a l concentration of adrenalin in hemolymph is 2 x 10"^ M. In both cases, i t i s estimated that the injected f l u i d increased the extracellular volume by about 18$. Since the capacity of the glass capillaries totalled 7 pi* several times throughout the collection period, the contents of the pipettes had to be expelled and the pipettes reinserted on the mouthparts of the ti c k . These periods are denoted on the curves by the dotted l i n e s . Occasional Imbibition of secreted saliva is evidenced by the reduction in cumulative volume at various points along the curve. Cumulative volume (microliters) TABLE X V I I THE EFFECT ON SALIVATION OF INJECTION OF ADRENALIN IN VIVO Time o f I n j e c t i o n I n j e c t i o n Medium Dosage p e r U n i t Wet Weight of T i c k (pl/mg) Number of T i c k s Volume o f S a l i v a (yd ± SE) $ of T o t a l Hemolymph Volume S e c r e t e d (± SE) Average Rate of S e c r e t i o n as P e r c e n t a g e Hemolymph Volume p e r Mi n u t e (± SE) A f t e r spontaneous s a l i v a t i o n B B e f o r e spontaneous s a l i v a t i o n R e - i n j e c t i o n o f t i c k s i n s e r i e s B a f t e r c e s s a t i o n o f i n d u c e d s a l i v a t i o n D N o n - i n j e c t e d c o n t r o l s medium 3 - 4 10 M a d r e n a l i n i n medium 3 1 pl/10 mg 4 1 pl/10 mg 3 1 u l / 2 0 mg 5 1 /al / 2 0 mg 5 1 . 2 $ N a C l -4 10 * M a d r e n a l i n i n 1 . 2 $ N a C l 1 0 " ^ M e q u i v a l e n t a d r e n a l i n t o t o t a l i n volume s e c r e -1 . 2 $ N a C l t e d i n s e r i e s B 0 0 2 . 5 + 0.8 4 . 6 + 1.8 3 . 4 + 3 . 1 k.2 ± 3 . 7 1 9 . 6 + 4 . 1 3 ^ ± 7 . 3 0 . 3 ± 0 . 1 0 . 1 + 0 . 1 1 . 5 ± 0 . 3 2 . 9 ± 1 . 5 ^ . 6 + 2 . 4 0 . 3 ± 0 . 1 3 . 0 + 1 . 1 8 . 4 + 3 . 1 0 . 6 + 0 . 2 140 s a l i v a r y e p i t h e l i u m a s y m m e t r i c a l l y (S/H r a t i o = 1 . 1 ) j moreover, i t s c o n c e n t r a t i o n i n t h e s a l i v a d i d n o t f l u c t u a t e s i g n i f i c a n t l y w i t h t h e p r o g r e s s i o n o f f e e d i n g ( C h a p t e r Two). S i m i l a r l y , t h e S/H r a t i o f o r c h l o r i d e I n v i t r o i s g r e a t e r t h a n one ( F i g u r e 1 4 ) . The f o l l o w i n g e x p e r i m e n t s were d e s i g n e d t o t e s t whether s e c r e -t i o n depended upon t h e p r e s e n c e o f c h l o r i d e i n t h e b a t h i n g medium, and whether t h e r a t e o f f l u i d s e c r e t i o n was c o r r e l a t e d w i t h t h e r a t e o f c h l o r i d e s e c r e t i o n . W i t h t h i s i n mind, t h e e f f e c t s o f s u b s t i t u t i n g n i t r a t e , a c e t a t e , and bromide f o r c h l o r i d e i n t h e b a t h i n g medium were t e s t e d . T i c k s were d i s s e c t e d i n medium 1; t h e c o n t r o l medium i n e a c h c a s e was a c h l o r i d e R i n g e r s o l u t i o n a c c o r d i n g t o t h e scheme shown i n t h e Methods s e c t i o n o f t h i s c h a p t e r . I n t h e s e e x p e r i m e n t s t h e m a i n t a i n e d dosage p r o t o c o l (see Methods) u s i n g -4 10 M a d r e n a l i n was f o l l o w e d . When t h e c h l o r i d e i n t h e b a t h i n g medium was r e p l a c e d w i t h i n c r e a s i n g l e v e l s o f n i t r a t e , t h e r a t e o f f l u i d s e c r e t i o n d e c r e a s e d . I n pure n i t r a t e R i n g e r s o l u t i o n ( i . e . no c h l o r i d e ) , t h e s e c r e t o r y r a t e was r e d u c e d t o 5% o f the r a t e i n pure c h l o r i d e R i n g e r s o l u t i o n ( F i g u r e 1 8 ) . T h i s i n h i b i t i o n c o u l d be p a r t i a l l y r e v e r s e d when t h e g l a n d s were o f f e r e d c h l o r i d e a g a i n , so i t was u n l i k e l y t h a t n i t r a t e p e r se was t o x i c ( F i g u r e 1 7 ) . T h i s i s f u r t h e r i l l u s t r a t e d l a t e r i n F i g u r e 23 w h i c h i l l u s t r a t e s t h a t a d d i n g 47 m e q / l i t e r o f NaNO^ t o medium 3 had t h e same e f f e c t as a d d i n g t h e e q u i v a l e n t o s m o t i c c o n c e n t r a t i o n o f N a C l . The e f f e c t o f r e p l a c i n g c h l o r i d e w i t h a c e t a t e was q u a l i -t a t i v e l y s i m i l a r t o t h e e f f e c t o f r e p l a c i n g c h l o r i d e w i t h 141 F i g u r e 1 7 . The r e c o v e r y o f g l a n d s b a t h e d i n a c e t a t e and n i t r a t e media when r e t u r n e d t o c h l o r i d e media ( m a i n t a i n e d -4 dosage o f 10 M a d r e n a l i n ) C o n t r o l c h l o r i d e medium ( O ) j pure n i t r a t e medium ( O l 2 0 i 8 0 m i x t u r e o f c h l o r i d e and n i t r a t e media ( A ) ; pure a c e t a t e medium ( a)• Arrows show when t h e g l a n d s were changed f r o m one medium t o a n o t h e r . The p a r t i a l r e c o v e r y on r e t u r n i n g g l a n d s t o c h l o r i d e medium i s a l m o s t immediate. (NOTE: A t l e a s t p a r t o f t h e r e a s o n t h a t r e c o v e r y i s o n l y p a r t i a l i s due t o t h e f a c t t h a t even i n pure c h l o r i d e R i n g e r s o l u t i o n , s e c r e t o r y a c t i v i t y decays s t e a d i l y w i t h t i m e . ) Cumulative volume (microliters) O — to W *>• tn 0» io o o 143 F i g u r e 18. R e l a t i o n s h i p between c h l o r i d e c o n c e n t r a t i o n i n t h e b a t h i n g medium and f l u i d s e c r e t o r y r a t e . V e r t i c a l b a r s denote SE of t h e mean. C h l o r i d e r e p l a c e d by a c e t a t e ( O ) o r n i t r a t e ( A ), t h e t o t a l m o l a r i t y b e i n g c o n s t a n t . F i g u r e 19. C h l o r i d e c o n c e n t r a t i o n of s a l i v a i n v i t r o as a f u n c t i o n of e x t e r n a l c h l o r i d e c o n c e n t r a t i o n . The c h l o r i d e c o n c e n t r a t i o n of t h e b a t h i n g medium was l o w e r e d by r e p l a c i n g c h l o r i d e w i t h a c e t a t e ( O ) o r r a i s e d by a d d i t i o n s of N a C l t o t h e medium ( • ) . V e r t i c a l b a r s denote SE of t h e mean u n l e s s t h e SE i s I n c l u d e d i n t h e p o i n t i t s e l f . The e q u a t i o n f o r th e r e g r e s s i o n c u r v e t h r o u g h t h e e x p e r i m e n t a l p o i n t s i s Y = 88 + 0.92 X. The s l o p e o f t h i s c u r v e i s I n s i g n i f i c a n t l y d i f f e r e n t f r o m t h a t o f t h e i s o t o n i c l i n e ( i . e . , 1.0). [c\ ] in medium (meq/liter) 145 F i g u r e 20. The r e l a t i o n s h i p between r a t e o f c h l o r i d e and f l u i d s e c r e t i o n . D a t a t a k e n f rom F i g u r e s 18 and 19. G l a n d s b a t h e d i n medium 3 ( O ), medium 9 ( n i t r a t e medium - • ) , v a r i o u s m i x t u r e s o f media 7 and 10 ( c h l o r i d e and a c e t a t e - A ), o r medium 3 w i t h added N a C l ( • ) . The e q u a t i o n f o r t h e r e g r e s s i o n c u r v e i s Y = -0.6 + 6.2 X. Rate of chloride secretion (neq/min) n i t r a t e . Pure a c e t a t e R i n g e r s o l u t i o n however was u n a b l e t o s u p p o r t s a l i v a r y s e c r e t i o n a t a l l ( F i g u r e 1 8 ) . T h i s i n h i b i t i o n was a l s o r e v e r s i b l e so t h a t t h e e f f e c t a g a i n was u n l i k e l y t o be one o f permanent t o x i c i t y ( F i g u r e 1 7 ) . Bromide was a b l e t o s u p p o r t s e c r e t i o n as w e l l as c h l o r i d e . The r a t e o f s e c r e t i o n o f f i v e g l a n d s i n c h l o r i d e R i n g e r s o l u t i o n was 2 3 + 3 n l / m i n (mean + S E ) , and t h a t i n bromide R i n g e r s o l u -t i o n was 2 1 + 3 n l / m l n . A p a i r s t - t e s t (Simpson e t a l . , i 9 6 0 ) showed t h a t t h i s d i f f e r e n c e was n o t s t a t i s t i c a l l y s i g n i f i c a n t . The c o n c e n t r a t i o n o f c h l o r i d e I n t h e s a l i v a was l i n e a r l y r e l a t e d t o and was c o n s i s t e n t l y h i g h e r (by a p p r o x i m a t e l y 80 meq/ l i t e r ) t h a n t h a t o f t h e b a t h i n g medium o v e r a t e n f o l d e x t e r n a l c o n c e n t r a t i o n range ( F i g u r e 1 9 ) • Moreover, t h i s h e l d t r u e r e g a r d l e s s o f t h e r a t e of f l u i d s e c r e t i o n , o r w h e t h e r t h e f i n a l c h l o r i d e c o n c e n t r a t i o n i n t h e medium was a r r i v e d a t by s u b s t i t u -t i o n o f c h l o r i d e w i t h a c e t a t e o r s i m p l y by a d d i n g N a C l . The s u r p r i s i n g a s p e c t of t h e r e s u l t s i n F i g u r e 1 9 I s t h a t t h e d i f -f e r e n c e between c h l o r i d e c o n c e n t r a t i o n i n s a l i v a and R i n g e r s o l u t i o n i s so much g r e a t e r t h a n t h e d i f f e r e n c e o b s e r v e d i n v i v o . The S/H r a t i o i n v i v o was 1 . 1 ( C h a p t e r Two) compared t o 1 , 6 I n v i t r o . The dependence o f f l u i d s e c r e t i o n on t h e c h l o r -i d e c o n c e n t r a t i o n o f t h e medium and t h e h i g h S/H r a t i o f o r c h l o r i d e a t a l l c o n c e n t r a t i o n s b o t h s u g g e s t t h a t f l u i d movement i s a consequence o f c h l o r i d e s e c r e t i o n . Moreover, t h e r e s u l t s i n F i g u r e 20 show a d i r e c t r e l a t i o n s h i p between t h e r a t e o f c h l o r i d e and f l u i d s e c r e t i o n ? I . e . , t h e r e i s n e t s e c r e t i o n o f ab o u t 6 n l o f f l u i d f o r each n a n o e q u i v a l e n t o f c h l o r i d e t r a n s -p o r t e d . 148 5) E f f e c t o f E x t e r n a l Sodium and P o t a s s i u m on S a l i v a t i o n The r a t e o f s a l i v a r y s e c r e t i o n i s dependent upon t h e p r e s e n c e o f c h l o r i d e . S i n c e c h l o r i d e i s p r o b a b l y t r a n s p o r t e d a g a i n s t an e l e c t r o c h e m i c a l g r a d i e n t (see P o t e n t i a l D i f f e r e n c e M easurements), t h i s t r a n s p o r t may be c o n s i d e r e d a c t i v e . The q u e s t i o n now a r i s e s , "by what mechanisms a r e t h e c a t i o n s t r a n s -p o r t e d I n o r d e r t o a c h i e v e e l e c t r o n e u t r a l l t y ? " I f e i t h e r s odium o r p o t a s s i u m i s a c t i v e l y c o - t r a n s p o r t e d w i t h c h l o r i d e , t h e r a t e o f s a l i v a r y s e c r e t i o n s h o u l d be s e n s i t i v e t o t h e N a i K r a t i o . On t h e o t h e r hand, i f t h e c a t i o n s s i m p l y d i f f u s e non-s p e c i f i c a l l y t h r o u g h t h e g l a n d u l a r e p i t h e l i u m down an e l e c t r o -c h e m i c a l g r a d i e n t e s t a b l i s h e d by t h e a c t i v e t r a n s p o r t o f c h l o r -i d e , t h e n t h e s a l i v a r y g l a n d might be r e l a t i v e l y i n d i f f e r e n t t o t h e p r e s e n c e of a s p e c i f i c c a t i o n . The r a t e o f f l u i d s e c r e t i o n may be p r o p o r t i o n a l t o t h e d i f f u s i o n r a t e of t h e accompanying c a t i o n s , w h i c h i n t u r n i s t y p i c a l l y r e l a t e d t o t h e I o n ' s h y d r a t e d s i z e . To t e s t t h e s e h y p o t h e s e s , g l a n d s were exposed t o media of v a r y i n g N a i K r a t i o s (Na + K b e i n g c o n s t a n t ) a l t h o u g h a l l o t h e r components of t h e medium ( i n c l u d i n g o s m o t i c p r e s s u r e ) were h e l d c o n s t a n t . The e x p e r i m e n t a l p r o t o c o l i n v o l v e d t h e P u l s e Method u s i n g 10"^ M a d r e n a l i n . F i g u r e 21 shows t h a t t h e r a t e o f f l u i d s e c r e t i o n was n o t d i r e c t l y p r o p o r t i o n a l t o t h e c o n c e n t r a t i o n o f e i t h e r c a t i o n , b u t was dependent on t h e r a t i o o f t h e two c a t i o n s . The c o n t r o l medium ( n o r m a l R i n g e r s o l u t i o n ) c o n t a i n e d 10 m e q / l i t e r K + and 210 m e q / l i t e r Na +» t h e g l a n d s s e c r e t e d most q u i c k l y i n t h i s medium. When the p o t a s s i u m i n t h e c o n t r o l medium was c o m p l e t e l y r e p l a c e d w i t h sodium, the g l a n d s s e c r e t e d a t about 20$ t h e Figure 21. E f f e c t of the Na*K r a t i o on secretory rate. The potassium concentration of the medium was ra i s e d by progressive replacement of sodium. V e r t i c a l bars denote the SE of the mean unless i t i s included within the point. Normal Ringer solu t i o n ( • ), experimental media with a l t e r e d N a i K r a t i o s ( O ). Figure 22. E f f e c t of Na»K r a t i o i n the bathing medium on concentration of cations i n the s a l i v a . A) Relationship between concentration of sodium i n the medium and s a l i v a when glands were bathed i n v i t r o i n medium 3 ( • ) or media having varying r a t i o s of sodium and potas-sium ( O ). B) Same as "A" f o r potassium. V e r t i c a l and ho r i z o n t a l bars denote SE of the means unless the SE i s included i n the point I t s e l f . \Ua*] in medium (meq/liter) 0 10 20 30 40 50 60 [ K + ] in medium (meq/liter) 152 maximum r a t e . However when t h e K c o n c e n t r a t i o n was r a i s e d above 10 m e q / l i t e r ( w i t h a concommittent d e c r e a s e i n sodium c o n c e n t r a t i o n ) , t h e r a t e o f f l u i d s e c r e t i o n f e l l d r a s t i c a l l y , r e a c h i n g z e r o when t h e medium c o n c e n t r a t i o n o f b o t h c a t i o n s was 110 m e q / l i t e r . To t e s t w hether t h e i n h i b i t i o n was c a u s e d by h i g h p o t a s s i u m p e r se ( r a t h e r t h a n t h e concommittent low s o d i u m ) , g l a n d s were exposed t o v a r i o u s media h a v i n g a c o n s t a n t sodium c o n c e n t r a t i o n (100 m e q / l i t e r ) , b u t s u c c e s s i v e l y h i g h e r KCI c o n c e n t r a t i o n s . To t e s t t h e e f f e c t o f e l e v a t e d o s m o t i c p r e s s u r e ( a s d i s t i n c t f r o m e l e v a t e d KCI c o n c e n t r a t i o n ) , t h e same g l a n d s were b a t h e d i n media t o w h i c h s u c r o s e was added. The e x p e r i -m e n t a l p r o t o c o l was t h e P u l s e Method u s i n g 10""^  M a d r e n a l i n . F i g u r e 23 (ahead) shows t h a t h i g h c o n c e n t r a t i o n s o f KCI had a c o n s i d e r a b l e e f f e c t on s e c r e t o r y r a t e . The i n h i b i t i o n c o u l d be o n l y p a r t i a l l y due t o t h e i n c r e a s e d o s m o t i c p r e s s u r e o f t h e KCI media, s i n c e t h e s u c r o s e media ( h a v i n g t h e same o s m o t i c p r e s s u r e s as t h e i r c o r r e s p o n d i n g KCI media) e l i c i t e d a much s m a l l e r i n h i b i t o r y e f f e c t . I n c o n c l u s i o n , when t h e sodium c o n c e n t r a t i o n o f t h e b a t h i n g medium I s h e l d c o n s t a n t , b o t h e l e v a t e d o s m o t i c p r e s s u r e and h i g h p o t a s s i u m c o n c e n t r a t i o n i n h i b i t f l u i d s e c r e t i o n . The S/H r a t i o s f o r sodium and p o t a s s i u m i n v i t r o were s l i g h t l y g r e a t e r t h a n 1.0 o v e r t h e complete range o f medium c o n c e n t r a t i o n t e s t e d ( F i g u r e 22), whereas i n v i v o t h e y were n o t s i g n i f i c a n t l y d i f f e r e n t f r o m 1.0 ( C h a p t e r Two). 6) Magnesium C o n c e n t r a t i o n i n Medium and S a l i v a S i n c e t h e media used i n p r e v i o u s e x p e r i m e n t s c o n t a i n e d 153 a high concentration of magnesium, and since evidence from the last chapter suggested that the gland excluded non-charged molecules which are larger than glucose (hydrated diameter = 7.2 A), i t was of interest to see whether magnesium (hydrated diameter = 1 0 . 8 A) could traverse the epithelium. The effect on secretory rate of varying the magnesium concentration was not examined. The concentration of magnesium in normal Ringer solution was 3 0 . 8 + 1.2 meq/liter (mean + SE, N = 5) and in the saliva 2.9 + 0 . 4 meq/liter (N » 1 2 ) . The S/H ratio was close to 0 . 1 , suggesting that the gland i s either relatively impermeable to magnesium, or else that magnesium Is reabsorbed back from the primary secretion to the hemolymph side. 7) Electropotentlal Differences Across  the Salivary Gland Electropotentlal differences were measured in several ways. In method A, the glands were exposed in si t u and trans-acinar potential differences were measured with glass micro-electrodes. In method B, the glands were prepared for secretion in v i t r o , and the potential differences between the secreted droplet and the bathing medium (normal Ringer solution) were measured with KCl-agar gel bridges. In method C, the glands were prepared as in method B, but transacinar potentials ( i . e . hemocoele to acinar lumen) and potentials between the secreted droplet and the bathing medium were measured with glass micro-electrodes. Further details are provided in the Methods sec-tion of this chapter. 154 The experiments involving method A were carried out "before i t was discovered that topical application of adrenalin is required before the glands of dissected ticks become activej most probably then, the measurements obtained by Method A were for resting glands. Consequently, before measurements were made using Methods B and C, i t was f i r s t ascertained that the glands were secreting. The results for the three methods are presented in Table XVIII. TABLE XVIII POTENTIAL MEASUREMENTS ACROSS THE SALIVARY GLANDS Method Number of Ticks or Glands Mean Potential Difference (mV) of Saliva Relative to Hemolymph Side for Each Tick or Gland Mean Potential Difference (mV) + SE Transacinar resting potentialsi 11 ticks glands iii s i t u B Potentials between secreted droplet 7 glands and bathing medium in v i t r o - ^ 7 . -15, - 3 0 , -20, -14, -12, - 4 , - 9 , - 6 , - 6, -12 +3.5. +3.8, +4.2, +1.2, +2.9, +3.2, +2.0 -16 + 4 +3.0 + 0 . 4 Transacinar potentials in v i t r o 2 glands - 2 5 , - 2 0 , - 3 0 , - 2 5 , - 7 5 , - 1 5 , - 3 0 , - 3 5 , - 2 0 . - 5 0 , -75 -36.4 + 6.4 Potentials between secreted droplet 2 glands and bathing medium from in v i t r o using above glass electrodes 0 to + 5 155 The p o t e n t i a l differences across secreting glands measured by glass microelectrodes (inserted i n the acinar lumen) were c o n s i s t e n t l y negative whether or not adrenalin was present, however, the secretory potentials (Method C) were on the average twice as great as the r e s t i n g potentials (Method A). When the pote n t i a l s were measured between the secreted droplet and the bathing medium, e i t h e r by means of KCl-agar g e l bridges or by glass microelectrodes, the readings were always zero or a few m i l l i v o l t s p o s i t i v e . The discrepancy between these r e s u l t s and the transacinar measurements can be explained i n several ways. I t i s possible that the current flowing between the s a l t bridges i n Method B was s h o r t - c i r c u i t e d through a surface f i l m of e l e c t r o l y t e s o l u t i o n along the outside of the duct. Although properly dissected ducts are not s u f f i c i e n t l y h ydrophllic to permit the secreted droplet to flow baok along the duct into the bathing medium, i t cannot be ruled out that the e l e c t r i c a l resistance along the duct's basal surface i s low enough to short c i r c u i t the p o t e n t i a l difference across the epithelium. A l t e r n a t i v e l y , i t should be pointed out that the s a l i v a r y gland i s not a simple tubular epithelium possessing a single c e l l type (see Chapter Four). The gland i s composed of three types of a c i n i interconnected by ducts. There i s no reason to assume a p r i o r i that the p o t e n t i a l differences are s i m i l a r i n a l l regions. Consequently i n the t i c k s a l i v a r y gland, one may not be measuring the same p o t e n t i a l when pierci n g an acinus as when using s a l t bridges. Since there i s reasonably good evidence that the s i t e of primary secretion i s the acinus I t s e l f (see Chapter Four), I t i s l i k e l y that the transacinar p o t e n t i a l 156 d i f f e r e n c e s r e f l e c t more c l o s e l y t h e e l e c t r i c a l g r a d i e n t a s s o c i -a t e d w i t h p r i m a r y s e c r e t i o n . Prom t h e N e r n s t e q u a t i o n , i t can be shown t h a t , i n t h e absence o f n e t f l u x , a 2.4 mV n e g a t i v e p o t e n t i a l c a n s u p p o r t t h e o b s e r v e d d i f f e r e n c e i n sodium c o n c e n t r a t i o n between t h e s a l i v a and medium, and a 21 mV n e g a t i v e p o t e n t i a l can s u p p o r t t h e d i f f e r e n c e I n p o t a s s i u m c o n c e n t r a t i o n . S i n c e t h e o b s e r v e d n e g a t i v e p o t e n t i a l i s 35 mV, one need n o t i n v o k e an a c t i v e mechanism t o a c c o u n t f o r t h e d i s t r i b u t i o n o f sodium and p o t a s s i u m a c r o s s t h e s a l i v a r y e p i t h e l i u m . By a s i m i l a r argument, c h l o r i d e must be a c t i v e l y t r a n s p o r t e d , s i n c e I t s movement i s a g a i n s t b o t h c o n c e n t r a t i o n and e l e c t r i c a l g r a d i e n t s . F o r t h e sake o f t h e above c a l c u l a t i o n s t h e a s s u m p t i o n s were madei (1) t h a t t h e a c i n i a r e th e o n l y s i t e s o f f l u i d s e c r e t i o n (see u l t r a s t r u c t u r e , C h a p t e r F o u r ) , and (2) t h a t t h e c o m p o s i t i o n o f s a l i v a i s n o t r a d i c a l l y a l t e r e d d u r i n g passage down t h e d u c t s y s t e m , an a s s u m p t i o n w h i c h may n o t be j u s t i f i e d , 8) The E f f e c t o f Ouabain on S e c r e t o r y Rate A l t h o u g h sodium and p o t a s s i u m were n o t o b s e r v e d t o move a g a i n s t t h e i r e l e c t r o c h e m i c a l g r a d i e n t s i n t h e p r e s e n t e x p e r i -ments, t h i s does n o t e x c l u d e a c t i v e t r a n s p o r t as t h e mechanism of t r a n s f e r . E v i d e n c e i n F i g u r e 21 i n d i c a t e s t h a t sodium and p o t a s s i u m p l a y v e r y s p e c i f i c r o l e s i n the s e c r e t o r y p r o c e s s ; sodium i s r e q u i r e d a t r e l a t i v e l y h i g h c o n c e n t r a t i o n s and p o t a s -sium a t r e l a t i v e l y low c o n c e n t r a t i o n s f o r maximum s e c r e t o r y r a t e . The r e l a t i o n s h i p o b s e r v e d i n F i g u r e 21 I s s i m i l a r t o t h a t w i d e l y o b s e r v e d f o r »pump A T P a s e s * . The c a r d i a c g l y c o s i d e 157 o u a b a i n , i n r e l a t i v e l y low c o n c e n t r a t i o n s (10"' t o 10 M), i n h i b i t s t h e a c t i v e t r a n s p o r t o f sodium and 'pump ATPase' a c t i v i t y i n many t i s s u e s (Skou, 1965). I t was t h u s of i n t e r e s t t o t e s t t h e s e n s i t i v i t y of t h e t i c k s a l i v a r y g l a n d t o low c o n -c e n t r a t i o n s o f o u a b a i n . T i c k s were d i s s e c t e d i n medium 2 and -3 -5 t h e n s u b j e c t e d t o e x p e r i m e n t a l media c o n t a i n i n g 10 ^ M, 10 J M o r 10""0" M o u a b a i n ( S t r o p h a n t h i n G, C a l b i o c h e m Corp.) a f t e r e s t a b l i s h i n g t h e c o n t r o l r a t e i n n o r m a l R i n g e r s o l u t i o n . The e f f e c t o f i n c u b a t i n g t h e g l a n d s I n o u a b a i n on t h e subsequent s e c r e t o r y r a t e i s shown i n T a b l e X I X . The p r o t o c o l employed was t h e P u l s e Method w i t h 10"-* M a d r e n a l i n . TABLE X I X EFFECT OF OUABAIN ON RATE OF SALIVATION IN VITRO Rate of R a t e of S e c r e t i o n S e c r e t i o n * i n Normal i n Normal I n c u b a t i o n R i n g e r R i n g e r Time i n C o n c e n t r a t i o n S o l u t i o n S o l u t i o n Ouabain of Ouabain P l u s Ouabain G l a n d ( n l / m l n ) ( m i n u t e s ) ( M / l l t e r ) ( n l / m i n ) 1 47 270 10-3 0 2 41 270 it 0 3 32 125 it 0 4 33 105 1 0 " 5 0 5 139 105 II 0 6 57 80 I O " 6 0 7 82 80 It 0 *The c o n t r o l r a t e s p r e s e n t e d h e r e were a d j u s t e d downward f r o m t h e i n i t i a l r a t e s t o a c c o u n t f o r t h e d e t e r i o r a t i o n o f s e c r e t o r y r a t e w i t h t i m e ( F i g u r e 12) e x p e c t e d f o r I n v i t r o g l a n d s f u n c t i o n -i n g i n n o r m a l R i n g e r s o l u t i o n . 158 Ouabain completely i n h i b i t e d s a l i v a r y s e c r e t i o n a t a concentra-t i o n of 10-^ M. In one case (gland 5)» the i n h i b i t i o n was p a r t i a l l y reversed a f t e r Incubation i n normal Ringer s o l u t i o n f o r 85 minutes; the r a t e of s e c r e t i o n a f t e r recovery was 5 n l / min. I n c o n c l u s i o n , the r e s u l t s suggest t h a t f l u i d s e c r e t i o n i s dependent on a o u a b a i n - s e n s i t i v e 'pump ATPase'. The s p e c i f i c requirement of h i g h sodium and low potassium may r e f l e c t the presence of a sodium pump r e q u i r i n g low l e v e l s of potassium to f u n c t i o n . The nature of the l i n k a g e between sodium movement and c h l o r i d e t r a n s p o r t has not been t e s t e d . 9) The E f f e c t of Osmotic Pressure on S e c r e t i o n A l l experiments were conducted according t o the Pulse Method u s i n g 10""^  M a d r e n a l i n . Thus f o r comparing the e f f e c t s of NaCl and sucrose or NaNO^, the former medium served as the c o n t r o l and one of the l a t t e r two as the experimental. L i k e w i s e , sucrose media served as the c o n t r o l when t e s t i n g the e f f e c t s of K C l . The r a t e s of s e c r e t i o n from a l l these treatments were compared t o those of a separate s e r i e s of glands pulsed i n medium 3. F i g u r e s 23 and 24 show the e f f e c t s of osmotic pressure on s a l i v a t i o n . The e f f e c t of i n c r e a s i n g the osmotic pressure from 300 t o 400 mOsm/liter w i t h NaCl or sucrose was a s l i g h t a c c e l e r -a t i o n of s e c r e t o r y r a t e ; the e f f e c t of K C l , however, was i n h i b i -t o r y . A t 475 mOsm/liter, NaCl a c c e l e r a t e d (but sucrose diminished) the r a t e of s e c r e t i o n . Beyond 475 mOsm/liter, the r a t e was dimin-i s h e d by both NaCl and sucrose, though glands c o u l d s t i l l s e crete 159 Figure 23. Effect of osmotic pressure on salivation. The relation between osmotic pressure of the medium and secre-tory rate when the osmotic pressure of medium 3 (O) was raised by adding sucrose ( A ) , NaCl ( • ) , KCI ( • ) or NaNO^ ( • ). The ver t i c a l bars denote SE of the mean except when the SE is i n -cluded in the point i t s e l f , or for the sake of c l a r i t y when considerable overlap occurred ( 3 points at 390 mOsm/liter). Figure 24. The relationship between the osmotic pressure of the medium and osmotic pressure of the saliva when the osmotic pressure of medium 3 ( o) was raised by adding sucrose ( A) or NaCl ( • ). Individual values are plotted. 160 300 500 700 900 Osmotic pressure of medium (mOsm/ liter) Osmotic pressure of medium (mOsm/ liter) 161 i n NaCl Ringer s o l u t i o n reasonably well at 9 2 0 mOsm/liter. Since the NaCl curve p a r a l l e l e d that f o r sucrose above 4-75 mOsm/liter, t h i s suggests that a f t e r i n i t i a l stimulation by NaCl, only the osmotic e f f e c t of the l a t t e r substance was f e l t thereafter. In addi t i o n , part of t h i s i n h i b i t i o n may have resulted from the s t e a d i l y increasing N a t K r a t i o i n the medium concommittent with the a d d i t i o n of NaCl (see Figure 2 1 , rate of secretion In K-free medium)• The s a l i v a was c o n s i s t e n t l y hypo-osmotic by 3 0 - 3 5 mOsm/ l i t e r when the bathing medium ranged from 3 0 0 to 6 7 5 mOsm/liter, but s a l i v a was hypo-osmotic by 9 5 mOsm/liter when the bathing medium was 9 2 0 mOsm/liter (Figure 2k). 162 DISCUSSION 1) The I n - V l t r o P r e p a r a t i o n One p o s s i b l e r e a s o n t h a t t h e s a l i v a r y g l a n d s of Dermacen-t o r have p r o v e n l e s s h a r d y t h a n i n s e c t M a l p i g h i a n t u b u l e s when b a t h e d i n n o r m a l R i n g e r s o l u t i o n , i s t h a t t h e l a t t e r was o r i g -i n a l l y d e v e l o p e d by B e r r i d g e f o r t h e t u b u l e s o f an i n s e c t , C a l l l p h o r a t perhaps th e medium l a c k s c e r t a i n s u b s t a n c e s a t c o n -c e n t r a t i o n s r e q u i r e d by t h e t i c k s a l i v a r y g l a n d f o r e x t e n d e d p e r i o d s o f a c t i v i t y . A l t h o u g h some of t h e r e q u i r e m e n t s f o r m aximal s e c r e t i o n by g l a n d s o f Dermacentor ( N a + , C l " , K + , a d r e n a l i n ) have been e s t a b l i s h e d , t h e e f f e c t s o f o t h e r components i n t h e medium a r e unknown, and Indeed the l e v e l s o f some of t h e components may be d e t r i m e n t a l . F o r i n s t a n c e , t h e magnesium l e v e l I n B e r r i d g e ' s medium i s 10 t o 15 t i m e s t h a t i n t h e hemo-lymph of t h e t i c k . I n o r g a n i c p h o s p h a t e , a t l e v e l s q u i t e common i n t h e body f l u i d s o f many a r t h r o p o d s ( F l o r k i n and J e u n i a u x , 1 9 6 4 ) , a p p e a r s t o i n h i b i t t h e f u n c t i o n i n g of t i c k s a l i v a r y g l a n d s i n v i t r o . I t may a l s o be t h a t w h i l e t r i g g e r i n g o f s a l i -v a r y s e c r e t i o n does n o t r e q u i r e e n d o c r i n e f a c t o r s , t h e m a i n t e n -ance o f c e l l u l a r a c t i v i t y d o e s . F u r t h e r e x p e r i m e n t s c o n c e r n e d w i t h t h e c o m p o s i t i o n o f media r e q u i r e d t o m a i n t a i n s e c r e t o r y a c t i v i t y w o u l d be w o r t h w h i l e . 2) The C o n t r o l of S e c r e t i o n The h y p o t h e s i s was advanced e a r l i e r t h a t t h e g l a n d 163 o p e r a t e s by a s e c r e t o r y mechanism w h i c h i s t r i g g e r e d by an a d r e n o m i m e t i c a g e n t . I a l s o p r o p o s e d t h a t t h i s a gent l i k e l y r e a c h e d t h e r e c e p t o r s i t e v i a n e r v e s , r a t h e r t h a n v i a t h e c i r c u l a t i n g hemolymph. (That t h e a c i n i and d u c t s o f t h e s a l i -v a r y g l a n d do r e c e i v e I n n e r v a t i o n w i l l be shown i n t h e f o l l o w i n g c h a p t e r . ) The r e c e p t o r s i t e i s most s e n s i t i v e t o a l p h a - a d r e n e r -g i c s t i m u l a n t s , though t h e b e t a - a d r e n e r g i c d r u g i s o p r o t e r e n o l s t i m u l a t e s s e c r e t i o n a t h i g h e r c o n c e n t r a t i o n s . E i t h e r t h e l a t t e r i s a c t i n g a t the same s i t e s as t h e a l p h a - a d r e n e r g i c d r u g s , b u t w i t h l e s s p o t e n c y , o r e l s e t h e s e c r e t o r y e p i t h e l i u m p o s s e s s e s a t l e a s t two t y p e s o f r e c e p t o r . S e r o t o n i n s t i m u l a t e d s e c r e t i o n o n l y a t doses above 1 0 M, s u g g e s t i n g t h a t i t s e f f e c t may be p h a r m a c o l o g i c a l r a t h e r t h a n p h y s i o l o g i c a l . I t i s known t h a t i n v e r t e b r a t e s y s t e m s , th e g r e a t e s t sympathomimetic a c t i v i t y i s c o n f e r r e d upon c a t e c h o l a m i n e s when two c a r b o n atoms s e p a r a t e t h e a r o m a t i c n u c l e u s f r o m th e amino group (Innes and N i c k e r s o n , 1 9 6 5 ) ; p o s s i b l y t h e s t r u c t u r a l resemblance of t h e a l i p h a t i c p o r t i o n t o t h a t o f c a t e c h o l a m i n e s ( s p e c i f i c a l l y dopamine) r e n d e r s s e r o t o n i n c a p a b l e o f a c t i n g a t t h e a d r e n e r g i c r e c e p t o r s i t e . I t t a k e s a d r e n a l i n a p p r o x i m a t e l y 1 1 m i n u t e s t o s t i m u l a t e t h e s a l i v a r y g l a n d i n v i t r o t o t h e h a l f - m a x i m a l r e s p o n s e ( F i g u r e 1 3 ) . On r e m o v a l o f a d r e n a l i n f r o m t h e b a t h i n g medium, i t t a k e s t h e g l a n d o n l y two m i n u t e s t o r e t u r n t o the h a l f - m a x i m a l r e s p o n s e . T h i s c o u l d be e x p l a i n e d i f a d r e n a l i n f i r s t has t o d i f f u s e t o the r e c e p t o r s i t e s ( a c r o s s b a r r i e r s w h i c h a r e n o t n o r m a l l y encoun-t e r e d d u r i n g n e u r a l r e l e a s e o f t h e n a t u r a l t r a n s m i t t e r ) i n o r d e r 164 t o e x e r t i t s e f f e c t . A f t e r r e m o v a l of a d r e n a l i n f r o m t h e b a t h i n g medium ( i . e . f r o m t h e b a s a l membrane), t h e a d r e n a l i n w i t h i n the c e l l c a n c o n t i n u e e x e r t i n g i t s e f f e c t u n t i l i t i s removed from t h e r e c e p t o r s i t e s . I n o r d e r t o a c c e p t t h a t a g i v e n compound i s a r e a s o n a b l e c a n d i d a t e f o r a n e u r o t r a n s m i t t e r s u b s t a n c e , s i x c r i t e r i a s h o u l d be s a t i s f i e d ( E c c l e s , 1964). One o f t h e s e i s t h a t an i n a c t i v a -t i n g enzyme s h o u l d be l o c a l i z e d i n t h e r e g i o n o f t h e r e c e p t o r s i t e . The more r a p i d decay o f s e c r e t o r y a c t i v i t y o f t h e t i c k s a l i v a r y g l a n d a f t e r r e m o v a l o f a d r e n a l i n f r o m t h e b a t h i n g medium s u g g e s t s t h e pr e s e n c e o f such an i n a c t i v a t i n g enzyme. The demon-s t r a t i o n o f perhaps a monoamine o x i d a s e o r c a t e c h o l - O - m e t h y l t r a n s f e r a s e i n t h e t i c k s a l i v a r y g l a n d w o u l d f u l f i l l one c r i t e r -i o n i n e s t a b l i s h i n g t h a t an a d r e n a l i n - l i k e s u b s t a n c e i s t h e n a t u r a l t r i g g e r i n g agent i n v i v o . A l t h o u g h some c a t e c h o l a m i n e s have been w e l l e s t a b l i s h e d as t r a n s m i t t e r s u b s t a n c e s i n v e r t e b r a t e s , t h e i r r o l e as p o t e n t i a l m e d i a t o r s i n i n s e c t s i s l e s s w e l l documented. A l t h o u g h t h e n e r v o u s and m u s c u l a r systems o f a number o f i n v e r t e b r a t e s a r e s e n s i t i v e t o low c o n c e n t r a t i o n s o f a d r e n a l i n (Welsh, 1939} Davenport e t a l . , 1940» D a v e n p o r t , 19^9* Davey, 1964j Gahery and B o i s t e l , 1965; B o l s t e l , 1968), i t has been p o i n t e d o u t (Davey, 1964) t h a t i n many c a s e s , t h e s e same systems a r e s e n s i t i v e t o a wide v a r i e t y o f n o n - r e l a t e d p h a r m a c o l o g i c a l and e n d o c r i n o l o g i c a l f a c t o r s . T h i s s i t u a t i o n compounds t h e d i f f i c u l t y i n d i f f e r e n t i -a t i n g between p h y s i o l o g i c a l and p h a r m a c o l o g i c a l e f f e c t s . That t h e s a l i v a r y g l a n d o f Dermacentor, however, seems t o be s e n s i t i v e 165 t o o n l y a s m a l l number of c l o s e l y r e l a t e d m o l e c u l e s , r a i s e s hopes t h a t i d e n t i f i c a t i o n o f the n a t u r a l t r a n s m i t t e r s u b s t a n c e may be r e a l i z e d i n t h e n o t - t o o - d i s t a n t f u t u r e . C y c l i c AMP has l o n g been i m p l i c a t e d as a l i n k between the p r i m a r y hormonal message a r r i v i n g a t the c e l l and t h e e f f e c t o r s y stem i t s e l f . C y c l i c AMP can mimic the e f f e c t o f a wide v a r i e t y o f hormones w h i c h n o r m a l l y s t i m u l a t e o n l y s p e c i f i c t a r g e t o r g a n s , a l t h o u g h t h e p r e c i s e mechanism whereby c y c l i c AMP e x e r t s i t s e f f e c t i s s t i l l unknown ( O r l o f f and H a n d l e r , 1963? B e r r i d g e , 1 9 7 0 ) . S i n c e c y c l i c AMP i s b e l i e v e d t o m e d i a t e i n t h e s e c r e t o r y e v e n t s o f t h e s a l i v a r y g l a n d s of t h e I n s e c t , C a l l i p h o r a ( B e r -r i d g e , 1970) and o f t h e M a l p i g h i a n t u b u l e s o f t h e i n s e c t s R hodnlus and C a r a u s i u s ( M a d d r e l l e t a l . , 1 9 7 1 ) . i t was o f I n t e r -e s t t o o b s e r v e i t s e f f e c t on t h e s a l i v a r y g l a n d o f Dermacentor. - 2 Exogenous c y c l i c AMP a s h i g h a s 10 M had no s t i m u l a t o r y e f f e c t on s a l i v a r y s e c r e t i o n . E i t h e r i t p l a y s no r o l e as a s e c o n d a r y messenger i n t h e manner d e s c r i b e d above, o r e l s e i n t h i s i n s t a n c e i t i s u n a b l e t o r e a c h t h e r e c e p t o r s i t e s . The b a s a l plasma membrane of C a l l i p h o r a s a l i v a r y g l a n d s i s b e l i e v e d t o be o n l y _2 s l i g h t l y permeable t o c y c l i c AMP, a l t h o u g h a t 10 M, t h e s e c r e -- 9 t o r y r e s p o n s e p a r a l l e l s t h a t c a u sed by s e r o t o n i n a t 5 x 10 7 M ( B e r r i d g e , 1 9 7 0 ) . W e l l s (1967) r e p o r t s t h a t t h e s a l i v a r y g l a n d s o f r a t s , and i n d e e d most t i s s u e s , a r e a l s o o n l y s l i g h t l y permeable t o c y c l i c AMP. I t i s q u i t e p o s s i b l e t h e n , t h a t t h e b a s a l s e c r e t o r y membrane i n t h e s a l i v a r y g l a n d o f Dermacentor. a l s o r e s t r i c t s t h e a c c e s s of c y c l i c AMP t o t h e c e l l . F i n a l l y , t h e i n a b i l i t y o f c h o l i n e r g i c d r ugs ( a c e t y l c h o -1 6 6 l i n e and p i l o c a r p i n e ) t o s t i m u l a t e s e c r e t i o n o f t h e t i c k s a l i -v a r y g l a n d i n v i t r o i s a t v a r i a n c e w i t h t h e f i n d i n g s o f o t h e r a u t h o r s who use p i l o c a r p i n e r o u t i n e l y t o i n c r e a s e t h e y i e l d of s a l i v a i n v i v o * However t h e s e a u t h o r s i n j e c t e d e x c e s s i v e c o n -c e n t r a t i o n s of p i l o c a r p i n e i n t o t i c k s . On t h e a s s u m p t i o n t h a t t h e hemolymph volume r e p r e s e n t s a p p r o x i m a t e l y 20% o f t h e body w e i g h t , one c a n c a l c u l a t e t h a t t h e f i n a l c o n c e n t r a t i o n o f p i l o -c a r p i n e was 4 t o 6 x 1 0 " ^ M i n t h e hemolymph of O r n l t h o d o r o s  s a v l g n y l ( H o w e l l , 1 9 6 6 ) , 8 x 1 0 ~ 2 M I n t h e hemolymph o f B o o p h l l u s  m l c r o p l u s ( T a t c h e l l , 1 9 6 7 a ) , and 4 t o 12 x 1 0 ~ 2 M i n the hemo-lymph o f R h l p l c e p h a l u s a p p e n d l c u l a t u s ( P u r n e l l e t a l . , 1 9 6 9 ) . I n t h e p r e s e n t s t u d y , t h e h i g h e s t c o n c e n t r a t i o n o f p i l o c a r p i n e and a c e t y l c h o l i n e a p p l i e d t o t h e e x c i s e d g l a n d s o f Dermacentor was 10"*^ M. Moreover, i t i s n o t c l e a r a t w h i c h l e v e l t h e c h o l i n e r g i c d r ugs a c t i n v i v o . On t h e b a s i s o f the p r e s e n t o b s e r v a t i o n s t h e y a r e u n l i k e l y t o a f f e c t t h e s e c r e t o r y membrane i t s e l f . P o s s i b l y , p i l o c a r p i n e a c t i v a t e s neurones i n t h e c e n t r a l n e r v o u s system w h i c h t h e n s t i m u l a t e e f f e r e n t axons i n n e r v a t i n g t h e s a l i v a r y g l a n d ; a l t e r n a t i v e l y , t h i s d r u g may s t i m u l a t e the a b s o r p t i o n o f f l u i d f r o m t h e g u t w h i c h I n t u r n might i n d i r e c t l y t r i g g e r t h e e v e n t s l e a d i n g t o s e c r e t i o n . F i n a l l y , t h e r e q u i r e -ment o f such l a r g e doses o f p i l o c a r p i n e may s i m p l y r e f l e c t t h e p r e s e n c e o f a p o w e r f u l enzyme i n t h e hemolymph o f t h e s e t i c k s w h i c h r a p i d l y d e s t r o y s t h e i n j e c t e d d r u g * I n c o n c l u s i o n , I would propose t e n t a t i v e l y t h a t t h e a c t of s a l i v a t i o n I s c o n t r o l l e d a t two l e v e l s . ( 1 ) When s t i m u l a t e d , e f f e r e n t n e r v e f i b r e s t o t h e s a l i v a r y g l a n d i n i t i a t e t h e s e c r e -t o r y p r o c e s s by r e l e a s i n g an a l p h a - a d r e n e r g i c agent a t r e c e p t o r 167 s i t e s on t h e s a l i v a r y e p i t h e l i u m . ( 2 ) E x p u l s i o n of s a l i v a i s p r o b a b l y augmented by t h e c o n t r a c t i o n of a b d o m i n a l m u s c l e s . The v a l v e l o c a t e d a t t h e j u n c t u r e of each a c i n u s and i t s e f f e r e n t d u c t ( C h a p t e r F o u r ) and t h e v a l v e - l i k e arrangement a n t e r i o r t o t h e e n t r a n c e of each main s a l i v a r y d u c t (Gregson, i 9 6 0 ) p r o b a b l y e n s u r e t h e u n i d i r e c t i o n a l f l o w o f s a l i v a t o w a r d th e b u c c a l c a v i t y . S i n c e t h e s a l i v a and t h e meal occupy t h e b u c c a l c a v i t y a t d i f f e r e n t t i m e s , o p e r a t i o n o f t h e p h a r y n g e a l pump m u t u a l l y e x c l u d e s t h e e x p u l s i o n o f s a l i v a ( B a l a s h o v , 1 9 6 5 ) . 3) Mechanism of F l u i d S e c r e t i o n and I o n T r a n s f e r a) A n i o n s I t was p o i n t e d out e a r l i e r t h a t because s a l i v a t i o n c a n p r o c e e d i n v i t r o , t h i s i s c l e a r e v i d e n c e t h a t a s e c r e t o r y r a t h e r t h a n a f i l t r a t i o n mechanism i s o p e r a t i n g . The s a l i v a r y g l a n d r e q u i r e s t h e p r e s e n c e of c h l o r i d e i n t h e b a t h i n g medium i f f l u i d s e c r e t i o n i s t o p r o c e e d a t a l l , and t h e r a t e o f f l u i d s e c r e t i o n i s p r o p o r t i o n a l t o t h e c h l o r i d e c o n c e n t r a t i o n . Moreover, t h e r a t e o f f l u i d s e c r e t i o n i s a l i n e a r f u n c t i o n o f t h e r a t e o f c h l o r i d e s e c r e t i o n . These r e s u l t s i n d i c a t e t h a t f l u i d movement i s dependent on i o n t r a n s p o r t ? i . e . , t h e y I n d i c a t e s e c o n d a r y t r a n s p o r t o f w a t e r . A c e t a t e and n i t r a t e cannot r e p l a c e c h l o r i d e , b u t bromide c a n . S i n c e n i t r a t e i s o n l y 1 . 0 7 t i m e s t h e h y d r a t e d r a d i u s o f c h l o r i d e , t h e s e c r e t o r y p r o c e s s a p p e a r s t o have a s p e c i f i c r e q u i r e m e n t f o r a h a l i d e a n i o n r a t h e r t h a n f o r mono-v a l e n t a n i o n s of s i m i l a r h y d r a t e d s i z e . I t i s most r e a s o n a b l e t h e r e f o r e , t o propose t h a t c h l o r i d e t r a v e r s e s t h e s a l i v a r y e p i t h e l i u m v i a a s p e c i f i c c a r r i e r . The e l e c t r o n i c c o n f i g u r a t i o n 168 o f bromide e n a b l e s t h i s a n i o n t o compete w i t h c h l o r i d e f o r t h e c a r r i e r , b u t t h e c a r r i e r w i l l n o t r e c o g n i z e n i t r a t e , a c e t a t e , and p r o b a b l y o t h e r n o n - h a l i d e s . Such s p e c i f i c i t y has o f t e n been o b s e r v e d f o r o t h e r t i s s u e s w h i c h t r a n s p o r t c h l o r i d e ( e . g . , L u n d b e r g , 1957c» M a d d r e l l , 1969)* There i s e v i d e n c e s u g g e s t i n g t h a t i n t h e s a l i v a r y g l a n d o f Dermacentor the c h l o r i d e pump i s electrogenic» t h e r e s t i n g p o t e n t i a l d i f f e r e n c e a c r o s s t h e t i c k s a l i v a r y a c i n u s i s h y p e r p o l a r i z e d (by a f a c t o r o f two) d u r i n g s e c r e t i o n . T h i s c a n be e x p l a i n e d by p r o p o s i n g t h a t a p o t e n t i a l due t o t h e t r a n s p o r t o f an a n i o n ( I n i t i a l l y i n e x c e s s o f a c a t i o n ) i s s u p e r i m p o s e d upon t h e n o r m a l r e s t i n g p o t e n t i a l , r e g a r d l e s s how t h e l a t t e r may be c a u s e d . Lundberg (1957a, b, c) re a o h e d a s i m i l a r c o n c l u s i o n f o r t h e c a t s u b l i n g u a l g l a n d . The p r e s e n t d a t a on s e c r e t o r y p o t e n t i a l s s h o u l d be a c c e p t e d w i t h some c a u t i o n however, because t h e r e s t i n g p o t e n t i a l s (Method A) and t h e s e c r e t o r y p o t e n t i a l s (Method C) were measured under d i f f e r e n t c o n d i t i o n s . Phosphate was p r e s e n t i n t h e f i r s t mediumj s i n c e t h i s i o n was l a t e r shown t o inhabit f l u i d s e c r e t i o n , q u i t e p o s s i b l y t h e l o w e r p o t e n t i a l s r e c o r d e d i n t h i s c a s e stem f r o m t h e p r e s e n c e o f phosp h a t e , r a t h e r t h a n t h e i n a c t i v e c h l o r i d e pump. A l s o , t h e g l a n d s were m e r e l y exposed i n s i t u i n t h e f i r s t method b u t were e x c i s e d i n t h e t h i r d method. T h e r e f o r e , one s h o u l d r e p e a t t h e s e e x p e r i m e n t s u s i n g a s i n g l e p r o t o c o l . b) C a t i o n s The c o n c e n t r a t i o n d i f f e r e n c e o f sodium and p o t a s s i u m a c r o s s t h e s a l i v a r y e p i t h e l i u m can be e x p l a i n e d by t h e o b s e r v e d t r a n s a c i n a r p o t e n t i a l d i f f e r e n c e (35 mV, lumen n e g a t i v e ) . On 169 t h e b a s i s o f t h i s , one cannot e x c l u d e t h e p o s s i b i l i t y t h a t t h e s e i o n s move p a s s i v e l y a c r o s s t h e a c i n a r e p i t h e l i u m i n r e s p o n s e t o t h e a c t i v e t r a n s p o r t o f c h l o r i d e . But o t h e r e v i d e n c e i n d i c a t e s t h a t t h i s h y p o t h e s i s seems u n l i k e l y . F i r s t , p o t a s s i u m cannot r e p l a c e sodium i n t h e n u t r i e n t s o l u t i o n , even though t h e h y d r a t e d r a d i u s o f p o t a s s i u m i s s m a l l e r t h a n t h a t o f sodium. Second, t h e e f f e c t o f p o t a s s i u m i s c o n c e n t r a t i o n - d e p e n d e n t } when sodium i s h e l d c o n s t a n t , t h e a d d i t i o n o f a s m a l l amount o f p o t a s s i u m s t i m u l a t e s t h e r a t e o f f l u i d s e c r e t i o n f i v e f o l d , b u t h i g h e r c o n c e n t r a t i o n s I n h i b i t s f l u i d s e c r e t i o n t o an e x t e n t w h i c h c a n -n o t be a t t r i b u t e d t o an o s m o t i c e f f e c t a l o n e . None o f t h e s e e f f e c t s c a n be e x p l a i n e d on t h e b a s i s o f a l t e r e d c h l o r i d e c o n -c e n t r a t i o n o f t h e medium. C l e a r l y b o t h sodium and p o t a s s i u m p l a y f u n d a m e n t a l r o l e s i n f l u i d s e c r e t i o n , though t h e n a t u r e of t h e s e r o l e s i s u n c l e a r . However a c l u e may be g a i n e d f r o m t h e f o l l o w i n g . Skou (1965) r e v i e w s e v i d e n c e f a v o u r i n g t h e e x i s t e n c e i n a number o f b i o l o g i c a l membranes o f an enzyme system t h a t i s c a p a b l e o f t r a n s f o r m i n g energy f r o m ATP i n t o t h e movement o f sodium and p o t a s s i u m a g a i n s t t h e i r r e s p e c t i v e e l e c t r o c h e m i c a l g r a d i e n t s . T h i s enzyme system i s commonly c a l l e d t h e Mg -4* + dependent, Na , K - a c t i v a t e d ATPase, o r more s i m p l y , 'pump A T P a s e 1 . There a r e two s i t e s on the enzyme complex, one w i t h a h i g h a f f i n i t y f o r sodium r e l a t i v e t o p o t a s s i u m , and one w i t h r e v e r s e a f f i n i t i e s . Maximum a c t i v i t y o c c u r s when b o t h c a t i o n s a r e p r e s e n t i n s p e c i f i c r a t i o s . A t c o n c e n t r a t i o n s o f p o t a s s i u m w h i c h a r e h i g h r e l a t i v e t o sodium, p o t a s s i u m can d i s p l a c e 170 sodium f r o m i t s s p e c i f i c s i t e by c o m p e t i t i v e i n h i b i t i o n , and t h u s d e c r e a s e th e ATPase a c t i v i t y . Ouabain i n t e r f e r e s d i r e c t l y w i t h t h e »pump ATPase* (Skou, 1 9 6 5 ) . The g e n e r a l shape of t h e c u r v e i n F i g u r e 21 i s r e m i n i s -c e n t o f t h e c o m p e t i t i o n between sodium and p o t a s s i u m i n a c l a s s i c a l *pump-ATPase* system. I n sodium R i n g e r s o l u t i o n ( c o n t a i n i n g magnesium), s a l i v a t i o n o c c u r s , b u t a t a modest r a t e . A d d i t i o n o f p o t a s s i u m i n amounts s m a l l r e l a t i v e t o sodium, mark-e d l y i n c r e a s e s t h e s e c r e t o r y r a t e , b u t f u r t h e r i n c r e a s e o f p o t a s s i u m l e a d s t o i n h i b i t i o n . I t i s w o r t h r e c a l l i n g a t t h i s p o i n t t h a t once a s t e a d y l e v e l o f i o n c o n c e n t r a t i o n and o s m o t i c p r e s s u r e i s r e a c h e d i n t h e hemolymph (see C h a p t e r Two), t h e N a i K r a t i o o f t h e hemolymph i s 2 2 , t h a t o f t h e s a l i v a i s 18j b u t t h a t o f t h e meal (whole r a b b i t b l o o d ) i s o n l y 2 . 5 * I n c o m p a r i s o n , t h e N a i K r a t i o o f t h e medium w h i c h s u p p o r t s t h e maximum s e c r e t o r y r a t e s i n v i t r o ( i . e . , n o r m a l R i n g e r s o l u t i o n ) i s 1 9 . T h i s a g r e e -ment between t h e v a l u e f o r hemolymph and t h a t f o r maximum s e c r e -t o r y r a t e i n v i t r o s t r e n g t h e n s one*s case t h a t g e n e r a l c o n c l u s i o n s f r o m t h e i n - v l t r o e x p e r i m e n t s may be e x t r a p o l a t e d w i t h some c o n f i d e n c e t o t h e i n t a c t a n i m a l . S i n c e on t h e b a s i s o f F i g u r e 2 1 t h e N a i K r a t i o o f t h e b l o o d meal i s low enough t o c o n s i d e r a b l y i n h i b i t s a l i v a r y s e c r e t i o n , t h i s may be a r e a s o n t h a t t h e t i c k m a i n t a i n s low l e v e l s o f p o t a s s i u m i n t h e hemolymph by one o f t h e mechanisms enumerated i n C h a p t e r Two, A l t h o u g h movement o f sodium and p o t a s s i u m a g a i n s t t h e i r e l e c t r o c h e m i c a l g r a d i e n t s was n o t o b s e r v e d , t h i s may have been s i m p l y because th e g l a n d s were n o t exposed t o c o n d i t i o n s a p p r o -p r i a t e f o r d e m o n s t r a t i n g c o n c l u s i v e l y t h e a c t i v e t r a n s p o r t of 171 cations. Another p o s s i b i l i t y to consider i s that the chloride pump i n the s a l i v a r y gland gains i t s energy from an ATPase system which i s activated by sodium and potassium, but that sodium and potassium themselves move down an electrochemical gradient created by chloride transport. Since such a system would be unique amongst those described to date, i t would be wise to f i r s t exclude the p o s s i b i l i t y that sodium i s a c t i v e l y transported before examining i t more c l o s e l y . 4) The E f f e c t of Osmotic Pressure on Secretion The s a l i v a secreted by the glands i n v i t r o was s l i g h t l y , but s i g n i f i c a n t l y , hypo-osmotic to the bathing medium over the range 300 to 920 mOsm/liter. This would appear to c o n f l i c t with the e a r l i e r proposal that water movement was dependent on the transport of ions. For such a suggestion to be v a l i d , the secreted f l u i d should be hyperosmotic or iso-osmotic but not hypo-osmotic to the bathing medium. The simplest explanation f o r a hypo-osmotic s a l i v a i s to postulate that the primary f l u i d secreted Into the acinar lumen i s hyperosmotic (or i s o -osmotlc) but that solute i s subsequently reabsorbed back into the hemolymph i n excess of water. In the rat parotid gland, such reabsorption occurs i n the s t r i a t e d ducts (Mangos et a l . , 1966) and i n the rat submaxillary gland, reabsorption occurs i n the main excretory duct (Martinez e_t a l . , 19661 Young and Schogel, 1966). In the s a l i v a r y gland of Dermacentor. solute reabsorption could conceivably occur i n the acinus I t s e l f by c e l l s other than those which secrete the primary f l u i d , or along the duct system as occurs i n mammals. Further discus-sion of t h i s matter w i l l be delayed u n t i l the morphology and 1 7 2 u l t r a s t r u c t u r e of t h e s a l i v a r y g l a n d has been d e s c r i b e d (Chap-t e r F o u r ) . 5) Summary of C h a p t e r Three A) A method f o r e x c i s i n g t h e s a l i v a r y g l a n d s and s t i m u -l a t i n g them t o f u n c t i o n i n v i t r o was d e s c r i b e d . The maximum r a t e o f s e c r e t i o n by t h i s p r e p a r a t i o n i s g r e a t e r t h a n t h a t so f a r d e s c r i b e d f o r l n - v l t r o p r e p a r a t i o n s o f i n s e c t M a l p i g h i a n t u b u l e s and s a l i v a r y g l a n d s . However, the r a t e o f s e c r e t i o n s t e a d i l y d e cays w i t h t i m e , t h u s l i m i t i n g the p e r i o d a v a i l a b l e f o r e x p e r i m e n t i n g on a s i n g l e g l a n d t o a p p r o x i m a t e l y s i x h o u r s . B) I n t h e p r e s e n c e of a s u i t a b l e n u t r i e n t medium, the e x c i s e d g l a n d s r e q u i r e t o p i c a l a p p l i c a t i o n o f a s t i m u l a n t f o r s a l i v a t i o n t o o c c u r . N a t u r a l hemolymph from s a l i v a t i n g t i c k s , p i l o c a r p i n e , v a s o p r e s s i n , and c y c l i c AMP d i d n o t t r i g g e r s e c r e t i o n . S e r o t o n i n and i s o p r o t e r e n o l were r e l a t i v e l y weak s t i m u l a n t s , b u t a d r e n a l i n , n o r a d r e n a l l n and dopamine were h i g h l y e f f e c t i v e ; t h e t h r e s h o l d c o n c e n t r a t i o n f o r t h e l a t t e r t h r e e was no more t h a n 10~^ M. When a d r e n a l i n was i n j e c t e d i n t o l i v i n g t i c k s t o c r e a t e an e s t i m a t e d hemolymph c o n c e n t r a t i o n o f 2 x 10"^ M, t h e volume of s a l i v a s u b s e q u e n t l y s e c r e t e d ( i n terms of p e r c e n t a g e o f hemolymph volume) exceeded t h a t of s a l i n e -i n j e c t e d t i c k s e i g h t f o l d , and t h a t of n o n - i n j e c t e d t i c k s , f o u r -f o l d . From t h e above e v i d e n c e I proposed t h a t s a l i v a t i o n i s n o r m a l l y i n i t i a t e d by an a l p h a - a d r e n e r g i c m e d i a t o r . The media-t o r i s p r o b a b l y n o t r e l e a s e d f r e e i n the hemolymph, but r a t h e r a t neurone - s e c r e t o r y c e l l s y n a p s e s . From the t i m e - c o u r s e of h a l f - m a x i m a l s t i m u l a t i o n by a d r e n a l i n and of h a l f - m a x i m a l decay 173 on r e m o v a l of a d r e n a l i n , I s p e c u l a t e d t h a t an enzyme system c a p a b l e o f r a p i d l y i n a c t i v a t i n g a d r e n a l i n p o s s i b l y e x i s t s i n the s e c r e t o r y c e l l s . C) F l u i d movement does n o t o c c u r by f i l t r a t i o n , but i s c o u p l e d t o t h e a c t i v e t r a n s p o r t of c h l o r i d e and p o s s i b l y sodium. P o t a s s i u m i n s m a l l amounts s t i m u l a t e d s e c r e t i o n , b u t t h e r e a f t e r i t s e f f e c t was more i n h i b i t o r y t h a n c o u l d be a c c o u n t e d f o r on -6 th e b a s i s o f i t s o s m o t i c e f f e c t a l o n e . Ouabain a t 10~ M com-p l e t e l y a b o l i s h e d f l u i d s e c r e t i o n . The s i m p l e s t h y p o t h e s i s t o embrace t h e s e f i n d i n g s i s t h a t f l u i d s e c r e t i o n i s a l s o depen-+ + dent on a Na , K - a c t i v a t e d *pump ATPase'. J CHAPTER FOUR HISTOLOGY AND ULTRASTRUCTURE OF THE SALIVARY GLAND 17 k CHAPTER FOUR From t h e l a s t c h a p t e r we g a i n e d a deeper I n s i g h t I n t o t h e mechanism o f s a l i v a r y s e c r e t i o n . I t i s now a p p r o p r i a t e t o ask i f we can t r a c e t h e s e c r e t o r y p r o c e s s e s t o any w e l l d e f i n e d r e g i o n s o r even c e l l - t y p e s o f t h e s a l i v a r y g l a n d . A l t h o u g h i n e a r l i e r s e c t i o n s o f t h i s t h e s i s I spoke o f t h e s a l i v a r y " e p i t h e -l i u m " i n o r d e r t o s t r e s s t h e g l a n d ' s f u n c t i o n a l a t t r i b u t e s , t h e s t r u c t u r a l o r g a n i z a t i o n o f t h e g l a n d i s i n f a c t q u i t e complex. The f i r s t i m p o r t a n t works on t h e morphology o f t i c k s a l i v a r y g l a n d s a p p e a r e d e a r l y i n t h e p r e s e n t c e n t u r y . S i n c e 1943, t h e h i s t o l o g y o f t i c k s a l i v a r y g l a n d s has been s t u d i e d i n no f e w e r t h a n s even i x o d i d s p e c i e s . The s i m i l a r i t i e s among t h e s e s p e c i e s a r e f a r more s t r i k i n g t h a n t h e d i f f e r e n c e s . Such a s i t u a t i o n r a i s e s hopes t h a t t h e s p e c i f i c f i n d i n g s of t h i s t h e s i s may b r i n g t o l i g h t some g e n e r a l p r i n c i p l e s f o r t h e f a m i l y as a w h o l e . The two promine n t s a l i v a r y g l a n d s o f Dermacentor a n d e r -s o n i were f i r s t d e s c r i b e d by Douglas (19^3). Each g l a n d l i e s a l o n g t h e l a t e r a l p o r t i o n o f t h e body c a v i t y , and c o n s i s t s o f c l u s t e r s o f a c i n i . Each a c i n u s i s composed o f a l a y e r o f c e l l s w h i c h forms a s h e l l a r o u n d a more o r l e s s s p h e r i c a l lumen. The a c i n a r lumen opens by means o f a s h o r t e f f e r e n t d u c t t o a d u c t s y s t e m w h i c h e v e n t u a l l y l e a d s t o t h e b u c c a l c a v i t y . Douglas i d e n t i f i e d two t y p e s o f acini» t h e g r a n u l e - s e c r e t i n g ones 175 w h i c h a c c o u n t f o r t h e major p o r t i o n o f t h e g l a n d , and t h e " p y r a -m i d a l " ones w h i c h a r e r e s t r i c t e d t o t h e a n t e r i o r t h i r d o f t h e g l a n d . T i l l ( 1 9 6 1 ) r e c o g n i z e d t w o - t ypes of g r a n u l e - s e c r e t i n g a c i n i , a s w e l l as t h e p y r a m i d a l a c i n u s , i n t h e brown e a r t i c k R h l p l c e p h a l u s a p p e n d l o u l a t u s . T i l l i l l u s t r a t e d t h e d i f f e r e n c e s between t h e a d u l t male and fe m a l e g l a n d s , and p o i n t e d out t h a t t h e s e d i f f e r e n c e s become more pronounced d u r i n g f e e d i n g . More-o v e r , she o b s e r v e d t h a t o f t h e f i v e c e l l t y p e s i n t h e g r a n u l e -s e c r e t i n g a c i n i o f t h e f e m a l e , o n l y two ( t y p e s "b" and "e") e n l a r g e d c o n s i d e r a b l y d u r i n g t h e f e e d i n g p e r i o d . The p y r a m i d a l a c i n u s on t h e o t h e r hand d i d n o t e n l a r g e n o t i c e a b l y d u r i n g t h e f e e d i n g p e r i o d . So i t i s p e r t i n e n t t o t h e p r e s e n t s t u d y , t h a t d e s p i t e t h e a p p a r e n t d i v e r s i t y o f c e l l t y p e s i n t h e s a l i v a r y g l a n d , i n o n l y two t y p e s i s t h e c y t o l o g i c a l a c t i v i t y c o r r e l a t e d w i t h f e e d i n g a c t i v i t y . That t h i s c e l l u l a r a c t i v i t y s h o u l d a l s o p a r a l l e l d i s t e n s i o n o f t h e a c i n u s as a wh o l e , i m p l i c a t e s one o r b o t h o f t h e s e c e l l t y p e s t o be I n v o l v e d d i r e c t l y w i t h t h e s e c r e -t i o n o f a c o p i o u s f l u i d . G r e g s o n ( i 9 6 0 ) and C h i n e r y ( 1 9 6 5 ) show a v a l v e - l i k e s t r u c t u r e s e p a r a t i n g t h e lumen o f t h e a c i n u s f r o m t h a t o f the e f f e r e n t d u c t i n Dermacentor and H a e m a p h y s a l l s . r e s p e c t i v e l y . C h i n e r y o f f e r s t h e s u g g e s t i o n t h a t s i n c e t h e a c i n u s i s n o t p r o v i d e d w i t h m u s c l e s f o r p r o p e l l i n g s a l i v a , t h e e x p u l s i o n o f f l u i d f r o m t h e a c i n u s i s ca u s e d by d i l a t a t i o n o f t h e pharynx? t h e v a l v e e n s u r e s a u n i d i r e c t i o n a l f l o w a l o n g t h e d u c t s . U s e f u l as the l i g h t m i c r o s c o p e may be i n e l u c i d a t i n g m o r p h o l o g i c a l d e t a i l s and some c y t o l o g i c a l f e a t u r e s , i n o r d e r 176 to provide reasonable structural evidence in support of physio-logical phenomena one must turn to the electron microscope. It was primarily for the purpose of identifying the site of f l u i d secretion that the present study was undertaken. MATERIALS AND METHODS T i c k s were d i s s e c t e d and f l o o d e d w i t h f i x a t i v e w h i c h c o n s i s t e d o f 6% g l u t a r a l d e h y d e ( S a b a t i n i e t a l . , 1 9 6 3 ) i n 1/15 M S o r e n s e n ' s phosphate b u f f e r (pH 7 . 4 ? C u l l i n g , 1 9 6 3 ) w i t h k% s u c r o s e (w/v) added. The s m a l l p i e c e s o f t i s s u e were washed f o r 15 m i n u t e s w i t h t h e b u f f e r s o l u t i o n and p o s t - f i x e d w i t h 1% OsO^ i n phosphate b u f f e r . The t i s s u e was r a p i d l y dehy-d r a t e d I n a g r a d e d s e r i e s o f e t h a n o l s e n d i n g w i t h p r o p y l e n e o x i d e , and t h e n i n f i l t r a t e d w i t h Epon 812 f o r e i g h t h o u r s . o T h i n s e c t i o n s (about 8 0 0 A) were c u t on a "LKB U l t r o t o m e and p i c k e d up on u n c o a t e d c o p p e r g r i d s . S e c t i o n s were s t a i n e d f o r 1 5 m i n u t e s w i t h u r a n y l a c e t a t e ( s a t u r a t e d s o l u t i o n i n 7 0 $ e t h a n o l ) and f o r 30 m i n u t e s w i t h l e a d c i t r a t e ( R e y n o l d s , 1 9 6 3 ) . The s e c t i o n s were o b s e r v e d under t h e H i t a c h i HU-11 A e l e c t r o n m i c r o s c o p e . I n a d d i t i o n , t h i c k s e c t i o n s ( 1 m i c r o n ) o f epon-embedded t i s s u e were c u t and s t a i n e d w i t h a l k a l i n e t o l u i d i n e b l u e (Pease 1 9 6 4 ) f o r l i g h t m i c r o s c o p y . U n l e s s s t a t e d t o t h e c o n t r a r y , o b s e r v a t i o n s were made on g l a n d s f r o m t i c k s w h i c h had been a c t i v e l y f e e d i n g f o r a t l e a s t s i x days and w h i c h had a t t a i n e d a w e i g h t o f 2 0 0 t o 350 mg. 178 RESULTS I t i s n o t s u r p r i s i n g t o f i n d a number of c e l l t y p e s i n the s a l i v a r y g l a n d s of i x o d i d t i c k s . Many i x o d i d s s e c r e t e a cement w h i c h s e c u r e s t h e mouthparts t o the s k i n o f th e h o s t (Moorehouse, 1969). The enzymes and a n t i c o a g u l a n t s o f s a l i v a have been n o t e d f o r many y e a r s , and t h e p a r a l y t i c t o x i n s a r e a l s o b e l i e v e d t o be s e c r e t e d by (Gregson, 1957), and perhaps s y n t h e s i z e d by the s a l i v a r y g l a n d s . The p r e s e n t s t u d y s u p p o r t s t h e h y p o t h e s i s t h a t t h e s a l i v a r y g l a n d f u n c t i o n s as a w a t e r s e c r e t i n g o r g a n . T h e r e f o r e i t would be a s t o n i s h i n g i f a g l a n d w h i c h s e c r e t e d t h e s e d i v e r s e elements p r e s e n t e d I t s e l f as an e p i t h e l i u m o f a s i n g l e c e l l t y p e . The s a l i v a r y g l a n d o f Dermacentor a n d e r s o n l p o s s e s s e s t h r e e t y p e s o f a c i n u s : t h e p y r a m i d a l ( o r t y p e I ) a c i n u s , t h e g r a n u l e - s e c r e t i n g ( t y p e I I ) a c i n u s and t h e g r a n u l e - s e c r e t i n g ( t y p e I I I ) a c i n u s , a c c o r d i n g t o t h e n o m e n c l a t u r e o f T i l l (I96I). A p p a r e n t l y Douglas (19^ 3) d i d n o t r e c o g n i z e t h e t y p e I I a c i n u s . The p y r a m i d a l a c i n u s (33 ± 3 SE ^ 1 I n d i a m e t e r ) c o n s i s t s o f two c e l l t y p e s ( F i g u r e s 27, 2 8 ) , t h e t y p e I I a c i n u s (112 + 3 SE p. i n d i a m e t e r ) and t h e t y p e I I I a c i n u s (125 + 15 SE ^ 1 i n d i a m e t e r ) each c o n s i s t s o f f i v e c e l l t y p e s ( F i g u r e s 29A, 30). The d i f -f e r e n c e s between t h e s e t h r e e t y p e s o f a c i n i w i l l be i n d i c a t e d i n due c o u r s e . 1 7 9 1 ) The S a l i v a r y D ucts A r b i t r a r i l y , one may c l a s s i f y a d u c t a c c o r d i n g t o the degree o f b r a n c h i n g from t h e main d u c t . The s h o r t d u c t immedi-a t e l y d r a i n i n g t h e a c i n a r lumen i s u s u a l l y termed t h e e f f e r e n t d u c t . The e f f e r e n t d u c t s of many a c i n i empty i n t o a common t r u n k c a l l e d t h e s e c o n d a r y d u c t . The se c o n d a r y d u c t s u s u a l l y d r a i n i n t o one of two main c o l l e c t i n g d u c t s w h i c h i n t u r n j o i n t o f o r m t h e main e x c r e t o r y d u c t . The main e x c r e t o r y d u c t o f each g l a n d opens i n t o t h e s a l i v a r i u m i n d e p e n d e n t l y o f t h e o t h e r . The p y r a m i d a l a c i n i a r e un i q u e I n t h a t t h e y e n t e r t h e main e x c r e t o r y d u c t d i r e c t l y by means o f t h e i r s h o r t e f f e r e n t d u c t s . A few p y r a m i d a l a c i n i a l s o d r a i n i n t o t h e c o l l e c t i n g d u c t . The lumen o f t h e main e x c r e t o r y d u c t i s 4 4 + 3 u i n d i a m e t e r and t h e t h i c k n e s s o f t h e s u r r o u n d i n g c e l l l a y e r i s 1 5 ± 1 p» Two s t r i k i n g f e a t u r e s o f t h e w a l l o f the main e x c r e t o r y d u c t a r e t h e r e l a t i v e l y t h i c k basement membrane ( a p p r o a c h i n g 1 p) on t h e hemolymph s i d e , and t h e t h i c k c u t i c l e (3 t o 4 p) on t h e l u m i n a l s i d e ( F i g u r e s 2 7 . 28). The s e c o n d a r y and e f f e r e n t d u c t s a r e v e r y s i m i l a r t o t h e main e x c r e t o r y d u c t e x c e p t t h a t t h e y a r e s m a l l e r (lumen d i a m e t e r = 1 0 + 1 p; w a l l t h i c k n e s s = 1 0 + 1 p), and t h e c u t i c l e and b a s a l l a m i n a , though s t i l l p r o m i n e n t , do n o t make up q u i t e as l a r g e a p r o p o r t i o n of t h e w a l l t h i c k n e s s as t h e y do i n t h e main e x c r e t o r y d u c t s . The u l t r a s t r u c t u r e o f t h e s e c o n d a r y and main d u c t s i s p r e s e n t e d i n F i g u r e s 38 and 39. A t f a i r l y r e g u l a r i n t e r v a l s , t h e basement membrane p r o j e c t s i n t o t h e b a s a l i n f o l d i n g s and between t h e plasma membranes of a d j a c e n t c e l l s . Many b u n d l e s o f p a r a l l e l 180 m i c r o t u b u l e s a r e a r r a n g e d d i a g o n a l l y a c r o s s t h e c e l l f r o m t h e c u t i c l e a t t h e l u m i n a l b o r d e r t o t h e b a s a l and l a t e r a l plasma membranes. The l a t e r a l c e l l membranes r u n a t o r t u o u s r o u t e and o f t e n f o r m s e p t a t e desmosomes ( F i g u r e 39). The c y t o p l a s m i s q u i t e dense and c o n t a i n s a f a i r number of m i t o c h o n d r i a , a l t h o u g h t h e l a t t e r do n o t a p p e a r t o be v e r y l a r g e . 2) The P y r a m i d a l A c i n u s E a r l i e r a u t h o r s were a b l e t o d i s t i n g u i s h o n l y a s i n g l e c e l l t y p e ( f i b r i l l a r c e l l ) i n t h e p y r a m i d a l a c i n u s . So i t was somewhat s u r p r i s i n g t o f i n d a second c e l l t y p e i n t h e r e g i o n where one w o u l d e x p e c t t o see t h e a c i n a r lumen ( F i g u r e s 27. 28). That t h e c e n t r a l a c i n a r r e g i o n i s i n d e e d o c c u p i e d by a c e l l i s a l m o s t i m p o s s i b l e t o d e t e r m i n e from c o n v e n t i o n a l l i g h t m i c r o s c o p y s e c t i o n s , i s s t r o n g l y s u g g e s t e d i n t h e l i g h t m i c r o g r a p h s g a i n e d by t h e p r e s e n t t e c h n i q u e , and i s v e r i f i e d under t h e e l e c t r o n m i c r o s c o p e ( F i g u r e s 40A, 4 1 ) . There a r e o n l y two f i b r i l l a r c e l l s and one i n n e r c e l l p e r a c i n u s . The f i b r i l l a r c e l l s forms a s h e l l around t h e whole a c i n u s and shows an u l t r a s t r u c t u r e w h i c h i s c h a r a c t e r i s t i c of t r a n s -p o r t i n g c e l l s . The b a s a l l a m i n a i s i n f o l d e d e x t e n s i v e l y and i s a s s o c i a t e d w i t h a dense p o p u l a t i o n o f m l t o c h o n d r a j t h e m i t o -c h o n d r i a l c r l s t a e a r e r e g u l a r l y packed. The b a s a l l a m i n a w h i c h c o v e r s t h e s u r f a c e of t h e s e c e l l s does n o t ap p e a r t o e n t e r t h e i n f o l d i n g s . I t i s u n d o u b t e d l y t h e a r r a y of e l o n g a t e d m i t o c h o n -d r i a w h i c h i s r e s p o n s i b l e f o r t h e s t r i a t e d appearance o f t h e a c i n u s under t h e l i g h t m i c r o s c o p e . I t i s d i f f i c u l t t o d i s t i n -g u i s h t h e l a t e r a l plasma membranes. The a p i c a l plasma membrane 181 i s n o t i n f o l d e d , b u t r u n s p a r a l l e l t o t h e b a s a l membrane of the i n n e r c e l l (see b e l o w ) . The n u c l e u s i s s i t u a t e d n e a r t h e i n n e r b o r d e r o f t h e c e l l ( F i g u r e 40 A ) . The c h r o m a t i n a p p e a r s t o be condensed a r o u n d t h e n u c l e a r membrane and t h e n u c l e o l i a r e prom-i n e n t . The r e s t o f t h e n u c l e o p l a s m i s r e l a t i v e l y c l e a r - s t a i n i n g . I n some s e c t i o n s ( F i g u r e 40 B) s t r u c t u r e s v e r y r e m i n i s c e n t o f axons f i l l e d w i t h e l e c t r o n - d e n s e n e u r o s e c r e t o r y g r a n u l e s ( M a d d r e l l , 1 9 6 6 5 B r a d y and M a d d r e l l , 1 9 6 7 ) a r e seen a t t h e deep e r extremes o f t h e plasma membrane l n f o l d i n g s . The i n n e r c e l l s o f t h e p y r a m i d a l a c i n u s s t a n d i n marked c o n t r a s t t o t h e f i b r i l l a r c e l l s . The c y t o p l a s m i s c l e a r s t a i n -i n g and t h e r e a r e few i f any l n f o l d i n g s of t h e plasma membrane. One n o t i c e s a few randomly o r i e n t e d m i c r o t u b u l e s and o n l y t h e o c c a s i o n a l m i t o c h o n d r i o n . The plasma membrane o f t e n forms a r e a s of c l o s e a p p o s t l o n w i t h t h a t o f t h e f i b r i l l a r c e l l , b u t t h e mem-bra n e s do n o t i n t e r d i g i t a t e ( F i g u r e 41). Under t h e l i g h t m i c r o -s c o p e , one might e a s i l y c o n f u s e t h i s c e l l w i t h t h e lumen o f t h e a c i n u s . P r e v i o u s a u t h o r s have mentioned t h a t n u c l e i o f t h e f i b r i l l a r c e l l s o f t e n appear t o l i e f r e e i n t h e a c i n a r lumen ( D o u g l a s , 1943; T i l l , 1961). Perhaps i n many c a s e s , t h o s e i n q u e s t i o n wore s i m p l e y n u c l e i o f t h e u n r e c o g n i z e d i n n e r c e l l ( F i g u r e 2 8 ) . The a c i n u s I t s e l f does n o t app e a r t o have a lumen d i s -t i n c t f r o m t h a t o f t h e s h o r t e f f e r e n t d u c t l e a d i n g t o t h e main e x c r e t o r y d u c t . The p y r a m i d a l a c i n i a l w a y s J o i n t h e main e x c r e -t o r y d u c t o r c o l l e c t i n g d u c t s , but n o t t h e s e c o n d a r y d u c t s . 182 3 ) The G r a n u l e - S e c r e t i n g A c i n u s (Type I I I ) These a r e t h e most numerous o f t h e t h r e e t y p e s o f a c i n i . I n t h e l a s t c h a p t e r , p h y s i o l o g i c a l e v i d e n c e was p r e s e n t e d t h a t s a l i v a r y s e c r e t i o n was under ne r v o u s c o n t r o l ? n e r v e s have a l r e a d y been shown a s s o c i a t e d w i t h t h e d u c t s and t h e p y r a m i d a l a c i n i ( F i g u r e s 27, 28, 3 9 ) . I n F i g u r e Jk, a n e r v e a p p e a r s t o a p p r o a c h t h e b a s a l s u r f a c e of a t y p e I I I a c i n u s . To my knowledge, t h e s e a r e t h e f i r s t r e p o r t s o f n e r v e s a s s o c i a t e d w i t h t h e s a l i v a r y g l a n d I n an i x o d i d t i c k . T here a r e no f e w e r t h a n f i v e d i s t i n c t c e l l t y p e s making up t h e t y p e I I I a c i n u s . I have chosen t o name them as f o l l o w s t (a) n a k e d - g r a n u l e c e l l , (b) e n c a p s u l a t e d - g r a n u l e c e l l , ( c ) v a c u -o l a r c e l l , (d) w a t e r c e l l and (e) cap c e l l . A l l f i v e t y p e s a r e v i s i b l e i n F i g u r e 3 0 * They a r e a r r a n g e d i n t h e a c i n u s as f o l -l o ws J The two g r a n u l e - c e l l t y p e s s u r r o u n d t h e r e g i o n where the e f f e r e n t d u c t e n t e r s t h e a c i n u s ( F i g u r e 36)1 t h e l a t t e r r e g i o n may be r e f e r r e d t o as t h e " n o r t h p o l e " • There a r e u s u a l l y two e n c a p s u l a t e d - g r a n u l e c e l l s and f i v e n a k e d - g r a n u l e c e l l s . An e q u a t o r i a l s e c t i o n t h r o u g h t h e a c i n u s r e v e a l s t h e arrangement o f t h e t h r e e o t h e r c e l l t y p e s ( F i g u r e 3 5 ) . A t i e r o f f i v e o r s i x v a c u o l a r c e l l s a l t e r n a t e a r o u n d t h e e q u a t o r w i t h an e q u a l number o f w a t e r c e l l s , a l t h o u g h t h e b a s a l s u r f a c e o f t h e v a c u -o l a r c e l l s do n o t r e a c h t h e hemolymph s u r f a c e o f t h e a c i n u s . The hemolymph s u r f a c e o f t h e a c i n u s i s c o m p l e t e l y c o v e r e d by u m b r e l l a - l i k e e x t e n s i o n s o f the w a t e r c e l l s . I t a p p e a r s as i f t h e g r a n u l e - s e c r e t i n g c e l l s a r e a l s o l a r g e l y c u t o f f f rom d i r e c t c o n t a c t w i t h t h e hemolymph by t h e s e e x t e n s i o n s of the 183 w a t e r c e l l s ( F i g u r e s 33» 3 6 ) . S u r p r i s i n g l y enough, t h e a p i c a l s u r f a c e o f t h e w a t e r c e l l s i s n o t b a t h e d by t h e l u m i n a l f l u i d e x c e p t , as w i l l be shown l a t e r , p o s s i b l y a t c e r t a i n r e s t r i c t e d r e g i o n s . The cap c e l l s t r a d d l e s t h e a p i c a l s u r f a c e o f each w a t e r c e l l by b r i d g i n g t h e gap between two a d j a c e n t v a c u o l a r c e l l s ( F i g u r e 3 5 )» t h u s b l o c k i n g a c c e s s o f t h e lumen t o t h e w a t e r c e l l . A s c h e m a t i c d r a w i n g i n c o r p o r a t i n g t h e r e l a t i o n s h i p o f t h e c e l l s one t o t h e o t h e r i s p r e s e n t e d i n F i g u r e 2 5 . A l t o -g e t h e r t h e r e a r e a p p r o x i m a t e l y seven g r a n u l e - c e l l s , e i g h t o r n i n e w a t e r c e l l s , and e q u a l numbers o f v a c u o l a r and cap c e l l s , t o t a l l i n g 30 t o 35 c e l l s p e r a c i n u s . The i m p o r t a n t f e a t u r e s t o b e a r i n mind a r e t h a t t h e hemolymph s u r f a c e o f t h e a c i n u s i s c o v e r e d e n t i r e l y by t h e w a t e r c e l l s and t h e i r e x t e n s i o n s , and t h a t t h e l u m i n a l s u r f a c e i s c o v e r e d a l m o s t e n t i r e l y by v a c u o l a r c e l l s and cap c e l l s . The a p i c a l s u r f a c e of t h e w a t e r c e l l s i s b a t h e d by l u m i n a l f l u i d o n l y (1) a t p o i n t s where t h e b r i d g i n g o f t h e v a c u o l a r c e l l s by t h e cap c e l l s i s p o s s i b l y i n c o m p l e t e ( F i g u r e 32 a, b, d, e, f ) and (2) i n t h e s p h e r i c a l segment o f t h e a c i n u s d e f i n e d by t h e g r a n u l e - c e l l s ( F i g u r e 32 c, g ) . a) The n a k e d - g r a n u l e c e l l Under the l i g h t m i c r o s c o p e ( F i g u r e s 3 0 , 3 6 ) t h e c y t o p l a s m a p p e a r s homogeneous b u t f i l l e d w i t h d a r k - s t a i n i n g s p h e r i c a l g r a n u l e s a p p r o a c h i n g 6 p. i n d i a m e t e r . The n u c l e i (24 + 1 I n d i a m e t e r ) p o s s e s s l i g h t - s t a i n i n g n u c l e o p l a s m , a l a r g e n u c l e o l u s , and d i s c r e e t c o n d e n s t a t i o n s o f c h r o m a t i n t h r o u g h o u t t h e n u c l e o -p l a s m . Under t h e e l e c t r o n - m i c r o s c o p e ( F i g u r e 4 2 ) , t h e g r a n u l e s a p p e a r u n i f o r m l y e l e c t r o n - r a r e and t h e c y t o p l a s m i s f i l l e d w i t h 184 F i g u r e 25. A d i a g r a m a t i c r e c o n s t r u c t i o n o f the t y p e I I I a c i n u s showing th e s p a t i a l r e l a t i o n s h i p s among th e f i v e c e l l t y p e s . D Duct EG E n c a p s u l a t e d g r a n u l e c e l l V I V a l v e VC V a c u o l a r c e l l NG Naked g r a n u l e c e l l WC Water c e l l Cp Cap c e l l A l t h o u g h f o r t h e most p a r t t h e w a t e r c e l l i s c o m p l e t e l y c u t o f f f r o m t h e a c i n a r lumen by means of a d j a c e n t cap and v a c u o l a r c e l l s , t h e r e may be a few r e g i o n s where the membranes of t h e l a t t e r c e l l s a r e not c l o s e l y apposed ( a r r o w s ; c f . F i g u r e 3 2 ) . The a c t u a l e x t e n t o f t h e s e p o t e n t i a l c o r r i d o r s i s n o t known. N o t i c e t h a t t h e hemolymph s u r f a c e o f t h e a c i n u s i s c o m p l e t e l y c o v e r e d b y e x t e n s i o n s o f t h e w a t e r c e l l . 186 g r a n u l a r e n d o p l a s m i c r e t i c u l u m ( E R ) . The n u c l e u s c o n t a i n s c o a r s e l y p r e c i p i t a t e d clumps of c h r o m a t i n and a d i f f u s e n u c l e -o l u s . b) The e n c a p s u l a t e d - g r a n u l e c e l l Under t h e l i g h t m i c r o s c o p e ( F i g u r e J6) t h i s c e l l i s d i s t i n g u i s h e d f r o m t h e n a k e d - g r a n u l e c e l l on t h e b a s i s of g r a n u l e and n u c l e a r s t r u c t u r e , s i n c e t h e c y t o p l a s m i s n o t easy t o make out between t h e g r a n u l e s . Each g r a n u l e i n t h e p r e s e n t c e l l a p p e a r s s u r r o u n d e d by a l i g h t e r s t a i n i n g r e g i o n t h a t g i v e s i t t h e appearance of b e i n g e n c a p s u l a t e d . The c a p s u l e s a r e s p h e r i c a l b u t t h e clumps of g r a n u l e s u s u a l l y a ppear i r r e g u l a r l y shaped ( b e t t e r seen i n F i g u r e 29 B ) . Under t h e e l e c t r o n m i c r o -scope ( F i g u r e 4 2 ) , each c a p s u l e i s d e f i n e d by a membrane. The m a t r i x o f t h e c a p s u l e s t a i n s d e n s e l y b u t i s f i l l e d w i t h s t i l l d a r k e r g r a n u l e s t h a t do n o t appear t o be membrane-bound. The c y t o p l a s m i s s t u d d e d w i t h r i b o s o m e s i n t h e f o r m o f a g r a n u l a r ER, M i t o c h o n d r i a a r e p r e s e n t , b u t a r e n o t e x c e e d i n g l y numerous n o r p a r t i c u l a r l y d i s t e n d e d . The c r i s t a e a r e n o t p r o m i n e n t . The l a t e r a l membrane between a d j a c e n t g r a n u l e c e l l s a r e p a r a l l e l t h r o u g h o u t , b u t do n o t form many a r e a s o f c l o s e a p p o s i t i o n . c) The v a c u o l a r c e l l These c e l l s s t a i n d e e p l y w i t h t o l u i d i n e b l u e i n marked c o n t r a s t t o t h e a d j a c e n t w a t e r c e l l s ( F i g u r e 35). The n u c l e u s (16 + 1 JOL i n d i a m e t e r ) s i t s more o r l e s s c e n t r a l l y I n t h e c e l l . Condensed c h r o m a t i n i s u n i f o r m l y p r e c i p i t a t e d o v e r a m o d e r a t e l y dense n u c l e o p l a s m , b u t n u c l e o l i a r e n o t common. When p r e s e n t t h e y a r e e c c e n t r i c a l l y p l a c e d , and may be d i f f u s e and p a r t i c u -l a t e . A b r u s h b o r d e r a t t h e apex i s a prominent f e a t u r e o f t h i s c e l l . C l e a r v a c u o l e s ( a f t e r w h i c h t h e s e c e l l s a r e named) a r e abundant ( F i g u r e 37). Under the e l e c t r o n m i c r o s c o p e ( F i g u r e s 43, 44) t h e p r o m i n e n t f e a t u r e o f t h i s c e l l i s t h e abundance of g r a n u l a r ER, showing d i s t e n d e d p r o f i l e s f i l l e d w i t h a homo-geneous e l e c t r o n - d e n s e s u b s t a n c e , and t h e v a c u o l e s , c o n t a i n i n g a s e c r e t o r y p r o d u c t . M i t o c h o n d r i a a r e s m a l l and dense. The a p i c a l membrane b e a r s i r r e g u l a r m i c r o v i l l a e . The r e m a i n d e r of t h e l a t e r a l membrane ( i . e . where i t i s i n c o n t a c t w i t h t h e w a t e r c e l l ) , may show p r o t r u s i o n s w h i c h i n t e r d i g i t a t e w i t h s i m i l a r p r o t r u s i o n s of t h e w a t e r c e l l . T h i s i n t e r d i g i t a t i o n c o n t i n u e s a r o u n d t h e b a s a l membrane as w e l l ( F i g u r e 44)• d) The w a t e r c e l l The w a t e r c e l l t o g e t h e r w i t h t h e v a c u o l a r c e l l make up t h e m a j o r p o r t i o n o f t h e a c i n a r w a l l . E x c e p t f o r numerous d a r k -s t a i n i n g f i l a m e n t s ( F i g u r e 34) t h e ground c y t o p l a s m t a k e s up v e r y l i t t l e t o l u i d i n e b l u e . I n some s e c t i o n s , p a t c h e s o f t h e c e l l a p p e a r d e v o i d o f s t a i n e n t i r e l y ( F i g u r e 35) y e t t h e p a t c h e s a r e t o o i r r e g u l a r l y shaped t o be v a c u o l e s . C l e a r spaces between a d j a c e n t v a c u o l a r and w a t e r c e l l s a ppear t o be i n t e r c e l l u l a r c h a n n e l s ( F i g u r e 35)• Under t h e e l e c t r o n m i c r o s c o p e i t becomes a p p a r e n t t h a t t h e numerous d a r k - s t a i n i n g f i l a m e n t s a r e m i t o c h o n -d r i a , and t h e n o n - s t a i n i n g p o r t i o n s i n t h e l i g h t - m i c r o s c o p e s e c t i o n s a r e c l e a r c h a n n e l s w h i c h e x t e n d t h r o u g h o u t t h e c y t o -p l a s m ( F i g u r e s 43, 44), The c h a n n e l s a r e p r o b a b l y aqueous, and hence t h e name f o r t h i s c e l l t y p e . The c h a n n e l s a r i s e f r o m the 188 plasma membranes b o r d e r i n g t h e hemocoele and v a c u o l a r c e l l s . The degree of d i s t e n s i o n o f t h e s e w a t e r c h a n n e l s i s o f t e n marked, b u t t h i s may be an a r t i f a c t due t o d i f f e r e n c e s i n o s m o t i c p r e s -s u r e between t h e c e l l and t h e f i x a t i v e . The l a t e r a l membranes of e ach w a t e r c e l l and i t s a d j a c e n t v a c u o l a r c e l l s a r e s e p a r a t e d by an i n t e r c e l l u l a r c h a n n e l w i t h i n w h i c h the membranes o f b o t h c e l l s i n t e r d i g i t a t e , There a r e a few r e g i o n s however i n w h i c h t h e two membranes f o r m a r e a s o f c l o s e a p p o s i t i o n . I n c o n t r a s t t o t h e above, t h e a p i c a l membrane of t h e w a t e r c e l l i s s t r i c t l y p a r a l l e l t o t h a t o f t h e cap c e l l i n a l l r e g i o n s , and t h e mem-br a n e s show many a r e a s o f a p p a r e n t f u s i o n and s e p t a t e desmosomes. The numerous m i t o c h o n d r i a c o n t a i n d e n s e l y packed c r i s t a e i n p a r a l l e l a r r a y , and a r e o f t e n a s s o c i a t e d w i t h t h e l n f o l d i n g s o f t h e plasma membrane; t h e y a r e e v e n l y d i s t r i b u t e d t h r o u g h o u t t h e c e l l , b u t a r e n o t q u i t e as e l o n g a t e d as t h o s e of t h e f i b r i l -l a r c e l l i n t h e p y r a m i d a l a c i n u s ( c f F i g u r e 4 0 ) , The c y t o p l a s m c o n t a i n s r i b o s o m e s and some randomly o r i e n t e d m i c r o t u b u l e s . The n u c l e u s i s l a r g e l y e u c h r o m a t l c , i s s i t u a t e d n e a r t h e base of t h e c e l l and c o n t a i n s a s i n g l e e c c e n t r i c a l l y p l a c e d n u c l e o l u s , e) The cap c e l l Many o f t h e f e a t u r e s o f t h i s c e l l have a l r e a d y been p o i n t e d out i n p a s s i n g . I t i s an i r r e g u l a r l y shaped c e l l w h i c h u n d e r t h e l i g h t m i c r o s c o p e shows a b r u s h b o r d e r ( F i g u r e 3 5 ) . I t d e r i v e s i t s name f r o m s i t t i n g o v e r t h e a p i c a l s u r f a c e o f t h e w a t e r c e l l , t h u s l a r g e l y c o v e r i n g t h e l a t t e r f r o m t h e a c i n a r lumen. The c y t o p l a s m h a r d l y s t a i n s w i t h t o l u l d i n e b l u e ( F i g u r e 35) and a p p e a r s q u i t e s p a r s e under t h e e l e c t r o n m i c r o s c o p e 189 ( F i g u r e s 43, 44). I t p o s s e s s e s m l c r o v i l l a e , b u t n o t i n as dense an a r r a y as does t h e v a c u o l a r c e l l ( F i g u r e 43 I n s e t ) . The c y t o p l a s m c o n t a i n s o n l y few r i bosomes and o c c a s i o n a l m i t o -c h o n d r i a . But t h e r e e x i s t s a f a i r number o f p a r a l l e l m i c r o -t u b u l e s w h i c h a r e e s p e c i a l l y abundant n e a r th e c l o s e l y apposed plasma membranes of t h e cap and v a c u o l a r c e l l s ( F i g u r e 43). A l t h o u g h t h i s c e l l a p p e a r s r e l a t i v e l y i n a c t i v e , i t p o s s i b l y p l a y s a r o l e i n b i n d i n g the o t h e r c e l l s o f t h e a c i n u s t o g e t h e r -a r e a s o n a b l e p r o p o s i t i o n s i n c e t h e f l u i d p r e s s u r e s w i t h i n t h e a c i n u s d u r i n g a c t i v e s e c r e t i o n may be c o n s i d e r a b l e . 4) The G r a n u l e - S e c r e t i n g A c i n u s (Type I I ) The t y p e I I a c i n u s i s more abundant i n t h e a n t e r i o r h a l f o f t h e g l a n d t h a n i n t h e p o s t e r i o r h a l f ; i t i s b u i l t upon a somewhat s i m i l a r , b u t l e s s s t r a i g h t - f o r w a r d , p l a n as t h e t y p e I I I a c i n u s . I have n o t y e t worked out t h e p r e c i s e r e l a t i o n s h i p between t h e c e l l s o f t h i s a c i n u s . I n a l l , t h e t y p e I I a c i n u s i s composed o f c e l l s w h i c h can be d i v i d e d i n t o f i v e t y p e s . The lumen of t h e a c i n u s (28 + 3 p i n d i a m e t e r ) i s n e v e r as i n f l a t e d a s t h a t of t h e t y p e I I I a c i n u s (58 +15 p i n d i a m e t e r ) . The whole a c i n u s measures 112 + 3 p i n d i a m e t e r . There i s a v a l v e a t t h e o r i g i n o f t h e e f f e r e n t d u c t ( F i g u r e 29 A ) . The f i v e c e l l t y p e s a r e m o r e - o r - l e s s e q u i v a l e n t t o t h e f i v e c e l l t y p e s o f t h e t y p e I I I a c i n u s . a) E n c a p s u l a t e d - g r a n u l e c e l l ( F i g u r e s 29 B. 42) There a r e no o b v i o u s d i f f e r e n c e s between t h i s c e l l and i t s s i s t e r c e l l i n t h e t y p e I I I a c i n u s . 190 b) Naked - g r a n u l e c e l l ( F i g u r e 29 A) The c e l l s i n t h i s a c i n u s appear u l t r a s t r u c t u r a l l y s i m i l a r t o t h o s e i n the t y p e I I I a c i n u s , b u t t h e g r a n u l e s i z e s n e v e r a t t a i n t h e l a r g e s i z e s seen i n t h e type I I I a c i n u s . c) V a c u o l a r c e l l ( F i g u r e s 29 A. 45) T h i s i s t h e l a r g e s t c e l l o f t h e a c i n u s and c o m p r i s e s t h e ma j o r p o r t i o n o f t h e a c i n a r w a l l . A l t h o u g h i t n e v e r a p p e a r s t o c o n t a i n c l e a r v a c u o l e s under t h e l i g h t m i c r o s c o p e , i t sometimes c o n t a i n s g r a n u l e s . When d e v o i d o f g r a n u l e s , t h e c y t o p l a s m s t a i n s d e n s e l y w i t h t o l u i d i n e b l u e , t h u s r e s e m b l i n g t h e v a c u o l a r c e l l o f t h e t y p e I I I a c i n u s ? a b r u s h b o r d e r however cannot e a s i l y be made out a t t h e a p i c a l s u r f a c e o f t h e c e l l , and no c l e a r - s t a i n i n g v a c u o l e s can be se e n . The c y t o p l a s m does n o t s t a i n homogeneously however. R a t h e r , d a r k - s t a i n i n g t h r e a d - l i k e f i l a m e n t s t h a t a r e v i s i b l e under the l i g h t m i c r o s c o p e , a r e i d e n t i f i e d under t h e e l e c t r o n m i c r o s c o p e as m i t o c h o n d r i a ? t h e c y t o p l a s m i t s e l f I s tee m i n g w i t h r i b o s o m e s , and f i n a l l y , under t h e l i g h t m i c r o s c o p e t h e r e a r e c i r c u l a r l i g h t - s t a i n i n g p a t c h e s w h i c h do n o t resemble g r a n u l e s . Under t h e e l e c t r o n m i c r o s o c o p e i t i s c l e a r t h a t t h e s e p a t c h e s a r e p r o f i l e s o f d i s t e n d e d c i s t e r n a e of t h e en d o p l a s m i c r e t i c u l u m . The g r a n u l e s , when t h e y a p p e a r i n t h i s c e l l , s t a i n l i g h t e r w i t h t o l u i d i n e b l u e t h a n do t h o s e o f the n a k e d - g r a n u l e c e l l , b u t under t h e e l e c t r o n m i c r o s c o p e , t h e y a r e more e l e c t r o n dense t h a n t h o s e o f t h e n a k e d - g r a n u l e c e l l ? presumably t h e n , t h e s e two c e l l s a r e m a n u f a c t u r i n g d i f f e r e n t s e c r e t i o n s . 191 d) Water c e l l ( F i g u r e s 29 A. 45) The w a t e r c e l l o f t h e t y p e I I a c i n u s a l w a y s remains d i m l n u i t l v e , and a p p e a r s under t h e l i g h t m i c r o s c o p e o n l y as a stroma s u r r o u n d i n g t h e o t h e r c e l l s . From t h e appearance o f t h e n u c l e u s and the b a s i c u l t r a s t r u c t u r a l f e a t u r e s , t h e s i m i -l a r i t y t o t h e w a t e r c e l l o f t h e t y p e I I I a c i n u s i s n e v e r t h e l e s s c o n v i n c i n g . M i t o c h o n d r i a a r e p r e s e n t b u t c o n t a i n a c l e a r -s t a i n i n g m a t r i x and r e l a t i v e l y few c r l s t a e . e) Cap c e l l ( F i g u r e 46) T h i s c e l l has n o t been seen i n t h e l i g h t m i c r o s c o p e s e c t i o n s ; under t h e e l e c t r o n m i c r o s c o p e , i t p o s s e s s e s most o f t h e c h a r a c t e r i s t i c s o f i t s s i s t e r c e l l i n t h e t y p e I I I a c i n u s . 192 DISCUSSION 1) The D u c t s The u l t r a s t r u c t u r e o f t h e d u c t c e l l s s u g g e s t t h a t t h e y a r e s p e c i a l i z e d f o r r e n d e r i n g t h e d u c t s r i g i d . F i r s t t h e l a t e r a l membranes a r e e x t e n s i v e l y i n t e r d l g i t a t e d and a r e f r e q u e n t l y j o i n e d t o f o r m s e p t a t e desmosomes. Second, t h e r e l a t i v e abundance o f p a r a l l e l - o r i e n t e d m i c r o t u b u l e s i s b e l i e v e d t o m e c h a n i c a l l y s t r e n g t h e n t h e c e l l as a whole ( F a w c e t t , 1966). F i n a l l y , t h e r e l a t i v e l y t h i c k c u t i c l e f u r t h e r s u g g e s t s t h a t the d u c t s f o r t h e most p a r t a r e s i m p l y s t r e n g t h e n e d t u b e s f o r c a r -r y i n g f l u i d . The abundance o f r i b o s o m e s , m i t o c h o n d r i a , and a p i c a l m i c r o v i l l a e a r e u l t r a s t r u e t u r a l f e a t u r e s t h a t may be i n v o l v e d w i t h t h e s e c r e t i o n o f c u t i c l e and t h e t h i c k b a s a l l a m i n a . A l t h o u g h i t was p o s t u l a t e d i n C h a p t e r Two t h a t t h e d u c t s may be t h e s i t e f o r r e a b s o r p t l o n o f some s o l u t e s e c r e t e d i n t h e p r i m a r y f l u i d , t h e u l t r a s t r u c t u r e of the d u c t c e l l s does n o t o v e r w h e l m i n g l y s u p p o r t t h i s h y p o t h e s i s . But s i n c e t h e f i n a l s a l i v a i s o n l y s l i g h t l y (5 t o 10$) hypo-osmotic t o t h e b a t h i n g medium o v e r t h e f u l l c o n c e n t r a t i o n r a n g e , perhaps t h e d u c t c e l l s need n o t be e x t e n s i v e l y s p e c i a l i z e d f o r s o l u t e t r a n s p o r t . A l t e r n a t i v e l y , t h e r e a b s o r p t l o n o f s o l u t e f r om t h e p r i m a r y s e c r e t i o n may be l o c a l i z e d e l s e w h e r e (see b e l o w ) . 193 2 ) The P y r a m i d a l A c i n u s I t w ould a p p e a r f r o m t h e u l t r a s t r u c t u r e t h a t t h e f i b r i l l a r c e l l i s m e t a b o l i c a l l y v e r y a c t i v e . The numerous m i t o c h o n d r i a w i t h dense c r l s t a e , s t a c k e d among t h e v e r i t a b l e maze o f i n f o l d -i n g s o f t h e b a s a l plasma membrane a r e a l l f e a t u r e s a s s o c i a t e d w i t h t h e a c t i v e t r a n s p o r t o f i o n s ? t h e g e n e r a l l a c k of chroma-t i n i n t h e n u c l e u s a l s o on o c c a s i o n i n d i c a t e s a m e t a b o l i c a l l y a c t i v e c e l l ( F a w c e t t , I966), The i n n e r c e l l by c o n t r a s t pos-s e s s e s none o f t h e u l t r a s t u r c t u r a l f e a t u r e s one a s s o c i a t e s w i t h an a c t i v e c e l l . The plasma membrane i s n o t i n f o l d e d and t h e r e a r e h a r d l y any o f t h e u s u a l c e l l i n c l u s i o n s i n t h e c y t o p l a s m . A s i m i l a r c e l l i s d e p i c t e d by Co p e l a n d and F i t z j a r e l l (1968) among t h e s a l t - a b s o r b i n g c e l l s i n t h e g i l l s o f t h e b l u e c r a b ; t h e y c o n s i d e r i t a moribund c e l l . S i n c e t h e p y r a m i d a l a c i n i e x i s t s i n r e l a t i v e l y low numbers, o n l y a t t h e a n t e r i o r p o r t i o n o f t h e g l a n d , do n o t p o s s e s s a lumen, and do n o t f l u c t u a t e s i g n i f i c a n t l y i n s i z e t h r o u g h o u t the f e e d i n g c y c l e ( T i l l , 1961), i t seems u n l i k e l y t h a t t h e s e a c i n i a r e r e s p o n s i b l e f o r t h e s e c r e t i o n o f t h e c o p i o u s s a l i v a r e p r e s e n t i n g t h e e x c e s s f l u i d o f t h e m e a l . Moreover, t h e u l t r a s t u r c t u r e of t h e f i b r i l l a r c e l l i s n o t s i m i l a r t o t h a t o f o t h e r c e l l s w h i c h a r e known t o s e c r e t e c o n s i d e r a b l e volumes o f f l u i d , such as t h e p r i m a r y c e l l s i n t h e M a l p i g h i a n t u b u l e s o f i n s e c t s ( B e r r i d g e and Oschman, 1969). C o u l d i t be t h a t t h e c o m p o s i t i o n of t h e f l u i d s e c r e t e d by a c i n i i n t h e d i s t a l p o r t i o n o f t h e g l a n d i s m o d i f i e d i n t h e main d u c t , n o t by t h e d u c t c e l l s t h e m s e l v e s , b u t by f i b r i l l a r c e l l s o f t h e p y r a m i d a l a c i n i ? The p o s i t i o n o f t h e s e a c i n i 194 d i r e c t l y a round t h e main d u c t , and t h e i r j u n c t u r e w i t h t h e l a t t e r by means o f s h o r t e f f e r e n t d u c t s ( t o b r i n g the a c i n u s i n c l o s e r c o n t a c t w i t h t h e s a l i v a r y f l u i d ? ) a r e c o n s i s t e n t w i t h such an h y p o t h e s i s . M o reover, t h e u l t r a s t r u c t u r e o f t h e f i b r i l l a r c e l l s shows s i g n i f i c a n t p a r a l l e l s w i t h t h a t of t h e s t r i a t e d d u c t c e l l i n t h e p a r o t i d g l a n d of t h e mouse and r a t ( P a r k s , 1961). The l a t t e r c e l l has an u n f o l d e d b a s a l plasma membrane w i t h many m i t o c h o n d r i a a l l i g n e d between t h e l e a f l e t s . The a p i c a l p o r t i o n o f t h i s c e l l p o s s e s s few s h o r t m i c r o v i l l a e . I t I s w e l l known, fr o m t h e e a r l i e r m entioned m i c r o p u n c t u r e s t u d i e s , t h a t t h e s e c e l l s exchange t h e sodium i n t h e p r i m a r y s a l i v a f o r p o t a s s i u m , and r e a b s o r b s o l u t e i n e x c e s s o f w a t e r t o r e n d e r t h e f i n a l s a l i v a h y p o t o n i c . I t would be i n t e r e s t i n g t o t e s t t h e l a t t e r h y p o t h e s i s by c o m p a r i n g t h e o s m o t i c p r e s s u r e o f f l u i d c o l l e c t e d i n t h e main d u c t d i s t a l t o t h e r e g i o n o f t h e p y r a m i d a l a c i n i w i t h t h a t o f f l u i d c o l l e c t e d p r o x i m a l t o t h a t r e g i o n . One cannot r u l e o u t , however, t h a t t h e f i b r i l l a r c e l l i s s p e c i a l i z e d f o r s e c r e t i o n o f some component o f t h e s a l i v a r a t h e r t h a n f o r r e a b s o r p t l o n . The f u n c t i o n of t h e i n n e r c e l l r e mains o b s c u r e . 3) The G r a n u l e - S e c r e t i n g A c i n u s (Type I I I ) The f u n c t i o n s o f t h e f i v e c e l l s i n t h i s a c i n u s have a l r e a d y been a l l u d e d t o i n t h e names chosen f o r them. S i n c e by f a r t h e b u l k o f t h e g l a n d i s composed of t h e s e a c i n i , one would r e a s o n a b l y e x p e c t t o f i n d the w a t e r - s e c r e t i n g c e l l s i t u a t e d h e r e . A l l t h e f e a t u r e s o f the c e l l w h i c h I have d e s i g n a t e d t h e "water c e l l " , e s p e c i a l l y the c l e a r , e l o n g a t e d 195 c h a n n e l s , a r e c h a r a c t e r i s t i c o f t h e c e l l s I n o t h e r w a t e r -t r a n s p o r t i n g e p i t h e l i a ( W i g g l e s w o r t h and S a l p e t e r , 1 9 6 2 ; A n d e r s o n and H a r v e y , 1 9 6 6 ; Tormey and Diamond, 1 9 6 7 ; B e r r i d g e and G u p t a , 1 9 6 7 ; C o p e l a n d and F l t z j a r e l l , 1 9 6 8 ; B e r r i d g e and Oschman, 1 9 6 9 ; Oschman and B e r r i d g e , 1 9 7 0 ; P i l c h e r , 1 9 7 0 ) . P a r k s ( 1 9 6 2 ) o b s e r v e d t h e f o r m a t i o n o f c l e a r v a c u o l e s i n t h e p a r o t i d g l a n d s o f mice and r a t s w h i c h had r e c e i v e d p i l o c a r p i n e I n j e c t i o n s , o r w h i c h were f e d a f t e r a 24-hour f a s t . B o t h of t h e s e t r e a t m e n t s s t i m u l a t e t h e s e c r e t i o n o f a w a t e r y s a l i v a . P a r k s was a l s o of t h e o p i n i o n t h a t t h e s e v a c u o l e s formed by t h e s w e l l i n g o f p r e - e x l s t e n t membranous v e s i c l e s by t h e i m b i b i t i o n of w a t e r , and t h u s he a t t r i b u t e d t o t h e s e v a c u o l e s a measure o f permanence i n t h e c e l l . Such, s w o l l e n v a c u o l e s were n o t seen i n s t a r v e d a n i m a l s . A l t h o u g h P a r k s named t h e s e v e s i c l e s , " v a c u o l e s " , p r e sumably t h e y b e a r t h e same s i g n i f i c a n c e t o t h e p a r o t i d s a l i -v a r y g l a n d as do t h e o t h e r aqueous c h a n n e l s enumerated above. Such c l e a r c h a n n e l s a r e now b e l i e v e d t o be i m p o r t a n t i n e n a b l i n g t h e i s o - o s m o t i c t r a n s p o r t of f l u i d (see below) and p o s s i b l y f o r s e g r e g a t i n g t h e s e c r e t e d f l u i d f r o m t h e c y t o p l a s m i c c o n t e n t s o f t h e c e l l . I t w o u l d be d i f f i c u l t f o r a c e l l t o m a i n t a i n i t s i n t e g r i t y i n t h e f a c e o f s e c r e t i n g many t i m e s i t s own w e i g h t of f l u i d i n a r e l a t i v e l y s h o r t p e r i o d o f t i m e , i f t h a t f l u i d were a l l o w e d t o mix f r e e l y w i t h the c y t o p l a s m i t s e l f . A s i m i l a r phenomenon was o b s e r v e d by Harvey and Z e r a h n ( I 9 6 9 ) i n t h e i s o l a t e d midgut of t h e C e c r o p i a s i l k w o r m . P o t a s s i u m t h a t i s t r a n s p o r t e d by t h e e p i t h e l i u m does n o t mix a p p r e c i a b l y w i t h c e l l p o t a s s i u m . Even i f one assumes the s a l i v a r y g l a n d t o be composed e n t i r e l y o f w a t e r c e l l s , one a r r i v e s a t a f i g u r e f o r w a t e r s e c r e t i o n i n v i t r o o f c l o s e t o t w i c e t h e c e l l volume p e r h o u r ; c l e a r l y t h i s i s a c o n s e r v a t i v e f i g u r e s i n c e t h e r a t e o f f l u i d s e c r e t i o n i n v i v o exceeds t h a t i n v i t r o up t o f i v e f o l d , and o b v i o u s l y t h e s a l i v a r y g l a n d i s n o t composed c o m p l e t e l y of w a t e r c e l l s . The u m b r e l l a - l i k e e x t e n s i o n s o f t h e b a s a l s u r f a c e o f t h e w a t e r c e l l c o m p l e t e l y e n v e l o p e s t h e hemolymph s u r f a c e of t h e a c i n u s ; t h i s w o u l d appear t o be a m o d i f i c a t i o n o f c e l l s t r u c t u r e e l e g a n t l y d e s i g n e d t o i n c r e a s e t h e s u r f a c e a r e a a v a i l a b l e f o r s e c r e t i o n t o o c c u r . There i s f u r t h e r c i r c u m s t a n -t i a l e v i d e n c e t h a t c a n be br o u g h t t o b e a r s u g g e s t i n g t h e w a t e r c e l l s e l a b o r a t e t h e e x c e s s f l u i d f r o m t h e m e a l . A r g a s i d t i c k s , w h i c h employ t h e c o x a l g l a n d f o r t h e l a t e r f u n c t i o n , have s a l i -v a r y g l a n d s composed o n l y o f g r a n u l e - c e l l s ( T i l l , 1 9 6 1 ; C h i n e r y 1 9 6 5 ; D z h a f a r o v , 1 9 6 5)1 i . e . , t h e r e a r e no c e l l s s p e c i a l i z e d f o r c o p i o u s f l u i d s e c r e t i o n . Second, t h e fundus c e l l s of t h e t y p e I I I a c i n u s d e s c r i b e d by T i l l and C h i n e r y c o r r e s p o n d t o -i n d e e d p r o b a b l y a r e homologous t o - t h e v a c u o l a r c e l l and the w a t e r c e l l i n Dermacentor; however i n male i x o d i d t i c k s (which a r e n o t b e l i e v e d t o t r a n s m i t p a r a l y s i s o r s a l i v a t e c o p i o u s l y ) , t h e f u ndus c e l l s of t h e a c i n u s do n o t i n c r e a s e i n s i z e s i g n i f -i c a n t l y d u r i n g f e e d i n g . The l a t t e r s u g g e s t s t h a t i n t h e female the e n l a r g e d fundus c e l l s a r e a consequence of a c t i v e f l u i d s e c r e t i o n . The s m a l l volume o f s a l i v a s e c r e t e d by males f u n c -t i o n s as a l u b r i c a n t e n s u r i n g a smooth t r a n s f e r o f t h e sperma-t o p h o r e f r o m t h e male g e n i t a l a p e r t u r e t o t h a t o f t h e female (Feldman-Muhsam e t a l . , 1 9 7 0 ) . 197 I f t h e w a t e r c e l l i s r e s p o n s i b l e f o r t h e b u l k o f w a t e r s e c r e t i o n by t h e s a l i v a r y g l a n d , i t i s i m p o r t a n t t o d e t e r m i n e a t w h i c h p o i n t s s e c r e t e d f l u i d e n t e r s t h e lumen. (1) The cap c e l l i n most l i g h t s e c t i o n s seems t o be c o m p l e t e l y i n t e r p o s e d between t h e apex of t h e w a t e r c e l l and the a c i n a r lumen ( F i g u r e 35). But i n some s e c t i o n s , t h e cap c e l l seems n o t t o c o v e r t h e w a t e r c e l l , o r e l s e a t l e a s t becomes e x t r e m e l y t h i n ( F i g u r e 32). U n f o r t u n a t e l y , I have not y e t seen an EM m i c r o g r a p h s e c t i o n e d a t t h e same l e v e l s as t h o s e shown i n F i g u r e 32. However, such l o c a l c o r r i d o r s r e p r e s e n t a p o t e n t i a l s i t e o f d r a i n a g e . (2) Under t h e e l e c t r o n microscope, t h e cap c e l l a p p e a r s c l o s e l y apposed t o t h e plasma membranes of b o t h th e v a c u o l a r c e l l s and the w a t e r c e l l s , b u t c l e a r l y t h e mem-br a n e s do n o t f o r m a t i g h t j u n c t i o n i n t h e r e g i o n where t h e y s e p a r a t e t h e i n t e r c e l l u l a r space f r o m t h e a c i n a r lumen ( F i g u r e 43» i n s e t ) . Perhaps I f t h e s e narrow channels r e m a i n open a r o u n d a c o n s i d e r a b l e a r e a o f c e l l s u r f a c e , i t would be r e a l -i s t i c t o propose t h a t t h e s e c r e t e d f l u i d can r e a c h t h e lumen v i a t h e s e c h a n n e l s . (3) A l t e r n a t i v e l y , a l t h o u g h t h e cap c e l l may e f f e c t t h e complete m o r p h o l o g i c a l i s o l a t i o n o f t h e w a t e r c e l l f r o m t h e lumen, t h e cap c e l l may be v e r y " l e a k y " i n terms of p e r m i t t i n g t h e passage o f f l u i d t h r o u g h i t s c y t o p t a s m t o the lumen. Some c o m b i n a t i o n of t h e above-mentioned t h r e e s u g g e s t i o n s p r o b a b l y a c c o u n t f o r t h e d r a i n i n g of s e c r e t e d f l u i d f r o m t h e w a t e r c e l l . The i s o - o s m o t i c t r a n s p o r t of f l u i d a c r o s s c e r t a i n e p i t h e -l i a c a n u s u a l l y be e x p l a i n e d i n terms o f t h e double-membrane 198 t h e o r y ( C u r r a n and M a c i n t o s h , 1962; C u r r a n , 1965) o r t h e s t a n d -i n g o s m o t i c g r a d i e n t h y p o t h e s i s (Diamond and Tormey, 1966; Diamond and B o s s e r t , 1967), t h e l a t t e r b e i n g a more f u l l y -d e v e l o p e d model of t h e f o r m e r . I n b r i e f , t h e t h e o r e t i c a l c o n -s i d e r a t i o n s of Diamond and B o s s e r t show how l o n g , dead-end chan-n e l s ( s u c h as i n t e r - and i n t r a c e l l u l a r s p a ces) c o u l d dev&lope a s t a n d i n g g r a d i e n t o f d e c r e a s i n g o s m o l a r i t y f r o m t h e b l i n d end t o the open end. The c h a n n e l i t s e l f p r o v i d e s an e n c l o s e d , non-s t i r r e d compartment t h a t h i n d e r s r a p i d d i f f u s i o n o f s o l u t e away f r o m t h e t r a n s p o r t s i t e . S o l u t e i s pumped a c t i v e l y i n t o t h e c h a n n e l making t h e f l u i d h y p e r o s m o t i c . As t h e s o l u t e d i f f u s e s down t h e c h a n n e l toward t h e open mouth, w a t e r e n t e r s t h e c h a n n e l f r o m t h e c e l l by o s m o s i s , r e s u l t i n g i n a h y d r o s t a t i c p r e s s u r e t h a t moves f l u i d a l o n g t h e c h a n n e l . S i n c e t h e two p r o c e s s e s (pumping o f s o l u t e and osmosis o f w a t e r ) a r e c o n t i n u o u s l y o c c u r -r i n g , a s t e a d y s t a t e i s e v e n t u a l l y r e a c h e d w i t h t h e f l u i d more h y p e r o s m o t i c i n t h e r e g i o n o f t h e s o l u t e pumps, and l e s s so as the f l u i d r e a c h e s t h e open end of t h e c h a n n e l . Depending on what p a r a m e t e r s a r e chosen f o r t h e model ( r a d i u s o f c h a n n e l , l e n g t h o f c h a n n e l , w a t e r p e r m e a b i l i t y o f t h e membranes, e t c . ) , the f l u i d l e a v i n g t h e c h a n n e l ranges f r o m h y p e r o s m o t i c t o l s o -o s m o t i c (but n e v e r h y p o - o s m o t i c ) ; t h e s e parameters a r e t h o r o u g h l y d i s c u s s e d by Diamond and B o s s e r t (I967K The model a l s o r e q u i r e s t h a t t h e c h a n n e l be c l o s e d a t the compartment f r o m w h i c h f l u i d i s a b s o r b e d and open a t the compartment t o w a r d w h i c h f l u i d i s t r a n s p o r t e d . The p o p u l a r i t y o f t h e s t a n d i n g - g r a d i e n t h y p o t h e s i s stems l a r g e l y f r o m demon-s t r a t i o n s t h a t t h e u l t r a s t r u c t u r e of many t r a n s p o r t i n g e p i t h e l i a 199 conforms w e l l t o t h e g e o m e t r i c a l r e q u i r e m e n t s o f t h e model, and t h a t i n some c a s e s , t h e degree of c h a n n e l d i s t e n s i o n c a n be c o r r e l a t e d w i t h t h e r a t e o f f l u i d t r a n s p o r t (Tormey and Diamond, I967). I n a d d i t i o n , i t has r e c e n t l y been shown i n t h e c o c k r o a c h by m i c r o p u n c t u r e , t h a t t h e f l u i d i n t h e i n t e r c e l l u l a r spaces of t h e r e c t a l pads i s c o n s i s t e n t l y more c o n c e n t r a t e d t h a n t h e f l u i d i n t h e r e c t a l lumen p r o p e r ; t h i s i s d i r e c t e x p e r i m e n t a l e v i d e n c e t h a t t h e model f o r l o c a l osmosis may have a p p l i c a b i l i t y i n b i o -l o g i c a l systems ( W a l l e t a l , , 1970). A l t h o u g h t h e s t a n d i n g - g r a d i e n t h y p o t h e s i s has been u s e f u l f o r e x p l a i n i n g w a t e r - t o - s o l u t e c o u p l i n g i n r e a b s o r p t i v e e p i t h e -l i a , some s e c r e t o r y e p i t h e l i a ( c h o r o i d p l e x u s , M a l p i g h i a n t u b u l e s , e lasmobranch r e c t a l gands) p o s s e s s "backwards" c h a n n e l s , i . e . , t h o s e w h i c h open i n t h e d i r e c t i o n f r o m w h i c h f l u i d i s b e i n g t r a n s p o r t e d (Diamond and B o s s e r t , I968). The l a t t e r p a p e r shows t h e o r e t i c a l l y how t h e s e backwards c h a n n e l s c a n a l s o s u p p o r t s t a n d i n g g r a d i e n t s , b u t w i t h t h e b l i n d end o f t h e c h a n n e l h y p o - o s m o t i c t o t h e open end. Most o f t h e e lements of t h i s model a r e s i m i l a r t o t h e o r i g i n a l one, a l t h o u g h c e r t a i n l i m i t a t i o n s n o t imposed on " f o r w a r d s " c h a n n e l s s h o u l d be c o n -s i d e r e d ; t h e s e a r e d i s c u s s e d by Diamond and B o s s e r t (I968). I t w ould appear t h a t w a t e r - t o - s o l u t e c o u p l i n g i n t h e w a t e r c e l l o f t h e t y p e I I I a c i n u s c o u l d a l s o be e x p l a i n e d i n terms o f the s t a n d i n g g r a d i e n t h y p o t h e s i s ( F i g u r e 26), s i n c e t h e c e l l p o s s e s s e s t o r t u o u s i n t e r c o n n e c t i n g c h a n n e l s o r i g i n a t i n g as b a s a l , l a t e r a l and a p i c a l l n f o l d i n g s . The m u l t i p l i c i t y o f m i t o c h o n d r i a a s s o c i a t e d w i t h t h e i n t r a c e l l u l a r c h a n n e l s i s a l s o 200 F i g u r e 26. A h i g h l y s c h e m a t i c r e p r e s e n t a t i o n of how t h e s t a n d -i n g - g r a d i e n t h y p o t h e s i s c o u l d e x p l a i n w a t e r - t o - s o l -u t e c o u p l i n g i n t h e w a t e r c e l l of the ty p e I I I a c i n u s . The b a s a l l a m i n a I s g e n e r a l l y assumed t o f i l t e r out o n l y l a r g e m o l e c u l e s such as p r o t e i n s ( c i r c l e s a t r i g h t of d i a g r a m ) , but n o t s m a l l e r s o l u t e s such as i o n s ( s t i p p l i n g ) . The d e n s i t y o f t h e s t i p p l i n g d e n o t e s t h e degree o f i o n c o n c e n t r a t i o n i n any r e g i o n . * The i o n pumps a r e b e l i e v e d t o e x i s t i n t h e membranes o f th e i n f o l d i n g s , and t h e d i r e c t i o n of pumping i s den o t e d by t h e s o l i d a r r o w s . The a c t i v e pumping o f s o l u t e out o f the b a s a l i n f o l d i n g s i n i t i a l l y c r e a t e s an a r e a o f low s o l u t e c o n c e n t r a t i o n i n the c h a n n e l , and h i g h c o n c e n t r a t i o n i n t h e c e l l . Water e n t e r s t h e c e l l down an o s m o t i c g r a d i e n t (open a r r o w s ) and hemolymph w a t e r moves down t h e c h a n n e l t o t a k e i t ' s p l a c e . When t h e s t e a d y s t a t e i s r e a c h e d , t h e r e s u l t i s a s t a n d i n g g r a d i e n t i n a "backwards" c h a n n e l (see t e x t ) . O t h e r i o n pumps i n i t i a l l y c r e a t e a h i g h s o l -u t e c o n c e n t r a t i o n i n t h e a p i c a l i n f o l d i n g s ( s o l i d a r r o w s ) and w a t e r l e a v e s t h e c e l l i n re s p o n s e t o t h i s g r a d i e n t (open a r r o w s ) . The r e s u l t i n g h y d r o s t a t i c p r e s s u r e i n t h e c h a n n e l f o r c e s f l u i d t o f l o w t o w a r d t h e lumen, and the s t e a d y s t a t e a g a i n r e f l e c t s a s t a n d i n g g r a d i e n t down a " f o r w a r d s " c h a n n e l . Thus f l u i d a p p e a r s t o f l o w between two e q u i - o s r a o l a r s o l u t i o n s , b u t I n r e a l i t y i t i s f l o w i n g a l o n g l o c a l o s m o t i c g r a d i e n t s w i t h i n the c e l l ( c f . D i a -mond and B o s s e r t , 1967, 1968? B e r r i d g e and Oschman, 1969)• *The p a t t e r n of s t i p p l i n g i s i n t e n d e d n o t t o r e p r e s e n t a c t u a l p r o f i l e s of i o n d e n s i t y , b u t m e r e l y t o i n d i c a t e s c h e m a t i c a l l y how the model i s b e l i e v e d t o work. b.l. 202 a f e a t u r e t h e c e l l has i n common w i t h many s e c r e t o r y and a b s o r p -t i v e e p i t h e l i a . T h i s arrangement o f m i t o c h o n d r i a b r i n g s the ene r g y s t o r e s o f t h e c e l l c l o s e t o t h e membrane i n f o l d i n g s and hence t o t h e h y p o t h e s i z e d i o n pumps t h a t i n i t i a t e t r a n s p o r t . Of c o u r s e s i n c e the a c i n u s t r a n s p o r t s f l u i d f r o m t h e hemolymph t o t h e lumen, and s i n c e t h e b a s a l i n f o l d i n g s o f t h e w a t e r c e l l open t o t h e b a s a l l a m i n a , t h e i n t r a c e l l u l a r s p a ces of t h e w a t e r c e l l c o r r e s p o n d t o backwards c h a n n e l s . I f t h e w a t e r c e l l i s i n d e e d r e s p o n s i b l e f o r f l u i d s e c r e -t i o n , t h e n i t s a p i c a l s u r f a c e s h o u l d make d i r e c t c o n t a c t w i t h t h e l u m i n a l f l u i d . However t o a l a r g e measure, t h e a p i c a l s u r -f a c e o f the w a t e r c e l l i s d i s t i n c t l y i s o l a t e d f r om t h e a c i n a r lumen by t h e cap c e l l . There appear t o be o n l y few c o r r i d o r s l e a d i n g d i r e c t l y t o t h e lumen. What t h e f u n c t i o n a l s i g n i f i c a n c e i s o f t h i s r e s t r i c t e d c o n t a c t by t h e a p i c a l membrane w i t h t h e a c i n a r lumen re m a i n s t o be s e e n . An i n t e r e s t i n g p a r a l l e l t o t h i s arrangement a p p e a r s i n t h e r e c t a l p a p i l l a e o f C a l l l p h o r a . The i n t e r c e l l u l a r c h a n n e l s o f t h e a p i c a l membrane i n t e r c o n n e c t t o f o r m a s i n u s ; t h e l a t t e r opens t o the hemolymph n e a r t h e apex o f t h e p a p i l l a a t a s i n g l e p o i n t (Gupta and B e r r i d g e , 1966). Thus t h e h y p o t h e s i z e d s e c r e t i o n of KCI i n t o t h e i n t e r c e l l u l a r s i n u s c o u l d e a s i l y d e v e l o p e the l o c a l g r a d i e n t n e c e s s a r y t o e x t r a c t w a t e r f r o m t h e r e c t a l lumen. As mentioned e a r l i e r , t h e s t a n d i n g g r a d i e n t h y p o t h e s i s w i l l n o t a l l o w t h e emergent f l u i d t o be h y p o - o s m o t i c , y e t t h e s a l i v a emerging from t h e t i c k s a l i v a r y g l a n d i s s t a t i s t i c a l l y s i g n i f i c a n t l y h y p o - o s m o t i c t o t h e b a t h i n g medium. I t s h o u l d 203 be emphasized t h e r e f o r e , t h a t t h e o s m o l a r i t y o f t h e s a l i v a e m erging f r o m t h e main e x c r e t o r y d u c t might n o t be s i m i l a r t o t h a t i n t h e a c i n a r lumen. The e l e g a n t m i c r o p u n c t u r e e x p e r i m e n t s of Martenez e t a l . (1966), Young and S c h o g e l (1966) and Mangos e t a l . (I966) d e m o n s t r a t e d t h a t t h e f l u i d i n t h e i n t e r c a l a t e d d u c t s j u s t p r o x i m a l t o t h e a c i n u s i t s e l f was e s s e n t i a l l y an u l t r a f i l t r a t e o f plasma. T h i s f l u i d becomes h y p o - o s m o t i c w h i l e p a s s i n g t h r o u g h t h e s t r i a t e d and e x c r e t o r y d u c t s . So t o a p p l y t h e s t a n d i n g g r a d i e n t h y p o t h e s i s t o any s e c r e t o r y e p i t h e l i u m , one must c o n s i d e r o n l y t h e c o m p o s i t i o n o f t h e p r i m a r y f l u i d and no t t h a t o f t h e emerging f l u i d . F o r example, t h e t u b u l a r s a l i v a r y g l a n d o f C a l l i p h o r a i s r e g i o n a l l y s p e c i a l i z e d i n t o s e c r e t o r y and r e a b s o r p t i v e r e g i o n s . When s e t up i n v i t r o and s t i m u l a t e d w i t h c y c l i c AMP, the . i n t a c t g l a n d e l a b o r a t e s a h y p o - o s m o t i c s a l i v a . However, when t h e s e c r e t o r y r e g i o n a l o n e i s p r e p a r e d f o r i n v i t r o s a l i v a t i o n , t h e s e c r e t e d f l u i d i s s l i g h t l y h y p e r o s m o t i c (Oschman and B e r r i d g e , 1970). We must a w a i t w i t h a n t i c i p a t i o n t h e s u c c e s s f u l m i c r o -p u n c t u r e o f t h e t y p e I I I a c i n u s f o r c o n f i r m a t i o n t h a t t h e p r i m a r y s e c r e t i o n i s s i m i l a r l y i s o - o s m o t i c o r h y p e r o s m o t i c t o the b a t h i n g medium. The f u n c t i o n of t h e cap c e l l s t i l l r emains l a r g e l y a m a t t e r f o r c o n j e c t u r e . I t i s h i g h l y u n l i k e l y f r om the u l t r a -s t r u c t u r e t h a t i t i s engaged i n a c t i v e s e c r e t i o n o r r e a b s o r p t i o n . The p r e s e n c e o f o r i e n t e d m i c r o t u b u l e s n e a r t h e r e s t r i c e d o p e ning between a d j a c e n t v a c u o l a r and cap c e l l s s u g g e s t s t h a t i t h e l p s t o h o l d the a c i n u s t o g e t h e r d u r i n g s e c r e t i o n when the l u m i n a l 204 h y d r o s t a t i c p r e s s u r e i s l i k e l y t o be c o n s i d e r a b l e . C l e a r l y t h e w a t e r c e l l i t s e l f becomes d i s t o r t e d d u r i n g f l u i d s e c r e t i o n . I n F i g u r e 30, t h e a p i c a l - b a s a l a x i s i s l o n g compared t o the l a t e r a l a x i s j t h i s i s c h a r a c t e r i s t i c o f a " d e f l a t e d " a c i n u s . But i n F i g u r e 33, t h e a b i l i t y f o r t h e b a s a l s u r f a c e o f t h e w a t e r c e l l t o c o n form i t s shape t o t h e expanded a c i n a r w a l l i s a p p a r e n t . The a p i c a l b o r d e r o f the w a t e r c e l l i n F i g u r e 33, i s d i s t o r t e d t o a much l e s s e r d e g r e e , presumably due t o t h e r e s t r a i n i n g i n f l u e n c e o f t h e cap c e l l . The two g r a n u l e - s e c r e t i n g c e l l s show no a p p r e c i a b l e h i s t o l o g i c a l changes t h r o u g h o u t the f e e d i n g p e r i o d ( T i l l , 1961; C h i n e r y , 1965). A c c o r d i n g t o t h e l a t t e r a u t h o r s , i t i s o n l y a f t e r t h e g o r g e d f e m a l e has l e f t t h e h o s t t h a t t h e g r a n u l e s a p p e a r t o be r e l e a s e d . I would p r o p o s e , from s e v e r a l l i n e s o f e v i d e n c e , t h a t one o r b o t h of t h e s e c e l l s s e c r e t e s t h e cement by w h i c h th e t i c k s e c u r e s i t s m o u t h p a r t s t o the h o s t s k i n . F i r s t , s i n c e cement i s o n l y s e c r e t e d f o r a b r i e f p e r i o d a t t h e commencement of f e e d i n g , t h i s w ould e x p l a i n t h e l a c k of g r a n u l e -c e l l a c t i v i t y d u r i n g most o f t h e f e e d i n g p e r i o d ; t h e r e l e a s e a t t h e end o f f e e d i n g i s p r o b a b l y a r e s u l t o f t h e g e n e r a l d i s s o l -u t i o n o f t h e s a l i v a r y t i s s u e t h a t i s known t o o c c u r a t t h a t t i m e . S e c o n d l y , Moorehouse (1969) r e p o r t e d t h a t t h e cement i t s e l f and t h e c e m e n t - s e c r e t i n g c e l l s s t a i n s t r o n g l y w i t h e o s i n . A l t h o u g h he showed no f i g u r e d e n o t i n g t h e p o s i t i o n of t h e s e c e l l s i n t h e a c i n u s , Douglas (19^3). T i l l (1961), and C h i n e r y (I965) a l l r e p o r t t h a t t h e s e c r e t o r y g r a n u l e s of t h e g r a n u l e c e l l s a r e i n t e n s e l y e o s i n o p h i l i c . 205 A p a r t from t h e appearance o f the g r a n u l e s t h e m s e l v e s , t h e u l t r a s t r u c t u r e o f t h e naked- and e n c a p s u l a t e d - g r a n u l e c e l l s i n Dermacentor i s v e r y s i m i l a r . The l a t t e r s u g g e s t s t h a t b o t h c e l l t y p e s may i n r e a l i t y be one, b u t a t d i f f e r e n t s t a g e s of s e c r e t i o n o r s y n t h e s i s . F o r example, i t may be t h a t t h e encap-s u l a t e d g r a n u l e s r e p r e s e n t the a t t a c h i n g cement, o r a p r e c u r s o r of t h i s s u b s t a n c e , and t h e naked g r a n u l e s a r e s i m p l y t h e c a p -s u l e s r e m a i n i n g a f t e r s e c r e t i o n of cement o r b e f o r e i t s s y n t h e s i s . I f s o , i t i s somewhat s u r p r i s i n g t h a t I have a l w a y s seen two e n c a p s u l a t e d g r a n u l e c e l l s and about f i v e n aked-g r a n u l e c e l l s p e r a c i n u s . One would have e x p e c t e d the r a t i o of e n c a p s u l a t e d - t o n a k e d - g r a n u l e c e l l s t o have been more v a r i a b l e f rom a c i n u s t o a c i n u s i f one was d e a l i n g s i m p l y w i t h a s i n g l e c e l l t y p e a t d i f f e r e n t s t a g e s o f a c t i v i t y . N e v e r t h e -l e s s , i t would be i n t e r e s t i n g t o see how t h e f r e q u e n t r e m o v a l of a t i c k f r om and r e p l a c e m e n t on the h o s t would a f f e c t t h e appearance o f t h e g r a n u l e c e l l s . The v a c u o l a r c e l l s a r e a l s o a c t i v e l y s e c r e t i n g as e v i d e n c e d by (1) t h e moderate a r r a y o f a p i c a l m i c r o v l l l a e j i n some c a s e s t h e l a t t e r i s a s s o c i a t e d w i t h s e c r e t o r y as opposed t o r e s o r p t i v e a c t i v i t y ( F a w c e t t , 1966), (2) t h e generous endowment o f dense m i t o c h o n d r i a , (3) t h e d i s t e n d e d p r o f i l e s of the g r a n u l a r ER, and (4) t h e v a c u o l e s t h e m s e l v e s w h i c h p r o b a b l y c o n t a i n t h e s e c r e t e d p r o d u c t . S i n c e g r a n u l a r ER i s a c h a r a c -t e r i s t i c f e a t u r e o f g l a n d u l a r c e l l s w h i c h s y n t h e s i z e a p r o t e i n -r i c h s e c r e t i o n ( F a w c e t t , 1966), i t i s r e a s o n a b l e t o sug g e s t t h a t t h e s e c r e t i o n o f t h i s c e l l may be p r o t e i n a c e o u s as w e l l . 2 0 6 However whether t h i s c e l l i s r e s p o n s i b l e f o r s e c r e t i n g the p a r a l y t i c t o x i n , an a n t i c o a g u l a n t , o r an enzyme, cannot be d e c i d e d f r o m t h e p r e s e n t d a t a . 4) The G r a n u l e - S e c r e t i n g A c i n u s (Type I I ) The c e l l s o f t h e t y p e I I a c i n u s a r e somewhat s i m i l a r t o t h o s e of t h e t y p e I I I a c i n u s and l i t t l e f u r t h e r comment need be made h e r e , e x c e p t t o re-emphasize t h a t t h e w a t e r c e l l i s p r o b a b l y l e s s a c t i v e i n t h e t y p e I I t h a n i n the t y p e I I I a c i n u s . Presumably however, i t s e c r e t e s s u f f i c i e n t f l u i d t o wash t h e s e c r e t o r y p r o d u c t s of t h e c e l l s i n t o t h e main stream of t h e d u c t s y s t e m . However, I would suggest t h a t i t s s e c r e -t i o n p r o b a b l y does n o t a c c o u n t f o r a major p r o p o r t i o n o f t h e s a l i v a volume. The v a c u o l a r c e l l p o s s e s s e s most of t h e t y p i c a l c h a r a c -t e r i s t i c s o f c e l l s w h i c h a r e engaged i n p r o t e i n s y n t h e s i s , namely an abundance of g r a n u l a r ER w i t h p r o f i l e s d i s t e n d e d and of c o u r s e the s e c r e t e d p r o d u c t i t s e l f I n v e s i c l e f o r m ( F a w c e t t , 1966). C o n s e q u e n t l y , much of t h e argument on t h e v a c u o l a r c e l l o f t h e t y p e I I I a c i n u s a p p l i e s e q u a l l y w e l l h e r e . I n c o n c l u s i o n , t h e r e have been enough independent s t u d i e s on t i c k s a l i v a r y g l a n d s i n t h e p a s t t o e n a b l e some s o r t o f c o m p i l a t i o n o f t h e v a r i o u s f i n d i n g s . A l t h o u g h t h e s i m i l a r i t i e s among i x o d i d s p e c i e s have f o r t u n a t e l y l e d t o l i t t l e c o n f u s i o n , p r e v i o u s a u t h o r s have been l a r g e l y u n a b l e t o r e l a t e t h e c y t o -l o g i c a l f i n d i n g s t o p h y s i o l o g i c a l p r o c e s s e s . I t has been p o s s i b l e t o a g r e a t e r degree i n t h i s s t u d y t o c o n s i d e r c e l l s t r u c t u r e f r o m a more f u n c t i o n a l v i e w p o i n t . Perhaps more 207 i n f o r m a t i o n on t h e f u n c t i o n o f each c e l l t y p e c o u l d be g a i n e d by comparing the u l t r a s t r u c t u r e of each c e l l t y p e a t d i f f e r e n t s t a g e s o f t h e l i f e c y c l e . I t i s n e v e r t h e l e s s a p p r o p r i a t e a t t h i s p o i n t t o c o r r e l a t e as f a r as p o s s i b l e t h e c e l l t y p e s r e c o g n i z e d w i t h t h o s e d e s c r i b e d by T i l l (1961) and C h i n e r y (1965) f o r o t h e r s p e c i e s ( T a b l e X X ) . One s h o u l d n o t however i n f e r f r om T a b l e XX t h a t e q u i v a l e n t c e l l t y p e s a r e n e c e s s a r i l y s y n t h e s i z i n g i d e n t i c a l s u b s t a n c e s i n t h e t h r e e s p e c i e s . TABLE XX CORRELATION OF THE CELL TYPES FOR THREE SPECIES OF IXODID TICKS S a l i v a r y g l a n d c e l l s i d e n t i f i e d i n Dermacentor  a n d e r s o n i ( t h i s s t u d y ) Type I acinus> F i b r i l l a r c e l l I n n e r c e l l Type I I a c i n u s > E n c a p s u l a t e d - g r a n u l e c e l l N a k e d - g r a n u l e c e l l V a c u o l a r c e l l Water c e l l Cap c e l l Type I I I a c i n u s 1 E n c a p s u l a t e d - g r a n u l e c e l l N a k e d - g r a n u l e c e l l V a c u o l a r c e l l Water c e l l C o r r e s p o n d i n g c e l l s d e s c r i b e d f o r R h l p l c e p h a l u s a p p e n d l c u l a t u s ( T i l l , 1961) and Hae m a p h y s a l l s  s p l n l g e r a ( C h i n e r y aemapny; , 1965) The s i n g l e c e l l t y p e i n t h e t y p e I a c i n u s Lumen o f t y p e I a c i n u s c o n t a i n i n g n u c l e i p r o t r u d i n g f r o m t h e s i n g l e c e l l t y p e The " a " c e l l i n t h e ty p e I I a c i n u s The "b" c e l l i n t h e t y p e I I a c i n u s Not r e c o g n i z e d P r o b a b l y " c " c e l l i n type I I I a c i n u s P r o b a b l y "d" c e l l i n t y p e I I I a c i n u s Do n o t r e c o g n i z e two c e l l t y p e s i n the fundus o f t h e a c i n u s . P r o b a b l y t h e "e" c e l l c o r r e s p o n d s t o the two c e l l s i n Dermacentor, though f r o m t h e d e s c r i p t i o n o f t h i s c e l l , i t app e a r s t o c o r r e s p o n d more c l o s e l y t o t h e v a c u o l a r c e l l , i n which c a s e , t h e w a t e r c e l l was n o t r e c o g n i z e d . Cap c e l l Not r e c o g n i z e d 208 5) Summary of C h a p t e r F o u r A) The morphology and h i s t o l o g y o f t h e s a l i v a r y g l a n d was d e s c r i b e d . The g l a n d c o n s i s t s o f t h r e e a c i n a r t y p e s ( I , I I , I I I ) and a d u c t system w h i c h c o n n e c t s each a c i n u s u l t i m a t e l y w i t h t h e o r a l c a v i t y . Nerves a r e a s s o c i a t e d w i t h t h e d u c t s , t h e t y p e I a c i n u s and the t y p e I I I a c i n u s , but t h e y were n o t n o t i c e d t o be i n c l o s e c o n n e c t i o n w i t h the t y p e I I a c i n u s . The t y p e I a c i n u s i s composed of two f i b r i l l a r c e l l s and one i n n e r c e l l . The t y p e I I I a c i n u s i s composed of two encap-s u l a t e d - g r a n u l e c e l l s and f i v e n a k e d - g r a n u l e c e l l s l o c a t e d a r o u n d t h e o r i g i n o f t h e e f f e r e n t d u c t , and a c h a r a c t e r i s t i c arrangement o f e i g h t o r n i n e v a c u o l a r c e l l s . w a t e r c e l l s and cap c e l l s i n t h e fundus o f t h e a c i n u s ? t h e t o t a l number of c e l l s composing the a c i n u s i s 30 t o 35• The t y p e I I a c i n u s a l s o c o n -t a i n s f i v e c e l l t y p e s w h i c h by.-and-large a r e e q u i v a l e n t t o t h o s e of t h e t y p e I I I a c i n u s , a l t h o u g h t h e i r c y t o l o g y i s d i s -t i n c t and t h e i r arrangement i n t h e a c i n u s i s n o t c l e a r . The e x i s t e n c e o f i n n e r v a t i o n and s e v e r a l o f the s a l i v a r y c e l l t y p e s a r e d e s c r i b e d a p p a r e n t l y f o r t h e f i r s t t i m e . B) On t h e b a s i s o f t h e u l t r a s t r u c t u r e , I proposed t h a t s a l i v a i s s e c r e t e d i n i t i a l l y by the w a t e r c e l l o f t h e t y p e I I I a c i n u s , and t h a t subsequent r e a b s o r p t i o n of s o l u t e i n e x c e s s of w a t e r o c c u r s I n t h e d u c t s e i t h e r by the d u c t c e l l s t h e m s e l v e s , o r c o n c e i v a b l y by t h e f i b r i l l a r c e l l s o f the t y p e I a c i n u s . The vacuolar c e l l s i n the t y p e I I and t y p e I I I a c i n i p r o b a b l y s e c r e t e p r o t e i n a c e o u s s u b s t a n c e s , and t h e g r a n u l e c e l l s o f t h e s e a c i n i a r e p r o b a b l y a s s o c i a t e d w i t h cement s e c r e t i o n . The cap c e l l i s l i k e l y r e s p o n s i b l e f o r m a i n t a i n i n g the i n t e g r i t y of the a c i n u s 209 d u r i n g s a l i v a t i o n , f o r a t t h i s t i m e , the h y d r o s t a t i c f l u i d p r e s s u r e w i t h i n the a c i n a r lumen i s p r o b a b l y c o n s i d e r a b l e . List of Abbreviations for Figures 27 to 46 aq aqueous c h a n n e l bb b r u s h b o r d e r b l b a s a l l a m i n a Cp cap c e l l c u c u t i c l e D d u c t c e l l E f e f f e r e n t d u c t EG e n c a p s u l a t e d g r a n u l e c e l l ER e n d o p l a s m i c r e t i c u l u m Fb f i b r i l l a r c e l l g r g r a n u l e hem hemolymph IC i n n e r c e l l Lu lumen m m i t o c h o n d r i o n mv m i c r o v i l l a e NG naked g r a n u l e c e l l NS n e u r o s e c r e t o r y axon nu n u c l e u s SD s e p t a t e desmosome t n t a e n i d i u m t r t r a c h e a vac v a c u o l e VC v a c u o l a r c e l l WC w a t e r c e l l 211 F i g u r e s 27 and 28. C r o s s s e c t i o n t h r o u g h t h e main e x c r e t o r y d u c t and l o n g i t u d i n a l s e c t i o n t h r o u g h some t y p e I a c i n i . Nerve b u n d l e s r u n a d j a c e n t t o t h e a c i n i ( F i g u r e 27) and appear t o i n n e r v a t e t h e a c i n i ( F i g u r e 2 8 ) . I n F i g u r e 28 ( l e f t hand a c i n u s ) t h e two n u c l e i o f t h e f i b r i l l a r c e l l s a r e seen a t t h e i n n e r b o r d e r o f t h e c e l l s . The l i g h t e s t r e g i o n o f t h e a c i n u s I s t h e i n n e r c e l l and i t s n u c l e u s i s a l s o v i s i b l e i n F i g u r e 28. M a g n i f i c a t i o n i F i g u r e 27, 2100 X F i g u r e 28, 1406 X 213 F i g u r e 29 A. A p o l a r s e c t i o n t h r o u g h a t y p e I I a c i n u s . N o t i c e t h a t one o f t h e v a c u o l a r c e l l s c o n t a i n s g r a n u l e s of s e c r e t e d p r o d u c t w h i c h s t a i n l i g h t e r t h a n t h o s e o f t h e naked-g r a n u l e c e l l . The w a t e r c e l l s f o r m a s troma a r o u n d t h e o t h e r c e l l s . The v a l v e i s seen i n o b l i q u e s e c t i o n t h r o u g h th e e f f e r e n t d u c t . A s e c o n d a r y d u c t I s a l s o seen I n c r o s s s e c t i o n a t t h e t o p . M a g n i f i c a t i o n : 857 X F i g u r e 29 B. A l i g h t l y s t a i n e d s e c t i o n o f t h e e n c a p s u l a t e d -g r a n u l e c e l l showing more c l e a r l y t h e s t r u c t u r e o f t h e g r a n u l e s . M a g n i f i c a t i o n : 2182 X 2 1 5 F i g u r e 3 0 . S e c t i o n t h r o u g h a " d e f l a t e d " t y p e I I I a c i n u s showing t h e f i v e c e l l - t y p e s . M a g n i f i c a t i o n 1 9 7 0 X F i g u r e 3 1• A s e c o n d a r y d u c t . C r o s s s e c t i o n t h r o u g h a s e c o n d a r y d u c t n e a r p o i n t o f e n t r y o f t y p e I I I a c i n u s showing t h e c u t i c l e , b a s a l l a m i n a and l a r g e v a c u o l e s i n t h e d u c t c e l l s . The v a l v e a t t h e base o f t h e e f f e r e n t d u c t i s c l e a r l y seen w i t h s e c r e t o r y p r o d u c t b e h i n d i t i n t h e a c i n a r lumen. M a g n i f i c a t i o n ! 2 2 3 1 X F i g u r e 3 2 . S e v e r a l s e c t i o n s i n w h i c h t h e w a t e r c e l l a p p e a r s t o make c o n t a c t w i t h t h e a c i n a r lumen (arrowheads) M a g n i f i c a t i o n * a) 1846 X b) 2080 X c) 1933 X d) 1742 X e) 1908 X f ) 1684 X g) 2 0 0 0 X 2 1 9 Figure 33* Section parallel to the equator of an "inflated" type III acinus. Notice the considerable extent of the basal portion of each water c e l l , and that the granule ce l l s are surrounded by water-cell extensions. Magnification i 903 X 221 Figure 3k, Innervation of the type III acinus. A nerve approaches the basal surface of a water c e l l in the type III acinus. The mitochondria of the water c e l l show up very clearly as black rods. The nucleus of a water c e l l i s seen in the subjacent acinus. Magnification t 2021 X Figure 35. A particularly lucid view of the fundus cells of a type III acinus in equatorial section. The brush border of the dark vacuolar ce l l s i s clearly v i s i b l e and extends over the apical surface of the white cap cel l s as well. Notice that the water cel l s cover the entire hemolymph surface of the acinus, but are cut off from the luminal sur-face by the cap c e l l s . The clear patches in the water cel l s (large black arrows) are expanded channels of the plasma membrane lnfoldings (as can be seen from Figures 4 3 , 4 4 ) . The intercellular channel (white arrow) can be seen between adjacent water and vacuolar c e l l s . Magnifications 91k X 223 F i g u r e 36. S e c t i o n a c r o s s t h e " n o r t h p o l e " r e g i o n of a t y p e I I I a c i n u s j u s t below th e r e g i o n o f the e n t r y of the e f f e r e n t d u c t . Two e n c a p s u l a t e d - g r a n u l e and f i v e n a k e d - g r a n u l e c e l l s s u r r o u n d t h e p o l a r a r e a and a r e c l e a r l y s e t o f f one from t h e o t h e r by e x t e n s i o n s o f t h e w a t e r c e l l s between each g r a n u l e c e l l . The w a t e r c e l l s a l s o b l o c k a c c e s s o f t h e g r a n u l e c e l l s t o t h e lumen ( c e n t r a l a r r o w ) . M a g n i f i c a t i o n t 933 X F i g u r e 37. The v a c u o l a r c e l l , t y p e I I I a c i n u s . S e c t i o n t h r o u g h a t y p e I I I a c i n u s s i m i l a r t o F i g u r e 33 b u t showing c l e a r l y the v a c u o l e s of t h e v a c u o l a r c e l l s ; i n t h i s c a s e , t h e v a c u o l e s a r e c o n c e n t r a t e d n e a r t h e b a s a l r e g i o n o f t h e c e l l . S t a i n e d w i t h t o l u i d i n e b l u e and photographed under phase c o n t r a s t o p t i c s . M a g n i f i c a t i o n 1 880 X 225 Figure 38. Cross section through the wall of the main excretory duct. Notice the thick cuticle (bordering the lumen) and thick basal lamina. Taenidial growths are apparent at the inner surface of the cuticle and numerous microvillae are also seen in that region. Mitochondria are f a i r l y common. Further character-i s t i c s are better seen in Figure 39* Magnification1 14,436 X 22? Figure 39. Cross section through the wall of a secondary-duct. Parallel-arrayed microtubules (black stars) run In groups from the taenidia to regions of the plasma membranes where the basal lamina lnvaginates (i. e . probably between the lateral plasma membranes). The tortuous course of the lat e r a l plasma membrane and septate desmosomes are seen at the right hand side of the micrograph. In the inset, a nerve can be seen embedded in the basal lamina of a secondary duct. The nerve appears in the region where the basal lamina lnvaginates between the lat e r a l plasma membranes of adjacent duct c e l l s . Magnification! 27,450 X InsetJ 28,400 X F i g u r e 40 A. S e c t i o n t h r o u g h t h e p y r a m i n a l a c i n u s showing th e f i b r i l l a r c e l l and the i n n e r c e l l . N o t i c e i n t h e f i b r i l l a r c e l l t h e d e n s e l y packed l e a f l e t s o f t h e b a s a l l n f o l d i n g s o f t h e plasma membrane, and t h e l o n g i -t u d i n a l l y a r r a n g e d m i t o c h o n d r i a s t a c k e d amongst them. The n u c l e u s i s seen a t t h e i n n e r b o r d e r of t h e f i b r i l l a r c e l l . M a g n i f i c a t i o n ! 7,362 X F i g u r e 40 B. N e u r o s e c r e t o r y axon i n t h e t y p e I a c i n u s . S e c t i o n t h r o u g h t h e a p i c a l r e g i o n o f t h e f i b r i l l a r c e l l show-i n g c r o s s - s e c t i o n s t h r o u g h two s t r u c t u r e s h i g h l y r e m i n i s c e n t of n e u r o s e c r e t o r y axons ( c f . M a d d r e l l , 1966? B r a d y and M a d d r e l l , 196?; J a r l a l and S c u d d e r , 1970). These axons s i t a t t h e a p i c e s o f t h e b a s a l l n f o l d i n g s o f t h e plasma membrane. The n u c l e i o f t h e two a d j a c e n t f i b r i l l a r c e l l s a r e seen a t t h e b o t t o m of t h e m i c r o g r a p h . The n e u r o s e c r e t o r y g r a n u l e s a p p r o a c h 750 A i n d i a m e t e r . M a g n i f i c a t i o n ! 18,808 X 231 Figure 41. Section through the Inner c e l l of the type I acinus. Notice the general lack of intracellular structure except for the few mitochondria and random microtubules. The plasma membrane i s not infolded, but does form many areas of close apposition with that of the f i b r i l l a r c e l l (arrows). Magnification i 13,331 X 233 Figure 42. Section through a naked-granule c e l l and an encapsulated-granule c e l l of a type II or I I I acinus from an unfed t i c k . The p a r a l l e l l a t e r a l membranes between these two c e l l s can be followed (begin at arrow). The naked granules are d i s t i n c t from the encapsulated granules, although the cytoplasm i n both c e l l s i s s i m i l a r , showing a dense network of granular ER. Occasionally the cisternae are r e g u l a r l y oriented around the granules themselves. In the lower region of the micrograph, the l i g h t - s t a i n i n g duct c e l l i n the region of the valve (cross-section) stands out c l e a r l y against the darker granule c e l l s . Microtubules of the duct c e l l s are seen i n the lower l e f t corner of the micrograph ( s t a r ) . Magnification t 23,9^7 X 235 F i g u r e 43. C r y p t r e g i o n o f t y p e I I I a c i n u s . S e c t i o n t h r o u g h t h e l u m i n a l s u r f a c e o f a " d e f l a t e d t y p e I I I a c i n u s showing p o r t i o n s o f a d j a c e n t w a t e r , cap, and v a c u o l a r c e l l s . I r r e g u l a r m i c r o v i l l a e appear on t h e l u m i n a l s u r f a c e o f t h e v a c u o l a r and cap c e l l s . When t h e a c i n u s i s d e f l a t e d , c r y p t s f o r m i n t h e lumen d e l i m i t e d by t h e l u m i n a l s u r f a c e s o f t h e cap and v a c u o l a r c e l l s j t h e c r y p t s a r e f i l l e d w i t h t h e a p i c a l m i c r o v i l l a e of t h e s e c e l l s . The prominent f e a t u r e s o f t h e v a c u o l a r c e l l a r e t h e d i s t e n d e d p r o f i l e s o f t h e g r a n u l a r ER c o n t a i n i n g a m a t r i x w h i c h i s r e l a t i v e l y e l e c -t r o n - d e n s e compared t o t h e c y t o p l a s m . N o t i c e t h a t t h e v a c u o l e s t h e m s e l v e s a r e much l i g h t e r t h a n t h e m a t r i x of t h e ER. The m i c r o t u b u l e s o f t h e cap c e l l a r e o r i e n t e d i n p a r a l l e l a r r a y i n t h e r e g i o n where t h e cap and v a c u o l a r c e l l s meet ( w h i t e a r r o w ) . A l t h o u g h t h e membranes o f t h e s e c e l l s a r e p a r a l l e l i n t h i s r e g i o n , a t i g h t J u n c t i o n i s n o t formed ( w h i t e a rrow i n i n s e t ) . I t i s c o n c e i v a b l e t h a t f l u i d s e c r e t e d by t h e w a t e r c e l l may d r a i n i n t o t h e a c i n a r lumen t h r o u g h t h i s narrow c h a n n e l (see t e x t ) . The i n s e t i s a s e c t i o n t h r o u g h t h e cap c e l l i n an i n f l a t e d a c i n u s , so no c r y p t i s v i s i b l e . T i g h t j u n c t i o n s between t h e cap and w a t e r c e l l s ( b l a c k a r r o w s i n i n s e t ) s u p p o r t t h e h y p o t h e s i s t h a t t h e cap c e l l ' s f u n c t i o n i s t o m a i n t a i n t h e i n t e g r i t y o f the a c i n u s i n t h e f a c e o f t h e h y d r o s t a t i c p r e s s u r e s g e n e r a t e d i n f l u i d s e c r e t i o n . M a g n i f i c a t i o n * 5 0 , 6 2 5 X I n s e t i 2 8 , 0 6 0 X F i g u r e 44. S e c t i o n t h r o u g h t h e b a s a l end o f t h e same w a t e r c e l l shown i n t h e p r e v i o u s f i g u r e . The i n t e r d i g i t a t i o n s between t h e w a t e r c e l l and t h e v a c u o l a r c e l l a r e e x t e n s i v e a t t h e b a s a l r e g i o n o f t h e l a t t e r ( b l a c k t r i a n g l e ) . C l o s e j u n c t i o n s a r e seen between the w a t e r c e l l and v a c u o l a r c e l l ( w h i t e a r r o w s ) . N o t i c e a l s o t h e a d h e s i o n s between t h e membranes of t h e w a t e r c e l l and cap c e l l ( b l a c k a r r o w s ) , and t h e dense p a r a l l e l c r i s t a e i n t h e m i t o c h o n d r i a o f t h e w a t e r c e l l . Passage o f f l u i d f r o m th e b a s a l t o a p i c a l s u r f a c e s o f t h e w a t e r c e l l i s b e l i e v e d t o o c c u r p r i m a r i l y t h r o u g h t h e aqueous c h a n n e l s . A t s e v e r a l p o i n t s , t h e c h a n n e l s open d i r e c t l y i n t o t h e space b e n e a t h t h e b a s a l l a m i n a ( b l a c k s t a r s ) . The i n s e t shows a s e c t i o n t h r o u g h th e n u c l e u s of a w a t e r c e l l . The n u c l e u s i s l a r g e l y e u c h r o m a t l c , b u t t h a t c h r o m a t i n w h i c h i s p r e s e n t i s p r e c i p i t a t e d around th e n u c l e a r membrane ( n o t shown v e r y w e l l i n t h i s i n s e t ) . M a g n i f i c a t i o n t 14,891 X I n s e t t 1,846 x Figure 4 5 . Section through a type II acinus showing a water c e l l and vacuolar cells devoid of granules. The inset shows part of a vacuolar c e l l which contains an abundance of granules. The cisternae of the granular ER are considerably distended (arrows) in the vacuolar c e l l of the type II acinus. Magnification1 2 6 , 3 ^ 1 X Inset* 9 , 5 0 0 X 2 4 l Figure 46. The cap c e l l , type II acinus. Section through the cap c e l l region of a type II acinus showing the arrangement of the vacuolar, cap and water c e l l s that i s s i m i l a r l y c h a r a c t e r i s t i c of the type III acinus. The vacuolar c e l l s i n t h i s section are packed with granules. Magnification: 15,306 X 243 CHAPTER FIVE Summary Remarks It had been suggested that the permeability increase to water of the c u t i c l e i n engorged ixodid t i c k s was s u f f i c i e n t to account f o r the evaporation of excess water (Lees, 1946a, 1947). There the matter rested u n t i l Gregson i n I967 suggested a h i t h e r t o unsuspected route f o r water excretion. Ticks were known to transmit various disease-causing agents i n the s a l i v a , and d i r e c t observations on Dermacentor suggested that the s a l i v a may be secreted i n copious amounts. T a t c h e l l (1967b) substan-t i a t e d Gregson»s suggestion by demonstrating i n Boophllus  mloroplus that a s i g n i f i c a n t proportion of water injected into the hemocoele of the t i c k eventually found i t s way into the ti s s u e s and excreta of the host, presumably v i a the s a l i v a r y glands. The goal of the second chapter i n t h i s thesis was to present quantitative estimates of the f l u i d passing through the s a l i v a r y gland i n Dermacentor andersoni. My findings sug-gested that 75% of the t o t a l water loss during the feeding period occurred v i a the s a l i v a r y glands, less than 5% was evaporated from the integument, and the remainder was probably excreted with the feces. Since the evidence at hand suggested that the s a l i v a r y glands played a major role i n the excretion of excess water, the next problem was to determine the general mechanism (1) by 244 w h l c h f l u i d e x c r e t i o n o c c u r r e d ( i . e . by f i l t r a t i o n o r a c t i v e s e c r e t i o n ) and (2) by w h i c h i t was c o n t r o l l e d ( i . e . hormonal o r n e u r a l ) . P r e l i m i n a r y e v i d e n c e ( p e r m e a b i l i t y c h a r a c t e r i s t i c s o f t h e s a l i v a r y g l a n d ) f a v o u r e d a s e c r e t o r y mechanism, and t h i s was c o n f i r m e d by d e m o n s t r a t i n g t h e s a l i v a r y g l a n d ' s a b i l i t y t o f u n c t i o n i n v i t r o i n t h e absence o f an a p p l i e d h y d r o s t a t i c p r e s s u r e g r a d i e n t a c r o s s t h e e p i t h e l i u m . S e c r e t i o n was u n l i k e l y t o be t r i g g e r e d by t h e r e l e a s e i n t o t h e hemolymph o f a hormone a n a l o g o u s t o t h e d i u r e t i c hormone d e s c r i b e d f o r Rhodnlus ( M a d d r e l l , 1962-66); r a t h e r , t h e c a t e c h o l a m i n e s a d r e n a l i n , n o r a d r e n a l i n , and dopamine were a l l a b l e t o t r i g g e r i n - v i t r o s e c r e t i o n w i t h a t h r e s h o l d o f no g r e a t e r t h a n 10"^ Mj a d r e n a l i n a l s o s t i m u l a t e d s a l i v a r y s e c r e t i o n i n v i v o . On t h e o t h e r hand, t h e c h o l i n o m i m e t i c d r u g p i l o c a r p i n e had no e f f e c t on t h e i s o -l a t e d g l a n d even though o t h e r s had shown i t s e f f i c a c y i n s t i m u -l a t i n g s a l i v a t i o n i n v i v o . On t h e b a s i s of t h e s e o b s e r v a t i o n s , i t seemed r e a s o n a b l e t o propose t h a t s a l i v a r y s e c r e t i o n was t r i g g e r e d d i r e c t l y by means of e f f e r e n t a d r e n e r g i c n e r v e f i b r e s , and t h a t p i l o c a r p i n e e x e r t e d i t s e f f e c t o n l y i n d i r e c t l y . The p r e s e n c e o f n e r v e axons c l o s e l y a s s o c i a t e d w i t h t h e v a r i o u s c e l l t y p e s o f t h e s a l i v a r y g l a n d was de m o n s t r a t e d m o r p h o l o g i c a l l y and u l t r a s t r u c t u r a l l y . F l u i d s e c r e t i o n a p p e a r s t o be c o u p l e d t o t h e n e t f l u x of N a C l . C h l o r i d e was d e m o n s t r a t e d t o be, and sodium s u s p e c t e d t o be, a c t i v e l y t r a n s p o r t e d . There was i n d i c a t i o n t h a t f l u i d t r a n s p o r t g a i n e d i t s energy f r o m a N a + f K + - a c t i v a t e d 'pump ATPase'. Water t r a n s p o r t was i n h i b i t e d by h i g h o s m o t i c p r e s s u r e and h i g h p o t a s s i u m c o n c e n t r a t i o n . From ultrastructural evidence I proposed that f l u i d secretion occurred primarily through the "water c e l l " of the type III acinus. This c e l l showed many of the characteristics found in other f l u i d secreting c e l l s . Also, the ultrastructure of the water c e l l was compatible with the local osmosis scheme for water-to-solute coupling proposed by Diamond and Bossert (I968). The latter model can only generate a hyperosmotic to iso-osmotic secretion, whereas the saliva secreted from the tick salivary gland was slightly (but consistently) hypo-osmotic to the hemolymph. This discrepancy could be explained by invoking some reabsorptlon of solute from the primary secre-tion as the latter passed from the acinar lumen to the exterior. Such reabsorptlon i s known to occur in the salivary ducts of mammalian salivary glands, but the site(s) of reabsorptlon was not determined for the tick salivary gland. Tentatively, I suggested that this reabsorptlon occurs either via the salivary ducts as in mammals, or conceivably via the type I acinus. The mechanism of f l u i d secretion in ixodid ticks is thus different from that in argasld ticks. In the argasid Ornltho-doros moubata, excess ions and water are excreted via two coxal glands which open to the exterior between the f i r s t and second coxae. The coxal gland functions by f i l t r a t i o n followed by reabsorptlon, in a manner analogous to that visualized for the vertebrate glomerular nephron and the crustacean antennal gland (Kaufman, 1971). 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