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Ion and water regulation during feeding in the female tick Ornithodorus moubata Kaufman, Susan Elaine 1971

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i ION AND WATER REGULATION DURING FEEDING IN THE FEMALE TICK ORNITHODORUS HOUBATA SUSAN E. KAUFMAN B.Sc., U n i v e r s i t y C o l l e g e , London, 1965 H.Sc, M c G i l l U n i v e r s i t y , M o n t r e a l , 1967 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n the Department of ZOOLOGY We accept t h i s t h e s i s as conforming t o the r e q u i r e d standard The U n i v e r s i t y of B r i t i s h Columbia October 1971 In p r e s e n t i n g t h i s t h e s i s i n 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 a t the U n i v e r s i t y of B r i t i s h Columbia, I agree t h a t 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 r e f e r e n c e and study. I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g 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 g r a n t e d 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 . I t i s understood t h a t c o p y i n g or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be allowed without my w r i t t e n p e r m i s s i o n . Department o f The U n i v e r s i t y o f B r i t i s h Columbia Vancouver 8, Canada Date OcJj- 2 6 i i ABSTRACT Females of the s o f t t i c k Ornithodorus moubata were f e d a r t i f i c i a l l y on blood meals of v a r y i n g i o n i c and osmotic c o m p o s i t i o n s . C h l o r i d e and sodium were a c t i v e l y t r a n s p o r t e d from gut to hemolymph. Potassium i o n s may have been a c t i v e l y t r a n s p o r t e d from hemolymph to gut or p a s s i v e l y d i s t r i b u t e d across a gut e p i t h e l i u m of low p e r m e a b i l i t y . Water movement acro s s the gut was dependent on the t o t a l sodium and c h l o r i d e t r a n s p o r t e d and on the osmotic p r e s s u r e d i f f e r e n c e a c r o s s the gut w a l l . Constant sodium c h l o r i d e c o n c e n t r a t i o n s and osmotic p r e s s u r e s were maintained i n the hemolymph when t i c k s were f e d meals o f d i f f e r i n g compositions p r o v i d e d these meals were i s o s m o t i c w i t h normal b l o o d . Otherwise the osmotic p r e s s u r e of the hemolymph p a r a l l e l e d t h a t of the meal. The f o l l o w i n g evidence l e d me t o conclude t h a t the c o x a l f l u i d was produced by u l t r a f i l t r a t i o n . The c o x a l gland never produced f l u i d which was h y p e r t o n i c t o the hemolymph. I n u l i n showed a c o x a l f l u i d : hemolymph r a t i o o f u n i t y over a wide range of i n u l i n c o n c e n t r a t i o n s i n the hemolymph. H y d r o s t a t i c pressure i n the hemolymph i n -creased d u r i n g c o x a l f l u i d p r o d u c t i o n and the r a t e of i i i p r o d u c t i o n was pressure s e n s i t i v e . The s i t e of u l t r a -f i l t r a t i o n was demonstrated v/hen f l u o r e s c e i n - l a b e l l e d albumin was trapped i n the t h i n membraneous s t r u c t u r e e n v e l o p i n g the t u b u l a r p a r t of the gland. The u l t r a -s t r u c t u r e of t h i s membrane was v e r y s i m i l a r t o t h a t of o t h e r t i s s u e s engaged i n f i l t r a t i o n . Sodium was a c t i v e l y reabsorbed and c h l o r i d e moved p a s s i v e l y down an e l e c t r o p o t e n t i a l g r a d i e n t across the r e s o r p t i o n t u b u l e o f the c o x a l g l a n d . Potassium was a c t i v e l y t r a n s p o r t e d from hemolymph to r e s o r p t i o n c e l l s and p a s s i v e l y d i f f u s e d i n t o the c o x a l f l u i d . There was some r e s o r p t i v e c a p a c i t y f o r amino a c i d s but they were not r e g u l a t e d to the same degree as i n o r g a n i c i o n s . The u l t r a s t r u c t u r e of the r e s o r p t i o n c e l l s was t y p i c a l o f t h a t found i n c e l l s engaged i n i o n and water t r a n s p o r t . i v TABLE OF CONTENTS CHAPTER PAGE I GENERAL INTRODUCTION 1 I I ANATOMY, GENERAL METHODS AND GENERAL OBSERVATIONS 13 A) ANATOMY 14 1. General anatomy 14 2. Techniques f o r studying anatomy of coxal gland 16 3. -Anatomy of coxal gland 17 4. Discussion 20 B) GENERAL METHODS AND OBSERVATIONS 20 1 . Rearing and feeding procedure 20 2. Time sequence of feeding and excretion 23 I I I OSMOTIC AND IONIC REGULATION 24 A) INTRODUCTION 25 B) METHODS 27 1 . Preparation of blood meal 27 2. C o l l e c t i o n of body f l u i d s and rate measurements 28 3. Chemical measurements 29 4. Volume measurements 30 3. Measurement of osmotic pressure 32 6. Measurement of e l e c t r o p o t e n t i a l difference across the gut 32 C) RESULTS 34 V CHAPTER 1 PAGE 1. Change i n d i s t r i b u t i o n of body-f l u i d s d u r i n g f e e d i n g and c o x a l f l u i d p r o d u c t i o n 34 2. D i s t r i b u t i o n of i o n s i n body f l u i d s 4-0 3. Balance sheet f o r t o t a l body c h l o r i d e 58 4. Time sequence of i o n c o n c e n t r a t i o n s i n body f l u i d s d u r i n g c o x a l f l u i d p r o d u c t i o n 59 5. Rate of c o x a l f l u i d p r o d u c t i o n 66 6. Osmotic p r e s s u r e s of body f l u i d s 6? 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 across the gut 77 D) DISCUSSION 78 IV MECHANISM OF COXAL FLUID FORMATION 100 A) ' INTRODUCTION 101 B) METHODS 104 14 1. Clearance of i n u l i n - c a r b o x y l - C 104 14 2. Reabsorption of i n u l i n - c a r b o x y l - C 104 3. H i s t o l o g y of the c o x a l gland 105 4. U l t r a s t r u c t u r e of the c o x a l gland 106 5. S i t e of f i l t r a t i o n 106 6. Pr e s s u r e dependence of c o x a l f l u i d f o r m a t i o n 107 C) RESULTS 110 14 1. Re a b s o r p t i o n of i n u l i n - c a r b o x y l - C 110 14 2. Clearance of i n u l i n - c a r b o x y l - C 111 3. H i s t o l o g y and u l t r a s t r u c t u r e of the c o x a l gland 113 4. S i t e of f i l t r a t i o n 113 v i CHAPTER PAGE 5. P r e s s u r e dependence of c o x a l f l u i d f o r m a t i o n 117 D) DISCUSSION 122 V REABSORPTION IN THE COXAL GLAND 127 . A) INTRODUCTION 128 B) METHODS 131 1. H i s t o l o g y and u l t r a s t r u c t u r e of the r e s o r p t i o n t u b u l e 131 2. A n a l y s i s of amino a c i d s i n body f l u i d s 131 14 3. Clearance of p r o l i n e - C and a s p a r t i c - a c i d - C l ^ - 132 4. 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 s a c r o s s the c o x a l gland 132 5- I n t r a c e l l u l a r potassium c o n c e n t r a t i o n of the c o x a l gland 134 6. pH of body f l u i d s 134 C) RESULTS 136 1. H i s t o l o g y and u l t r a s t r u c t u r e of the r e s o r p t i o n t u b u l e 136 2. A n a l y s i s of amino a c i d s i n body f l u i d s 140 14 3. Clearance of p r o l i n e - C and a s p a r t i c - a c i d - C l 4 140 4. 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 s a c r o s s the c o x a l gland 143 5- I n t r a c e l l u l a r potassium c o n c e n t r a t i o n of the c o x a l gland 147 6. pH of body f l u i d s 147 D) DISCUSSION 148 v i i CHAPTER PAGE VI SUMMARY AND FINAL DISCUSSION 158 REFERENCES CITED 167 LIST OP TABLES TABLE PAGE I I I I I I IV V VI VII VIII IX X XI XII XIII XIV Volumes of body f l u i d s during and a f t e r feeding 37 Concentration f a c t o r of gut contents two hours a f t e r feeding, by three independent methods 38 Chloride concentrations i n body f l u i d s during and a f t e r feeding 4-3 C a l c u l a t i o n of c h l o r i d e concentration of gut absorbate 44 Sodium concentrations i n body f l u i d s during and a f t e r feeding 4-5 C a l c u l a t i o n of sodium concentration of gut absorbate 4-6 Potassium concentrations i n body f l u i d s during and a f t e r feeding 4-7 C a l c u l a t i o n of potassium concentration of gut absorbate 4-7 Increase i n t o t a l body c h l o r i d e (Mean - SE) 59 Chloride concentration i n hemolymph when coxal f l u i d f i r s t appeared and a f t e r coxal f l u i d production had ceased (Mean ± SE) 63 Ion concentrations i n i n i t i a l drops of coxal f l u i d and i n samples pooled over secretory p e r i o d (Mean ± SE; 64 Chloride concentration i n whole gut f l u i d when coxal f l u i d f i r s t appeared and a f t e r coxal f l u i d production had ceased (Mean ± SE) 65 Osmotic pressures of gut f l u i d and hemo-lymph two hours a f t e r feeding (Mean ± SE) 7O Amino a c i d a n a l y s i s of f i n a l hemolymph and pooled coxal f l u i d (Mean ± SE) 141 i x LIST OF FIGURES FIGURE PAGE 1 Morphology o f the c o x a l gland 18 2 Arrangement f o r f e e d i n g t i c k on human blood 22 3 R e l a t i o n between volume o f gut absorbate and volume of c o x a l f l u i d 39 4 R e l a t i o n between volume absorbed a c r o s s gut w a l l (as percent of i n g e s t e d f l u i d ) and t o t a l (Na + C l ) i o n c o n c e n t r a t i o n of b l o o d meal 40 5 R e g u l a t i o n of c h l o r i d e c o n c e n t r a t i o n i n hemolymph 50 6 R e g u l a t i o n o f sodium c o n c e n t r a t i o n i n hemolymph 51 7 R e l a t i o n between mean c h l o r i d e con-c e n t r a t i o n s o f gut absorbate and c o x a l f l u i d 53 8 R e l a t i o n between mean sodium concen-t r a t i o n s o f gut absorbate and c o x a l f l u i d 53 9 R e l a t i o n between t o t a l c h l o r i d e ab-sorbed across gut w a l l and t o t a l c h l o r i d e e x c r e t e d i n c o x a l f l u i d p l u s i n c r e a s e i n hemolymph c h l o r i d e d u r i n g two hour experimental p e r i o d 55 10 R e l a t i o n between t o t a l sodium absorbed acro s s gut w a l l and t o t a l sodium e x c r e t e d i n c o x a l f l u i d p l u s i n c r e a s e i n hemo-lymph sodium d u r i n g two hour e x p e r i -mental p e r i o d 56 11 R e l a t i o n between t o t a l c h l o r i d e and t o t a l sodium absorbed across gut v/all d u r i n g two hour experimental p e r i o d 57 12 T y p i c a l time sequence study of c h l o r i d e c o n c e n t r a t i o n i n c o x a l f l u i d from a t i c k f e d human blo o d 60 X FIGURE PAGE 13 Some t y p i c a l time sequences of c h l o r i d e c o n c e n t r a t i o n s i n the c o x a l f l u i d and hemolymph from t h r e e t i c k s f e d human bl o o d 61 14- Rate and accumulative volume of c o x a l f l u i d p r o d u c t i o n 67 15 Rate and accumulative volume of c o x a l f l u i d p r o d u c t i o n 68 16 R e l a t i o n between osmotic p r e s s u r e s of i n g e s t e d f l u i d and hemolymph 71 17 R e l a t i o n between osmotic p r e s s u r e s o f gut f l u i d and hemolymph and r a t e o f c o x a l f l u i d p r o d u c t i o n ( t i c k s f e d h.b.) 74-18 R e l a t i o n between osmotic p r e s s u r e s o f gut f l u i d and hemolymph and r a t e of c o x a l . . f l u i d p r o d u c t i o n ( t i c k s f e d h.b. + NaCl) 75 19 R e l a t i o n between osmotic d i f f e r e n c e across gut w a l l and r a t e o f f l o w o f gut absorbate 76 20 R e l a t i o n between mean r a t e of c h l o r i d e a b s o r p t i o n a c r o s s gut w a l l and median c h l o r i d e c o n c e n t r a t i o n i n gut f l u i d 81 21 R e l a t i o n between mean r a t e o f sodium a b s o r p t i o n across gut w a l l and median sodium c o n c e n t r a t i o n i n gut f l u i d 82 22 R e l a t i o n between t o t a l c h l o r i d e and t o t a l f l u i d absorbed acr o s s gut w a l l d u r i n g two hour experimental p e r i o d 86 23 R e l a t i o n between median c h l o r i d e con-c e n t r a t i o n i n gut f l u i d and mean r a t e o f f l u i d a b s o r p t i o n a c r o s s gut w a l l d u r i n g two hour experimental p e r i o d 87 24- R e l a t i o n between median sodium concen-t r a t i o n i n gut f l u i d and mean r a t e o f f l u i d a b s o r p t i o n across gut w a l l d u r i n g two hour experimental p e r i o d 88 25 R e g u l a t i o n of c h l o r i d e c o n c e n t r a t i o n i n hemolymph 93 FIGURE x i PAGE 26 R e l a t i o n between mean r a t e o f pr o -d u c t i o n of c o x a l f l u i d and d i f f e r e n c e i n i o n c o n c e n t r a t i o n between c o x a l f l u i d and hemolymph 97 u 27 C o n c e n t r a t i o n s of i n u l i n carboxyl-C i n hemolymph and c o x a l f l u i d d u r i n g f e e d i n g 112 28a F i l t r a t i o n membrane of c o x a l gland 114 28b F i l t r a t i o n membrane showing l a r g e n ucleus 114 29 General appearance of f i l t r a t i o n membrane 115 50 D e t a i l o f f i l t r a t i o n membrane showing nature of c e l l processes 116 31a Coxal gland showing d i s t r i b u t i o n of albumin - f l u o r e s c e i n conjugate 118 31b F i l t r a t i o n membrane showing albumin-f l u o r e s c e i n conjugate trapped i n pockets 118 32 H y d r o s t a t i c p r e s s u r e i n hemolymph d u r i n g f e e d i n g and c o x a l f l u i d p r o d u c t i o n 119 33 E f f e c t of hemorrhaging on r a t e of co x a l f l u i d p r o d u c t i o n i n f o u r t i c k s 121 34 General appearance of r e s o r p t i o n t u b u l e 137 35 P a r t o f r e s o r p t i o n t u b u l e b e f o r e f e e d i n g 138 36 P a r t of r e s o r p t i o n t u b u l e d u r i n g c o x a l f l u i d p r o d u c t i o n 139 37 R e l a t i o n between amino a c i d concen-t r a t i o n i n b l o o d meal and r a t i o of c o n c e n t r a t i o n s i n co x a l f l u i d : hemolymph 142 14 38 C o n c e n t r a t i o n s of p r o l i n e - C i n hemo-lymph and c o x a l f l u i d d u r i n g f e e d i n g 144 C o n c e n t r a t i o n s of a s p a r t i c acid-C i n hemolymph and c o x a l f l u i d d u r i n g f e e d i n g Model f o r l o c a t i o n of t r a n s p o r t mechanisms i n c o x a l gland d e r i v e d from c o n d i t i o n s e x i s t i n g i n unfed^ t i c k x i i l ACKNOWLEDGEMENTS I would l i k e to thank Dr. J. E. P h i l l i p s f o r h i s supervision of t h i s research and assistance i n the preparation of t h i s t h e s i s . I acknowledge with appreciation Miss Joan Meredith f o r preparing the el e c t r o n micrographs, Dr. A. Perks f o r running the amino acid analyses and Mr. L. Sharman f o r preparing the l i g h t micrographs. My thanks are due also to my husband B i l l f o r h i s encouragement and advice and to my son Oren f o r sleeping through the nigh t s . CHAPTER I GENERAL INTRODUCTION -2-T i c k s share w i t h o t h e r b l o o d s u c k i n g arthropods the p e c u l i a r problem of coping w i t h an excess of water d u r i n g and immediately a f t e r f e e d i n g . At o t h e r times, however, they face the t a s k c h a r a c t e r i s t i c of t e r r e s t r i a l organisms o f c o n s e r v i n g water. These problems are some-what d i f f e r e n t f o r the t h r e e f a m i l i e s of t i c k s namely the Argasidae or s o f t t i c k s , the l x o d i d a e or hard t i c k s and the N u t t a l l i e l l i d a e r e presented by a s i n g l e s p e c i e s . Hard t i c k s d i f f e r from s o f t t i c k s not o n l y i n t h e i r appear-ance but a l s o i n f e e d i n g h a b i t s , the former f e e d i n g over a p e r i o d o f s e v e r a l days and the l a t t e r engorging w i t h i n one hour. The t a s k of e l i m i n a t i n g water r a p i d l y i s o b v i o u s l y o f g r e a t e r importance to the s o f t t i c k s and, as s h a l l be seen, t h i s i s r e f l e c t e d i n t h e i r anatomy and p h y s i o l o g y . O r n i t h o d o r u s moubata Murray i s a s o f t t i c k d i s t r i b u t e d w i d e l y i n A f r i c a . T h i s t h e s i s i s concerned w i t h water and i o n r e g u l a t i o n d u r i n g the f e e d i n g c y c l e . I hoped to e l u c i -date the mechanisms by which the r e g u l a t o r y organs operated and t h e i r c a p a c i t y under c o n d i t i o n s of i o n i c and osmotic s t r e s s . I n crustaceans u r i n e i s formed from the hemolymph by f i l t r a t i o n through a membrane f o l l o w e d by s e l e c t i v e r e a b s o r p t i o n of e s s e n t i a l molecules. By c o n t r a s t , a s e c r e t -o r y mechanism i s found i n the M a l p i g h i a n t u b u l e s of i n s e c t s . Work on these animals has been reviewed by K i r s c h n e r (1967) - 3 -However, v e r y l i t t l e study has been made of the a r a c h n i d s . I t i s thus of p a r t i c u l a r i n t e r e s t from a p h y l o g e n e t i c v i e w p o i n t t o i n v e s t i g a t e the processes o c c u r r i n g i n the c o x a l gland of a t i c k . A m o r p h o l o g i c a l study of a r a c h n i d s , e s p e c i a l l y s p i d e r s and s c o r p i o n s , suggested t h a t the c o x a l gland i s concerned w i t h e x c r e t i o n (Buxton, 1917). The ar a c h n i d c o x a l gland u s u a l l y possesses t h r e e component p a r t s ; a t e r m i n a l sac o r s a c c u l e , the w a l l s of which c o n s i s t of v e r y d e l i c a t e c u b i c a l or f l a t t e n e d e p i t h e l i u m , opens t o a c o l l e c t i n g t u b u l e . T h i s i n t u r n l e a d s t o the l a b y r i n t h i n some cases v i a a sac l i n e d w i t h s e c r e t o r y e p i t h e l i u m . The l a b y r i n t h u s u a l l y c o n s i s t s of a s i n g l e c o i l e d tube whose w a l l s are l i n e d w i t h " ... e x c r e t o r y e p i t h e l i u m having the u s u a l s t r i a t e d base i n d i c a t i v e o f e x c r e t o r y f u n c t i o n s . " (Buxton, 1917). S o l i d p a r t i c l e s such as carmine were shown to pass from the hemolymph across the t e r m i n a l sac but never ac r o s s the w a l l s of the l a b y r i n t h (Buxton, 1917). C h r i s t o p h e r s ( 1 9 0 6 ) f i r s t d e s c r i b e d the c o x a l gland i n the s o f t t i c k Ornithodorus s a v i g n y i ; t h i s gland opened v i a a s m a l l pore between the f i r s t and second coxae and ex-c r e t e d a copious c l e a r f l u i d a f t e r f e e d i n g . The c o x a l g l a n d was l a t e r d e s c r i b e d by Robinson and Davidson ( 1913) as having an a l v e o l a r s t r u c t u r e not u n l i k e t h a t of a mam-mal i a n s a l i v a r y g l a n d . These w i d e l y d i f f e r i n g concepts o f c o x a l gland s t r u c t u r e and f u n c t i o n remained unchallenged u n t i l Bone (194-3) made an i n t e n s i v e study of water and i o n r e g u l a t i o n i n Ornithodorus moubata. Bone showed t h a t the c o x a l gland c o n s i s t e d of a tube c o i l e d s e v e r a l times upon i t s e l f . Near the c o x a l o r i f i c e a s m a l l accessory gland j o i n e d the c o x a l gland. Presumably i t was t h i s accessory gland which Robinson and Davidson (1913) had mistaken f o r the c o x a l gland. Bone o b t a i n e d some p r e l i m i n a r y evidence t h a t the t i c k was capable o f r e g u l a t i n g the osmotic pressure of the hemo-lymph even when i n g e s t i n g f i v e t o seven times i t s own weight of mammalian b l o o d . When the c o m p o s i t i o n of the meal was v a r i e d Bone showed t h a t c h l o r i d e r e g u l a t i o n was p o s s i b l e p r o v i d e d osmotic p r e s s u r e o f the meal v/as not a l t e r e d . The t i c k never produced c o x a l f l u i d h a v i n g a h i g h e r c h l o r i d e c o n c e n t r a t i o n than t h a t of the hemolymph. Measurements were made three hours a f t e r f e e d i n g at which time the observed c h l o r i d e c o n c e n t r a t i o n (the o n l y i o n s t u d i e d ) was lower i n the gut than i n the hemolymph. At t h i s time a l s o t h e r e was l i t t l e d i f f e r e n c e i n osmotic p r e s s u r e o f hemolymph and gut f l u i d . A v e r y low t i t r e of n i t r o g e n o u s m a t e r i a l i n the c o x a l f l u i d i n d i c a t e d t h a t the c o x a l gland d i d not p l a y a p a r t i n the e x c r e t i o n of n i t r o g e n o u s waste. I n summary, Bone regarded the c o x a l gland as the predominant organ of i o n i c and osmotic r e g u l -a t i o n d u r i n g and immediately a f t e r f e e d i n g . I t was thus analagous f u n c t i o n a l l y t o the 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 and the antennal gland of c r u s t a c e a n s . - 5 -Lees (1946) i n an independent study v e r i f i e d most of Bone's r e s u l t s . He showed i n a d d i t i o n t h a t the M a l p i g h i a n t u b u l e s o f the t i c k d i d not become a c t i v e u n t i l about one hour a f t e r f e e d i n g . They produced a s l u r r y of guanine h a v i n g a c h l o r i d e c o n c e n t r a t i o n too low t o be measured. The q u a n t i t y of water thus e x c r e t e d was v e r y s m a l l and o n l y s u f f i c i e n t t o f l u s h out the t u b u l e s . C l e a r l y the M a l p h i g h i a n t u b u l e s p l a y no p a r t i n the ex-c r e t i o n of c h l o r i d e or water d u r i n g and immediately a f t e r f e e d i n g . Since these p r e l i m i n a r y s t u d i e s , t h e r e has been no f u r t h e r examination of osmoregulation and c o x a l gland f u n c t i o n i n the s o f t t i c k s . These authors had not con-s i d e r e d i o n i c and osmotic changes d u r i n g the f e e d i n g c y c l e . Thus I decided t o c a r r y out a more e x t e n s i v e i n v e s t i g a t i o n w i t h r e g a r d t o the number of parameters measured and the v a r i e t y of experimental c o n d i t i o n s s t u d i e d . A major p o i n t of disagreement arose between Bone and Lees as t o the s t r u c t u r e of the c o x a l gland. Whereas Bone r e p o r t e d i t t o be a simple c o i l e d t u b u l e ^ Lees des-c r i b e d a membraneous s t r u c t u r e communicating w i t h the tubule at a s i n g l e point.. He a s c r i b e d t o the former, the f u n c t i o n o f a f i l t r a t i o n membrane whose lumen co u l d be h e l d open by numerous small muscles. T h i s would permit the c r e a t i o n of a n e g a t i v e i n t e r n a l pressure w i t h i n the lumen thus pro--6-v i d i n g a d r i v i n g f o r c e f o r f i l t r a t i o n . The evidence presented f o r f i l t r a t i o n was t h a t ( i ) substances i n j e c t e d i n t o the hemolymph appeared r a p i d l y i n the c o x a l f l u i d and ( i i ) hemoglobin, a f a i r l y l a r g e molecule, c o u l d be e l i m -i n a t e d v i a the c o x a l g l a n d . However, no q u a n t i t a t i v e estimate o f c o n c e n t r a t i o n s i n c o x a l f l u i d and hemolymph was made. The h i g h r a t e of c o x a l f l u i d p r o d u c t i o n and the anatomical o b s e r v a t i o n by l i g h t microscopy of a v e r y t h i n membraneous s t r u c t u r e which might serve as a f i l t r a t i o n membrane completed Lees' evidence f o r f i l t r a t i o n . He p r o -posed t h a t f i l t r a t e passed from the f i l t r a t i o n chamber i n t o the t u b u l e where e s s e n t i a l .molecules were s e l e c t i v e l y r e -absorbed. T h i s t u b u l e was d e s c r i b e d , not as a simple un-branched system, but as be i n g i n t e r c o n n e c t e d so t h a t f l u i d might circumvent p a r t of the t o r t u o u s route through the t u b u l e . Despite these seemingly c o n c l u s i v e r e p o r t s of the nature and f u n c t i o n of the c o x a l g l a n d , a paper appeared more r e c e n t l y ( S i d o r o v , I960) i n which, on the b a s i s of a mor p h o l o g i c a l examination o f hemolymph and c o x a l f l u i d , the c o n c l u s i o n was drawn t h a t c o x a l f l u i d appears as a r e -s u l t of b l e e d i n g . Remy (1922) had reached a s i m i l a r con-c l u s i o n on the same evidence. When the pres e n t study was undertaken i t thus remained undecided as t o whether the c o x a l gland operated by s e c r e t i o n (Bone, 194-3), by f i l -t r a t i o n and r e a b s o r p t i o n (Lees, 194-6) or by simple b l e e d i n g ( S i d o r o v , i 9 6 0 ) . While Lees' h y p o t h e s i s was most w i d e l y - 7 -accepted, h i s p r i n c i p l e evidence was by no means c o n c l u s i v e or i n c o n s i s t e n t w i t h a s e c r e t o r y mechanism. Thus one of the p r i n c i p l e o b j e c t i v e s of t h i s t h e s i s was to o b t a i n r i g o r o u s evidence f o r or a g a i n s t a f i l t r a t i o n mechanism. K i r s c h n e r ( 1 9 6 7 ) reviewed f i v e c r i t e r i a which have g e n e r a l l y been used as evidence f o r a f i l t r a t i o n mechanism of e x c r e t i o n : (a) e x c r e t i o n of polymers i n u r i n e (b) e f f e c t of i n h i b i t o r s on e x c r e t i o n of glucose (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 of f i l t r a t i o n s i t e (d) a n a l y s i s of t u b u l a r f l u i d from f i l t r a t i o n s i t e (e) p r e s s u r e s e n s i t i v i t y of t u b u l a r f l u i d f o r m a t i o n . I f a f i l t r a t i o n membrane were present q u i t e l a r g e molecules would be expected to pass through the membrane from the hemolymph i n t o the u r i n e . Thus i t has been found i n some mol l u s c s and some crustaceans t h a t i n u l i n (molecular weight 5000) may be e x c r e t e d ( K i r s c h n e r , 1 9 6 7 ) . Unfortun-a t e l y , i n some cases i n u l i n c l e a r a n c e was measured simply by i t s disappearance from the hemolymph of the ani m a l . Though i t i s l i k e l y t h a t the l o s s r e s u l t e d from e x c r e t i o n i n the u r i n e i t i s by no means proven. N e i t h e r does i n u l i n e x c r e t i o n per se e l i m i n a t e the p o s s i b i l i t y of e x c r e t i o n by p i n o c y t o s i s or a c t i o n s e c r e t i o n . For example, M a l p i g h i a n t u b u l e s of i n s e c t s have been shown to excrete i n u l i n i n c o n c e n t r a t i o n s which are low r e l a t i v e to the hemolymph (Ramsay and R e i g e l , 1 9 6 1 ) . Therefore the c r i t i c a l d i s --8-t i n c t i o n i s the q u a n t i t a t i v e r e l a t i o n s h i p between concen-t r a t i o n s i n plasma and u r i n e , a f u r t h e r d i s c u s s i o n of which i s found i n Chapter IV. T h i s q u a n t i t a t i v e assay was not made by Lees thus r e n d e r i n g h i s evidence f o r f i l t r a t i o n i n c o n c l u s i v e . S tronger evidence f o r a f i l t r a t i o n mechanism has been presented f o r some organisms by t h e i r r e n a l h a n d l i n g of glucose ( K i r s c h n e r , 1 9 6 7 ) . When the c o n c e n t r a t i o n of glucose i n the hemolymph i s e l e v a t e d , g l y c o s u r i a r e s u l t s thus s u g g e s t i n g a t h r e s h h o l d above which glucose i s l o s t i n t o the u r i n e . I n a d d i t i o n , when glucose t r a n s p o r t i s a b o l i s h e d by p h l o r i z i n , the c o n c e n t r a t i o n of glucose i n the u r i n e r i s e s . T h i s response t o p h l o r i z i n i s c o n t r a r y t o t h a t expected f o r a s e c r e t o r y mechanism where the concen-t r a t i o n of glucose might be expected to f a l l p r o v i d e d f l u i d s e c r e t i o n i s u n a f f e c t e d by the i n h i b i t o r . I n some mollu s c s i n c l u d i n g the octopus, the p e r i -c a r d i a l membrane has been i m p l i c a t e d as the s i t e of f i l -t r a t i o n ( K i r s c h n e r , 1 9 6 7 ) . I n cr u s t a c e a n s , the coelomosac has been suggested as the s i t e f o r primary f i l t r a t i o n . K i r s c h n e r and Wagner ( 1 9 6 5 ) attempted t o l o c a l i z e the s i t e of f i l t r a t i o n d i r e c t l y by means of f l u o r e s c e i n - l a b e l l e d g l o b u l i n . Although accumulations of p r o t e i n were found, i n the coelomosac, the f l u o r e s c e n t dye appeared t o be d i s p e r s e d throughout the p e r i t u b u l a r c e l l s r a t h e r than c o n f i n e d t o the v a s c u l a r spaces b a t h i n g them. This suggests a d i f f e r e n t - 9 -f i l t r a t i o n mechanism from t h a t i n the v e r t e b r a t e nephron. To date, analyses of p e r c a r d i a l f l u i d i n molluscs and f l u i d from the coelomosac of crustaceans have been c o n s i s -t e n t w i t h the concept of u l t r a f i l t r a t i o n . S l i g h t d i s c r e p -a n c i e s r e p o r t e d i n s o l u t e c o n c e n t r a t i o n between hemolymph and primary f i l t r a t e may be e x p l a i n e d by simple b i n d i n g of s o l u t e t o p r o t e i n thus r a i s i n g the apparent i o n concen-t r a t i o n i n the hemolymph ( K i r s c h n e r , 1 9 6 7 ) . Pressure s e n s i t i v i t y of the f o r m a t i o n of u r i n e has on l y been i n v e s t i g a t e d i n the v e r t e b r a t e s and i n a few m o l l u s c s . However, the r e s u l t s have always supported the theory of f i l t r a t i o n (Reviews by P i t t s , 1968 and K i r s c h n e r , 1 9 6 7 ) . V i r t u a l l y a l l the s t u d i e s of f i l t r a t i o n i n the i n -v e r t e b r a t e s have been c a r r i e d out on a q u a t i c organisms and o f t e n the evidence f o r f i l t r a t i o n i s not c o n v i n c i n g . The i n h e r e n t l i m i t a t i o n s of the a q u a t i c environment have l e d to a l a c k of measurements of the r a t e of u r i n e f o r m a t i o n . T h i s data has g e n e r a l l y been o b t a i n a b l e o n l y by i n d i r e c t methods such as i n u l i n c l e a r a n c e . Where attempts have been made to measure the r a t e d i r e c t l y l a r g e d i s c r e p a n c i e s have been observed ( P i c k e n , 1 9 3 7 ; Vorwohl, 1 9 6 1 ) . S i m i l a r l y the q u e s t i o n of c o n t r o l of f i l t r a t i o n has v i r t u a l l y been i g n o r e d . Thus there was a need f o r a d e t a i l e d study of a t e r r e s t r i a l i n v e r t e b r a t e having a f i l t r a t i o n type of ex--10-c r e t o r y system. One o b j e c t i v e of t h i s t h e s i s was to o b t a i n c o n v i n c i n g and unambiguous evidence, f o r or a g a i n s t a f i l t r a t i o n and r e a b s o r p t i o n mechanism i n the r e n a l organ of a t i c k . Furthermore, the system i n t h i s i n v e r t e b r a t e was p a r t i c u l a r l y s u i t e d t o an i n v e s t i g a t i o n of f a c t o r s i n f l u e n c i n g r a t e s o f u r i n e p r o d u c t i o n and of p o s s i b l e con-t r o l mechanisms. Since i o n and water economy of the whole organism was s t u d i e d , i t was i n e v i t a b l e t h a t some a t t e n t i o n should be p a i d to the passage of these elements acr o s s the gut w a l l . I t became apparent t h a t p e r t u r b a t i o n s o f the hemo-lymph are not d i r e c t l y r e l a t e d to the f l u i d i n g e s t e d by the t i c k but r a t h e r t o the composition of absorbate p a s s i n g a c r o s s the gut w a l l . Thus the s t i m u l u s f o r p r o d u c t i o n of c o x a l f l u i d was more l i k e l y dependent on the dynamics of f l u i d movement acro s s the gut than on the r a t e of i n g e s t i o n and c o m p o s i t i o n of the b l o o d meal. Passage of water across the w a l l of the midgut has been s t u d i e d i n s e v e r a l i n s e c t s both by the use of dyes as i n d i c a t o r s o f gut volume change and by g a i n i n weight of a q u a t i c organisms from which a l a r g e p r o p o r t i o n of hemolymph has been removed (Wigglesworth, 1933; Hobson, 1931; Shaw, 1955; O'Riorden, 1969). The l a s t r e f e r e n c e suggests t h a t water can be absorbed by simple osmosis from d i l u t e s o l u t i o n s and a d d i t i o n a l l y by a secondary t r a n s p o r t mechanism t h a t i s p r o b a b l y l i n k e d t o the a c t i v e t r a n s p o r t o f sodium i o n s . A c t i v e t r a n s p o r t o f sodium, potassium and c h l o r i d e across - l i -the midgut w a l l has been demonstrated i n v a r i o u s i n s e c t s . However, i n any g i v e n s p e c i e s the movement of a l l t h r e e i o n s i s not n e c e s s a r i l y a c t i v e (Reviewed by Treherne, 1 9 6 7 ) . Apart from the p r e l i m i n a r y evidence on c h l o r i d e concen-t r a t i o n and osmotic p r e s s u r e i n the gut c o n t e n t s of the t i c k t h r e e hours a f t e r f e e d i n g (Bone, 194-3) t h e r e i s n o t h i n g p u b l i s h e d on i o n and water t r a n s p o r t a c r o s s the gut e p i t h -e l i u m o f a r a c h n i d s . An a d d i t i o n a l o b j e c t i v e o f t h i s t h e s i s was t h e r e f o r e t o o b t a i n i n f o r m a t i o n on the p r o p e r t i e s and mechanisms o f s o l u t e and water movement acro s s the gut w a l l and i n p a r t i c u l a r t o determine whether f l u i d t r a n s p o r t was due t o osmosis, secondary t r a n s p o r t l i n k e d t o i o n s o r t o both mechanisms. I n summary, t h i s t i c k was known to a s s i m i l a t e and e x c r e t e s e v e r a l times i t s own o r i g i n a l content o f water and v a r i o u s s o l u t e s . The o b j e c t i v e s of t h i s t h e s i s were: ( i ) t o e s t a b l i s h t o what extent c o m p o s i t i o n and volume of hemolymph are a l t e r e d d u r i n g a normal f e e d i n g c y c l e and t o determine the r o l e o f the gut and c o x a l gland i n r e g u l a t i o n of these parameters; ( i i ) t o f u r t h e r determine l i m i t s o f the i o n i c and osmotic r e g u l a t o r y c a p a c i t y o f these organs by imposing p e r t u r b a t i o n s on the t i c k through changes i n the composition o f the i n g e s t e d f l u i d ; ( i i i ) t o e l u c i d a t e the mechanisms of f l u i d and s o l -ute t r a n s f e r a c r o s s the c o x a l gland and gut w a l l both by the response of these organs to the p e r t u r b a t i o n s imposed i n -12-( i i ) and by a s e r i e s of more s p e c i f i c experiments; and ( i v ) t o o b t a i n some p r e l i m i n a r y c l u e s as t o the f a c t o r s c o n t r o l l i n g the i n i t i a t i o n , t e r m i n a t i o n and r a t e of a b s o r p t i o n i n the gut and e x c r e t i o n by the c o x a l g l a n d . CHAPTER I I ANATOMY, GENERAL METHODS AND GENERAL OBSERVATIONS' A. ANATOMY 1• General Anatomy A d u l t females of Ornithodorus moubata are about 8 mm l o n g by 7 mm broad when unengorged. The gut i s c h a r a c t e r i z e d by l o n g d i v e r t i c u l a e extending t o every p a r t o f the body. T h i s p a r t i c u l a r s p e c i e s of t i c k i s p e c u l i a r i n t h a t the midgut ends b l i n d l y , there b e i n g no connection w i t h the h i n d g u t . The hindgut thus r e c e i v e s o n l y the sec-r e t i o n s of the two M a l p i g h i a n t u b u l e s which meander throughout the body coming i n t o c l o s e c o n t a c t w i t h every organ. The b r a i n , which l i e s v e n t r a l t o the gut, i s a s i n g l e g a n g l i o n i c mass p e r f o r a t e d by the oesophagus. On e i t h e r s i d e of the b r a i n and seemingly connected t o i t by a l a r g e muscle, i s a c o x a l gland measuring about 1 mm across and 0.5 mm t h i c k . T h i s g l a n d , which appears t o c o n s i s t o f a c o i l e d t u b u l e , i s w e l l s u p p l i e d w i t h t r a c h a e . The s t r u c t u r e o f the gut of Ornithodorus s a v i g n i has been d e s c r i b e d by C h r i s t o p h e r s (1906): "The s t r u c t u r e o f the sac and i t s d i v e r t i c u l a e i s i d e n t i c a l . The c a v i t y i s l i n e d by a s i n g l e l a y e r of l a r g e c e l l s r e s t i n g upon a t h i n basement membrane. E x t e r n a l l y t h e r e are v e r y l a r g e s i n g l e muscular f i b r e s o f a p e c u l i a r n a t u r e , arranged c i r c u l a r l y and l o n g i t u d i n a l l y . These form an open meshwork w i t h square meshes as i n the mosquito. The l i n i n g e p i t h e l i a l c e l l s are l a r g e c e l l s w i t h r e t i c u l a r protoplasm and l a r g e v e s i c u l a r n u c l e i . Some of them are seen p r o j e c t i n g f r e e l y i n t o the lumen. Such c e l l s are of e s p e c i a l l y l a r g e s i z e , and have t h e i r i n n e r p o r t i o n s much sw o l l e n and v a c u o l a t e d , and they may c o n t a i n g l o b u l e s of a dense b l a c k nature as w e l l as red c e l l s i n v a r i o u s stages of i n t r a c e l l u l a r d i g e s t i o n . I n a d d i t i o n to these l a r g e p r o j e c t i n g c e l l s t h e r e are s m a l l e r c e l l s , whose n u c l e i are s i t u a t e d n e a r e r t o the basement membrane. P r a c t i c a l l y a l l the c e l l s of the sac c o n t a i n s m a l l densely b l a c k g r a n u l e s , e v i d e n t l y d e r i v e d from the d i g e s t i o n of the blood i n the lumen. I n undistended d i v e r t i c u l a the e p i t h e l i u m may form a more o r ' l e s s continuous l i n i n g to the tube, but i n the d i s t e n d e d tube the c e l l s become v e r y unevenly d i s t r i b u t e d , b e i n g almost absent i n some p l a c e s , w h i l s t i n others they form v e r y s t r i k i n g p r o j e c t i n g masses." The d i g e s t i v e processes have been d e s c r i b e d by H o o g s t r a a l ( 1 9 5 6 ) . Twenty f o u r hours a f t e r the meal, the gut i s d i s t e n d e d and c o n t a i n s a s o f t c o a g u l a t i o n of undigested red blood c e l l s . Over a p e r i o d of weeks the meal i s grad-u a l l y taken up by wandering c e l l s which detach from the w a l l s of the gut. These c e l l s grow u n t i l they are v i s i b l e t o the naked eye. Hemoglobin i s absorbed by the e n d o t h e l i a l c e l l s of the gut where i t i s broken down to prothematin and appears as a l k a l i n e hematin i n the hemolymph (Wigglesworth, 1 9 4 3 ) . Since the midgut i s not c o n f l u e n t w i t h the rectum, any undigested c o n s t i t u e n t s of the blood meal must remain -16-i n the gut u n t i l the death o f the t i c k ( H o o g s t r a a l , 1956). Lees' ( 1946 ) evidence f o r a f i l t r a t i o n mechanism i n the c o x a l gland r e l i e d l a r g e l y on h i s o b s e r v a t i o n of a t h i n membraneous s t r u c t u r e c o n f l u e n t w i t h the t u b u l e . T h i s s t r u c t u r e , he p o s t u l a t e d , was the l o c u s of u l t r a -f i l t r a t i o n . Since Bone (19^3) made no mention of such a membrane, i t v/as i m p e r a t i v e t o c a r r y out a c a r e f u l h i s t o -l o g i c a l study of the c o x a l gland t o a s c e r t a i n whether o r not t h i s s t r u c t u r e e x i s t s . Another p o i n t of disagreement arose between these two authors when Bone d e s c r i b e d the tu b u l e as b e i n g simple and unbranched whereas Lees found t h a t the c o i l s o f the t u b u l e were i n t e r c o n n e c t e d . An attempt was t h e r e f o r e made t o r e s o l v e t h i s c o n f l i c t both h i s t o -l o g i c a l l y and by a d i r e c t o b s e r v a t i o n of the path taken by dye when i t was i n j e c t e d i n t o the t u b u l e . 2 . Techniques f o r s t u d y i n g anatomy o f c o x a l gland D i s s e c t i o n s were c a r r i e d out from the d o r s a l s u r f a c e on t i c k s c o n s t r a i n e d by p r e s s i n g them i n t o a melted mixture o f beeswax and r e s i n . F i n e s t r a i g h t j e w e l l e r s f o r c e p s and t i n y s c a l p e l s i n a r a z o r blade h o l d e r were the p r i n c i p a l d i s s e c t i n g t o o l s . A t i s s u e c u l t u r e medium developed by Rehacek and B r z o s t o w s k i ( 1 9 6 9 ) was used as a Ringer s o l -u t i o n ( c o m p o s i t i o n on p. 1 3 2 ) . F o r h i s t o l o g i c a l s t u d i e s , c o x a l glands were f i x e d i n Baker's. formaldehyde c a l c i u m ( P a n t i n , 1 9 6 4 ) , s e r i a l l y dehydrated i n a l c o h o l s and vacuum embedded i n p a r a f f i n wax. -17-S e c t i o n s were cut at t e n microns and s t a i n e d w i t h hematoxylin and e o s i n . Methylene blue was i n j e c t e d i n t o the lumen of c o x a l glands v i a a f i n e g l a s s needle i n s e r t e d near the c o x a l o r i f i c e . The needle was attached t o an ' A g l a 1 micrometer s y r i n g e so t h a t a slow, c o n t r o l l e d f l o w of dye c o u l d be m a i n t a i n e d . 3 . Anatomy of the c o x a l gland S e r i a l s e c t i o n s showed the gland t o c o n s i s t of a wide t u b u l e bent s e v e r a l times upon i t s e l f . One end of t h i s t u b u l e opened to the o u t s i d e through an o r i f i c e between the f i r s t and second coxae. The o t h e r end of the t u b u l e changed q u i t e a b r u p t l y i n t o a t h i n membraneous s t r u c t u r e which covered and was i n i n t i m a t e c o n t a c t w i t h much of the t u b u l a r p a r t of the gland ( F i g . 1). The t u b u l e w a l l s were one c e l l l a y e r t h i c k and d i s p l a y e d a brush border on the lumen s i d e . Techniques used i n t h i s study r e v e a l e d o n l y one c e l l type i n the t u b u l e w a l l . The s m a l l accessory gland c o n s i s t e d of groups of a c i n i whose ducts j o i n e d a l a r g e r duct which then anastomosed w i t h the duct of the c o x a l gland near the c o x a l o r i f i c e . I n g e n e r a l appearance i t resembled a s a l i v a r y g l a n d . The path taken by methylene blue when i t was i n -j e c t e d i n t o the duct of the c o x a l gland, v e r i f i e d the arrangement of t u b u l e s found by h i s t o l o g y . That i s , the FIGURE 1 MORPHOLOGY OF THE COXAL GLAND Legend: c o , c o x a l o r i f i c e a.g. accessory gland m.s. membraneous s t r u c t u r e t . t u b u l e m. muscle d. duct A } ) See t e x t B ) Sc a l e 10 cm. = 1 m.m. anterior -19-t u b u l e was simple and unbranched. Apparently a v a l v e e x i s t e d between the t u b u l a r and membraneous p a r t s of the gland s i n c e the gland s w e l l e d up when i t was i n j e c t e d but dye d i d not e n t e r the f i l t r a t i o n chamber. T h i s was assumed s i n c e no c o l o u r c o u l d be seen on the s u r f a c e of the tubule as would have been expected i f dye had p e n e t r a t e d the mem-braneous l a b y r i n t h . 4. D i s c u s s i o n The dimensions and g e n e r a l anatomy observed i n t h i s study are i n agreement w i t h those r e p o r t e d by A r t h u r (1962). The presence of a membraneous l a b y r i n t h i n the c o x a l gland i s i n agreement w i t h the morphology r e p o r t e d by Lees (1946) although Bone (1943) found no such s t r u c t u r e . However, the absence of any i n t e r c o n n e c t i o n of t u b u l e s A and B ( P i g . - 1) as r e v e a l e d by h i s t o l o g y and by the i n j e c t i o n of methylene blue would suggest t h a t i n t h i s case Bone was c o r r e c t . One can o n l y conclude t h a t the opening present i n Lees' s e c t i o n s may have been an a r t i f a c t . The f u n c t i o n o f the accessory gland which was mistaken f o r the c o x a l gland by Robinson and Davidson (1913), s t i l l i s not known. - 2 0 -B. GENERAL METHODS AND OBSERVATIONS !• Re a r i n g and f e e d i n g procedure The t i c k c o l o n y was r e a r e d from some specimens k i n d l y p r o v i d e d by Dr. G. Kohls of the Rocky Mountain L a b o r a t o r y , Montana. They were maintained at 28° C. and 5 0 $ r e l a t i v e h u m i d i t y and were f e d , c o n f i n e d i n c a p s u l e s , on c o n s t r a i n e d c h i c k e n s which were about f i v e weeks o l d . Approximately 1 0 days a f t e r f e e d i n g a female t i c k would l a y about 6 0 eggs which would hatch a f t e r a f u r t h e r 1 6 days. A f t e r p a s s i n g through a qui e s c e n t l a r v a l stage, f i v e i n s t a r s were observed i n the case of a female and f o u r f o r a male befor e the a d u l t emerged. Males and females remained t o g e t h e r f o r s e v e r a l days d u r i n g which time they mated. The females were then separated from the males and used o n l y a f t e r a p e r i o d of three weeks had elap s e d . T h i s time i n t e r v a l was important s i n c e the copious p r o -d u c t i o n of guanine a f t e r m o u l t i n g had then ceased making weight i n c r e a s e d u r i n g f e e d i n g p l u s t o t a l volume of c o x a l f l u i d a r e l i a b l e estimate of blood i n g e s t e d . Female t i c k s were capable of p a s s i n g through s e v e r a l such f e e d i n g and egg l a y i n g c y c l e s before d y i n g . For a l l experimental work the t i c k s were f e d on outdated, c i t r a t e d whole human blood through a washed c h i c k e n s k i n . The blood was maintained at 38° C i n a water bath; both blood and bath were s t i r r e d e l e c t r o m a g n e t i c a l l y . -21-Por ease of o b s e r v a t i o n and c o l l e c t i o n o f c o x a l f l u i d , the t i c k was t i l t e d up away from the membrane w i t h the a i d of t h i n adhesive s t r i p s ( F i g . 2 ) . The v e n t r a l s u r f a c e of the t i c k was then viewed through a d i s s e c t i n g microscope arranged w i t h i t s o p t i c a l path i n the h o r i z o n t a l p l a n e . There are t h r e e b i o l o g i c a l races of Ornithodorus  moubata d i f f e r i n g i n t h e i r h a b i t a t , choice of h o s t , and r e s i s t a n c e t o environmental change (Walton, 1957). The t i c k s used i n t h i s r e s e a r c h came o r i g i n a l l y from a mud hut on the coast of Mozambique. They are most l i k e l y t h a t race which p r e f e r s c h i c k e n s t o men o r hogs (Walton, 1957). Although they would feed r e a d i l y - o n human b l o o d and i n i t i a l l y produced v i a b l e eggs, p r o g r e s s i v e l y t h i s l a b o r a t o r y s t r a i n developed a s e n s i t i v i t y t o such a meal: the t i c k s turned p u r p l e and d i e d about a week a f t e r f e e d i n g . Such a phenomenon has been recorded i n Argas p e r s i c u s ( N u t t a l l et a l . , 1908) and i n Ornithodorus hermsi (Gregson, 1956). Since p a t h o l o g i c symptoms d i d not appear u n t i l s e v e r a l days a f t e r f e e d i n g and s i n c e the b e h a v i o r of the t i c k s d u r i n g the experimental p e r i o d was v e r y s i m i l a r t o t h a t of t i c k s f e d on c h i c k e n s , i t was decided t o continue r e s e a r c h u s i n g human b l o o d . T h i s f e e d i n g system was f a r more f l e x i b l e than one c o n f i n e d to l i v e c h i c k e n s . FIGURE 2 ARRANGEMENT FOR FEEDING TICK ON HUMAN BLOOD Legend: a.t. adhesive tape c . f . c o x a l f l u i d c. s. c h i c k e n s k i n h.b. human b l o o d w.b. water bath m.s. magnetic s t i r r e r d. m. d i s s e c t i n g microscope -23-2. Time sequence of f e e d i n g and e x c r e t i o n The t i c k became v e r y a c t i v e when p l a c e d on the c h i c k e n s k i n . A f t e r choosing a s i t e i t spat out a p o o l of s a l i v a onto the s k i n and probed w i t h the hyperstrome u n t i l , w i t h a prolonged t h r u s t , i t punctured the s k i n . At t h i s moment blood was observed b e i n g r a p i d l y pumped through the mouth p a r t s . A f t e r f e e d i n g f o r a p e r i o d of 2 t o 10 minutes, the t i c k s t a r t e d t o produce c o x a l f l u i d ; d r o p l e t s of about one-quarter m i c r o l i t e r appeared s i m u l t a n e o u s l y from each g l a n d . I f the t i c k was d i s t u r b e d p r o d u c t i o n of c o x a l f l u i d would sometimes suddenly cease suggesting t h a t i t s r e l e a s e was under nervous c o n t r o l . At the onset of c o x a l f l u i d p r o d u c t i o n the t i c k was r o u g h l y s p h e r i c a l . As f e e d i n g progressed and body s i z e i n c r e a s e d , i t became much f l a t t e n e d and even somewhat w r i n k l e d . A f t e r about 30 minutes of f e e d i n g the t i c k de-tached although c o x a l f l u i d c ontinued t o be produced f o r a f u r t h e r 30 minutes d u r i n g which time body s i z e decreased. CHAPTER I I I OSMOTIC AND IONIC REGULATION -25-A. INTRODUCTION Both Bone (194-3) and Lees (1946) found that the mean ch l o r i d e concentration of pooled coxal f l u i d was about 140 meq/liter and that of the hemolymph was 170 meq/liter. Bone fed h i s t i c k s on guinea-pig blood through a mouse s k i n . Thus, he was able to vary the i o n i c and osmotic composition of the meal. He found that, provided the meals were kept isosmotic, chloride concentrations i n the hemolymph remained constant when the c h l o r i d e concen-t r a t i o n s of the blood meals v a r i e d from 35 meq/liter to 145 meq/liter. Despite v a r i a t i o n s i n c h l o r i d e concentrations of the meal, the osmotic pressures of gut and hemolymph were very s i m i l a r to each other three hours a f t e r feeding. Bone only made observations on mean chl o r i d e concentration of pooled coxal f l u i d and c h l o r i d e concen-t r a t i o n s of hemolymph before and a f t e r feeding. I n i t i a l experiments were therefore undertaken to v e r i f y and extend these observations by studying sodium, potassium and c h l o r -ide concentrations of body f l u i d s throughout the normal feeding c y c l e . Measurements were made of the h i t h e r t o un-considered volume changes of gut contents, hemolymph and coxal f l u i d . Thus, I hoped to be able to draw up a more complete p i c t u r e of i o n i c and volume r e g u l a t i o n during feeding. Such a procedure was also followed when t i c k s were fed blood meals d i f f e r i n g i n t h e i r i o n i c and osmotic -26-c o m p o s i t i o n s . Some p r e l i m i n a r y i n s i g h t i n t o the mode o f a c t i o n of the c o x a l gland and gut was sought by s t u d y i n g the e f f e c t s o f these f e e d i n g regimes on the r a t e at which r e g u l a t i o n of i o n i c and osmotic c o m p o s i t i o n occurred i n the body f l u i d s . Sauer et a l . (1969) had i m p l i c a t e d the midgut of the cockroach as be i n g an extremely important osmo-r e g u l a t o r y organ i n the i n t a c t i n s e c t . They showed t h a t r a p i d s o l u t e a b s o r p t i o n c o u l d take p l a c e across the gut i n the absence of s i g n i f i c a n t net water movement and t h a t the mechanism r e s p o n s i b l e was s e n s i t i v e t o d i n i t r c p h e n o l . Harvey and Nedergaard (1964) d i s c o v e r e d an e l e c t r o g e n i c potassium, pump i n the l a r v a of the phytophagus i n s e c t Hyalophora c e c r o p i a . A l i n k e d Na- K pump has been l o c a l i z e d i n the cockroach midgut (O'Riordan, 1969) s u g g e s t i n g t h a t the mechanism i n v o l v e d i s p r o b a b l y v e r y s i m i l a r t o t h a t i n -v o l v e d i n the f r o g s k i n (Koefoed-Johnson and Ussing "1958). The e x i s t e n c e of a c t i v e processes f o r s o l u t e movement had thus been e s t a b l i s h e d i n the midgut of some i n s e c t s . By s t u d y i n g i o n i c and osmotic g r a d i e n t s across the gut w a l l and measuring the accompanying e l e c t r o p o t e n t i a l g r a d i e n t s , I hoped t o determine whether s i m i l a r a c t i v e processes played a p a r t i n the o v e r a l l homeostasis of the t i c k . P a r t i c u l a r l y I wished t o determine whether f l u i d t r a n s f e r was l a r g e l y due t o osmotic f l o w , t o secondary t r a n s p o r t coupled t o s o l u t e f l o w or t o both phenomena. B. METHODS !• P r e p a r a t i o n of blood meal In some experiments the i o n i c c o n c e n t r a t i o n of the b l o o d meal was reduced t o o n e - h a l f - o r one-quarter of the value f o r whole bl o o d " w h i l e the osmotic pressure was maintained c o n s t a n t . This was achieved by d i l u t i n g the blood w i t h an i s o s m o t i c s o l u t i o n of gl u c o s e . The osmotic p r e s s u r e was lowered by d i l u t i n g the blood meal w i t h d i s -t i l l e d water. U n f o r t u n a t e l y , the osmotic pressure c o u l d not be maintained constant when i n c r e a s e d i o n i c concentra t i o n s above normal l e v e l s were r e q u i r e d . I n these cases c o n t r o l experiments were designed i n which the osmotic p r e s s u r e of the meal was r a i s e d an equal amount by the a d d i t i o n of glucose t o the b l o o d . The a b b r e v i a t i o n s used t o designate the composition of these blood meals are shown below. h.b. Human blo o d as r e c e i v e d from the Red Cross. ACD s o l u t i o n added as a n t i c l o t t i n g agent. h.b. + NaCl Human blood p l u s sodium c h l o r i d e added at a c o n c e n t r a t i o n of 2.96 g / l i t e r whole bloo d . h.b. + g. Human blo o d p l u s glucose added at a concen-t r a t i o n of 31 g / l i t e r whole b l o o d . This ha the same osmotic pressure as h.b. + NaCl ( F r e e z i n g p o i n t d e p r e s s i o n = 0 . 8 7 ° C). h.b./D.W. Human blood d i l u t e d 50/50 w i t h d i s t i l l e d water. -28-h.b./g.50/50 Human blood d i l u t e d 50/50 w i t h an i s o s m o t i c s o l u t i o n of glucose c o n t a i n i n g 55.5 g / l i t e r glucose i n d i s t i l l e d water. h.b./g.25/75 Human blood d i l u t e d 25/75 w i t h an i s o s m o t i c s o l u t i o n of gl u c o s e . 2. C o l l e c t i o n of body f l u i d s and r a t e measurements Measurement of the r a t e of p r o d u c t i o n of c o x a l f l u i d and of the nature and q u a n t i t y of i o n s e x c r e t e d was achieved by c o l l e c t i n g the c o x a l f l u i d d i r e c t l y from the c o x a l o r i f i c e . A f i n e g l a s s m i c r o p i p e t t e was attached t o a Gilmont micrometer s y r i n g e and micrometer readings were taken at 5 second i n t e r v a l s . F or measurements o f compos-i t i o n w i t h r e s p e c t t o time , the samples were c o l l e c t e d i n 1 m i c r o l i t e r d i s p o s a b l e p i p e t t e s . Care was taken t o remove any excess c o x a l f l u i d immediately t o prevent e r r o r s from e v a p o r a t i o n . Samples were s t o r e d i n the p i p e t t e s under m i n e r a l o i l . Hemolymph was sampled by amputating a l e g and c o l l e c t i n g two a l i q u o t s i n 1 m i c r o l i t e r d i s p o s a b l e p i p e t t e s . Between sampling, the f l o w of hemolymph was h a l t e d w i t h a sm a l l s e r r e f i n e . Whole hemolymph was used f o r a n a l y s e s ; no problem w i t h c l o t t i n g was encountered when the samples were s t o r e d i n the p i p e t t e s under m i n e r a l o i l . Before gut samples were taken, the hemolymph was dr a i n e d by amputating the l e g s . When a f i n e i n c i s i o n ..was then made d o r s a l l y the gut pr o t r u d e d and cou l d be punctured and sampled. Gut contents were c o l l e c t e d i n hem a t o c r i t -29-tubes and, except where i n d i c a t e d t o the c o n t r a r y , the tubes were c e n t r i f u g e d f o r 5 minutes i n a c l i n i c a l microhemat-o c r i t c e n t r i f u g e . The plasma was then s t o r e d f r o z e n i n the s e a l e d tubes. 3 . Chemical measurements C h l o r i d e d e t e r m i n a t i o n s were c a r r i e d out accord-i n g t o the method of Ramsay et a l . (1955) whereby 1-micro-l i t e r samples were d i l u t e d i n 1 0 0 - m i c r o l i t e r s of 50$ a c e t i c a c i d and t i t r a t e d p o t e n t i o m e t r i c a l l y w i t h s i l v e r n i t r a t e . Sodium and potassium c o n c e n t r a t i o n s were measured by d i -l u t i n g the 1 - m i c r o l i t e r samples i n " 8 ml of g l a s s d i s t i l l e d water f o r sodium or 4- ml of 500 ppm. sodium swamp s o l u t i o n f o r potassium. The sodium swamp s o l u t i o n ensured t h a t i n t e r f e r e n c e of sodium remained constant d u r i n g the determ-i n a t i o n of potassium. Both i o n s were measured u s i n g e i t h e r a Unicam SP 900 or a Techtron AA 1120 flame spectrophoto-meter i n the e m i s s i o n mode o f o p e r a t i o n . T o t a l body c h l o r i d e was found by homogenizing the whole t i c k i n 4- ml of g l a s s d i s t i l l e d water. A 50-m i c r o l i t e r sample added t o 5 0 - m i c r o l i t e r s of g l a c i a l a c e t i c a c i d v/as then t i t r a t e d a g a i n s t s i l v e r n i t r a t e (Ramsay et a l . , 1955); t h i s p r o v i d e d an estimate of the c h l o r i d e content. -30-4. Volume measurements The t o t a l i n c r e a s e i n body volume d u r i n g f e e d i n g was measured by weighing the t i c k on an a n a l y t i c a l balance before f e e d i n g and again a f t e r c e s s 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 . I t was found t h a t 10 m i c r o l i t e r s of gut f l u i d had, t o two s i g n i f i c a n t f i g u r e s , the same weight as 10 m i c r o l i t e r s of water. Since the s p e c i f i c g r a v i t y was u n i t y , weight and volume i n c r e a s e were i n t e r c o n v e r t a b l e . The t o t a l b l o o d i n g e s t e d by the t i c k was e s t i m -ated by a s p e c t r o p h o t o m e t r y comparison of hemoglobin con-c e n t r a t i o n i n an homogenate of the whole t i c k w i t h standards prepared from the human b l o o d meal. A comparison of the t o t a l hemoglobin i n a f e d t i c k w i t h t h a t i n an unfed t i c k enabled the t o t a l b l ood volume i n g e s t e d by the t i c k t o be c a l c u l a t e d . The hemoglobin spectrum from the t i c k was the same as t h a t f o r human bl o o d ; t h i s i n d i c a t e d t h a t breakdown of hemoglobin had not occurred d u r i n g f e e d i n g . A Bauch and Laumb double beam scanning spectrophotometer was used f o r a l l measurements. The t o t a l b l ood i n g e s t e d was a l s o estimated by comparing the hematocrit of the gut contents of the f e d t i c k w i t h t h a t of the blood meal on which i t had been f e e d i n g . The gut f l u i d and blood meal were c o l l e c t e d i n microhemat-o c r i t tubes a f t e r c o x a l f l u i d p r o d u c t i o n had ceased. These were s e a l e d w i t h C r i t o c a p s ( C l a y Adams) and c e n t r i f u g e d f o r 5 minutes i n a Clay Adams c l i n i c a l microhematocrit c e n t r i --31-fuge. The h e m a t o c r i t c o u l d then he measured d i r e c t l y i n the tube. Only those f e e d i n g c o n d i t i o n s i n which hemolysis of the r e d blood c e l l s d i d not occur c o u l d be used i n t h i s e s t i m a t e . Thus the b l o o d meals having lowered i o n i c con-c e n t r a t i o n s c o u l d not be t r e a t e d i n t h i s manner s i n c e the gut contents were found t o be somewhat hemolysed. Hemolymph volumes were estimated by d i l u t i o n of a r a d i o a c t i v e t r a c e r . One m i c r o l i t e r of i n u l i n - c a r b o x y l -C 1^ s o l u t i o n (New England N u c l e a r ) was i n j e c t e d v i a a l e g i n t o the t i c k . The i n j e c t i o n procedure used was t h a t of P a t c h i n and Davey (1968) w i t h the e x c e p t i o n t h a t the l e g had t o be clamped t o prevent l o s s of f l u i d due t o the pos-i t i v e hemolymph p r e s s u r e i n t i c k s . I t was found t h a t 1 hour a f t e r i n j e c t i o n , samples of hemolymph taken from remote p o i n t s i n the body showed the same c o n c e n t r a t i o n . The assumption v/as thus made t h a t t h i s time i n t e r v a l was s u f -f i c i e n t f o r e q u i l i b r a t i o n . T w o - m i c r o l i t e r samples of hemo-lymph were then taken and mixed immediately w i t h 10 ml of Bray's s o l u t i o n (Bray, I 9 6 0 ) . The samples were counted on a N u c l e a r Chicago Mark I l i q u i d s c i n t i l l a t i o n counter u s i n g the channels r a t i o method of quench c o r r e c t i o n . When data were r e q u i r e d on hemolymph volumes a f t e r f e e d i n g , care v/as taken t o ensure t h a t c o x a l f l u i d p r o -d u c t i o n had indeed ceased before i n j e c t i o n of the r a d i o -a c t i v e t r a c e r was performed. I f n o t , i t was found t h a t an unknown p r o p o r t i o n o f the t r a c e r was immediately v/ashed out - 3 2 -i n any c o x a l f l u i d produced; t h i s l e d t o e r r o r s i n the e s t i m a t i o n of volume. For t h i s reason i t was necessary t o handle the t i c k s as g e n t l y as p o s s i b l e and t o d i s c a r d any t h a t d i d produce c o x a l f l u i d a f t e r i n j e c t i o n . From a knowledge of the t o t a l counts per minute i n j e c t e d i n t o the t i c k ' s hemolymph and the c o n c e n t r a t i o n of t r a c e r (counts per minute per m i c r o l i t e r ) a f t e r e q u i l i b r a t i o n i n the hemocoel, i t was p o s s i b l e t o c a l c u l a t e the volume of the hemolymph. 3. Measurement of osmotic pressure Osmotic p r e s s u r e s were measured by the method of f r e e z i n g - p o i n t d e p r e s s i o n (Ramsay, 194-9). However, the drops of sample were i n t e r s p e r s e d w i t h o i l i n 1 - m i c r o l i t e r p i p e t t e s i n s t e a d of the c a p i l l a r y tubes used by Ramsay. Gut samples were c e n t r i f u g e d and a drop of the supernatent was p l a c e d under m i n e r a l o i l . T iny d r o p l e t s c o u l d then be taken up from t h i s i n t o a m i c r o l i t e r p i p e t t e . Coxal f l u i d samples were t r e a t e d s i m i l a r l y but not c e n t r i f u g e d . U n f o r t u n a t e l y , hemolymph samples c l o t t e d when put under o i l and thus had t o be sampled d i r e c t l y from the s i t e of hemmorrhage i n t o the m i c r o l i t e r p i p e t t e . 6. Measurement of 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 the gut T i c k s were prepared i n the f o l l o w i n g manner f o r - 3 5 -measurement of the e l e c t r o p o t e n t i a l g r a d i e n t across the gut. A V-shaped i n c i s i o n was made i n a coxa. A lobe of gut and a p o o l of hemolymph then extruded through the i n -c i s i o n . The i n d i f f e r e n t e l e c t r o d e c o n s i s t e d of a s i l v e r / s i l v e r c h l o r i d e wire embedded i n a P a s t e u r p i p e t t e f i l l e d w i t h 1.5 molar potassium c h l o r i d e i n agar g e l . The t i p of t h i s p i p e t t e had been drawn out t o about 1 mm i n diameter and t h i s was touched t o the drop of hemolymph. The r e c o r d -i n g e l e c t r o d e was a f i n e g l a s s m i c r o p i p e t t e of the type used f o r i n t r a c e l l u l a r r e c o r d i n g (Kennard, 1958). T h i s was f i l l e d w i t h 1.5 molar potassium c h l o r i d e and h e l d over a s i l v e r / s i l v e r c h l o r i d e w i r e by f r i c t i o n . The micro-p i p e t t e s were made on a Palmer m i c r o e l e c t r o d e p u l l e r so t h a t t h e i r r e s i s t a n c e was about 10 M ohm. The r e c o r d i n g e l e c t r o d e was attached t o a M e d i s t o r cathode f o l l o w e r h e l d i n a P r i o r m i cromanipulator. The e l e c t r o d e was g e n t l y lowered i n t o the drop of hemolymph, the asymmetry p o t e n t i a l was backed-off and the p o t e n t i a l then measured as the e l e c t r o d e passed across the gut w a l l . The p o t e n t i a l s were observed on a T e c t r o n i x cathode r a y o s c i l l o s c o p e . -34-C. RESULTS 1. Change i n d i s t r i b u t i o n of body f l u i d s d u r i n g  f e e d i n g and c o x a l f l u i d p r o d u c t i o n I f the water r e g u l a t i o n of an organism i s t o be s t u d i e d i t i s necessary not only t o c o n s i d e r the gross p i c t u r e of water e n t e r i n g and l e a v i n g the system as a whole, but a l s o the d i s t r i b u t i o n of water w i t h i n the sub-compart-ments of t h a t system. The volume of c o x a l f l u i d produced was measured d i r e c t l y . The t o t a l volume i n c r e a s e i n the t i c k c o u l d a l s o be c a l c u l a t e d from a knowledge of the weight i n c r e a s e from b e f o r e f e e d i n g u n t i l c o x a l f l u i d p r o d u c t i o n had ceased. The sum of these f i g u r e s p l u s the water l o s s through the c u t i c l e , s a l i v a t i o n and e x c r e t i o n should then g i v e an estimate of the volume of blood meal i n g e s t e d . Water l o s s through the c u t i c l e c o u l d be d i s r e g a r d e d s i n c e Lees (1946) showed the volume l o s t by t h i s route t o be n e g l i g a b l e . I t was p o s s i b l e , when the t i c k was f e e d i n g through the c h i c k e n s k i n , to observe the f l o w of b l o o d through the mouth p a r t s . Except f o r the i n i t i a l p r o d u c t i o n of s a l i v a , presumably to f a c i l i t a t e p e n e t r a t i o n of the membrane, I never ob-served a break i n the one way passage of blood i n t o the t i c k . On t h i s evidence the l o s s of water through s a l i v -a t i o n was a l s o d i s r e g a r d e d . As d i s c u s s e d i n Chapter I , Lees (1946) showed t h a t there was no l o s s of water v i a the -35-M a l p i g h i a n t u b u l e s d u r i n g the p e r i o d of c o x a l f l u i d p r o d u c t i o n . Thus the volume of bl o o d meal i n g e s t e d c o u l d be computed from the volume of c o x a l f l u i d produced and the volume (weight) i n c r e a s e of the t i c k d u r i n g f e e d i n g . Hemolymph volumes were determined befo r e f e e d i n g and a g a i n two hours a f t e r f e e d i n g at which time c o x a l f l u i d p r o d u c t i o n had ceased. S i n c e the change i n hemolymph v o l -ume and the t o t a l volume i n c r e a s e of the t i c k d u r i n g the same p e r i o d was known, an estimate c o u l d be made of the i n c r e a s e i n gut volume. The assumption was made t h a t t i s s u e volume d i d not i n c r e a s e s i g n i f i c a n t l y r e l a t i v e t o the changes observed f o r gut and hemolymph volumes. T h i s i s j u s t i f i e d i n t h a t a 50$ i n c r e a s e i n t i s s u e volume of a 40 mg t i c k (hemolymph volume 15 m i c r o l i t e r s ) would o n l y account f o r a maximum i n c r e a s e of 12 m i c r o l i t e r s . Since by d i r e c t o b s e r v a t i o n the volume o f the gut be f o r e f e e d i n g appeared t o be v e r y small ( p r o b a b l y l e s s than 5 m i c r o l i t e r s ) and s i n c e the f i n a l volume of the gut was approximately 150-200 m i c r o l i t e r s , t h i s l a t t e r f i g u r e was, f o r the purpose of c a l c u l a t i o n s , taken t o be approx-i m a t e l y equal t o the i n c r e a s e i n gut volume. The hemolymph volume befor e f e e d i n g was 15.0 - 1.1 m i c r o l i t e r s (Mean - S.E., N = 10). The volume of the body f l u i d s d u r i n g and a f t e r f e e d i n g are shown i n Table I . The volume of f l u i d absorbed ac r o s s the gut d u r i n g the 2 hour expe r i m e n t a l p e r i o d was c a l c u l a t e d from the i n c r e a s e i n hemolymph volume p l u s the t o t a l c o x a l f l u i d produced. The f a c t o r by which the blood meal was concen-t r a t e d i n the gut by water a b s o r p t i o n was determined by t h r e e independent methods. The hemoglobin content of the gut and the h e m a t o c r i t of the gut f l u i d were measured 2 hours a f t e r f e e d i n g . The c o n c e n t r a t i o n f a c t o r s vrere com-pared w i t h those c a l c u l a t e d from the f l u i d volumes shown i n Table I . Table I I shows t h a t the v a l u e c a l c u l a t e d from the h e m a t o c r i t was low f o r t i c k s f e d on human b l o o d . T h i s i s t o be expected i f some hemolysis of the red blood c e l l s o c c u r r e d . Such a phenomenon i s l e s s l i k e l y t o be of concern i n the two blood meals which \\rere h y p e r t o n i c t o normal b l o o d . This i s r e f l e c t e d i n t h e i r c l o s e agreement w i t h the o t h e r method of c a l c u l a t i o n . Again, the v a l u e c a l c u l a t e d from the hemoglobin c o n c e n t r a t i o n might be expected t o be - low due t o some breakdown of hemoglobin i n the gut. However, d e s p i t e the i n h e r e n t e r r o r s , these independently measured c o n c e n t r a t i o n factors-" s t i l l agree s u f f i c i e n t l y w e l l t o p r o -v i d e a d d i t i o n a l weight t o the assumption t h a t a l l r o u t e s of water movement had been c o n s i d e r e d . Table I shows t h a t a d i r e c t r e l a t i o n s h i p e x i s t e d between the volume of c o x a l f l u i d produced and the volume of f l u i d absorbed across the gut w a l l ( F i g . 3 ) . Moreover, the displacement of t h i s l i n e from the l i n e f o r equal v o l -umes (y = x) r e p r e s e n t s the i n c r e a s e i n hemolymph volume. T h i s i n c r e a s e i n volume approaches a constant value (4-0 m i c r o l i t e r s ) as the volume of gut absorbate i n c r e a s e s . - 3 7 -TABLE 1 VOLUMES OP BODY FLUIDS DURING AND AFTER FEEDING T o t a l and f i n a l volumes r e f e r t o time i n t e r v a l o f two hours from s t a r t o f f e e d i n g . The t i c k s were s u b j e c t e d t o minimal d i s t u r b a n c e d u r i n g these measurements. Those v a l u e s which were measured d i r e c t l y are presented as the mean - SE. The o t h e r v a l u e s were c a l c u l a t e d . A l l volumes are expressed i n m i c r o l i t e r s . TABLE 1 Meal T o t a l v o l . T o t a l v o l . F i n a l v o l . V o l . g u t F i n a l v o l . T o t a l v o l . N inc r e a s e i n g e s t e d gut' absorbate hemolymph c o x a l f l u i d h.b. 235 ± 6.4 364 192 172 58 ± 3.7 129 ± 5.1 4-h.b. + NaCl 225 1 22 291 197 94 43 - 3.8 66 ± 2.9 10 h.b. + g. 237 ± 12 261 1 9 ? 64 55 ± 6.1 24 ± 1.6 6 h.b./DW 181 - 10 223 1 3 2 71 44 ± 5.9 42 ± 9.3 8 h.b./g. 50/50 177 1 8 202 1 6 0 42 32 - 2.8 25 1 2.1 14 h.b./g. 25/75 248 ± 20 267 P a n 27 23 ± 3.7 19 - 1.9 5 - 38 -TABLE I I CONCENTRATION FACTOR OF GUT CONTENTS TWO  HOURS AFTER FEEDING, BY THREE INDEPENDENT METHODS Those v a l u e s which were measured d i r e c t l y are p r e s -ented w i t h t h e i r standard e r r o r s . The o t h e r v a l u e s were c a l c u l a t e d . TABLE I I Meal From Table I Prom hemato c r i t Prom hemoglobin % agreement h.b. 1.89 1.61 - 0.02 1.72 - 0.05 15 (N = 4-) (N = 4-) (N = 5) , h.b. + NaCl 1.4-7 1.38 - 0.03 6 (N = 10) (N = 4-) h.b. + g. 1.32 1.38 - 0.03 4 (N = 6) (N = 4-) - 3 9 -FIGURE 3 RELATION BETWEEN VOLUME 03? GUT ABSORBATE AND VOLUME OF  COXAL FLUID The broken l i n e i s t h a t g r a d i e n t f o r equal volumes. Standard e r r o r s may be found i n Table I . Measurements were made over a 2 hour experimental p e r i o d . T i c k s were f e d h.b. Volume gut absorbate (A.) -40-The volume of f l u i d absorbed across the gut v a r i e d w i d e l y w i t h the nature of the blood meal even though the volume of blood i n g e s t e d had a maximal t w o f o l d v a r i a t i o n . F i g u r e 4- shows t h a t when the percent of i n g e s t e d f l u i d absorbed across the gut i s p l o t t e d a g a i n s t the t o t a l sodium p l u s c h l o r i d e c o n c e n t r a t i o n s of the blood meal, a l i n e a r r e l a t i o n s h i p emerges i f the osmotic p r e s s u r e i s h e l d c o n s t a n t . T h i s r e l a t i o n s h i p i s s h i f t e d t o the l e f t ( t h a t i s a h i g h e r percentage absorbed) f o r low osmotic p r e s s u r e and i s s h i f t e d t o the r i g h t f o r h i g h osmotic p r e s s u r e s . I n o t h e r words, at any gi v e n i o n c o n c e n t r a t i o n , the average volume of a b s o r p t i o n d u r i n g the f e e d i n g and ex-c r e t o r y p e r i o d i s i n v e r s e l y r e l a t e d t o the t o t a l osmotic c o n c e n t r a t i o n of the blood meal. The t o t a l weight i n c r e a s e was measured at the time when c o x a l f l u i d f i r s t appeared, t o see whether a c r i t i c a l volume e x i s t e d t o p r o v i d e a s t i m u l u s f o r the 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 . No such r e l a t i o n s h i p was found. 2. D i s t r i b u t i o n of i o n s i n body f l u i d s The i o n i c c o n c e n t r a t i o n s of the gut c o n t e n t s , hemolymph and pooled c o x a l f l u i d were measured 2 hours a f t e r f e e d i n g . The t i c k s were f e d blood meals w i t h d i f f e r e n t i o n c o n c e n t r a t i o n s and osmotic p r e s s u r e s . Analyses of blood meal and gut contents were made both on whole samples and on samples which had been c e n t r i f u g e d t o remove the red -41-FIGURE 4 RELATION BETWEEN VOLUME ABSORBED ACROSS GUT WALL (AS  PERCENT OF INGESTED FLUID) AND TOTAL (Ma + C l ) ION CON- CENTRATION OF BLOOD MEAL Measurements were made over a 2 hour experimental p e r i o d . Legend: • B l o o d meal 100$ osmotic p r e s s u r e of h.b. ( T i c k s f e d h.b., h.b./g.50/50 and h.b./g.25/75) O B l o o d meal 158$ osmotic pressure o f h.b. ( T i c k s f e d h.b. + NaCl and h.b. + g.) A Blood meal 50$ osmotic p r e s s u r e o f h.b. ( T i c k s f e d h.b./D.W.) Total ( N a + + C I ) ion concentrat ion ( m e q / l i t e r ) -42-blood c e l l s . The r e s u l t s are presented i n Tables I I I , V and VII. The i o n concentrations of the hemolymph before feeding are presented below as the mean - SE. Chloride concentration 200 i 2.0 meq/liter (N = 9) Sodium concentration 231 - 5.8 meq/liter (N = 4) Potassium concentration 11.6 - 0.7 meq/liter (N = 4) The concentration of the f l u i d passing across the gut ( i . e . absorbate) was c a l c u l a t e d i n the same way f o r each i o n . The equations and a sample c a l c u l a t i o n are shov/n below, c i A = Olj. - C1 R and C l j = [Ol] . . v . C 1 E - t 0 1 l R V E where C l ^ = t o t a l c h l o r i d e absorbed across gut i n 2 hr. Glj = t o t a l c h l o r i d e ingested C1R••:.= t o t a l c h loride remaining i n gut 2 hr. a f t e r feeding [Cl] j = chloride concentration of ingested f l u i d [ClJ R = chl o r i d e concentration of f l u i d remaining i n gut 2 hr. a f t e r feeding V-j- = Volume of ingested f l u i d V R = Volume of f l u i d remaining i n gut 2 hr. a f t e r feeding For t i c k s fed h.b. (Tables I and I I I ) : C1 A = (96 x 364) - (77 x 192) = 20.160 yueq (a) A l t e r n a t i v e l y t h i s may be c a l c u l a t e d from: c i A . Cl - Cljj + c i c d O and C 1 H 2 . '[ca] H 2 . V H 2 -43-TABLE I I I CHLORIDE CONCENTRATIONS IN BODY FLUIDS  DURING AND AFTER FEEDING T o t a l and f i n a l v a l u e s r e f e r t o time i n t e r v a l of two hours from s t a r t o f f e e d i n g . Those v a l u e s which were measured d i r e c t l y are pres e n t e d as the mean - SE w i t h the e x c e p t i o n of the i n -gested f l u i d s . These are s i n g l e v a l u e s s i n c e any s e r i e s o f experiments under a g i v e n f e e d i n g c o n d i t i o n was c a r r i e d out u s i n g the same bat c h of b l o o d . The v a l u e s f o r gut absorbate"were c a l c u l a t e d and hence do not bear standard e r r o r s . A l l c o n c e n t r a t i o n s are expressed as m e q / l i t e r . The two v a l u e s f o r mean gut absorbate were computed from the equations on pages 42 and 48. The c a l c u l a t i o n s are shown i n Table IV. TABLE I I I Meal Ingested f l u i d F i n a l gut contents Mean gut F i n a l nemo- Mean N Centrifuged Whole Centrifuged Whole absorbate lymph coxal f l u i d h.b. 116 96 49 + 1.2 77 + 2.0 117 - 127 170 + 2.0 117 + 5.3 6 h.b. + NaCl 162 142 70 + 2.6 88 + 5.7 230 - 255. 239 + 8.7 217 + 3.0 5 h.b. + g. 116 96 56 + 3-4- 58 + 2.1 200 - 214 206 + 3.9 187 5.3 5 h.b./D.W. 45 45 51 + 2.5 52 + 3.7 30 - 46 95 3.6 51 + 2.5 4 h.b./g. 50/50 4-5 45 26 + 1.7 37 + 0.4 75 - 103 173 + 3.0 70 + 2.1 5 h.b./g. 25/75 21 21 20 + 1.0 19 + 0.6 39 - 59 172 + 2.9 34 + 0.5 4 _44~ TABLE IV CALCULATION OF CHLORIDE CONCENTRATION  OF GUT ABSORBATE See e x p l a n a t i o n i n t e x t (page 42) TABLE IV Meal C h l o r i d e absorbed Volume of [Cl~] (a) ^ e q (b) ^ e q absorbate absorbate ( m i c r o l i t e r s ) m e q / l i t e r h.b. 20.160 21.953 172 117-127 h.b. + NaCl 23.986 21.599 94 230-255 h.b. + g. 13.630 12.818 64 200-214 h.b./D.W. 2.131 3-322 71 30-46 h.b./g.50/50 3.170 4.286 42 75-103 h.b./g.25/75 1.047 1.602 27' 29-59 -4-5-TABLE V SODIUM CONCENTRATIONS IN BODY FLUIDS  DURING AND AFTER FEEDING T o t a l and f i n a l v a l u e s r e f e r t o time i n t e r v a l of two hours from s t a r t of f e e d i n g . Those v a l u e s which were measured d i r e c t l y are presented as the mean - SE w i t h the e x c e p t i o n of the i n g e s t e d f l u i d s . These are s i n g l e v a l u e s s i n c e any s e r i e s of experiments under a g i v e n f e e d i n g c o n d i t i o n was c a r r i e d out u s i n g the same batch o f b l o o d . The v a l u e s f o r gut absorbate were c a l -c u l a t e d and hence do not bear standard e r r o r s . A l l c o n c e n t r a t i o n s are expressed as m e q / l i t e r . The two v a l u e s f o r gut absorbate were computed from the equations on pages 4-2 and 4-8 . The c a l c u l a t i o n s are shown i n Table V I . TABLE V Meal Ingested f l u i d F i n a l gut contents Mean gut F i n a l hemo- Mean N Ce n t r i f u g e d Whole C e n t r i f u g e d Whole absorbate lymph Coxal f l u i d h.b. 177 124 h.b. + NaCl 223 170 h.b. + g. 177 124 h.b./D.W. 62 62 h.b./g. 50/50 62 62 h.b./g. 25/75 31 31 197 + 0.7 95 + 2.0 115--156 218 + 4.8 . 139 + 2.4 235--261 135 + 3.7 89 + 3.7 232--236 73 + 3.3 73 + 3.3 38-- 4 8 51 + 1.7 49 + 1.5 109--109 33 + 1.2 — 154 + 3.1 111 + 3.1 4 237 + 13.9 217 + 2.0 4 239 8.0 225 + 3.9 5 108 + 13.0 75 -i. 7.6 3 169 + 2.6 105 + 6.8 4 TABLE VI CALCULATION OF SODIUM CONCENTRATION OF GUT ABSORBATE e x p l a n a t i o n i n t e x t (page 4 2 ) . TABLE VI Meal Sodium absorbed Volume of [Na+] (a) ^ Ueq (b) yUeq absorbate absorbate ( m i c r o l i t e r s ) m e q / l i t e r h.b. 26.896 19.786 172 115-156 h.b. + NaCl 22.087 22.596 94 235-261 h.b. + g. 14.831 15.080 64 232-236 h.b./D.W. 2.730 3.429 71 38-48 h.b./g. 50/50 4.604 4.568 42 108.5-109.5 -47-TABLE 711 POTASSIUM'CONCENTRATIONS IN BODY FLUIDS  DURING AND AFTER FEEDING T o t a l and f i n a l v a l u e s r e f e r t o time i n t e r v a l of two hours from s t a r t of f e e d i n g . Those v a l u e s which were measured d i r e c t l y are presented as the mean i S E w i t h the e x c e p t i o n of the i n g e s t e d f l u i d . T h i s i s a s i n g l e v a l ue s i n c e the same batch of blood was used f o r the e n t i r e experiment. The value f o r gut absorbate was c a l c u l a t e d and hence does not bear a standard e r r o r . A l l c o n c e n t r a t i o n s are expressed as m e q / l i t e r . The v a l u e f o r mean gut absorbate was computed from the equations on page 42. The c a l c u l a t i o n s are shown i n Table V I I . TABLE V I I I CALCULATION OF POTASSIUM CONCENTRATION  OF GUT ABSORBATE See e x p l a n a t i o n i n t e x t (page 42 ). •See page 49. TABLE VII Meal Ingested f l u i d P i n a l gut contents Mean gut F i n a l Mean N C e n t r i f u g e d Whole C e n t r i f u g e d Whole Absorbate hemo- Coxal lymph f l u i d h.b. 4.2 41 52 ± 1.6 61 .5 ± 2.9 4.4 5.0 ± 0 5.0 ± 0.8 4 TABLE V I I I Meal Potassium absorbed Volume o f (a) yUeq (b) yueq Absorbate absorbate ( m i c r o l i t e r s ) m e q / l i t e r h.b. 2.680 0.755 172 4.4 - (?) -48-o i H = [ c i ] H .V o 0 0 c l o - c . v c where Clrr = c h l o r i d e i n hemolymph two hours a f t e r r e a d i n g Clpj = c h l o r i d e i n hemolymph before f e e d i n g o C l + c h l o r i d e e x c r e t e d i n c o x a l f l u i d [Cl] o = c h l o r i d e c o n c e n t r a t i o n i n hemolymph 2 h r . 2 a f t e r f e e d i n g [Cl] TT = c h l o r i d e c o n c e n t r a t i o n i n hemolymph before o f e e d i n g [Cl] = c h l o r i d e c o n c e n t r a t i o n i n pooled c o x a l f l u i d VTT = hemolymph volume 2 h r . a f t e r f e e d i n g n 2 = hemolymph volume before f e e d i n g o V = t o t a l c o x a l f l u i d produced. For t i c k s f e d h.b. (Tables I and I I I ) : C 1 A = (170 x 58) - (200 x 15) + (117 x 129) = 21 .953 /aeq (b) These two independent methods of c a l c u l a t i o n agreed r e a s o n a b l y w e l l and thus p r o v i d e d an estimated range and a check on the experimental e r r o r . T h i s a l s o v a l i d a t e d the assumption t h a t any change i n the i o n content of the t i s s u e was n e g l i g i b l e . The volume of f l u i d p a s s i n g across the gut had p r e v i o u s l y been c a l c u l a t e d (Table I ) ; t h e r e f o r e the c o n c e n t r a t i o n o f f l u i d absorbed across the gut could be found. I n the example taken above ( a ) , the c h l o r i d e concen-t r a t i o n of the absorbate i s : [Cl] A = 20.160 = 117 m e q / l i t e r 172 The v a l u e s f o r t o t a l i o n s absorbed across the gut are shown i n Tables IV, VI and V I I I . -49-I t w i l l be n o t i c e d i n the case of potassium (Table V I I I ) t h a t the two v a l u e s (a) and (b) are not i n good agreement. Sin c e (a) r e s u l t e d from a s m a l l d i f f e r e n c e between two very l a r g e numbers ( (a) = 14500 - 11820 = 2680), i t was decided t h a t ( b ) , being computed from the a d d i t i o n and s u b t r a c t i o n of s m a l l numbers, was l i k e l y t o be the more accurate e s t i m a t e . The f i r s t q u e s t i o n t h a t might be d i r e c t e d t o t h i s data concerns the a b i l i t y of the t i c k t o r e g u l a t e the i o n i c c o n c e n t r a t i o n i n the hemolymph when f e d meals which vary g r e a t l y i n t h e i r i o n c o n t e n t . To t h i s end c h l o r i d e and sodium c o n c e n t r a t i o n s i n the hemolymph were p l o t t e d a g a i n s t c h l o r i d e and sodium c o n c e n t r a t i o n s i n the blood meal ( P i g s . 5 and 6 ) . A p p a r e n t l y r e g u l a t i o n of the i o n concen-t r a t i o n i n the hemolymph c o u l d o n l y occur i f the osmotic p r e s s u r e of the meal remained c o n s t a n t . I f the osmotic p r e s -sure of the b l o o d meal was i n c r e a s e d a r i s e i n the sodium c h l o r i d e c o n c e n t r a t i o n s i n the hemolymph r e s u l t e d i r r e s p e c -t i v e of whether the i n c r e a s e d osmotic pressure was due t o sodium c h l o r i d e or g l u c o s e . Conversely, when the osmotic pressure was reduced but the sodium c h l o r i d e c o n c e n t r a t i o n i n the b l o o d meal was kept constant a f a l l i n the sodium c h l o r i d e c o n c e n t r a t i o n of the hemolymph r e s u l t e d . The volume of f l u i d absorbed across the gut w a l l was shown t o be s e v e r a l times the hemolymph volume (Table I ) . C l e a r l y then, one must c o n s i d e r the absorbate and not the i n g e s t e d f l u i d when d i s c u s s i n g p e r t u r b a t i o n s of the hemo-- 5 0 -FIGURE 5 REGULATION OF CHLORIDE CONCENTRATION IN HEMOLYMPH Samples were taken two hours a f t e r feeding. Standard errors may be found i n Table I I I . Broken l i n e i s that gradient f o r equal concentrations. Legend: O Blood meal 100$ osmotic pressure of h .b . (Ticks fed h . b . , h .b . / g .50 /50 and h .b. /g .25 /75 6 Blood meal 158$ osmotic pressure of h.b. (Ticks fed h.b. + NaCl and h.b. + g. A Blood meal 50$ osmotic pressure of h.b. (Ticks fed h.b./D.W.) [ci ] blood meal (meq/l i ter) - 5 1 -FIGURE 6 REGULATION OF SODIUM CONCENTRATION IN HEMOLYMPH Samples were taken two hours a f t e r feeding. Standard e r r o r s may be found i n Table V. Broken l i n e i s that gradient f o r equal concentrations. Legend: 9 Blood meal 100$ osmotic pressure of h.b. (Ticks fed h.b., h.b./g.50/50 and h.b./g .25/75) O Blood meal 158$ osmotic pressure of h.b. (Ticks fed h.b. + NaCl and h.b. + g.) A Blood meal 50$ osmotic pressure of h.b. (Ti c k s fed h.b./D.W. 2 4 0 -i 2 2 0 H o -O , / 2 0 0 cr E 180 H 160 140 - \ 120 100 80 • : ' " f T 1 1 - i 1 1 1 1 20 4 0 6 0 80 100 120 140 160 180 2 0 0 2 2 0 240 [Na +] blood meal (meq/liter) - 5 2 -lymph. F i g u r e 3 shows the c o x a l gland t o have c o n s i d e r a b l e r e g u l a t o r y a b i l i t y w i t h r e s p e c t t o volume. An i n d i c a t i o n o f the i o n i c r e g u l a t i o n performed by t h i s gland c o u l d be s i m i l a r l y found by a comparison o f the i o n i c c o n c e n t r a t i o n s o f gut absorbate t o those of c o x a l f l u i d under d i f f e r e n t f e e d i n g regimes. The c o n c e n t r a t i o n s o f sodium and c h l o r i d e i n the two f l u i d s were found to be equal over a s i x f o l d range i n absorbate c o n c e n t r a t i o n ( F i g s . 7 and 8 ) . Note t h a t the t o t a l i o n s t r a n s f e r r e d from gut t o c o x a l f l u i d exceeded by s e v e r a l times the t o t a l i o n content of the hemo-lymph, although the i o n c o n c e n t r a t i o n o f the c o x a l f l u i d was lower than t h a t of the hemolymph (p. 4-7). These r e s u l t s showed the c o x a l gland t o be a powerful r e g u l a t o r y organ f o r i o n s . As might be expected f r o m ' F i g s . , 3 , 7 and.8, the t o t a l sodium c h l o r i d e t r a n s f e r r e d across the gut was equal t o the t o t a l sodium c h l o r i d e e x c r e t e d i n the c o x a l f l u i d p l u s the i n c r e a s e d sodium c h l o r i d e i n the hemolymph ( F i g s . 9 and 10 ) . T h i s a l s o i n d i c a t e d t h a t any i n c r e a s e i n t i s s u e i o n content d u r i n g f e e d i n g was n e g l i g i b l e . I n o r d e r t o g a i n some i n s i g h t i n t o the mechanisms i n v o l v e d i n the t r a n s f e r of these i o n s across the gut w a l l , the r e l a t i o n s h i p between t o t a l c h l o r i d e and t o t a l sodium absorbed was p l o t t e d ( F i g . 1 1 ). C l e a r l y c h l o r i d e and sodium were t r a n s p o r t e d across the gut i n equal amounts d e s p i t e a t e n f o l d v a r i a t i o n i n the t o t a l i o n s t r a n s p o r t e d and a s i x f o l d v a r i a t i o n i n the i o n c o n c e n t r a t i o n s o f the blood meals. - 5 3 -FIGURE 7 RELATION BETWEEN MEAN CHLORIDE CONCENTRATIONS OF GUT  ABSORBATE AND COXAL FLUID L i m i t s shown are from Table I I I . Broken l i n e i s t h a t g r a d i e n t f o r equal c o n c e n t r a t i o n s . Measurements were made over a 2 hour experimental p e r i o d . 240 -i 200 i / / *~ 160 H / ' / / 120 H / / / 80 H / — I / / 40 H / / / V  ~~I I 1 1 , 1 , 0 40 80 120 160 200 [cr] gut absorbate (meq/liter) -54-FIGURE 8 RELATION BETWEEN MEAN SODIUM CONCENTRATIONS OF GUT  ABSORBATE AND COXAL FLUID L i m i t s shown are from Table V. Broken l i n e i s t h a t g r a d i e n t f o r equal c o n c e n t r a t i o n s . Measurements were made over a 2 hour experimental p e r i o d . 240 200 -\ 160 120 H 80 H 40 H 1 I I 1 1 — 40 80 120 160 200 [Na+] gut absorbate (meq/liter) -55-FIGURE 9 RELATION BETWEEN TOTAL CHLORIDE ABSORBED ACROSS GUT WALL  AND TOTAL CHLORIDE EXCRETED IN COXAL FLUID PLUS INCREASE  IN HEMOLYMPH CHLORIDE DURING TWO HOUR EXPERIMENTAL PERIOD L i m i t s shown are from Table IV. Broken l i n e i s t h a t g r a d i e n t f o r equal q u a n t i t i e s o f c h l o r i d e . 2 4 -i Q. E C7 >» <u O 5, E ~ •=1 OT ro a> i u c OT 3 2 0 H 16 H 12 X I ' 3 8H to X O E co 4 H 12 16 2 0 2 4 2 8 Total Cl absorbed across gut (jueq) -56-FIGURE 10 RELATION BETWEEN TOTAL SODIUM ABSORBED ACROSS GUT WALL  AND TOTAL SODIUM EXCRETED IN COXAL FLUID PLUS INCREASE  IN HEMOLYMPH SODIUM DURING TWO HOUR EXPERIMENTAL PERIOD L i m i t s shown are from Table V I . ' Broken l i n e i s t h a t g r a d i e n t f o r equal q u a n t i t i e s o f sodium. 24 - i J C a . E >> mol eq) he =, ed + tn to a> V c tn _ 3 Q . + 1 0 Z X I [ 3 •— " r o X O 0 0 E 3 co 20 -16 12 8 -4 ~r 8 1 12 16 1— 20 24 I 28 Total Na absorbed across gut (jueq) - 5 7 -FIGURE 11 RELATION BETWEEN TOTAL CHLORIDE AND TOTAL SODIUM ABSORBED ACROSS GUT WALL DURING TWO HOUR EXPERIMENTAL PERIOD The v e r t i c a l l i m i t s are from Table IV and the h o r i z o n t a l l i m i t s from Table V I . The broken l i n e i s t h a t g r a d i e n t f o r equal q u a n t i t i e s o f sodium and c h l o r i d e . c r o> a. tn tn o i _ o <D X I <L> Si L— o tn Si to ro o 28 n 24 20 16 H 12-^  4 H 0 + 1 ' I 8 12 16 20 24 28 Total Na absorbed across gut (jueq) -58-3. Balance sheet f o r t o t a l body c h l o r i d e A back check on the v a l u e s p r e v i o u s l y obtained f o r the volumes of body f l u i d s , was made by two i n d e -pendent measurements of the f a c t o r by which the blood meal was c o n c e n t r a t e d i n the gut (Table I I ) . S i m i l a r l y I v e r i f i e d the c h l o r i d e d e t e r m i n a t i o n s by measuring d i r e c t l y the i n c r e a s e i n t o t a l body c h l o r i d e over the 2 hour ex-p e r i m e n t a l p e r i o d . This v a l u e c o u l d then be compared w i t h the estimate c a l c u l a t e d by s u b t r a c t i n g the c h l o r i d e ex-c r e t e d i n the c o x a l f l u i d from the t o t a l c h l o r i d e i n g e s t e d (Table I X ) . These f i g u r e s were judged t o be i n reasonable agreement. I t was suggested p r e v i o u s l y t h a t t i s s u e c h l o r i d e d i d not i n c r e a s e d u r i n g the experimental p e r i o d . Table IX i n d i c a t e d t h a t t h i s t i s s u e c h l o r i d e space must a l s o be r e l a t i v e l y s m a l l s i n c e most of the body c h l o r i d e can be accounted f o r by the gut and hemolymph con t e n t . 4. Time sequence of i o n c o n c e n t r a t i o n s i n body  f l u i d s d u r i n g c o x a l f l u i d p r o d u c t i o n Thus f a r , i o n c o n c e n t r a t i o n s had been r e p o r t e d o n l y f o r c o x a l f l u i d c o l l e c t e d and pooled over the e n t i r e e x c r e t o r y p e r i o d . I n or d e r t o understand more f u l l y the r e l a t i o n s h i p between c o n d i t i o n s i n the gut, hemolymph and c o x a l f l u i d , I decided t o measure the i o n c o n c e n t r a t i o n s i n each f l u i d w i t h time ( P i g s . 12 and 13). The c h l o r i d e TABLE IX INCREASE IN TOTAL BODY CHLORIDE (ME.AH - SE) T o t a l body c h l o r i d e was measured before f e e d i n g and 2 h r . a f t e r f e e d i n g . TABLE IX Meal D i r e c t l y measured Calculated value Queq) value (yueq) h.b. 18.2 ± 0.2 19.8 (N •= 5) . h.b./g. 50/50 5.5 1 0.3 (N = 4) 7.3 - 6 0 -FI GURE 12 TYPICAL TIME SEQUENCE STUDY OF CHLORIDE CONCENTRATION  IN COXAL FLUID FROM A TICK FED HUMAN BLOOD Legend: A T i c k was removed from membrane and c o x a l s e c r e t i o n stopped B T i c k r e a t t a c h e d and s t a r t e d to produce c o x a l f l u i d FIGURE 13 SOME TYPICAL TIME SEQUENCES OF CHLORIDE CONCENTRATIONS  IN THE COXAL FLUID AND HEMOLYMPH FROM THREE TICKS FED  HUMAN BLOOD Legend: hemolymph c o x a l f l u i d t time when c o x a l f l u i d appeared -62-c o n c e n t r a t i o n i n the hemolymph f e l l from an i n i t i a l h i g h v a l u e to a minimum; about t h i s time c o x a l f l u i d f i r s t appeared. T h i s minimum value was shown to be s i g n i f i c a n t l y d i f f e r e n t (95$ c . l . ) from the f i n a l e q u i l i b r i u m l e v e l a f t e r c o x a l f l u i d p r o d u c t i o n had ceased (Table X ) . The f i r s t drops of c o x a l f l u i d always showed a s i g -n i f i c a n t l y lower c h l o r i d e c o n c e n t r a t i o n than the mean value (95$ c . L ) under a v a r i e t y of f e e d i n g c o n d i t i o n s . However, sodium c o n c e n t r a t i o n s were not s i g n i f i c a n t l y d i f f e r e n t but potassium was s i g n i f i c a n t l y h i g h e r i n the f i r s t few drops (Table X I ) . To complete the p i c t u r e of c o n d i t i o n s at the time when c o x a l f l u i d f i r s t appeared, some t i c k s were s a c r i f i c e d at t h a t i n s t a n t and gut samples immediately taken. C h l o r i d e c o n c e n t r a t i o n s were measured on the whole sample and found i n the two i s o s m o t i c blood meals t o be not s i g n i f i c a n t l y d i f f e r e n t (95$ c . l . ) from the v a l u e s 2 hours a f t e r f e e d i n g (Table X I I ) . T h i s suggested t h a t the l a r g e g r a d i e n t a g a i n s t which c h l o r i d e was absorbed from the gut was f a i r l y constant d u r i n g the main a b s o r p t i o n p e r i o d ( i . e . d u r i n g c o x a l f l u i d p r o d u c t i o n ) . However, the gut f l u i d of t i c k s f e d h.b. + NaCl ( i . e . w i t h i n c r e a s e d i o n i c and osmotic c o n c e n t r a t i o n s ) , showed s i g n i f i c a n t l y h i g h e r c h l o r i d e c o n c e n t r a t i o n s at the s t a r t of c o x a l f l u i d p r o d u c t i o n compared w i t h the e q u i l -i b r i u m v a l u e . TABLE X CHLORIDE CONCENTRATION IN HEMOLYMPH WHEN COXAL FLUID  FIRST APPEARED AND AFTER COXAL FLUID PRODUCTION HAD  CEASED (MEAN - S E) A l l c o n c e n t r a t i o n s are expressed as m e q / l i t e r . Legend: a . c . f . p . at time when c o x a l f l u i d appeared e . c . f . p . two hours a f t e r f e e d i n g TABLE X Meal [Cl J in hemolymph a.c.f.p. e.c.f.p. h.b. 155 + 1.4 170 + 2.0 (N 9) (N = 6) h.b. + NaCl 190 + 0.3 239 8.7 (N = 6) (N = 5) h.b./g. 50/50 153 + 1.0 173 + 3.0 (N = 9) (N = 5) h.b./g. 25 /75 145 + 2.2 172 JL 2.9 (N = 9) (N = I n i t i a l [ci~] i n hemolymph 200 - 2.0 m e q / l i t e r . TABLE XI ION CONCENTRATIONS IN INITIAL DROPS OF COXAL FLUID  AND IN SAMPLES POOLED OVER SECRETORY PERIOD (MEAN t SE) A l l c o n c e n t r a t i o n s are expressed as m e q / l i t e r . TABLE XI Meal C h l o r i d e c o n c e n t r a t i o n Sodium c o n c e n t r a t i o n Potassium c o n c e n t r a t i o n I n i t i a l drops Mean I n i t i a l drops Mean I n i t i a l drops Mean h.b. 68 ± 7.3 (N = 8) h.b. + NaCl 132 ± 19 (N = 5) h.b. + g 65 ± 1.4 (N = 3) h.b./D.W. 16.0 - 5.7 (N = 5) 117 - 5.3 (N = 6) 217 1 3.0 CN = 5) 187 ± 5.3 (N = 5) 51 1 2.5 (N = 4) 112 ± 4.8 (N = 4) 111 ± 3.1 20 - 2.3 (N = 4 ) (N = 4) 5.0 - 0.8 (N = 4) TABLE X I I CPILORIDE CONCENTRATION IN WHOLE GUT FLUID WHEN COXAL  FLUID FIRST APPEARED AND AFTER COXAL FLUID PRODUCTION  HAD CEASED (MEAN ± S E) A l l c o n c e n t r a t i o n s are expressed as m e q / l i t e r . Legend: a.c.f.p . e . c . f . p . at time when c o x a l f l u i d appeared two hours a f t e r f e e d i n g . TABLE X I I Meal [Cl~] i n gut f l u i d a.c.f.p. e.c.f.p. h.b. 75 1 1.2 77 1 2.0 (N=9) (N*6) h.b. + NaCl 113 - 0.5 88 ± 5.7 (H-6) ' (N=5) h.b./g. 50/50 37 i 0.5 37 i 0.4 (N=9) (N=5) -66-5. Rate o f c o x a l f l u i d p r o d u c t i o n Measurements were made on r a t e s o f c o x a l f l u i d p r o -d u c t i o n when t i c k s were f e d b l o o d meals o f v a r i o u s com-p o s i t i o n . T h i s , I hoped, would y i e l d i n f o r m a t i o n on the f a c t o r s c o n t r o l l i n g c o x a l e x c r e t i o n . I n the i n i t i a l stages o f t h i s r e s e a r c h when ex-c e p t i o n a l l y l a r g e t i c k s ( 70 mg) were f e d d i r e c t l y on c h i c k e n s , r a t e s o f c o x a l f l u i d p r o d u c t i o n as h i g h as s i x m i c r o l i t e r s p e r minute were recorded. However, the f o l l o w -i n g d a t a were c o l l e c t e d u s i n g t i c k s which i n i t i a l l y weighed between 30 and 50 mg. The h i g h e s t r a t e o f p r o d u c t i o n of c o x a l f l u i d was seen i n i n i t i a l f l o w from t i c k s f e d h.b. T h i s was 3.3 m i c r o l i t e r s p e r minute but i t f e l l s t e a d i l y throughout t he e x c r e t o r y p e r i o d ( F i g . 14a). W i t h i n any s e r i e s o f i s o s m o t i c meals, the i n i t i a l and average r a t e s o f c o x a l f l u i d p r o -d u c t i o n f e l l w i t h d e c r e a s i n g c h l o r i d e c o n c e n t r a t i o n i n the b l o o d meal. The p e r i o d of e x c r e t i o n was unchanged at 60-80 minutes f o r meals i s o s m o t i c w i t h h.b. ( F i g s . 14a, 15b and 15c ) . When the osmotic pr e s s u r e of the blo o d meal was i n c r e a s e d , i n i t i a l and average r a t e s o f c o x a l f l u i d p r o d u c t i o n decreased r e g a r d l e s s of the s o l u t e added. How-ever, at any time d u r i n g the e x c r e t o r y p e r i o d , the r a t e was h i g h e r f o r t i c k s f e d b l o o d meals w i t h added sodium c h l o r i d e r a t h e r than glucose ( F i g s . 14b and 1 4 c ) . Those t i c k s which were f e d b l o o d meals c o n t a i n i n g 50$ c h l o r i d e FIGURE 14 RATE AND ACCUMULATIVE VOLUME OF COXAL FLUID PRODUCTION a) T i c k s f e d h.b. (N=6) b) T i c k s f e d h.b. + NaCl (N=5) c) T i c k s f e d h.b. + g. (N=5) V e r t i c a l l i m i t s show Mean - SE Legend: accumulative volume r a t e of c o x a l f l u i d p r o d u c t i o n Accumulative volume (X.) Rate of production ( X / m i n ) -68-FIGURE 15 RATE AND ACCUMULATIVE VOLUME OF COXAL FLUID PRODUCTION a) T i c k s f e d h.b./D.W. b) T i c k s f e d h.b./g. 50/50 c) T i c k s f ed h.b./g. 25/75 V e r t i c a l l i m i t s show Mean - SE Legend: accumulative volume r a t e of c o x a l f l u i d p r o d u c t i o n Time ( m i n s after coxal f luid a p p e a r e d ) -69-but d i f f e r i n g osmotic p r e s s u r e s (h.b./D.W. and h.b./g 50/50) showed the same average r a t e of c o x a l f l u i d p r o -d u c t i o n . However, the i n i t i a l r a t e s and time courses of p r o d u c t i o n were q u i t e d i f f e r e n t ( F i g s . 15a and 15h). The s h o r t e s t time of c o x a l f l u i d p r o d u c t i o n was observed when the blood meal was made hyperosmotic w i t h g l u c o s e , a molecule t h a t might be expected t o move more s l o w l y across the gut w a l l . The l o n g e s t time r e s u l t e d when t i c k s were fe d meals made hypo-osmotic w i t h d i s t i l l e d water ( F i g s . 14c and 15a). 6. Osmotic p r e s s u r e s of body f l u i d s One o f the aims of t h i s r e s e a r c h was t o e l u c i d a t e the nature of the f o r c e behind the movement of i o n s and water between compartments i n the t i c k ' s body. S i n c e , as was d i s c u s s e d e a r l i e r , osmotic-pressure g r a d i e n t s are o f t e n i m p l i c a t e d , these were measured d u r i n g and a f t e r the move-ment of water across the gut. A f t e r c o x a l f l u i d p r o d u c t i o n had ceased, the osmotic pressure of the hemolymph was not found t o be s i g n i f i c a n t l y d i f f e r e n t from t h a t o f the gut (Table X I I I ) . F i g u r e 16 shows t h a t t h e r e was a d i r e c t r e l a t i o n s h i p between the f i n a l hemolymph osmotic p r e s s u r e and t h a t of the i n g e s t e d f l u i d . I f the osmotic c o n c e n t r a t i o n of the meal was kept constant (e.g. = 0 .55°0) an i n c r e a s e i n sodium c h l o r i d e c o n c e n t r a t i o n i n the blood meal caused an i n c r e a s e i n the f i n a l hemolymph -70-TABLE X I I I OSMOTIC PRESSURES OF GUT FLUID AND HEMOLYMPH TWO  HOURS AFTER FEEDING (MEAN - SE) Osmotic p r e s s u r e s are expressed as f r e e z i n g - p o i n t de-p r e s s i o n i n °C. The v a l u e s f o r i n g e s t e d f l u i d are not presented w i t h standard e r r o r s s i n c e o n l y the osmotic pressure of h.b. was measured. The v a l u e s f o r the o t h e r meals were c a l -c u l a t e d from t h i s . Osmotic pressure ingested fluid ( A ° C ) -71-FIGURE 16 RELATION BETWEEN OSMOTIC PRESSURES OF INGESTED FLUID  AND HEMOLYMPH Samples were taken two hours a f t e r f e e d i n g . V e r t i c a l l i m i t s show Mean - SE Legend: Blood meal plasma c o n t a i n s : A 162 m e q / l i t e r c h l o r i d e , 223 m e q / l i t e r sodium (h.b. + NaCl) • 116 m e q / l i t e r c h l o r i d e , 177 m e q / l i t e r sodium (h.b. and h.b. + g.) O 4-5 m e q / l i t e r c h l o r i d e , 62 m e q / l i t e r sodium (h.b./g.50/50 and h.b./D.W.) • 2 l m e q / l i t e r c h l o r i d e , 31 m e q / l i t e r sodium (h.b./g.25/75) TABLE X I I I Meal Ingested f l u i d Gut f l u i d Hemolymph h.b. 0.55 0.89 + 0.02 0.85 + 0.01 (N = 5) (N 5) h.b. + NaCl 0.87 0.85 + 0.02 0.98 + 0.05 (N 4-) (N = 5) h.b. + g 0.87 0.87 0.02 0.88 + 0.05 - (N 4-) (N = 3) h.b./D.W. 0.26 0.4-0 + 0.02 0 . 5 0 + 0.02 (N = 4-) (N = 4-) h.b./g/ 50/50 0.55 0.67 + 0 . 0 1 (N 4-) h.b./g. 2 5 / 7 5 0.55 0.45 + 0.03 0.54- + 0.03 (N = 4) (N 3) -72-osraotic p r e s s u r e . The osmotic pressure of the hemolymph was not measured f o r the h.b./g. 50/50 meal; i t was assumed t o be equal t o t h a t of the gut f l u i d s i n c e Bone (194-3) found i t t o be so and s i n c e t h i s was the case i n a l l other f e e d -i n g c o n d i t i o n s . The f r e e z i n g p o i n t d e p r e s s i o n of pooled c o x a l f l u i d from t i c k s f e d h.b. was 0.59 - 0.02°C (Mean i SE, N = 5); t h i s was c o n s i d e r a b l y lower than the osmotic pressure of the hemolymph (Table X I I I ) . The f i r s t few drops of c o x a l f l u i d had a s t i l l lower osmotic pressure of 0.4-3 - 0.03 (Mean i SE, N = 5); t h i s was a n t i c i p a t e d from the data on c h l o r i d e c o n c e n t r a t i o n s (Table X I ) . The mean osmotic p r e s -sure of the c o x a l f l u i d approximately e q u a l l e d t h a t of the i n g e s t e d f l u i d . That i s , the t i c k r e g u l a t e d the osmotic pre s s u r e of i t s hemolymph by v o i d i n g a s o l u t i o n having the same osmotic pressure as t h a t of the s o l u t i o n i t was i n -g e s t i n g , when f e d h.b. Some measurements of osmotic pressures of gut f l u i d and hemolymph were made d u r i n g the p r o d u c t i o n of c o x a l f l u i d . F i g u r e 16 shows t h a t an i n i t i a l drop i n the hemolymph os-motic pressure preceded the 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 . T h i s suggests t h a t the gut absorbate i s hypo-osmotic w i t h r e s p e c t t o the hemolymph and t h a t the i n c r e a s e i n hemolymph volume d u r i n g f e e d i n g , which was r e p o r t e d e a r l i e r i n t h i s t h e s i s , might occur at t h i s t i m e . I f t h i s i s so, the r a t e of p r o d u c t i o n and osmotic c o n c e n t r a t i o n of the c o x a l f l u i d p robably r e f l e c t the r a t e of a b s o r p t i o n and - 7 3 -osmotic c o n c e n t r a t i o n of f l u i d absorbed across the gut. Fi g u r e s 17 and 18 show t h a t as the osmotic p r e s s u r e s of gut f l u i d and hemolymph approached each o t h e r , the r a t e of c o x a l f l u i d p r o d u c t i o n f e l l . T h i s r e s u l t suggests t h a t the r a t e of a b s o r p t i o n of f l u i d across the gut p a r -a l l e l s the osmotic-pressure g r a d i e n t , assuming t h a t the r a t e of c o x a l f l u i d p r o d u c t i o n r e f l e c t e d the r a t e of a b s o r p t i o n a c r o s s the gut. A f u r t h e r e f f o r t was made t o determine whether the r a t e of f l o w of absorbate across the gut was r e l a t e d t o the osmotic p r e s s u r e g r a d i e n t across the gut w a l l . I n the two Cases s t u d i e d , the osmotic pressure of the hemolymph f e l l r a p i d l y a f t e r f e e d i n g s t a r t e d ( F i g s . 17 and 18). I t was a l s o shown t h a t the c o n c e n t r a t i o n of c h l o r i d e i n the hemp-lymph e x h i b i t e d an i n i t i a l r a p i d f a l l to i t s f i n a l v a l u e under the f o u r f e e d i n g c o n d i t i o n s s t u d i e d (Table X ) . Since the sodium and c h l o r i d e c o n c e n t r a t i o n s i n the hemolymph accounted f o r 70$ of the observed osmotic p r e s s u r e , the assumption was made t h a t the osmotic p r e s s u r e o f the hemo-lymph recorded a f t e r c o x a l f l u i d p r o d u c t i o n has ceased, was t h a t v alue e x i s t i n g through most of the e x c r e t o r y p e r i o d . Gut f l u i d c o u l d not be t r e a t e d i n t h i s manner s i n c e the l i m i t e d data d i d suggest t h a t the two hour p o s t - p r a n d i a l v a l u e was a t t a i n e d more s l o w l y f o r gut f l u i d than f o r hemo-lymph. Thus the range of i n i t i a l t o f i n a l osmotic pressures was c o n s i d e r e d f o r the gut i n p l o t t i n g F i g . 19. T h i s t r e a t --74-FIGURE 17 RELATION BETWEEN OSMOTIC PRESSURES OF GUT FLUID AND HEMO-LYMPH AND RATE OF COXAL FLUID PRODUCTION (TICKS FED h.b.) Osmotic p r e s s u r e s are expressed as f r e e z i n g - p o i n t de-p r e s s i o n i n . °C. V e r t i c a l l i m i t s show Mean - SE. Legend: • hemolymph O gut f l u i d r a t e o f c o x a l f l u i d p r o d u c t i o n (from F i g . 14) t time at which c o x a l f l u i d appeared Osmot ic pressure ( A ° C ) o o p o o — Cn O N 00 , <> O 7- ' rO CO .U Cn o b b b o Rate of coxal fluid production ( A / m i n ) - 7 5 -FIGURE 18 RELATION BETWEEN OSMOTIC PRESSURES OF GUT FLUID AND HEMO- LYMPH AND RATE OF COXAL FLUID PRODUCTION (TICKS FED ' .." , . h.b. + NaCl) Osmotic p r e s s u r e s are expressed as f r e e z i n g - p o i n t d e p r e s s i o n i n °C. V e r t i c a l l i m i t s show Mean - SE. Legend: O hemolymph O gut f l u i d r a t e o f c o x a l f l u i d p r o d u c t i o n (from F i g . 14-) t time at which c o x a l f l u i d appeared. Time (min after start of feeding) -76-FIGURE 19 RELATION BETWEEN OSMOTIC DIFFERENCE ACROSS GUT WALL AND  RATE OF FLOW OF GUT ABSORBATE Osmotic p r e s s u r e s are expressed as f r e e z i n g - p o i n t de-p r e s s i o n i n °C. Arrows extend from i n i t i a l t o f i n a l v a l u e s o f osmotic g r a d i e n t a c r o s s gut. Rate o f a b s o r p t i o n i s mean f o r two hour experimental p e r i o d . Osmotic gradient ( A ° C ) p _ L _ O _ l _ p rO _ l _ O c o o _ l o Cn Cn ^ Cn -77-ment o f the data does not i n d i c a t e t h a t the r a t e of ab-s o r p t i o n a c r o s s the gut was s o l e l y i n f l u e n c e d by the magnitude o f the osmotic d i f f e r e n c e between gut f l u i d and hemolymph. 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 across the gut Both i o n i c c o n c e n t r a t i o n s and osmotic p r e s s u r e s o f hemolymph and gut f l u i d appeared t o i n f l u e n c e the passage o f i o n s and water acr o s s the gut w a l l . To see whether the movement o f i o n s was a c t i v e o r p a s s i v e , I measured 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 a c r o s s the gut w a l l . T i c k s were f e d a b l o o d meal i n the u s u a l e x p e r i -mental manner and, a f t e r c o x a l f l u i d p r o d u c t i o n had ceased, the p o t e n t i a l a c r o s s the gut w a l l was measured. I n a l l f i v e . c a s e s when t i c k s were f e d h.b., a r e p e a t a b l e p o t e n t i a l o f between 14 and lt5 mv was measured; the gut lumen was ne g a t i v e w i t h r e s p e c t t o the hemolymph. When t i c k s were fe d h.b./g.25/75 an e q u a l l y r e p r o d u c i b l e p o t e n t i a l o f between 14 and 16 mv was recorded but under these f e e d i n g c o n d i t i o n s , t h e gut lumen was p o s i t i v e w i t h r e s p e c t t o the hemolymph ( i . e . the p o t e n t i a l was now r e v e r s e d ) . D. DISCUSSION T i c k s were f e d blood meals w i t h sodium c h l o r i d e c o n c e n t r a t i o n s r a n g i n g from 31 t o 223 m e q / l i t e r sodium and 21 t o 162 m e q / l i t e r c h l o r i d e . The osmotic p r e s s u r e s v a r i e d from 0.26 t o 0.87°C. de p r e s s i o n of f r e e z i n g p o i n t . Despite these v a r i a t i o n s ( 8 - f o l d f o r i o n c o n c e n t r a t i o n s and 3 . 5 - f o l d f o r osmotic p r e s s u r e s ) the volume of blood meal i n g e s t e d showed a l e s s than t w o f o l d v a r i a t i o n . This sug-gest s t h a t i n the osmotic and i o n i c ranges s t u d i e d , the t i c k had l i t t l e a b i l i t y t o d i s t i n g u i s h the d i f f e r e n t con-d i t i o n s o r , i f i t c o u l d d i s t i n g u i s h them, i t showed no c l e a r p a t t e r n of a v e r s i o n t o s p e c i f i c meals. However, no blood meals having i o n i c or osmotic v a l u e s h i g h e r than those of the i n i t i a l t i c k ' s hemolymph were s t u d i e d due to t e c h n i c a l d i f f i c u l t i e s of b l o o d p r o t e i n s p r e c i p i t a t i n g out. The r e s u l t s i n d i c a t e t h a t the t i c k would have no r e g u l a t o r y c a p a b i l i t y f o r such a meal and i t i s p o s s i b l e t h a t engorge-ment would not proceed. Tables I I I and V show t h a t sodium and c h l o r i d e i o n s move from gut t o hemolymph a g a i n s t 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 g r a d i e n t s under some f e e d i n g c o n d i t i o n s . Since these con-c e n t r a t i o n s were measured under e q u i l i b r i u m c o n d i t i o n s a f t e r water movement across the gut had ceased (e v i d e n t -79-from the c e s s 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 ) , measure-ments of e l e c t r o p o t e n t i a l g r a d i e n t s across the gut e p i t h e l i u m were made under the same c o n d i t i o n s . Thus evidence c o u l d be gathered f o r the presence or absence of a c t i v e t r a n s p o r t of these i o n s . When t i c k s were f e d h.b./g. 25/75, c h r o r i d e moved i n t o the hemolymph a g a i n s t an e i g h t f o l d c o n c e n t r a t i o n g r a d i e n t and a g a i n s t an e l e c t r o -p o t e n t i a l g r a d i e n t of 15 mv. Moreover, a l a r g e concen-t r a t i o n g r a d i e n t e x i s t e d throughout the a b s o r p t i o n p e r i o d (Tables X and X I I ) . This i s s t r o n g evidence f o r a c t i v e t r a n s p o r t of c h l o r i d e a c r o s s the gut w a l l . When t i c k s were f e d h.b. the e l e c t r o p o t e n t i a l g r a d i e n t a c r o s s the gut w a l l was 15 mv (gut n e g a t i v e ) and the sodium c o n c e n t r a t i o n i n the gut f l u i d (2 h r . a f t e r f e e d i n g ) was 197 m e q / l i t e r . A c c o r d i n g t o the Nernst equation the hemolymph sodium c o n c e n t r a t i o n would be 110 m e q / l i t e r i f the i o n s were p a s s i v e l y d i s t r i b u t e d along the e l e c t r o -p o t e n t i a l g r a d i e n t . But the measured hemolymph concen-t r a t i o n was 154 m e q / l i t e r . This suggests t h a t sodium i o n s were be i n g pumped a g a i n s t the e l e c t r o p o t e n t i a l g r a d i e n t i n t o the hemolymph. I n d i r e c t evidence f o r a c t i v e t r a n s p o r t of sodium was a l s o o b t a i n e d when t i c k s were f e d h.b./g. 25/75. I n t h i s case the sodium c o n c e n t r a t i o n i n the hemo-lymph was not measured. However, i t may be reasonably e x t r a p o l a t e d from F i g . 6 t h a t , s i n c e the meal was i s o s m o t i c w i t h h.b., the hemolymph sodium c o n c e n t r a t i o n would be maintained at about 160 m e q / l i t e r . The observed e l e c t r o -p o t e n t i a l g r a d i e n t c o u l d o n l y support a hemolymph concen-t r a t i o n of 59 m e q / l i t e r (gut c o n c e n t r a t i o n 33 m e q / l i t e r ) . Thus the l i k e l i h o o d i s t h a t sodium i o n s were again b e i n g a c t i v e l y t r a n s p o r t e d across the gut e p i t h e l i u m . During the p e r i o d of a b s o r p t i o n across the gut, the potassium c o n c e n t r a t i o n i n the gut rose from 4- t o 52 m e q / l i t e r and the c o n c e n t r a t i o n g r a d i e n t i n c r e a s e d t e n -f o l d when'ticks were f e d h.b. (Table V I I ) . The measured e l e c t r o p o t e n t i a l g r a d i e n t of 15 mv. (gut n e g a t i v e ) c o u l d not account f o r t h i s c o n c e n t r a t i o n g r a d i e n t i f r a p i d f r e e d i f f u s i o n was assumed. This can mean e i t h e r t h a t potassium was b e i n g a c t i v e l y t r a n s p o r t e d from the hemolymph t o the gut as Harvey and Nedergaard found i n the C e c r o p i a moth (1964-) or t h a t the gut e p i t h e l i a l c e l l s had a v e r y low p e r m e a b i l i t y t o potassium. T h i s study y i e l d e d i n s u f f i c i e n t d ata t o d i s t i n g u i s h between these two mechanisms. An a c t i v e process such as i o n t r a n s p o r t would be expected to show evidence of s a t u r a t i o n of the c a r r i e r system as the c o n c e n t r a t i o n of the t r a n s p o r t e d i o n i s i n -creased. F i g u r e s 20 and 21 show t h a t data o b t a i n e d from the study on t r a n s p o r t across the t i c k gut are c o n s i s t e n t w i t h such s a t u r a t i o n k i n e t i c s thus adding weight to the evidence f o r a c t i v e t r a n s p o r t of sodium and c h l o r i d e i o n s . F u r t h e r evidence might, at f i r s t s i g h t , a r i s e from the one t o one uptake of sodium and c h l o r i d e across the gut - 8 1 -FIGURE 20 RELATION BETWEEN MEAN RATE OF CHLORIDE ABSORPTION ACROSS GUT WALL AND MEDIAN CHLORIDE CONCENTRATION IN GUT FLUID Gut f l u i d samples were taken two hours a f t e r f e e d i n g . Rate of a b s o r p t i o n i s mean f o r two hour experimental p e r i o d . V e r t i c a l l i m i t s show c a l c u l a t e d range from Table IV. 0.40 -I 1 1 1 1 1 1 0 20 40 60 80 100 120 M e d i a n [ci~] in gut f l u id ( m e q / l i t e r ) FIGURE 21 RELATION BETWEEN MEAN RATE OF SODIUM ABSORPTION ACROSS  GUT WALL AND MEDIAN SODIUM CONCENTRATION IN GUT FLUID Gut f l u i d samples were taken two hours a f t e r f e e d i n g . Rate o f a b s o r p t i o n i s mean f o r two hour experimental p e r i o d . V e r t i c a l l i m i t s show c a l c u l a t e d range from Table V I . Median [Na + ] in gut fluid (meq/ l i te r ) - 8 3 -w a l l ( F i g . 1 1 ) although potassium was present i n h i g h con-c e n t r a t i o n i n the gut. However, the suggestion was made t h a t gut e p i t h e l i a l c e l l s are impermeable t o potassium. Thus sodium and c h l o r i d e i o n s might be the o n l y major i o n s present t o ensure e l e c t r o n e u t r a l i t y . T h i s would e x p l a i n t h e i r equal t r a n s p o r t . The presence of these a c t i v e pumps i s not unex-pecte d i n l i g h t of s t u d i e s made on other arthropod systems. The cockroach midgut m a i n t a i n s , i n v i t r o , an e l e c t r o p o t e n t i a l g r a d i e n t o f about 1 2 mv; the lumen i s n e g a t i v e w i t h r e s p e c t to the hemolymph. A l i n k e d sodium-potassium pump has been proposed w i t h the p o t e n t i a l a r i s i n g from a lumen-side sodium d i f f u s i o n p o t e n t i a l and a hemolymph-side potassium p o t e n t i a l (O'Riorden, 1 9 6 9 ) . The l i m i t e d data o b t a i n e d from the a r a c h n i d i n t h i s p resent study appear to be sim-i l a r t o t h a t demonstrated i n i n s e c t s (see above) and i n v e r t e b r a t e e p i t h e l i a (Koefoed-Johnsen and U s s i n g , 1 9 5 8 ) . Although a c t i v e t r a n s p o r t of c h l o r i d e has not been demon-s t r a t e d i n the midgut of i n s e c t s , i t s presence i s w i d e l y d i s t r i b u t e d i n ot h e r t i s s u e s both of i n s e c t s and crustaceans (reviewed by K i r s c h n e r , 1 9 7 0 ) . The volume of b l o o d meal v a r i e d l e s s than two-f o l d over the range o f f e e d i n g c o n d i t i o n s i n v e s t i g a t e d . N e v e r t h e l e s s , the volume of f l u i d absorbed across the gut v a r i e d from 2 7 m i c r o l i t e r s to 1 7 2 m i c r o l i t e r s (Table I) and was dependent on the compo s i t i o n of the bl o o d meal -84-( P i g . 4 ) . What then was the nature o f the c o n t r o l over volume and r a t e o f passage of absorbate a c r o s s the gut w a l l ? Under a l l c o n d i t i o n s s t u d i e d there was an i n i t i a l osmotic g r a d i e n t f a v o r a b l e f o r simple osmotic movement o f water from gut t o hemolymph. An i n i t i a l h i g h r a t e o f water a b s o r p t i o n a c r o s s the gut was suggested by the i n i t i a l r a p i d f a l l i n hemolymph osmotic p r e s s u r e ( P i g . 17). T h i s h i g h r a t e o f water a b s o r p t i o n might have r e s u l t e d from a r e d u c t i o n i n osmotic p r e s s u r e of gut f l u i d by the incoming b l o o d meal. T h i s p r o p o s a l would e l i m i n a t e the need t o p o s t u l a t e a c o n t r o l mechanism f o r i n i t i a t i n g the movement of water across the gut e p i t h e l i u m . Hemolymph volume d i d not i n c r e a s e s u b s t a n t i a l l y compared w i t h the volume of f l u i d p a s s i n g through the t i c k . Moreover, osmotic p r e s s u r e and i o n c o n c e n t r a t i o n s of the hemolymph r a p i d l y reached an e q u i l i b r i u m v a l u e i n those c o n d i t i o n s s t u d i e d . Thus the r a t e o f c o x a l f l u i d p r o -d u c t i o n was assumed t o be equal t o the r a t e a t which f l u i d passed across the gut w a l l f o r most of the two hour e x p e r i -mental p e r i o d . On t h i s assumption, evidence was o b t a i n e d f o r two f e e d i n g c o n d i t i o n s t h a t the r a t e o f a b s o r p t i o n of f l u i d a c r o s s the gut p a r a l l e d the osmotic g r a d i e n t between gut and hemolymph ( P i g s . 17 and 18). T h i s i n d i c a t e d t h a t the r a t e o f water movement acro s s the gut w a l l was i n -f l u e n c e d by simple osmotic f o r c e s . However, there are some reasons to doubt t h a t t h i s - 8 5 -i s the complete s t o r y . When t i c k s were f e d h.b./D.W. ther e was good i n g e s t i o n of the meal and the most f a v o r -able osmotic g r a d i e n t f o r water movement from the gut t h a t was observed i n any experiment. N e v e r t h e l e s s , l e s s than h a l f the volume of water was absorbed across the gut i n t h i s case as when t i c k s were f e d h.b. (Table I ) . I n f a c t , even though the i n g e s t e d volumes were comparable, the volume of gut absorbate v/as v e r y s i m i l a r t o t h a t measured when t i c k s were f e d h.b. + NaCl or h.b. + g. where the i n i t i a l osmotic g r a d i e n t was c o n s i d e r a b l y s m a l l e r than i n t i c k s f e d h.b./D.W. Th i s c o u l d be e x p l a i n e d i f f l u i d up-take was somehow dependent on sodium c h l o r i d e movement across the gut w a l l . I n f a c t i t was shown t h a t the volume-of f l u i d absorbed bore a d i r e c t r e l a t i o n s h i p t o the t o t a l c h o r i d e i o n s absorbed ( F i g . 2 2). Seemingly then, the l e s s c h l o r i d e t h a t was t r a n s p o r t e d , the l e s s was the water ab-sorbed across the gut. I t may be t h a t i n the case of t i c k s fed h.b./D.W. the c h l o r i d e c o n c e n t r a t i o n i n the gut approached the lower l i m i t a g a i n s t which a b s o r p t i o n of the i o n c o u l d occur; hence water a b s o r p t i o n was low even i n the presence of a f a v o r a b l e osmotic g r a d i e n t . That water movement v/as i n some way c o r r e l a t e d w i t h i o n s was f u r t h e r s u b s t a n t i a t e d by the d i r e c t r e l a t i o n s h i p found between the r a t e of water a b s o r p t i o n across the gut and the median c h l o r i d e ( o r sodium) c o n c e n t r a t i o n i n the gut ( F i g s . 23 and 24). What then was the b a s i s of t h i s c o nnection between i o n s and water movement? I t may be t h a t -86-FIGURE 22 RELATION BETWEEN TOTAL CHLORIDE AND TOTAL FLUID ABSORBED  ACROSS GUT WALL DURING TWO HOUR EXPERIMENTAL PERIOD H o r i z o n t a l l i m i t s show c a l c u l a t e d range from Table IV. Legend: • bl o o d meal 100$ osmotic p r e s s u r e o f h.b. ( T i c k s f e d h.b., h.b./g.50/50 and h.b./g.25/75) O bl o o d meal 158$ osmotic p r e s s u r e o f h.b. ( T i c k s f e d h.b. + NaCl and h.b. + g.) A b l o o d meal 50$ osmotic p r e s s u r e o f h.b. ( T i c k s f e d h.b./D.W.) 200 150 3 cn 100 -\ 50 H 10 15 Total Cl absorbed across gut (jueq) -87-FIGURE 23 RELATION BETWEEN MEDIAN CHLORIDE CONCENTRATION IN GUT  FLUID AND MEAN RATE OF FLUID ABSORPTION ACROSS GUT WALL  DURING TWO HOUR EXPERIMENTAL PERIOD Legend: 9 b l o o d meal 100$ osmotic p r e s s u r e of h.b. ( T i c k s f e d h.b., h.b./g.50/50 and h.b./g.25/75 O b l o o d meal 158$ osmotic p r e s s u r e o f h.b. ( T i c k s f e d h.b. + NaCl and h.b. + g.) A b l o o d meal 50$ osmotic p r e s s u r e of h.b. ( T i c k s f e d h.b./D.W.) Median [Cf] in gut fluid (meq/liter) -88-FIGURE 24 RBLATION BETWEEN MEDIAN SODIUM CONCENTRATION IN GUT FLUID  AND MEAN RATE OF FLUID ABSORPTION ACROSS GUT WALL DURING  TWO HOUR EXPERIMENTAL PERIOD Legend: • b l o o d meal 100$ osmotic pressure of h.b. ( T i c k s f e d h.b., h.b./g.50/50 and h.b./g.25/75 O blood meal 158$ osmotic p r e s s u r e of h.b. ( T i c k s f e d h.b. + NaCl and h.b. + g.) A b l o o d meal 50$ osmotic pressure of h.b. ( T i c k s f e d h.b./D.W.) Median N a + in gut fluid ( m e q / l i t e r ) -89-a c t i v e uptake of sodium c h l o r i d e removed o s m o t i c a l l y a c t i v e s o l u t e from the gut lumen and thus maintained a f a v o r a b l e osmotic p r e s s u r e g r a d i e n t f o r water movement i n t o the hemolymph. I n t i c k s f e d h.b. + g., the accumulation of gl u c o s e , p o s s i b l y a more s l o w l y absorbed s o l u t e , would then decrease the osmotic-pressure g r a d i e n t across the gut w a l l and reduce a b s o r p t i o n . The c o r r e l a t i o n between i o n c o n c e n t r a t i o n s i n the gut f l u i d and the r a t e of f l u i d ab-s o r p t i o n could i n d i c a t e t h a t p e r m e a b i l i t y to water of the gut e p i t h e l i u m was i n some way dependent on the i o n c o n c e n — t r a t i o n of gut f l u i d . A l t e r n a t i v e l y , water may move by a mechanism of l o c a l osmosis. This i s s p e c u l a t i v e s i n c e data was not obt a i n e d i n t h i s study on i s o s m o t i c uptake of water nor on the u l t r a s t r u c t u r e of gut e p i t h e l i a l c e l l s . However, the l i m i t e d data t h a t i s a v a i l a b l e i s c o n s i s t e n t w i t h a process of l o c a l osmosis as d e s c r i b e d by Diamond (1965, 1968). Moreover, water movement i n s e v e r a l arthropods has been p o s t u l a t e d to occur i n t h i s way (B e r r i d g e and Gupta, 1967; B e r r i d g e and Oschman, 1969). The i n i t i a t i o n of water movement from the gut has been a t t r i b u t e d t o the osmotic g r a d i e n t s e t up by the i n -gested blood meal; but i t i s not c l e a r what causes t h i s a b s o r p t i o n t o cease. I t has been suggested t h a t water move-ment was dependent on the i o n c o n c e n t r a t i o n s i n the gut f l u i d . However, the f i n a l i o n c o n c e n t r a t i o n of gut f l u i d i n t i c k s f e d h.b. was g r e a t e r than the i n i t i a l i o n Concen-t r a t i o n i n gut f l u i d of t i c k s f e d h.b./g. 25/75 (Tables I I I -90-and V) and y e t , i n the l a t t e r case, water d i d move across the gut w a l l . S i m i l a r l y osmotic p r e s s u r e g r a d i e n t s have been i m p l i c a t e d i n water movement. N e v e r t h e l e s s , the f i n a l osmotic p r e s s u r e g r a d i e n t across the gut w a l l of t i c k s f e d h.b./D.W. appeared t o pro v i d e a d r i v i n g f o r c e f o r water movement, but none o c c u r r e d . I f , as suggested e a r l i e r , the p e r m e a b i l i t y t o water of the gut e p i t h e l i u m was dependent on the i o n c o n c e n t r a t i o n o f gut f l u i d , the decreased i o n c o n c e n t r a t i o n p r e s e n t i n h.b./D.W. cou l d have l i m i t e d and e v e n t u a l l y stopped the a b s o r p t i o n of water across the gut even though a f a v o r a b l e osmotic g r a d i e n t s t i l l e x i s t e d . C o n v e r s e l y , i n those t i c k s f e d h.b. + g. or h.b. + NaCl, a s u f f i c i e n t i o n c o n c e n t r a t i o n may have been present t o i n -crease the p e r m e a b i l i t y to water-of the gut w a l l . Water movement i n these cases might have been c u r t a i l e d by a b u i l d up of s l o w l y d i f f u s i b l e s o l u t e s i n the gut f l u i d t h a t a b o l i s h e d the osmotic g r a d i e n t across the gut w a l l . As a more orthodox h y p o t h e s i s one i s l e d t o suspect the e x i s t e n c e of hormonal or nervous c o n t r o l of water movement. This system c o u l d be more s e n s i t i v e t o the c o n d i t i o n s of the organism as a whole r a t h e r than m o n i t o r i n g and r e g u l a t i n g c o n d i t i o n s l o c a l i z e d at the c e l l u l a r l e v e l . I t i s u n l i k e l y t h a t t h i s nervous or hormonal c o n t r o l i s l i n k e d t o s t r e t c h r e c e p t o r s i n the gut s i n c e , i n t i c k s fed h.b./g. 2 5 / 7 5 , the f i n a l gut volume was h i g h e r than the t o t a l volume i n -gested by t i c k s f e d h.b./D.W. I n the l a t t e r case the gut -91-c o u l d not have been s t r e t c h e d t o a g r e a t e r extent than the f i n a l s t a t e of t i c k s f e d h.b./g. 25/75 and yet water move-ment was e l i c i t e d . Thus simple s t r e t c h i n g of the gut e p i t h e l i u m would not seem to p r o v i d e a c o n t r o l f o r water a b s o r p t i o n across the gut. The v e r y h i g h f i n a l c o n c e n t r a t i o n of potassium i n the gut f l u i d promotes s p e c u l a t i o n t h a t t h i s i o n may, i n hi g h enough c o n c e n t r a t i o n s , i n h i b i t sodium c h l o r i d e uptake or may decrease the p e r m e a b i l i t y t o water of the gut w a l l . T h i s argument i s not however v e r y p o w e r f u l when a p p l i e d t o t i c k s f e d h.b./g 25/75 where potassium i n t a k e and concen-t r a t i o n i n the gut was reduced thus making i t . u n l i k e l y t h a t a t h r e s h o l d c o u l d be reached. I t i s important t o r e a l i z e t h a t the c o m p o s i t i o n of the gut absorbate must be con s i d e r e d when measuring changes i n i o n i c and osmotic c o n c e n t r a t i o n s i n the hemolymph. Pro-v i d e d osmotic p r e s s u r e remained c o n s t a n t , the o v e r a l l r e g -u l a t o r y a b i l i t y o f the t i c k enabled a constant c h l o r i d e c o n c e n t r a t i o n t o be maintained i n the hemolymph when con-c e n t r a t i o n s i n the b l o o d meal v a r i e d from 21 m e q / l i t e r to 116 m e q / l i t e r ( F i g . 5). Bone (194-3) found t h a t above 14-5 m e q / l i t e r c h l o r i d e i n the blood meal, the c h l o r i d e concen-t r a t i o n i n the hemolymph r o s e . He s t a t e d t h a t the osmotic pressure o f the b l o o d meal was kept constant but i t i s not c l e a r from the paper how t h i s v/as achieved f o r c h l o r i d e c o n c e n t r a t i o n s h i g h e r than those of normal b l o o d . This -92-present study showed t h a t an i n c r e a s e of osmotic pr e s s u r e due t o glucose would cause the c h o r i d e c o n c e n t r a t i o n i n the hemolymph to r i s e ( F i g s . 5 and 25). Although the time sequence of osmotic c o n d i t i o n s was not measured i t appears p o s s i b l e from the low volume and h i g h i o n c o n c e n t r a t i o n s o f gut absorbate (Tables I , I I I and V) t h a t water was h e l d back i n the gut by a b u i l d up of osmotic p r e s s u r e i n the gut f l u i d due t o g l u c o s e . The i n g e s t i o n of blood meals w i t h h i g h c h l o r i d e c o n c e n t r a t i o n appears t o r e s u l t i n a s i m i l a r l y decreased volume and i n c r e a s e d i o n c o n c e n t r a t i o n of gut absorbate (Tables I , I I I and V ) . As d i s c u s s e d l a t e r , s i n c e the t i c k d i d not produce h y p e r t o n i c c o x a l f l u i d , the hemolymph c o n c e n t r a t i o n s c o u l d not be r e g u l a t e d i n face of t h i s i n f l u x from the gut of h y p e r t o n i c f l u i d . Thus the i n -a b i l i t y of the t i c k t o r e g u l a t e under c o n d i t i o n s of i n -creased osmotic pr e s s u r e i n the blood meal may l a r g e l y r e -s u l t from i t s i n a b i l i t y t o handle blood meals of i n c r e a s e d i o n c o n c e n t r a t i o n . R e g u l a t i o n of sodium c o n c e n t r a t i o n s i n the body f l u i d s f o l l o w e d the same p a t t e r n as f o r c h l o r i d e w i t h the n o t a b l e e x c e p t i o n of the gut f l u i d . A f t e r c o x a l f l u i d p r o d u c t i o n had ceased the c h l o r i d e c o n c e n t r a t i o n i n the gut was v e r y low but the sodium c o n c e n t r a t i o n remained about the same as i n the i n g e s t e d f l u i d . The c o x a l gland d i d not produce hyper-osmotic s o l -u t i o n s or indeed h y p e r - i o n i c s o l u t i o n s under any experimental c o n d i t i o n s even though t h i s r e s u l t e d sometimes i n a l a c k of -93-FIGURE 25 REGULATION OF CHLORIDE CONCENTRATION IN HEMOLYMPH Samples were taken tv/o hours a f t e r f e e d i n g . Standard e r r o r s may be found i n Table I I I . Broken l i n e i s t h a t g r a d i e n t f o r equal c o n c e n t r a t i o n s . Legend: • bl o o d meal 100$ osmotic p r e s s u r e of h.b. ( T i c k s f e d h.b., h.b./g.50/50 and h.b./g.25/75) O b l o o d meal 158$ osmotic p r e s s u r e of h.b. ( T i c k s f e d h.b. + NaCl and h.b. + g.) A b l o o d meal 50$ osmotic pressure of h.b. ( T i c k s f e d h.b./D.W.) [ d ~ ] hemolymph ( m e q / l i t e r _o,4~ hemolymph r e g u l a t i o n . The gland v/as o n l y capable of r e g -u l a t i n g i o n c o n c e n t r a t i o n s i n the hemolymph pr o v i d e d f l u i d e n t e r i n g from the gut had lower i o n c o n c e n t r a t i o n s than those p r e v a i l i n g i n the hemolymph. These data are i n g e n e r a l agreement w i t h those presented by Lees (1946) and Bone (1943). However, Lees r e p o r t e d volumes of c o x a l f l u i d t h a t were o n l y o n e - t h i r d t o one-half those found i n t h i s study. Moreover, h i s t i c k s i n g e s t e d e q u a l l y reduced volumes of b l o o d . T h i s suggests t h a t the f e e d i n g c o n d i t i o n s he used were not conducive t o a n a t u r a l u n d i s t u r b e d p a t t e r n of engorgement. Bone mentioned t h a t h i s t i c k s were e a s i l y d i s t u r b e d and would d e t a t c h when samples of hemolymph were b e i n g taken from them. No such d i f f i c u l t i e s v/ere encountered i n t h i s p r e s e n t study p o s s i b l y due t o the manner i n which the t i c k s were taped to the f e e d i n g membrane so t h a t samples of hemolymph and c o x a l f l u i d c o u l d be taken without moving them. No mention was made of such an arrangement i n the papers by Lees and Bone. The c h a r a c t e r i s t i c s of c o x a l f l u i d p r o d u c t i o n l e a d t o s p e c u l a t i o n as t o the probable mode of a c t i o n of the c o x a l gland. The r a t e s of c o x a l f l u i d p r o d u c t i o n v/ere h i g h e r than have been r e p o r t e d i n the l i t e r a t u r e f o r sec-r e t i o n ( M a d d r e l l , 1964). T h i s suggests t h a t a f i l t r a t i o n -r e s o r p t i o n system might be i n o p e r a t i o n . Other organisms which r e q u i r e a means of r a p i d l y e l i m i n a t i n g water e x h i b i t ample evidence of such systems ( K i r s c h n e r , 1967). Did - 9 5 -t h i s study of the c o x a l gland y i e l d any r e s u l t s which are i n c o n s i s t e n t w i t h such a model? The i n a b i l i t y t o produce hyperosmotic u r i n e i s c e r t a i n l y t o be expected. The morpho-l o g i c a l study of the gland (Chap. I I ) r e v e a l e d a membrane which c o u l d w e l l be used f o r f i l t r a t i o n . Moreover, the d i f f e r i n g i o n c o n c e n t r a t i o n s i n the i n i t i a l drops of c o x a l f l u i d from those of the pooled sample c o u l d be e x p l a i n e d i n the f o l l o w i n g manner. Since the f i l t r a t e f o r these i n -i t i a l drops had been co n t a i n e d s t a t i o n a r y w i t h i n the glands b e f o r e b e i n g r e l e a s e d , i t had time t o e q u i l i b r a t e w i t h the c e l l s of the r e s o r p t i o n t u b u l e . C h l o r i d e i o n s were reab-sorbed and some potassium d i f f u s e d from the c e l l s i n t o the f i l t r a t e ; t h i s changed the f i n a l c o m p o s i t i o n of the i n i t i a l drops of c o x a l f l u i d . N ormally d u r i n g the f r e e f l o w of c o x a l f l u i d such processes would occur but, because the f l u i d was moving r a p i d l y through the r e s o r p t i o n t u b u l e , not to the same e x t e n t . Thus a r e l a t i o n s h i p was sought between the i o n con-c e n t r a t i o n of the emerging c o x a l f l u i d and the r a t e at which t h a t f l u i d had passed through the gl a n d . Since the r a t e of s e c r e t i o n and the c h l o r i d e c o n c e n t r a t i o n had been measured w i t h time t h i s comparison could be made f o r t i c k s f e d h.b. I f g r e a t e r i o n r e a b s o r p t i o n occurred at low f l o w r a t e s , one would have observed the i o n c o n c e n t r a t i o n of the c o x a l f l u i d t o f a l l as the r a t e of c o x a l f l u i d p r o -d u c t i o n f e l l ( P i g s . 12 and 14a). However, no obvious r e -l a t i o n s h i p emerged. The r a t e of c o l l e c t i o n of c o x a l f l u i d -96-p o s s i b l y d i d not a c c u r a t e l y r e f l e c t the r a t e at which f l u i d had passed through the gland s i n c e , as was d e s c r i b e d i n Chapter I I , f l u i d was produced i n d i s c r e t e d r o p l e t s . The r e l a t i o n s h i p between r a t e of p r o d u c t i o n of c o x a l f l u i d and i o n r e a b s o r p t i o n c o u l d a l s o be c o n s i d e r e d by com-p a r i n g d i f f e r e n t f e e d i n g c o n d i t i o n s . For t h i s purpose the average r a t e of f l u i d p r o d u c t i o n and the mean c h l o r i d e c o n c e n t r a t i o n of pooled c o x a l f l u i d were used. I n seeking t h i s r e l a t i o n s h i p the assumption was made t h a t f l u i d r e -a b s o r p t i o n was n e g l i g i b l e (Chp. I V ) . Only those t i c k s f e d on b l o o d meals i s o s m o t i c w i t h h.b. were able t o r e g u l a t e t h e i r hemolymph c o n c e n t r a t i o n s s a t i s f a c t o r i l y ( F i g s . 5, 6 and 25). I f r e s u l t s from these c o n d i t i o n s are c o n s i d e r e d a c l e a r r e l a t i o n s h i p emerges t o show t h a t the degree of i o n r e a b s o r p t i o n ( t h i s was i n d i c a t e d by the d i f f e r e n c e i n sodium c h l o r i d e c o n c e n t r a t i o n s of hemolymph and c o x a l f l u i d ) was indeed dependent on the r a t e of c o x a l f l u i d p r o d u c t i o n ( F i g . 2 6 ) . Why then d i d the t i c k s f a i l t o r e g u l a t e when i n -g e s t i n g the other blood meals? In the two hyper-osmotic meals the i o n c o n c e n t r a t i o n of gut absorbate was g r e a t e r than or equal t o t h a t of the hemolymph. Thus, although volume r e g u l a t i o n c o u l d c o n t i n u e , there was no way i n which i o n r e g u l a t i o n c o u l d take p l a c e s i n c e the most concentrated c o x a l f l u i d t h a t c o u l d be produced was o n l y equal t o t h a t of the hemolymph. In the case of t i c k s f e d h.b./D.W. the - 9 7 -FIGURE 26 RELATION BETWEEN MEAN RATE OF PRODUCTION OF COXAL FLUID  AND DIFFERENCE IN ION CONCENTRATION BETWEEN COXAL FLUID  AND HEMOLYMPH T i c k s were f e d meals i s o s m o t i c w i t h h.b. Rate of c o x a l f l u i d p r o d u c t i o n was averaged over e n t i r e e x c r e t o r y p e r i o d . Mean c o x a l f l u i d c o n c e n t r a t i o n s were s u b t r a c t e d from f i n a l hemolymph c o n c e n t r a t i o n s . Legend: O sodium O c h l o r i d e 160 -i o H 1 1 1 1 1 1 1 i 1 i 0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 Rate of coxal fluid production (A/min) -98-i o n c o n c e n t r a t i o n of the c o x a l f l u i d was, as expected, low but not s u f f i c i e n t l y low t o ensure r e g u l a t i o n of the hemo-lymph i o n c o n c e n t r a t i o n . Since the organism was being s u b j e c t e d t o extreme s t r e s s c o n d i t i o n s , q u i t e p o s s i b l y the r e s o r p t i v e c a p a c i t y of the c o x a l gland was exceeded. The f o l l o w i n g scheme i s proposed f o r the r e g u l a t i o n of sodium c h l o r i d e c o n c e n t r a t i o n s and volumes of c o x a l f l u i d . Sodium c h l o r i d e was a c t i v e l y t r a n s p o r t e d from the gut and, as has been shown, the more sodium c h l o r i d e t h a t was t r a n s -p o r t e d , the g r e a t e r was the volume of water t h a t f o l l o w e d . The g e n e r a l decrease i n r a t e of c o x a l f l u i d p r o d u c t i o n w i t h meals of i n c r e a s e d osmotic pressure p o s s i b l y r e f l e c t e d a lower r a t e of osmotic f l o w across the gut w a l l due t o a decreased osmotic g r a d i e n t . The prolonged and g e n e r a l l y h i g h e r c o x a l f l u i d p r o d u c t i o n observed f o r h.b. + NaCl ( r e l a t i v e t o h.b. + g.) might r e f l e c t a g r e a t e r m o b i l i t y of sodium c h l o r i d e than glucose across the gut w a l l . The r a t e of c o x a l f l u i d p r o d u c t i o n c o u l d w e l l be dependent on the hemolymph volume. This c o u l d be mediated by s t r e t c h r e -c e p t o r s , by sense c e l l s m o n i t o r i n g the i o n i c or osmotic c o n d i t i o n s i n the hemolymph or d i r e c t l y by the i n c r e a s e d r a t e of f i l t r a t i o n r e s u l t i n g from an i n c r e a s e d h y d r o s t a t i c p r e s s u r e i n the hemolymph. Thus a h i g h r a t e of a b s o r p t i o n a c r o s s the gut would i n t u r n l e a d t o a h i g h r a t e of sodium c h l o r i d e l o s s due t o i n c r e a s e d r a t e of f i l t r a t i o n and de-creased i o n r e a b s o r p t i o n . Conversely, a low r a t e of sodium -99-c h l o r i d e t r a n s p o r t across the gut would l e a d t o a low r a t e of water a b s o r p t i o n . This i n t u r n would l e a d t o a low r a t e of c o x a l f l u i d p r o d u c t i o n p r o v i d i n g more time f o r sodium c h l o r i d e t o be reabsorbed and hence conserved. CHAPTER IV MECHANISM OF COXAL FLUID FORMATION -101-A. INTRODUCTION I n the p r e v i o u s chapter I showed t h a t the c o x a l gland was r e s p o n s i b l e f o r r e g u l a t i n g both volume and i o n i c c o n c e n t r a t i o n of the hemolymph. These s t u d i e s i n d i c a t e d t h a t the gland was i n c a p a b l e of producing a hyperosmotic s o l u t i o n . T h i s coupled w i t h the h i g h r a t e s of c o x a l f l u i d p r o d u c t i o n suggested t h a t a f i l t r a t i o n - r e s o r p t i o n system might be o p e r a t i v e . Moreover, h i s t o l o g i c a l s t u d i e s (Chap. I I ) r e v e a l e d a t h i n membraneous s t r u c t u r e (an e x c e l l e n t candidate f o r the s i t e of f i l t r a t i o n ) e n v e l o p i n g the t u b u l e . Bone (194-3) d e s c r i b e d the c o x a l gland as a t u b u l a r l a b y r i n t h but found no evidence o f , and d i d not p o s t u l a t e the e x i s t e n c e o f , a f i l t r a t i o n membrane. On the other hand Lees (1946) d e s c r i b e d a t h i n membrane, 1-2 microns t h i c k which enveloped the t u b u l e and was c o n f l u e n t w i t h i t at one p o i n t . He a s c r i b e d t o t h i s membrane the f u n c t i o n of a f i l t r a t i o n membrane. I n support of h i s h y p o t h e s i s he demon-s t r a t e d t h a t molecules l a r g e r than serum albumin would not pass from the hemolymph i n t o the c o x a l f l u i d . However, when s m a l l e r molecules such as hemoglobin were i n j e c t e d i n t o the hemolymph they appeared immediately i n the c o x a l f l u i d . There was, he concluded, too s h o r t a p e r i o d of time f o r the molecules to have been s e c r e t e d . Lees suggested t h a t the volume of the f i l t r a t i o n -102-chamber c o u l d be i n c r e a s e d by c o n t r a c t i o n of numerous s m a l l muscles he observed running from the membrane t o the body w a l l . By means of a s p h i n c t e r t e m p o r a r i l y i s o l a t i n g the chamber from the e x t e r n a l environment, a negative pressure r e l a t i v e t o the hemolymph c o u l d develop. This c o u l d pro-v i d e a d r i v i n g f o r c e f o r u l t r a f i l t r a t i o n through the mem-brane. Sin c e f i l t r a t i o n systems have been shown t o e x i s t among the art h r o p o d s , i t i s not unreasonable t o propose, i n l i g h t of Lees' evidence, t h a t the c o x a l gland of t h i s a r a c h n i d operates by f i l t r a t i o n . K i r s c h n e r (1967) presents c e r t a i n c h a r a c t e r i s t i c s which i n d i c a t e the e x i s t e n c e of a f i l t r a t i o n system and i t was w i t h these i n mind t h a t e x p e r i -ments were designed t o e l u c i d a t e the mechanism of e x c r e t i o n by the c o x a l g l a n d . When K i r c b n e r ' s c r i t e r i a were a p p l i e d t o Lees' r e s u l t s i t was c l e a r t h a t the case f o r f i l t r a t i o n was by no means proven. Although he found a l i k e l y s i t e f o r f i l t r a t i o n , he d i d not demonstrate d i r e c t l y t h a t f i l t r a t i o n o c curred through t h i s membrane. The r a p i d appearance of dyes and l a r g e r molecules i n the u r i n e , although h i g h l y suggestive of f i l t r a t i o n , d i d not r u l e out the p o s s i b i l i t y of t h e i r s e c r e t i o n by p i n o c y t o s i s or other mechanisms. However, a q u a n t i t a t i v e study of the c o n c e n t r a t i o n s of such molecules i n the c o x a l f l u i d and hemolymph ( i . e . U/P r a t i o s ) c o u l d p r o v i d e a more d e f i n i t i v e answer. A p r i n c i p a l o b j e c t i v e of the present study was t o o b t a i n such data f o r i n u l i n ex-c r e t i o n . A second approach was t o compare the u l t r a --103-s t r u c t u r e of the proposed f i l t r a t i o n membrane i n the c o x a l gland w i t h t h a t of the v e r t e b r a t e glomerulus to l o o k f o r s t r u c t u r a l s i m i l a r i t i e s t h a t might be i n d i c a t i v e of s i m i l a r f u n c t i o n . To extend t h i s an attempt was made to d i r e c t l y l o c a l i z e the s i t e of f i l t r a t i o n by t r a p p i n g w i t h i n i t p r o -t e i n whose presence c o u l d be r e v e a l e d by c o n j u g a t i o n w i t h a f l u o r e s c e n t dye ( K i r s c h n e r and Wagner, 1965). F i n a l l y , i n accordance w i t h one o f K i r s c h n e r ' s c r i t e r i a f o r f i l -t r a t i o n , a study was made of the pressure s e n s i t i v i t y of the r a t e o f c o x a l f l u i d p r o d u c t i o n . - 1 0 4 -B. METHODS 14 1• Clearance of i n u l i n - c a r b o x y l - C P r i o r t o f e e d i n g , the t i c k s were i n j e c t e d , as des-—8 c r i b e d i n Chapter I I I , w i t h 2 m i c r o l i t e r s (approx." 28 x 10"" mC.) of an aqueous s o l u t i o n of i n u l i n - c a r b o x y l - C " ^ ( s u p p l i e d by New England N u c l e a r ) . I t was found t h a t 1 hour s u f f i c e d t o d i s t r i b u t e the t r a c e r throughout the t i c k ' s hemocoel. One hour a f t e r i n j e c t i o n the t i c k s were f e d and s e r i a l samples of c o x a l f l u i d and hemolymph taken almost s i m u l -t a n e o u s l y as p r e v i o u s l y d e s c r i b e d (Chap. I I I ) . The f i r s t sample of hemolymph was taken as soon as the f i r s t m icro-l i t e r of c o x a l f l u i d had been produced and c o l l e c t e d . This whole procedure f o r sampling both f l u i d s o n l y took about 20 seconds. Twenty hours a f t e r f e e d i n g , hemolymph and gut samples were ta k e n . The f l u i d samples were d i s s o l v e d i n 10 ml Bray's s o l u t i o n (Bray, i960) and counted i n a Nuclear Chicago Mark I l i q u i d s c i n t i l l a t i o n counter u s i n g the channels r a t i o method f o r quench c o r r e c t i o n . 14 2. Reabsorption of i n u l i n - c a r b o x y l - C A f r a c t i o n of a m i c r o l i t e r of aqueous i n u l i n -14 carboxyl-C s o l u t i o n was i n j e c t e d through one c o x a l o r i f i c e i n t o the c o x a l gland u s i n g a f i n e g l a s s m i c r o p i p e t t e w i t h a f i r e p o l i s h e d t i p . The area was cleaned and s e a l e d w i t h a -105-melted beeswax/resin mi x t u r e . A o n e - m i c r o l i t e r sample of hemolymph was subsequently taken from a l e g adjacent to the c o x a l gland which had r e c e i v e d the r a d i o a c t i v e t r a c e r . T h i s was t e s t e d f o r r a d i o a c t i v i t y to determine whether the gland had been punctured d u r i n g i n j e c t i o n c a u s i n g a In-d i r e c t i n t r o d u c t i o n of i n u l i n - C i n t o the hemolymph. Those t i c k s which d i d show immediate a c t i v i t y i n the hemo-lymph were d i s c a r d e d . Two hours l a t e r the t i c k s were f e d and c o x a l f l u i d was c o l l e c t e d from the unsealed ( i . e . u n i n j e c t e d ) c o x a l gland f o r a p e r i o d of 10 minutes. As w i l l be seen from the i n u l i n c l e a r a n c e s t u d i e s , 90$ of a l l i n u l i n i s c l e a r e d from the hemolymph i n t h i s time. The sample o f c o x a l f l u i d from the u n i n j e c t e d gland would thus c o n t a i n 90$ of any i n u l i n reabsorbed from the i n j e c t e d c o x a l gland. The wax was then p u l l e d away from the o r i f i c e of the sealed ( i . e . i n j e c t e d ) c o x a l gland and the f i r s t 4 m i c r o l i t e r s of c o x a l f l u i d were c o l l e c t e d . Since the volume of the gland i s o n l y about one-auarter m i c r o l i t e r , t h i s 4 - m i c r o l i t e r sample should c o n t a i n the t o t a l i n u l i n remaining i n the c o x a l g l a n d . I n t h i s way the t o t a l a c t i v i t y i n j e c t e d i n t o the c o x a l gland and the t o t a l amount reabsorbed i n t o the hemo-lymph d u r i n g two hours c o u l d be c a l c u l a t e d . 3. H i s t o l o g y of the c o x a l gland Coxal glands were o u i c k l y d i s s e c t e d from unfed t i c k s and from t i c k s which were producing c o x a l f l u i d . They were -106-f i x e d f o r s e v e r a l hours i n Baker's formaldehyde calcium ( P a n t i n , 1964) or Eouin's f i x a t i v e , s e r i a l l y dehydrated i n a l c o h o l s and embedded i n wax. S e c t i o n s v/ere cut at three microns and s t a i n e d w i t h hemotoxylin and e o s i n . Some h a l f - m i c r o n s e c t i o n s were a l s o examined from t i s s u e prepared f o r e l e c t r o n microscopy as d e s c r i b e d below. 4. U l t r a s t r u c t u r e of the c o x a l gland Coxal glands v/ere d i s s e c t e d and f i x e d f o r 2 hours i n 4$ g l u t a r a l d e h y d e c o n t a i n i n g M/15 phosphate b u f f e r (pH 7.4) and 7$ sucrose ( o s m o l a l i t y 0.821). A f t e r they were washed i n b u f f e r e d sucrose s o l u t i o n , and p o s t f i x e d i n Vfo osmium, the t i s s u e s were r a p i d l y dehydrated and embedded i n Epon 812. The s e c t i o n s were doubly s t a i n e d w i t h u r a n y l a c e t a t e and l e a d c i t r a t e . Observations v/ere made on a H i t a c h i HU11A e l e c t r o n microscope. 5 . S i t e of f i l t r a t i o n The s i t e of f i l t r a t i o n was d e l i m i t e d by the technique of d i r e c t p r o t e i n t r a c i n g ( N a i r n , 1969). Albumin was known t o be m a r g i n a l l y r e t a i n e d by the c o x a l gland (Lees, 1946); t h i s p r o t e i n v/as conjugated t o f l u o r e s c e i n u s i n g a C e l i t e powder p r e p a r a t i o n ( f l u o r e s c e i n i s o t h i o c y a n a t e on c e l i t e 10$. Calbiochem.) a c c o r d i n g to the method of Rinderknecht (1962). T i c k s were f e d and, a f t e r the appearance of c o x a l -107-f l u i d , 1 - m i c r o l i t e r i n j e c t i o n s of a l b u m i n - f l u o r e s c e i n con-jugate i n aaueous s o l u t i o n were made every minute f o r 10 minutes. The i n j e c t i o n technioue i s d e s c r i b e d i n Chapter I I I . The t i c k s were i n t e r r u p t e d from the blo o d meal and d i s s e c t e d i n Baker's formaldehyde c a l c i u m ( P a n t i n , 1964-). A f t e r f i x a t i o n f o r 18 hours the glands were t r a n s f e r r e d d i r e c t l y to 50$ a l c o h o l , r a p i d l y dehydrated and vacuum em-bedded i n p a r a f f i n wax. S e c t i o n s were cut at 8 microns and mounted i n n o n - f l u o r e s c e n t mounting medium. Great care had t o be taken i n h a n d l i n g the s e c t i o n s so as not t o wash the f l u o r e s c e n t conjugate from the t i s s u e . The s e c t i o n s were examined under a Z e i s s f l u o r e s c e n t microscope. Photo-graphs had to be taken as soon as p o s s i b l e w i t h a h i g h speed f i l m (ASA 125) si n c e the f l u o r e s c e n c e i r r e v e r s i b l y faded upon exposure t o u l t r a v i o l e t r a d i a t i o n . 6. Pressure dependence of c o x a l f l u i d f o r m a t i o n Pressure measurements of the hemolymph were made u s i n g a Statham low volume displacement t r a n s d u c e r (Model No. P 2 3 Gb) l i n k e d to a G i l s o n polygraph. The t i c k s were cannulated d i r e c t l y i n t o the body c a v i t y w i t h a l e n g t h of p e r f o r a t e d PE 10 t u b i n g ( C l a y Adams. C a l i f o r n i a ) f i l l e d w i t h s a l i n e . The p o s t e r i o r r i m of the t i c k s body was clamped w i t h a s m a l l s e r r e f i n e so t h a t the gut was d i s p l a c e d towards the head. An i n c i s i o n was made p o s t e r i o r to the clamp and the PE t u b i n g pushed g e n t l y i n t o the hemocoel so -108-t h a t no a i r bubbles were trapped i n the pressure l i n e . The area was c a r e f u l l y d r i e d and s e a l e d w i t h a melted bees-wax-resin m i x t u r e . A s u c c e s s f u l c a n n u l a t i o n was i n d i c a t e d by the f l o w of hemolymph up the t u b i n g when the s e r r e f i n e was removed. C a n n u l a t i o n by t h i s technioue was found t o be l e s s s u s c e p t i b l e t o blockage than was c a n n u l a t i o n i n t o the l e g . I t seemed i n the l a t t e r case t h a t muscle fragments became r e a d i l y trapped i n the t u b i n g . Pressure v/as recorded d u r i n g the e n t i r e c y c l e of attachment, engorgement, pro-d u c t i o n of c o x a l f l u i d and detachment. U n f o r t u n a t e l y i t was v e r y d i f f i c u l t t o m a i n t a i n a pressure t i g h t s e a l on the c a n n u l a t i o n due t o s w e l l i n g and s t r e t c h i n g of the t i c k ' s body. Thus, although s e v e r a l t r a c e s were recorded up t o and i n c l u d i n g the s t a r t of c o x a l f l u i d p r o d u c t i o n , o n l y one complete r e c o r d r i g h t through u n t i l detachment was procured. I n a d d i t i o n the e f f e c t on the r a t e of c o x a l f l u i d p r o d u c t i o n o f a sudden r e d u c t i o n i n hemolymph pressure v/as observed v/hen the f e e d i n g t i c k was hemorrhaged. A l e g v/as clamped and then cut d i s t a l l y t o the clamp. This served t o check v/hether the nervous shock from c u t t i n g the l e g would i t s e l f i n i t i a t e p r e s s u r e changes or an a l t e r a t i o n i n the s e c r e t o r y r a t e . Next the clamp was removed and the l e g b l e d p r o f u s e l y . F i n a l l y the l e g was again clamped. A f t e r each step of t h i s procedure the r a t e of c o x a l f l u i d p r o -d u c t i o n v/as measured. An attempt v/as made to measure d i r -e c t l y the drop i n hemolymph pre s s u r e which r e s u l t e d when -109-hemorrhaging o c c u r r e d . However, as e x p l a i n e d p r e v i o u s l y , i t was v e r y d i f f i c u l t to o b t a i n a continuous r e c o r d of pressure once c o x a l f l u i d p r o d u c t i o n had s t a r t e d . I n a d d i t i o n , d u r i n g c o x a l f l u i d p r o d u c t i o n the hemolymph p r e s -sure was a l r e a d y f a l l i n g and i t was not easy to d i s t i n g u i s h a change from normal i n the r a t e of f a l l v/hen hemorrhaging o c c u r r e d . A q u a n t i t a t i v e measurement of the f a l l i n p r e s -sure was t h e r e f o r e deemed i m p r a c t i c a b l e . Since the s p h i n c t e r at the c o x a l o r i f i c e was observed t o open and c l o s e r e g u l a r l y d u r i n g c o x a l f l u i d p r o d u c t i o n , an experiment was designed t o see whether t h i s s p h i n c t e r c o n t r o l l e d the r a t e of c o x a l f l u i d p r o d u c t i o n . The r a t e of f l u i d p r o d u c t i o n was measured from a c o x a l gland w i t h the o r i f i c e h e l d open. I achieved t h i s by i n t r o d u c i n g a f i n e g l a s s cannula d i r e c t l y i n t o the o r i f i c e . Great care was . needed not t o puncture the g l a n d . Such a mishap was c l e a r l y e v i d e n t when i t o c c u r r e d , by the f l o w of hemolymph through the o r i f i c e . Rates of c o x a l f l u i d p r o d u c t i o n were measured s i m u l t a n e o u s l y from the normal and s u c c e s s f u l l y cannulated glands of the same t i c k . -110-C. RESULTS 14-1. R e a b s o r p t i o n of i n u l i n - c a r b o x y l - C Before i n u l i n c o u l d be used as an i n d i c a t o r of c l e a r a n c e i t had t o be shown t h a t the t u b u l a r p a r t of the c o x a l gland was r e l a t i v e l y impermeable to t h i s molecule. Otherwise i t would not be known whether r e s o r p t i o n or p a s s i v e i n f l u x of i n u l i n had occurred i n t h i s segment. The t o t a l a c t i v i t y of the hemolymph f o l l o w i n g i n -j e c t i o n of i n u l i n i n t o the c o x a l glands of f i v e t i c k s was 1 - 1.5 (mean - SE.) counts per minute. A f t e r two hours i t was 8 - 3 . 1 counts per minute. T h i s change v/as not s i g n i f i c a n t at the 95$ l e v e l of c o n f i d e n c e . On the o t h e r hand, the a c t i v i t y i n the 4- m i c r o l i t e r s from the i n j e c t e d c o x a l gland (which i s a measure of the i n u l i n i n j e c t e d but not reabsorbed) was 189 - 69 counts per minute. Even i f the change i n the a c t i v i t y of the hemolymph from 1 t o 8 counts per minute had been s i g n i f i c a n t i t would re p r e s e n t a r e a b s o r p t i o n o f o n l y 3$ of the i n j e c t e d i n u l i n d u r i n g 2 hours. I n a system where the t o t a l volume of the c o x a l gland i s c l e a r e d every 10 seconds d u r i n g c o x a l f l u i d p r o -d u c t i o n t h i s r a t e of r e a b s o r p t i o n would be extremely s m a l l . I t was concluded t h a t r e a b s o r p t i o n or net p a s s i v e i n f l u x of i n u l i n v i a the t u b u l e w a l l of the c o x a l gland v/as n e g l i g i b l e . -111-2 . Clearance of i n u l i n - c a r b o x y l - C 14-The time sequence of i n u l i n - c a r b o x y l - C concen-t r a t i o n s i n the hemolymph and c o x a l f l u i d was s t u d i e d a f t e r t r a c e r had been i n j e c t e d i n t o the hemolymph. A U / P r a t i o f o r i n u l i n of much l e s s than u n i t y might be expected f o r a f l u i d s e c r e t o r y mechanism ( i f i n u l i n moved p a s s i v e l y ) whereas a r a t i o of u n i t y o r g r e a t e r would be expected f o r a f i l t r a t i o n - r e s o r p t i o n mechanism ( K i r s c h n e r , 1967). F i g u r e 27 shows t h a t i n u l i n was not r e t a i n e d by the c o x a l gland and t h a t a f t e r o n l y 5 minutes of c o x a l f l u i d p r o d u c t i o n , the c o n c e n t r a t i o n of i n u l i n i n the hemolymph was reduced by h a l f . A f t e r 10 minutes i t was reduced by 90$ and had v i r t u a l l y been e l i m i n a t e d a f t e r a p e r i o d o f 20 hours. At t h i s time no r a d i o a c t i v i t y was found i n the gut i n d i c a t i n g t h a t i n u l i n d i d not pass across the gut w a l l . The c o x a l f l u i d to hemolymph r a t i o ( U / P ) was not s i g n i f i c a n t l y d i f f e r e n t from one over the e n t i r e course of the experiment. T h i s was so even at time 5 minutes .(95$ confidence l i m i t s ) although the l a c k of s i g -n i f i c a n c e p o s s i b l y r e s u l t e d from the use of o n l y f o u r t i c k s . On the o t h e r hand s l i g h t d i s c r e p a n c i e s at t h i s p o i n t could r e s u l t from the time l a p s e between c o l l e c t i o n of the samples of hemolymph and c o x a l f l u i d s i n c e t i m i n g was. extremely c r i t i c a l d u r i n g t h i s p e r i o d o f r a p i d change. I n any case, c l e a r l y at no time was the U / P r a t i o much g r e a t e r than one -112-FIGURE 2 7 CONCENTRATIONS OF INULIN CARBOXYL-C 1 4 IN HEMOLYMPH AND  COXAL FLUID DURING FEEDING F l u i d s v/ere sampled s i m u l t a n e o u s l y from i n d i v i d u a l t i c k s f e e d i n g on h.b. V e r t i c a l l i m i t s show Mean - SE (N=4) Legend: hemolymph c o x a l f l u i d and i n no case was i t l e s s than one. 3. H i s t o l o g y and u l t r a s t r u c t u r e of the c o x a l gland A d e t a i l e d examination of the membraneous s t r u c t u r e r e v e a l e d i n the m o r p h o l o g i c a l study showed i t t o c o n s i s t of a maze of i n t e r c o n n e c t i n g chambers ( F i g . 28a). Large n u c l e i w i t h prominent n u c l e o l i were v i s i b l e i n the mem-brane ( F i g . 28b). Examination of the membrane under the e l e c t r o n micro-scope r e v e a l e d a convoluted s t r u c t u r e composed of two l a y e r s . The w e l l d e f i n e d and continuous basement membrane was c l o s e l y a s s o c i a t e d w i t h d i s c o n t i n u o u s c e l l processes forming a s i e v e - l i k e s t r u c t u r e ( F i g . 29). I n o b l i q u e s e c t i o n s the c e l l processes appeared not as round i s l a n d s but as l o n g p a r a l l e l f i n g e r - l i k e p r o j e c t i o n s ( F i g . 30). I n many i n s t a n c e s a t h i n diaphram c o u l d be seen i n the spaces between the c e l l p r o c e s s e s . 4. S i t e of f i l t r a t i o n Evidence was presented by Lees (1946) t h a t albumin would sometimes pass from the hemolymph i n t o the c o x a l f l u i d . T h i s l e d t o s p e c u l a t i o n t h a t albumin might be of such a s i z e and shape as t o be trapped i n the pores of the f i l -t r a t i o n membrane. I t was hoped by r e v e a l i n g the presence of t h i s molecule w i t h the a i d of a f l u o r e s c e n t dye ( i . e . f l u o r e s c e i n ) t h a t the s i t e of f i l t r a t i o n might be apparent. FIGURE 28 FILTRATION MEMBRANE OF COXAL GLAND a) General appearance b) D e t a i l showing l a r g e nucleus F i x e d i n Baker's formaldehyde c a l c i u m . S e c t i o n e d at 3 microns. S t a i n e d w i t h hematoxylin and e o s i n . Mag. x 160 Legend: f.m. f i l t r a t i o n membrane r . t . r e s o r p t i o n t u b u l e n. nucleus 1. lumen -115-FIGURE 29 GENERAL APPEARANCE OP FILTRATION MEMBRANE St a i n e d w i t h u r a n y l a c e t a t e and l e a d c i t r a t e Mag. x 25,000 Legend: b. m. basement membrane c. p. c e l l processes d. diaphram between c e l l processes -116-FIGURE 30 DETAIL OF FILTRATION MEMBRANE SHOWING NATURE OF CELL PROCESSES S t a i n e d w i t h u r a n y l acetate and l e a d c i t r a t e . Mag. x ?3,000 Legend: c.p.c. c e l l processes i n c r o s s s e c t i o n c.p.o. c e l l processes i n o b l i q u e s e c t i o n b.m. basement membrane - 1 1 7 -S e c t i o n s of gland which had been t r e a t e d w i t h a l -bumin- f l u o r e s c e i n conjugate showed, when examined under u l t r a - v i o l e t i l l u m i n a t i o n , a b r i l l i a n t f l u o r e s c e n c e i n the p e r i p h e r a l r e g i o n of the suspected f i l t r a t i o n membrane. The r e s t of the gland was weakly a u t o f l u o r e s c e n t ( F i g . 3 1 a ) . On c l o s e examination i t was p o s s i b l e t o see f l u o r e s c e n c e of the accumulated a l b u m i n - f l u o r e s c e i n conjugate a c t u a l l y trapped w i t h i n pockets of the f i l t r a t i o n membrane ( F i g . 3 1 b ) . 5. Pressure dependence of c o x a l f l u i d f o r m a t i o n A t r a c e f o r hemolymph pressure d u r i n g f e e d i n g and c o x a l f l u i d p r o d u c t i o n i s shown i n F i g . 3 2 . As men-t i o n e d i n the s e c t i o n on methods, t h i s was the o n l y complete t r a c e o b t a i n e d . However, i n the f i v e s u c c e s s f u l c a n n u l a t i o n s which d i d y i e l d p r e s s u r e records at the 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 , the hemolymph p r e s s u r e s were found to measure 3 9 - 4 mm Hg (Mean - SE). The sudden t r a n s i e n t p r e s s u r e i n c r e a s e recorded when the t i c k a ttached was con-s i s t e n t l y observed and was seen to be a s s o c i a t e d w i t h the e j a c u l a t i o n of a p o o l of s a l i v a . I n v a r i a b l y pressure r e -t u r n e d t o atmospheric a f t e r attachment. During the appear-ance of p r e s s u r e peaks immediately p r e c e d i n g and f o l l o w i n g the onset of c o x a l f l u i d p r o d u c t i o n , the t i c k appeared to be f l e x i n g the l a r g e i n t e r c o x a l muscles. Note the i n c r e a s e i n o v e r a l l hemolymph pres s u r e l e a d i n g up t o c o x a l f l u i d p r o d u c t i o n and i t s g r a d u a l r e t u r n t o atmospheric pressure -118-FIGURE 31a COXAL GLAND SHOWING DISTRIBUTION OF ALBUMIN - FLUORESCEIN  CONJUGATE Fi x e d i n Baker's formaldehyde c a l c i u m . S e c t i o n e d at 8 microns. Observed under u l t r a - v i o l e t microscope (X 320). Legend: f.m. f i l t r a t i o n membrane FIGURE 31 b FILTRATION MEMBRANE SHOWING ALBUMIN-FLUORESCEIN CONJUGATE  TRAPPED IN POCKETS F i x e d i n Baker's formaldehyde cal c i u m Sectioned at 8 microns Observed under u l t r a - v i o l e t microscope (x 640) FIGURE 32 HYDROSTATIC PRESSURE IN HEMOLYMPH DURING FEEDING AND  COXAL FLUID PRODUCTION T i c k s were f e d h.b. The d u r 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 reduced because the t i c k was d i s t u r b e d by the presence of the cannula. Legend: A t i c k a t t a c h e d B c o x a l f l u i d appeared C c o x a l f l u i d p r o d u c t i o n ceased Hemolymph pressure (mm Hg ) -120-by the time c o x a l f l u i d p r o d u c t i o n had ceased. F i g u r e 14a showed t h a t the r a t e of c o x a l f l u i d p r o d u c t i o n a l s o f e l l w i t h time. F i g u r e 33 shows the e f f e c t on the r a t e of c o x a l f l u i d p r o d u c t i o n of a sudden r e d u c t i o n i n hemolymph p r e s -sure due t o e x s a n g u i n a t i o n of the t i c k . The o p e r a t i o n of clamping and c u t t i n g the l e g d i d not a f f e c t the r a t e of s e c r e t i o n . However, when f r e e hemorrhage occurr e d , the r a t e of c o x a l f l u i d p r o d u c t i o n f e l l r a p i d l y . When the f l o w of hemolymph from the l e g was c u r t a i l e d , the r a t e i n -creased again i n three of the f o u r cases although recovery t o the normal r a t e was not observed. Since the c o x a l o r i f i c e was seen to open and c l o s e r h y m i c a l l y d u r i n g c o x a l f l u i d p r o d u c t i o n , the e f f e c t on r a t e of c o x a l f l u i d p r o d u c t i o n was i n v e s t i g a t e d when one c o x a l o r i f i c e was cannulated. The r a t e of c o x a l f l u i d p r o d u c t i o n from the normal gland was found t o be 0.72 - 0.07 m i c r o l i t e r s p e r minute (Mean - SE, N = 6 ) . That f o r can-n u l a t e d glands of the same t i c k s was twice as h i g h (1.38 - 0.18 m i c r o l i t e r s per minute, N = 6 ) . These r a t e s d i f f e r s i g n i f i c a n t l y at the 9 5 $ l e v e l o f conf i d e n c e . -121-FIGURE 33 EFFECT OF HEMORRHAGING ON RATE OF COXAL FLUID PRODUCTION  IN FOUR TICKS T i c k s were f e d h.b. The i n i t i a l r a t e s o f c o x a l f l u i d p r o d u c t i o n were sometimes lower than normal because the t i c k was d i s t u r b e d by the o p e r a t i v e procedure. Legend: l e g clamped and cut d u r i n g t h i s p e r i o d l e g undamped and hemorrhaging normal r a t e of c o x a l f l u i d p r o d u c t i o n T i m e a f te r coxa l f lu id appeared ( m i n ) -122-D. DISCUSSION E x c r e t o r y systems which are known t o u t i l i z e a f l u i d s e c r e t o r y mechanism have t o date been observed t o have a very low U/P r a t i o of i n u l i n c l e a r a n c e . For example, the M a l p i g h i a n t u b u l e s of the i n s e c t Carausius morosus ex c r e t e i n u l i n at such a low r a t e t h a t the U/P r a t i o i s o n l y 0.046 (Ramsay and R i e g e l , 1961). Thus the observed U/P r a t i o i n the t i c k of c l o s e t o u n i t y over the e n t i r e e x c r e t o r y p e r i o d i s d i f f i c u l t t o r e c o n c i l e w i t h a s e c r e t o r y mechanism e s p e c i a l l y s i n c e i n u l i n c o n c e n t r a t i o n s i n the hemolymph f e l l 1 0 0 - f o l d d u r i n g t h i s time. An a c t i v e sec-r e t i o n of i n u l i n would not be expected to show t h i s r e l a t i o n s h i p over such a wide c o n c e n t r a t i o n range. Moreover, s i n c e i n u l i n was shown not t o be reabsorbed by the t u b u l e , any p a s s i v e e n t r y of i n u l i n i n t o the gland must have been v i a the f i l t r a t i o n membrane. I f i t i s assumed t h a t the i n u l i n c o n c e n t r a t i o n of the i n i t i a l f i l t r a t e was equal t o t h a t of the hemolymph as i n other f i l t r a t i o n systems ( v e r t e b r a t e k i d n e y , P i t t s , 1968; f r e s h w a t e r gastropod, L i t t l e , 1965; c r a y f i s h , R i e g e l , 1965)» the c o n c e n t r a t i o n i n 'the e x c r e t e d c o x a l f l u i d must have been approximately equal t o t h a t of the primary u l t r a f i l t r a t e . The s u g g e s t i o n then i s t h a t very l i t t l e , i f any, f l u i d r e a b s o r p t i o n occurred i n the t u b u l e of the c o x a l g l a n d . For f i l t r a t i o n t o occur there must be a d r i v i n g - 1 2 3 -f o r c e i n the form of a h y d r o s t a t i c pressure g r a d i e n t a c r o s s the f i l t r a t i o n membrane. I n the t i c k , a pressure o f about 40 mm Hg. r e l a t i v e t o atmospheric was shown to e x i s t i n the hemolymph although the pressure g r a d i e n t across the f i l t r a t i o n membrane i t s e l f was not measured. Thus i f the f i l t r a t i o n membrane could i n some way be h e l d r i g i d , t h i s f o r c e would be a v a i l a b l e to d r i v e f l u i d through the p o r e s . F u r t h e r evidence f o r a h y d r o s t a t i c d r i v i n g f o r c e v/as p r o v i d e d by the c o r r e l a t i o n between the g r a d u a l f a l l i n hemolymph pr e s s u r e and the f a l l i n r a t e of c o x a l f l u i d p r o d u c t i o n d u r i n g a normal p a t t e r n of ex-c r e t i o n . T h i s h y p o t h e s i s was f u r t h e r r e i n f o r c e d by the o b s e r v a t i o n t h a t r e d u c t i o n i n the hemolymph pres s u r e due t o b l e e d i n g caused a sudden drop i n c o x a l s e c r e t i o n r a t e . The r a t e of c o x a l f l u i d p r o d u c t i o n v/as p a r t i a l l y r e s t o r e d when hemorrhaging v/as c u r t a i l e d . Thus i t seems t h a t when the l e g v/as reclamped a f t e r b l e e d i n g the hemolymph pressure i n c r e a s e d as a b s o r p t i o n of f l u i d from the gut proceeded. However, s i n c e the hemolymph volume had a l s o been decreased, the r a t e of c o x a l f l u i d p r o d u c t i o n v/as lower than normal. I attempted to i n c r e a s e the hemolymph pres s u r e by c a n n u l a t i n g a l e g t o a measured and v a r i a b l e head of water. U n f o r t u n -a t e l y , j u s t as Lees (1946) r e p o r t e d when he t r i e d t o e l i c i t c o x a l f l u i d p r o d u c t i o n by squeezing the t i c k , no c o n c l u s i v e r e s u l t s were o b t a i n e d . P o s s i b l y a nervous c o n t r o l becomes the dominant i n h i b i t o r y f a c t o r when the t i c k i s s u b j e c t e d t o such treatment. -124-As mentioned above, t h i s h y d r o s t a t i c d r i v i n g f o r c e can o n l y be e f f e c t i v e i f the f i l t r a t i o n membrane i s h e l d r i g i d so t h a t a pressure g r a d i e n t can form across i t . Lees (1946) suggested t h a t t h i s was achieved when pockets i n the membrane were evert e d by numerous s m a l l muscles con n e c t i n g the c o x a l gland to the body w a l l . H i s hyp o t h e s i s t h a t a n e g a t i v e h y d r o s t a t i c pressure was formed w i t h i n the f i l t r a t i o n chamber n e c e s s i t a t e s the presence of a s p h i n c t e r t o i s o l a t e t h i s chamber from the e x t e r n a l atmosphere. Since the r a t e of c o x a l f l u i d p r o d u c t i o n i n c r e a s e d when the c o x a l o r i f i c e was h e l d open, c l e a r l y t h i s s p h i n c t e r does not serve t h i s purpose. Indeed, i t may be unnecessary to invoke a n e g a t i v e p r e s s u r e i n the f i l t r a t i o n chamber, si n c e a l a r g e p o s i t i v e h y d r o s t a t i c p r e s s u r e , r e l a t i v e t o atmos-p h e r i c p r e s s u r e , was demonstrated i n the hemolymph. Opening of the s p h i n c t e r at the c o x a l o r i f i c e and the consecment escape of f l u i d c o u l d then permit maintainance of a l a r g e pressure g r a d i e n t across the f i l t r a t i o n membrane and hence i n c r e a s e d r a t e of f i l t r a t i o n . Conversely, c l o s u r e of the s p h i n c t e r and subsequent b u i l d up of h y d r o s t a t i c pressure w i t h i n the c o x a l gland would l e a d t o a decrease i n the pressure g r a d i e n t across the f i l t r a t i o n membrane and de-creased r a t e of f i l t r a t i o n . Therefore, t h i s s p h i n c t e r may w e l l have p a r t i a l c o n t r o l over the r a t e of c o x a l f l u i d p r o d u c t i o n . The p o s s i b i l i t y cannot be r u l e d out t h a t the v a l v e between the f i l t r a t i o n membrane and the r e s o r p t i o n - 1 2 5 -t u b u l e (Lees, 1946) does not a l s o i n f l u e n c e the r a t e of c o x a l f l u i d p r o d u c t i o n . T h i s c o n t r o l c o u l d be e x e r c i z e d independently or co u l d be l i n k e d t o the v a l v e at the c o x a l o r i f i c e . As d i s c u s s e d i n the f o l l o w i n g chapter, 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 developed across a c t i v e glands lends weight t o the l a t t e r h y p o t h e s i s . The presence of f l u o r e s c e i n l a b e l l e d albumin trapped w i t h i n pockets of the t h i n membraneous s t r u c t u r e suggests t h a t d u r i n g c o x a l f l u i d p r o d u c t i o n there was a f l o w of f l u i d through t h i s membrane. Thus i t seems t h a t the mem-brane was f r e e l y permeable t o water but not t o l a r g e r mol-e c u l e s such as albumin. These p r o p e r t i e s p o i n t t o t h i s membrane as being the s i t e f o r f o r m a t i o n of the primary u l t r a f i l t r a t e . The u l t r a s t r u c t u r e of the f i l t r a t i o n membrane bore a s t r i k i n g resemblance t o t h a t of the gl o m e r u l a r c a p i l l a r y wall- of the v e r t e b r a t e kidney. I n the l a t t e r t i s s u e the c a p i l l a r y w a l l c o n s i s t s of th r e e d i s t i n c t l a y e r s , namely an innermost endothelium, a basement membrane and p e r i -p h e r a l l y a v i s c e r a l e p i t h e l i u m . The endothelium c o n t a i n s numerous f e n e s t r a e which are occluded by a s i n g l e l a y e r e d membrane 60 - 70 A* i n t h i c k n e s s . The basement membrane i s a homogeneous, electron-dense l a y e r w i t h l i g h t e r areas immediately adjacent t o the endothelium and e p i t h e l i u m . The e p i t h e l i a l l a y e r c o n s i s t s of processes put out by the main c e l l body such t h a t they appear i n c r o s s - s e c t i o n s to be i s l a n d s of membrane bound cytoplasm a p p l i e d adjacent to the basement membrane. The spaces between these f i n g e r - l i k e -126-processes are a l s o c l o s e d by a s i n g l e l a y e r e d membrane of 50 - 70 8 i n t h i c k n e s s (Jorgensen, 1966). The f i l t r a t i o n membrane of the c o x a l gland had a s t r u c t u r e t h a t was analagous t o the glomerular c a p i l l a r y w a l l except t h a t the l a y e r corresponding t o the e n d o t h e l i a l sheet was absent. Cytoplasmic processes of the ' e p i t h e l i a l ' l a y e r were apparent as were the t h i n membranes o c c l u d i n g the f e n e s t r a e i n t h i s l a y e r . To date t h i s s t r u c t u r e has not been s a t i s f a c t o r i l y c o r r e l a t e d w i t h the th e o r y of f i l t r a t i o n . I t i s improbable t h a t the e n d o t h e l i a l f e n e s t r a t i o n s are s t a t i c s t r u c t u r e s . 1 Rather, i t has been suggested t h a t they are a s h i f t i n g network of s i t e s w i t h i n a continuous e n d o t h e l i a l sheet (Farquhar et a l , 1961). In c o n c l u s i o n , the s t r u c t u r a l sim-i l a r i t i e s between the f i l t r a t i o n membrane of the c o x a l gland and t h a t of the glo m e r u l a r c a p i l l a r y w a l l l e a d one to pos-t u l a t e t h a t the mechanism of f i l t r a t i o n i s s i m i l a r i n each case. CHAPTER V REABSORPTION IN THE COXAL GLAND -128-A. INTRODUCTION Evidence i s presented i n Chapter IV t h a t water i s e l i m i n a t e d from the t i c k by a process of u l t r a f i l t r a t i o n . T h i s mechanism adequately serves the t i c k ' s need t o r a p i d l y e x c r e t e a l a r g e volume of f l u i d . However, as i s apparent from the c l e a r a n c e s t u d i e s w i t h i n u l i n , t h i s r e s u l t s i n an i n i t i a l l o s s of a l l but the l a r g e r molecules from the hemolymph of the t i c k . Since the t o t a l b l ood volume i s f i l t e r e d through the c o x a l gland approximately every 5 minutes, i t i s e s s e n t i a l t h a t t h e r e be a mechanism f o r r e c o v e r i n g v a l u a b l e o r g a n i c and i o n i c molecules so as t o m a i n t a i n the hemolymph c o n c e n t r a t i o n s constant. The s t u d i e s on i o n i c r e g u l a t i o n i n Chapter I I I i n d i c a t e d t h a t such" a r e c o v e r y system f o r i o n s does indeed occur i n the c o x a l g l a n d . I t was p o s t u l a t e d t h e r e f o r e t h a t e s s e n t i a l o r g a n i c molecules must s i m i l a r l y be conserved. I n the v e r t e b r a t e kidney nephron, f i l t r a t e from the glomerulus f l o w s f i r s t through the p r o x i m a l t u b u l e where g l u c o s e , amino a c i d s , some p r o t e i n s and i o n s are a c t i v e l y reabsorbed. M o r p h o l o g i c a l l y the analogous s t r u c t u r e i n the c o x a l gland i s the wide c o i l e d tubule d e s c r i b e d i n Chapter I I . S e c t i o n s of the t u b u l e were thus examined under l i g h t and e l e c t r o n microscopes so t h a t comparisons might be made between these c e l l s and o t h e r c e l l s known t o be a c t i v e l y engaged i n r e a b s o r p t i o n . Although an -129-u l t r a s t r u c t u r a l study had been made of the s c o r p i o n c o x a l gland (Rasmont, i960) no such i n v e s t i g a t i o n had been made of the t i c k c o x a l gland. I n u l i n , the o n l y o r g a n i c molecule thus f a r i n v e s t i g -ated, was c l e a r l y not reabsorbed i n the c o x a l gland (Chapter I V ) . T h i s molecule i s not, however, one which might be co n s i d e r e d e s s e n t i a l t o the t i c k ' s metabolism. Therefore I wished to determine whether amino a c i d s were reabsorbed i n the gland o r whether they were maintained i n dynamic e q u i l i b r i u m by t r a n s p o r t from the gut i n t o the hemolymph at the same r a t e as e x c r e t i o n i n the c o x a l f l u i d . C o n c e n t r a t i o n s of amino a c i d s were measured i n hemolymph a f t e r f e e d i n g and i n pooled c o x a l f l u i d so t h a t the U/P r a t i o s o b tained might be compared t o t h a t of i n u l i n where r e a b s o r p t i o n was known not t o occur. Since l a r g e volumes were needed f o r t h i s study i t was not f e a s i b l e t o measure a time course of amino a c i d c l e a r a n c e from the hemolymph. Thus a separate study was made of the c l e a r a n c e of two r a d i o a c t i v e l a b e l l e d amino a c i d s , namely p r o l i n e and a s p a r t i c a c i d , d u r i n g c o x a l f l u i d p r o d u c t i o n . Ions were found t o be reabsorbed a g a i n s t a concen-t r a t i o n g r a d i e n t (Chapter I I I ) . I n an attempt t o b u i l d a c e l l model of the processes i n v o l v e d , the e l e c t r o p o t e n t i a l p r o f i l e of the r e s o r p t i o n t u b u l e was measured. A minimum estimate of the i n t r a c e l l u l a r potassium c o n c e n t r a t i o n v/as a l s o made. Thus knov/ing the l u m i n a l and s e r o s a l i o n i c con-c e n t r a t i o n s , a model of the a c t i v e and p a s s i v e processes -130-r e s p o n s i b l e f o r i o n r e a b s o r p t i o n across the t u b u l e c o u l d be proposed. The pH of c o x a l f l u i d and hemolymph were measured; I was h o p e f u l t h a t t h i s would r e v e a l whether a mechanism e x i s t e d f o r the c o n t r o l of u r i n a r y pH such as the t r a n s -aminase r e a c t i o n found i n the v e r t e b r a t e kidney ( P i t t s et a l . , 1 9 6 3 ) . -131-B. METHODS 1. H i s t o l o g y and u l t r a s t r u c t u r e of the r e s o r p t i o n  t u b u l e The h i s t o l o g i c a l techniaues f o r both l i g h t and e l e c t r o n microscopy are d e s c r i b e d i n Chapter IV. 2. A n a l y s i s of amino a c i d s i n body f l u i d s Groups of 15 t i c k s were fed on human blood and 1 0 0 - m i c r o l i t e r samples of pooled hemolymph (2 hours a f t e r f e e d i n g ) and c o x a l f l u i d were taken from them. Although 15 t i c k s were needed to c o l l e c t s u f f i c i e n t hemolymph, the 1 0 0 - m i c r o l i t e r sample of c o x a l f l u i d was r e a d i l y ob-t a i n a b l e from about f i v e t i c k s . Since these two samples were more c l o s e l y r e l a t e d t o each o t h e r than to samples taken from t i c k s which were r e a r e d , f e d , sampled and assayed s e v e r a l weeks l a t e r , they were c o n s i d e r e d as p a i r s when computing the U/P r a t i o s . The samples were mixed w i t h about 0.5 ml . methanol, the white p r e c i p i t a t e s c e n t r i f u g e d down and the supernatent f l u i d s r e t a i n e d . The p e l l e t s were washed twice w i t h f u r t h e r 0.5 ml samples of methanol and, a f t e r c e n t r i -f u g a t i o n , the three supernatent f r a c t i o n s were pooled. T h i s d e p r o t e i n a t e d s o l u t i o n was evaporated to dryness i n a f l a s h evaporator o p e r a t i n g at 35°C. The r e s i d u e was -132-resuspended i n approximately 0.5 ml. g l a s s d i s t i l l e d water and again evaporated t o dryness t o d r i v e o f f any remaining methanol. I t was then d i s s o l v e d i n a b u f f e r which had been s p e c i a l l y prepared f o r the amino a c i d a n a l -y s e r . A 'Biocal' amino a c i d a n a l y s e r (Model BC20) was used i n the mode of o p e r a t i o n designed f o r p h y s i o l o g i c a l f l u i d s . The analyses were compared w i t h standard s o l u t i o n s of amino a c i d s ; t h i s p e r m i t t e d q u a n t i t a t i v e e s t i m a t e s t o be made. 14- 14-3. Clearance of p r o l i n e - C and a s p a r t i c acid-C The technique used was t h a t d e s c r i b e d f o r i n u l i n -14-carboxyl-C i n Chapter IV. Chemicals were s u p p l i e d by New England N u c l e a r . 4-. 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 s across the c o x a l  gland 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 across the c o x a l gland was measured between a drop of hemolymph from an amputated l e g and a drop of c o x a l f l u i d at the c o x a l o r i f i c e . The apparatus used was i d e n t i c a l t o t h a t d e s c r i b e d i n Chapter IV. I n some cases the p o t e n t i a l v/as measured v/hile the c o x a l o r i f i c e was h e l d open by a f i n e g l a s s cannula. As noted e a r l i e r , great care was needed v/hen the c a n n u l a t i o n was performed not t o puncture the w a l l s of the c o x a l gland. -133-The e l e c t r o p o t e n t i a l p r o f i l e o f the t u b u l a r c e l l s o f the c o x a l gland was a l s o measured. The gut, r e p r o -d u c t i v e organs and s a l i v a r y glands were d i s s e c t e d from the unfed t i c k ; care was taken not t o touch the c o x a l g l a n d s . The body c a v i t y was f i l l e d w i t h a t i s s u e c u l t u r e medium developed f o r t i c k s by Rehacek and B r z o s t o w s k i (1969). The c o n c e n t r a t i o n s o f the major i o n s i n t h i s medium were: sodium (14-0 m e q / l i t e r ) , potassium (16 m e q / l i t e r ) and c h l o r i d e (110 m e q / l i t e r ) . The i n d i f f e r e n t e l e c t r o d e was touched to the po o l o f t i s s u e c u l t u r e medium and the r e c o r d i n g e l e c t r o d e lowered i n t o the drop so t h a t asymmetry c o r r e c t i o n s might be made. The r e c o r d i n g e l e c t r o d e was g e n t l y lowered w i t h i t s t i p a g a i n s t the wide r e s o r p t i o n t u b u l e u n t i l i t suddenly p i e r c e d the s u r f a c e . T r a n s i e n t p o t e n t i a l s were i g n o r e d and o n l y s t a b l e , r e p r o d u c i b l e r e a d i n g s were recorded. I f the e l e c t r o d e was advanced s t i l l f u r t h e r i n t o the gland i t was o f t e n p o s s i b l e t o r e c o r d a l u m i n a l p o t e n t i a l and then an i n t r a -c e l l u l a r p o t e n t i a l a g a i n on the remote s i d e o f the t u b u l e . F u r t h e r advancement would cause the e l e c t r o d e t o bend a g a i n s t the c u t i c l e . T h i s was rec o g n i z e d by d i s t o r t i o n of the t r a c e on the o s c i l l o s c o p e . The t i p of the r e c o r d i n g e l e c t r o d e was l o c a l i z e d i n some experiments by e l e c t r o p h o r e t i c i n j e c t i o n of a dye. Gl a s s m i c r o p i p e t t e s were f i l l e d w i t h a s o l u t i o n of potassium c h l o r i d e and amaranth. Immediately a f t e r the p o t e n t i a l -134-had been r e c o r d e d , a p o t e n t i a l d i f f e r e n c e of 100 V was a p p l i e d between the e l e c t r o d e s . T h i s r e s u l t e d i n movement of dye from the m i c r o p i p e t t e out i n t o the surrounding f l u i d . I f the t i p was w i t h i n a c e l l the c o l o u r was con-f i n e d t o a s m a l l area even when the gland was p a l p i t a t e d . However, when the t i p was i n the lumen, such treatment r e s u l t e d i n the dye r a p i d l y d i f f u s i n g away. Thus poten-t i a l s c o u l d be a c c u r a t e l y assigned t o a p a r t i c u l a r p a r t of the r e s o r p t i o n t u b u l e . 5. I n t r a c e l l u l a r potassium c o n c e n t r a t i o n of the  c o x a l gland Coxal glands were d i s s e c t e d from unfed t i c k s , q u i c k l y r i n s e d i n i s o t o n i c glucose s o l u t i o n and b l o t t e d d r y on absorbent paper. They were weighed on t a r e d p l a t i n u m t r a y s (wet w e i g h t ) , d r i e d o v e r n i g h t at 60°C. and reweighed (dry w e i g h t ) . Thus the amount of t i s s u e water c o u l d be measured. Samples were ashed at 900°C. The p l a t i n u m t r a y s p l u s ashed samples were then each dropped i n t o 2 ml s l i g h t l y a c i d -i f i e d (HC1) sodium c h l o r i d e s o l u t i o n . T h i s sodium swamp (500 ppm) was used to e l i m i n a t e i n t e r f e r e n c e of sodium dur-i n g the flame photometric d e t e r m i n a t i o n of potassium con-c e n t r a t i o n ( p r e v i o u s l y d e s c r i b e d i n Chapter I I I ) . 6.. pH of body f l u i d s F r e s h or f r e s h l y f r o z e n s e a l e d samples of hemolymph -135-and c o x a l f l u i d v/ere used f o r d e t e r m i n a t i o n s of pH. A Radiometer pH meter (Model PHM25a) w i t h an expanded s c a l e (Model P H A 9 2 5 a ) was used. The e l e c t r o d e s (Radiometer nos. K150 and G252c) had been e s p e c i a l l y designed f o r sm a l l volumes (5-100 m i c r o l i t e r s ) of f l u i d . D u p l i c a t e readings v/ere made on each sample of c o x a l f l u i d but su f -f i c i e n t hemolymph was a v a i l a b l e f o r s i n g l e readings o n l y . P r i o r t o measuring any sample; I r i n s e d the e l e c t r o d e s w i t h a l i t t l e of t h a t sample. This e l i m i n a t e d any d r i f t i n the measured pH. -136-C. RESULTS 1. H i s t o l o g y and u l t r a s t r u c t u r e of the r e s o r p t i o n  t u b u l e The t u b u l e w a l l s from c o x a l glands of s t a r v e d t i c k s measured 14- i 1.3 microns (Mean i SE) t h i c k . T h e i r n u c l e i were s c a t t e r e d and f a i n t l y s t a i n e d and no c e l l boundaries were v i s i b l e ( F i g . 34a). L u r i n g c o x a l f l u i d p r o d u c t i o n the t u b u l e c e l l s became s w o l l e n (27 i 1.4 microns t h i c k ) o f t e n t o such an extent t h a t the lumen was p a r t i a l l y o ccluded or appeared as a s e r i e s of v a c u o l e s . I n t h i s s t a t e the n u c l e i , which now s t a i n e d much more s t r o n g l y , were s i t u a t e d on the l u m i n a l border of the c e l l s ( F i g . 34b). Both before f e e d i n g and d u r i n g c o x a l f l u i d p r o d u c t i o n , t u b u l e c e l l s showed s t r i a t i o n s and a d i s t i n c t l u m i n a l brush border. Vacuoles were o f t e n apparent on the l u m i n a l s i d e of the c e l l s . The u l t r a s t r u c t u r a l i n v e s t i g a t i o n showed t h a t the l u m i n a l border of t u b u l e c e l l s from both s t a r v e d and ex-c r e t i n g t i c k s possessed numerous c l o s e l y packed m i c r o v i l l i . Large m i t o c h o n d r i a w i t h prominent c r i s t a e were s i t u a t e d between e x t e n s i v e i n f o l d i n g s of the b a s a l and l a t e r a l mem-branes. Septate desmosomes were c l e a r l y v i s i b l e between c e l l s ( F i g s . 35 and 36). One s t r i k i n g d i f f e r e n c e between the t u b u l e c e l l s before f e e d i n g and those d u r i n g c o x a l f l u i d p r o d u c t i o n was t h a t the l a t e r a l i n t e r c e l l u l a r spaces and -137-FIGURE 34 GENERAL APPEARANCE 07 RESORPTION TUBULE a) Before f e e d i n g b) During c o x a l f l u i d p r o d u c t i o n ( T i c k s f e d h.b.) Fi x e d i n Baker's formaldehyde c a l c i u m . S e c t i o n e d at 3 microns S t a i n e d w i t h hematoxylin and e o s i n . Mag. x 160 Legend: 1. lumen t.w. t u b u l e w a l l n. nucleus -138-FIGURE 35 PART OP RESORPTION TUBULE BEFORE FEEDING S t a i n e d w i t h u r a n y l a c e t a t e and l e a d c i t r a t e . Mag. x 19 ,900 Legend: m. m i t o c h o n d r i a b . i . b a s a l i n f o l d i n g s m.v. m i c r o v i l l i b.m. basement membrane L . i . l a t e r a l i n f o l d i n g s mv -139-FIGURE 36 PART OF RESORPTION TUBULE DURING COXAL FLUID PRODUCTION S t a i n e d w i t h u r a n y l a c e t a t e and l e a d c i t r a t e . Mag. x 19,900 Legend: m. m i t o c h o n d r i a b . i . b a s a l i n f o l d i n g s m.v. m i c r o v i l l i b.m. basement membrane s.d. septate desmosome L . i . l a t e r a l i n f o l d i n g s -140-b a s a l i n f o l d i n g s were v e r y d i s t e n d e d i n the l a t t e r case. 2. A n a l y s i s of amino a c i d s i n body f l u i d s T h i s a n a l y s i s showed t h a t u n l i k e i n o r g a n i c i o n s , amino a c i d , u r e a and ammonium i o n c o n c e n t r a t i o n s v a r i e d w i d e l y from one group of t i c k s t o another (Table X I V ) . The o n l y amino a c i d s which c o n s i s t e n t l y had a U/P r a t i o o f l e s s than u n i t y were asparagine and glutamine (combined peak), t h r e o n i n e , t a u r i n e , a r g i n i n e , methionine and c y s t i n e . Only one amino a c i d was c h a r a c t e r i z e d by a c o n s i s t e n t U/P r a t i o o f g r e a t e r than u n i t y and t h a t was glu t a m i c a c i d . A p l o t was made of mean U/P r a t i o s f o r each amino a c i d a g a i n s t the n a t u r a l c o n c e n t r a t i o n o f t h a t amino a c i d i n the b l o o d meal ( P i g . 37) . A p o s i t i v e c o r r e l a t i o n was found between these parameters ( p r o b a b i l i t y of 0.008 t h a t slope o f l i n e was z e r o ) . 14 14 3 . Clearance o f p r o l i n e - C and a s p a r t i c acid-C Time sequence measurements of p r o l i n e and a s p a r t i c a c i d c o n c e n t r a t i o n s i n hemolymph and co x a l f l u i d were made f o l l o w i n g i n j e c t i o n of these l a b e l l e d molecules i n t o the hemolymph. Thus an i n d i c a t i o n of r e s o r p t i v e a c t i v i t y i n the gland at v a r i o u s stages of the e x c r e t o r y p e r i o d c o u l d be s t u d i e d . T h i s study was not p o s s i b l e u s i n g the amino a c i d a n a l y s e r because o f the r e l a t i v e l y l a r g e samples needed f o r a n a l y s i s . TABLE XIV AMINO ACID ANALYSIS OP FINAL HEMOLYMPH AND POOLED COXAL FLUID (MEAN - SE) The abs o l u t e c o n c e n t r a t i o n s of some amino a c i d s are not pr e s e n t e d s i n c e standards v/ere not run i n these cases. However, r a t i o s of c o x a l f l u i d : hemolymph were measured i n a l l cases u s i n g the same column and are r e p o r t e d as the mean va l u e of p a i r e d samples. These r a t i o s f o r p a i r e d samples do not n e c e s s a r i l y agree w i t h r a t i o s estimated from the means f o r a l l d e t e r m i n a t i o n s . A l l c o n c e n t r a t i o n s are expressed as y u M / l i t e r . TABLE XIV Amino F i n a l hemolymph Pooled c o x a l R a t i o N A c i d c o n c e n t r a t i o n f l u i d con- c . f . / h . l . c e n t r a t i o n Asp 20 + 13 24 Thr 94 + 45 67 Ser 178 84 75 Pro 46 + 13 46 Glu 100 + 47 156 G l y 223 + 107 125 A l a 256 + 91 173 V a l 177 + 62 94 Cys 48 + 36 7 Met 25 + 6 8 i- L e u 89 + 4 29 Leu 180 + 11 75 Tyr 172 + 118 68 Phe 206 + 174 55 N V Lys H i s 30 + 10 32 Arg 158 + 47 30 Tau Urea Asp-NH 2 ) Glu-NH 2 T o t a l 2002 + 11 1.5 + 0.9 3 + 15 0.7 + 0.1 3 + 20 0.5 + 0.3 3 + 5 1.1 + 0.3 2 + 55 2.1 + 1.0 4 + 45 0.8 + 0.4 4 + 52 0.8 + 0.2 4 + 28 0.7 + 0.2 4 + 5 0.2 + 0.2 3 i 1 0.4 + 0.2 4 + 14 0.6 + 0.4 4 + 23 1.0 + 0.4 4 + 32 1.1 + 0.7 4 + 25 0.6 + 0.4 3 0.8 + 0.2 2 0.7 + 0.4 2 + 13 0.9 + 0.1 4 10 0.3 + 0.1 4 0.4 + 0.06 4 1.0 + 0.1 3 0.1 + 0.07 4 FIGURE 37 RELATION BETWEEN AMINO ACID CONCENTRATION IN BLOOD MEAL  AND RATIO OF CONCENTRATIONS IN COXAL FLUID: HEMOLYMPH Ti c k s were f e d h.b. R a t i o s were taken from Table XIV.' Co n c e n t r a t i o n s i n blood meal v/ere taken from the Handbook of B i o c h e m i s t r y ( P u b l . Chemical Rubber Co., 1968). I Amino acid concentration in human plasma (juM /liter) -143-F i g u r e s 38 and 39 i n d i c a t e t h a t t h e r e was some r e -t e n t i o n o f both l a b e l l e d p r o l i n e and a s p a r t i c a c i d by the c o x a l gland. A f t e r 25 minutes the a c t i v i t y of r a d i o -a c t i v e p r o l i n e i n the hemolymph had f a l l e n by o n l y 59$ and t h a t o f a s p a r t i c a c i d by 79$: t h i s should be compared w i t h the i n u l i n a c t i v i t y which had f a l l e n by 95$ d u r i n g the same p e r i o d . Student's t - t e s t was performed on these curv e s . For p r o l i n e , a c t i v i t i e s i n the hemolymph and c o x a l f l u i d were found t o be s i g n i f i c a n t l y d i f f e r e n t (95$ c o n f i d e n c e l i m i t s ) at p o i n t s A, B and C but not D. The a c t i v i t i e s were found to be s i g n i f i c a n t l y d i f f e r e n t f o r a l l f o u r p o i n t s f o r a s p a r t i c a c i d . The U/P r a t i o at 15 minutes was c a l c u l a t e d t o be 0 .65 f o r both p r o l i n e and a s p a r t i c a c i d as compared t o a va l u e of u n i t y f o r i n u l i n . The v a l u e s i n d i c a t e t h a t r e a b s o r p t i o n of these r a d i o a c t i v e amino a c i d s p r o b a b l y o c c u r r e d . 4. 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 s a c r o s s the c o x a l  g l and The p o t e n t i a l a c r o s s an e x c r e t i n g c o x a l gland f l u c -t u a t e d at about 65 p u l s e s per minute. The a c t u a l p o t e n t i a l v a r i e d from a low o f 0 -5 mv t o a h i g h of 30-40 mv; the lumen o f the c o x a l gland always remained n e g a t i v e w i t h r e s p e c t t o the hemolymph. C i r c u i t r e s i s t a n c e was found t o p u l s e s i m i l a r l y . The c o x a l o r i f i c e was examined c l o s e l y and found t o open and c l o s e r h y m i c a l l y w i t h about the same frequency. When the v a l v e i n the c o x a l o r i f i c e was h e l d -144-FIGURE 38 CONCENTRATIONS OF PROLINE-C 1^ IN HEMOLYMPH AND COXAL  FLUID DURING FEEDING F l u i d s were sampled s i m u l t a n e o u s l y from i n d i v i d u a l t i c k s f e e d i n g on h.b. V e r t i c a l l i m i t s show Mean - SE (N=4) Legend: O hemolymph • c o x a l f l u i d 3500 i A B C D 0 5 10 15 20 25 30 Time after start of feeding (min) -145-FIGURE 39 CONCENTRATIONS OF ASPARTIC ACID-C X" IN HEMOLYMPH AND  COXAL FLUID DURING FEEDING F l u i d s were sampled s i m u l t a n e o u s l y from i n d i v i d u a l t i c k s f e e d i n g on h.b. V e r t i c a l l i m i t s show Mean ± SE (N=4) Legend: O hemolymph O c o x a l f l u i d 3000 2500 H 2000 1500 1000 500 Time after start of feeding (min) -14-6-open by c a n n u l a t i o n , the p o t e n t i a l across t h i s gland was a b o l i s h e d . I n the i n t a c t exposed c o x a l gland from an unfed t i c k an e l e c t r o p o t e n t i a l p r o f i l e could be observed as the r e c o r d i n g e l e c t r o d e passed down through the gland and a g a i n as i t r e t u r n e d . The mean v a l u e s (- SE) were 0 mv-^(-) 10 - 0 mv (N=8) (-) 4-4- ± 4- mv (N=8) — (-) 10 - 0 mv (N=6). When the p i p e t t e t i p was i n the lumen, the p o t e n t i a l recorded i n d i f f e r e n t glands ranged from - 3 5 t o -60 mv but w i t h i n the same gland repeated impalements y i e l d e d a constant p o t e n t i a l t o w i t h i n 1 or 2 mv. There was no measureable v a r i a n c e f o r the i n t r a c e l l u l a r p o t e n t i a l of t u b u l e c e l l s ( i . e . l e s s than - 1 mv). Two experiments were performed to check the l o c a t i o n o f the r e c o r d i n g e l e c t r o d e . F i r s t i t was found t h a t when the t u b u l e was cut open the p o t e n t i a l d i f f e r e n c e p r e v i o u s l y a ssigned to the lumen was a b o l i s h e d . However, the i n t r a -c e l l u l a r p o t e n t i a l of - 10 mv c o u l d s t i l l be recorded at a s i t e remote from the i n c i s i o n . Secondly the t i p was l o c a l i z e d by the e l e c t r o p h o r e t i c i n j e c t i o n of amaranth. The s i t e which y i e l d e d a p o t e n t i a l d i f f e r e n c e of - 3 5 t o -60 mv was shown by t h i s technique to be the lumen (the dye r a p i d l y d i f f u s e d away from the t i p of the e l e c t r o d e ) . S i m i l a r l y the suspected i n t r a c e l l u l a r p o t e n t i a l was assoc-i a t e d w i t h a s i t e i n x^hich the dye remained l o c a l i z e d ( i . e . w i t h i n the c e l l ) . -147-5. I n t r a c e l l u l a r potassium c o n c e n t r a t i o n of the  c o x a l gland T i s s u e potassium v/as found t o have a c o n c e n t r a t i o n of 33 - 3.1 meq/liter.(Mean - SE, N=4). Some of the weight l o s s on d r y i n g was due, not t o i n t r a c e l l u l a r water, but t o e x t r a c e l l u l a r water and d r o p l e t s adhering t o the out-s i d e of the g l a n d . Since t h i s water had v e r y low potassium c o n c e n t r a t i o n , the v a l u e o b t a i n e d r e p r e s e n t s a minimum estimate of i n t r a c e l l u l a r potassium c o n c e n t r a t i o n of c o x a l gland t i s s u e . I t i s moreover an approximate estimate s i n c e i t was v e r y d i f f i c u l t t o b l o t excess water from such a t i n y p i e c e of t i s s u e . 6. pH of body f l u i d s The pH of hemolymph sampled 2 hours a f t e r the i n i t -i a t i o n of f e e d i n g was 6.82 - 0.05 (Mean - SE, N=4) and t h a t of pooled c o x a l f l u i d was 7.40 - 0.07 (Mean ± SE, N=4). These v a l u e s are s i g n i f i c a n t l y d i f f e r e n t at the 95$ l e v e l of c o n f i d e n c e . -148-D. DISCUSSION I n g e n e r a l appearance and u l t r a s t r u c t u r e , t u b u l e c e l l s of the c o x a l glands possessed c h a r a c t e r i s t i c s t y p i c a l of a c t i v e l y r e s o r b i n g c e l l s . More s p e c i f i c a l l y they bore a s t r i k i n g resemblance t o c e l l s of the p r o x i m a l convoluted t u b u l e of the v e r t e b r a t e kidney ( S j o s t r a n d and Rhodin, 1953). However, septate desmosomes, which are commonly found i n arthropod t i s s u e s (Locke, 1965), were evi d e n t i n the j u n c t i o n a l complexes. These s t r u c t u r e s have been c o r r e l a t e d w i t h a h i g h r e s i s t a n c e t o i o n i c d i f -f u s i o n a c r o s s the e p i t h e l i a l l a y e r and a low r e s i s t a n c e be-tween adjacent c e l l s (Loewenstein and Kanno, 1964). The brush border on the l u m i n a l s i d e of the c e l l was c h a r a c t e r -i s t i c of many a c t i v e l y r e s o r b i n g or s e c r e t i n g e p i t h e l i a ; e.g. i n s e c t M a l p i g h i a n t u b u l e s (Tsubo and Brandt, 1962), p r o x i m a l t u b u l e of v e r t e b r a t e kidney ( S j o s t r a n d and Rhodin, 1953) and the i n s e c t midgut (Anderson and Harvey, 1966). E x t e n s i v e i n f o l d i n g s of the b a s a l membrane are commonly found i n r e s o r b i n g and s e c r e t i n g c e l l s * , e.g. the p r o x i m a l t u b u l e of the v e r t e b r a t e kidney ( S j o s t r a n d and Rhodin, 1953), and p a p i l l a e of mosauitoes (Copeland, 1964 P h i l l i p s and M e r e d i t h , 1969), M a l p i g h i a n t u b u l e s °^ O a l l i p h o r a ( B e r r i d g e and Oschman, 1969) and r e s o r p t i o n t u b u l e s of the s c o r p i o n c o x a l gland (Rasmont, I960). -14-9-D i s t e n s i o n o f these i n f o l d i n g s observed i n a c t i v e l y ex-c r e t i n g c o x a l glands may have been a f i x a t i o n a r t e f a c t . However, t h i s might account f o r the g e n e r a l s w e l l i n g of these c e l l s observed by l i g h t microscopy. Such s t r u c t u r a l changes have i n s e v e r a l cases been c o r r e l a t e d w i t h p h y s i o -l o g i c a l measurements of i o n and water t r a n s p o r t r a t e s (Diamond and Tormey, 1966). S w e l l i n g of b a s a l i n f o l d i n g s and l a t e r a l i n t e r c e l l u l a r spaces has a l s o been observed i n c e l l s of the p r o x i m a l t u b u l e ( B e n t z e l , Parsa and Hare, 1969). I t would appear from the e x t e n s i v e s u r f a c e area and numerous mitochondria t h a t these c e l l s are capable of r e s o r p t i v e a c t i v i t y b e f o r e f e e d i n g as w e l l as d u r i n g c o x a l f l u i d p r o d u c t i o n . The h i g h l y v a r i a b l e c o n c e n t r a t i o n s of amino a c i d s found i n hemolymph of t h i s t i c k were not unexpected s i n c e t h i s i s g e n e r a l l y the case i n i n s e c t s (Gilmour, 1961). I t was i n i t i a l l y d i f f i c u l t t o account f o r the h i g h U/P r a t i o (2.1) f o r g l u t a m i c a c i d and the low r a t i o (0.1) f o r glutamine i n terms of m e t a b o l i c needs of the t i c k . However, i t should be r e c a l l e d t h a t the major source of ammonia i n the human kidney i s glutamine. An enzyme, glutaminase I , i s capable of s p l i t t i n g glutamine t o y i e l d glutamate and ammonia ( P i t t s et a l . , 1963). This r e a c t i o n would e x p l a i n the U/P r a t i o s observed f o r these two amino a c i d s i n the t i c k and the h i g h e r pH of c o x a l f l u i d r e l a t i v e t o hemolymph. I t i s reasonable t h e r e f o r e to propose t h a t such an enzyme may w e l l be a c t i v e i n the c o x a l g l a n d . -150-As might be p r e d i c t e d there was a t r e n d f o r those amino a c i d s which were i n low c o n c e n t r a t i o n s i n the blood meal t o be reabsorbed to a g r e a t e r extent i n the c o x a l gland. N a t u r a l l y though, h a n d l i n g of s p e c i f i c amino a c i d s might be expected to depend on the metabolic requirements of the t i c k . Any i n t e r p r e t a t i o n of the above data should be made w i t h r e a l i z a t i o n of the f o l l o w i n g l i m i t a t i o n . Since i t was not p o s s i b l e to c o l l e c t s u f f i c i e n t hemolymph f o r an a n a l y s i s of amino a c i d c o n c e n t r a t i o n s before and d u r i n g f e e d i n g , i t was necessary t o compare f i n a l hemolymph con-c e n t r a t i o n s w i t h pooled c o x a l f l u i d c o n c e n t r a t i o n s . The assumption i s thus made t h a t the f i n a l hemolymph concen-t r a t i o n i s the same as t h a t v a l u e which e x i s t e d v/hile c o x a l f l u i d was b e i n g produced. This was found t o be so f o r i n o r g a n i c i o n s . I n a d d i t i o n i t i s encouraging t o note t h a t urea., which i s a h i g h l y d i f f u s i b l e molecule and which one would expect to f i n d i n eaual Q u a n t i t i e s i n hemolymph and c o x a l f l u i d , does i n f a c t show a c o n s i s t e n t U/P r a t i o of u n i t y . T h i s lends weight t o any r e l a t i o n s h i p s which emerge from the comparison of f i n a l hemolymph t o pooled c o x a l f l u i d c o n c e n t r a t i o n s . On average the U/P r a t i o s were l e s s than u n i t y f o r most amino a c i d s . This suggests t h a t r e a b s o r p t i o n a g a i n s t a c o n c e n t r a t i o n g r a d i e n t d i d occur. I n the few cases d i s -cussed e a r l i e r , U/P r a t i o s which vrere i n v a r i a b l y l e s s than u n i t y might be i n t e r p r e t e d as s t a t i s t i c a l l y s i g n i f i c a n t - 1 5 1 -cases of r e a b s o r p t i o n . I n t e r e s t i n g l y , most of these were sul p h u r c o n t a i n i n g amino a c i d s . The analyses showed t h a t l a r g e Quantities of amino a c i d s were l o s t i n the c o x a l f l u i d . T h i s might at f i r s t seem to be disadvantageous t o the t i c k , but may be an un-avo i d a b l e consequence of a f i l t r a t i o n mechanism which r e -moves excess f l u i d r a p i d l y , but does not reabsorb ad-e q u a t e l y . However, s i n c e the b u l k of amino a c i d s remain i n the gut as undigested p r o t e i n , amino a c i d s i n the hemo-lymph may be r e a d i l y r e p l e n i s h e d . The two r a d i o a c t i v e l a b e l l e d amino a c i d s ( p r o l i n e and a s p a r t i c a c i d ) showed a f a i r l y constant U/P r a t i o w i t h time throughout c o x a l f l u i d p r o d u c t i o n . The U/P r a t i o s o f 0.65 were not s u f f i c i e n t l y low t o show u n e q u i v o c a l l y t h a t r e a b s o r p t i o n d i d occur. T h i s value c o u l d r e s u l t from exchange w i t h u n l a b e l l e d amino a c i d s i n the t u b u l e c e l l s but i f t h i s were the case the r a t i o might have been ex-pected t o r i s e d u r i n g c o x a l f l u i d p r o d u c t i o n . A l t e r n a t i v e l y the r a t i o may r e f l e c t the dynamics of amino a c i d movement through the hemolymph. I n Chapter I I I I demonstrated t h a t under a l l f e e d i n g c o n d i t i o n s s t u d i e d , sodium and c h l o r i d e were reabsorbed i n the c o x a l gland a g a i n s t a c o n c e n t r a t i o n g r a d i e n t . When t i c k s were f e d h.b./g. 2 5 / 7 5 c h l o r i d e was found to be r e -absorbed a g a i n s t a f i v e f o l d g r a d i e n t d u r i n g f r e e f l o w of c o x a l f l u i d . A n a l y s i s of the i n i t i a l drops of c o x a l -152-f l u i d from t i c k s f e d h.b./D.W. showed t h a t c h l o r i d e r e -a b s o r p t i o n must have occurred a g a i n s t a c o n c e n t r a t i o n g r a d i e n t of at l e a s t s i x f o l d . Potassium was not found to be reabsorbed d u r i n g f r e e f l o w of c o x a l f l u i d from t i c k s fe d h.b. (the o n l y c o n d i t i o n s t u d i e d f o r t h i s i o n ) . I n f a c t the i n i t i a l drops of c o x a l f l u i d from these t i c k s showed a potassium c o n c e n t r a t i o n f o u r times t h a t of the hemolymph. Thus there was s t r o n g evidence f o r r e a b s o r p t i o n of sodium and c h l o r i d e but no i n d i c a t i o n of t h i s f o r potassium. Measurements of p o t e n t i a l across the c o x a l gland both i n i n t a c t t i c k s which were producing c o x a l f l u i d and i n d i s s e c t e d glands from unfed t i c k s showed t h a t the lumen of the gland was n e g a t i v e w i t h r e s p e c t t o the hemolymph. The p o t e n t i a l measured i n i n t a c t t i c k s showed a c y c l i c o s c i l l a t i o n which may have been due t o the opening and c l o s i n g of a s p h i n c t e r . I t i s p o s s i b l e t h a t zero p o t e n t i a l s which were measured s p o r a d i c a l l y across n o r m a l l y o p e r a t i n g glands and i n v a r i a b l y across cannulated g l a n d s , arose from a short c i r c u i t through the f i l t r a t i o n membrane. Presumably some type of v a l v e such as t h a t suggested by Lees (1946) might e x i s t between the r e s o r p t i o n tubule and the f i l t r a t i o n membrane. Movements of t h i s v a l v e would e x p l a i n the p u l s e s of i n c r e a s e d r e s i s t a n c e and p o t e n t i a l . I n cannulated glands where the v a l v e at the o r i f i c e was h e l d open t h i s other v a l v e may have been unable t o c l o s e ( i . e . the two v a l v e s worked i n synchrony). -153-The e l e c t r o p o t e n t i a l p r o f i l e of r e s o r p t i o n t u b u l e c e l l s was measured i n glands from unfed t i c k s . C o r r e l a t i o n of t h i s w i t h i o n i c c o n c e n t r a t i o n s i n the c u l t u r e medium (Rehacek and B r z o s t o w s k i , 1969) and i n the i n i t i a l drops of c o x a l f l u i d (Table XI) enabled a h y p o t h e t i c a l model f o r i o n t r a n s p o r t across the r e s o r p t i o n t u b u l e of the c o x a l gland t o be p o s t u l a t e d . The i n i t i a l drop of c o x a l f l u i d was chosen s i n c e i t was assumed t h a t t h i s f l u i d had been c o n t a i n e d i n the gland f o r a c o n s i d e r a b l e time and thus had reached e q u i l i b r i u m w i t h the c e l l s of the r e s o r p t i o n t u b u l e . C h l o r i d e c o n c e n t r a t i o n s were lower i n t h i s i n i t i a l drop than i n the pooled sample of c o x a l f l u i d (Table X I ) . Since s e v e r a l times the volume of the gland were needed i n order t o measure i o n i c c o n c e n t r a t i o n s , "the assumption was made t h a t the c h l o r i d e c o n c e n t r a t i o n of f l u i d o r i g i n a l l y con-t a i n e d w i t h i n the gland was lower s t i l l than t h a t measured i n the i n i t i a l drop. By the same r e a s o n i n g , the potassium c o n c e n t r a t i o n i n the s t o r e d f l u i d was probably h i g h e r than t h a t measured i n the i n i t i a l drop whereas sodium concen-t r a t i o n s were the same. Sodium was shown t o be absorbed from f l u i d s t o r e d i n the c o x a l gland of an unfed t i c k a g a i n s t a 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 (Chapters I I I and V ) . T h i s i n -d i c a t e s t h a t under these c o n d i t i o n s sodium was a c t i v e l y t r a n s p o r t e d . I f the i n t r a c e l l u l a r sodium c o n c e n t r a t i o n was low i n the r e s o r p t i o n c e l l s , as i t i s i n most c e l l s , the - I m -probable l o c a t i o n of t h i s sodium pump was the s e r o s a l membrane. The e l e c t r o p o t e n t i a l g r a d i e n t measured across r e -s o r p t i o n c e l l s from an unfed t i c k w a s ' c a l c u l a t e d t o be g r e a t e r than v/as r e q u i r e d t o support the minimum estimate of the c h l o r i d e c o n c e n t r a t i o n g r a d i e n t . T h i s g r a d i e n t was a minimum estimate because, as d e s c r i b e d e a r l i e r , the c h l o r i d e c o n c e n t r a t i o n measured i n the f i r s t drop of c o x a l f l u i d v/as p robably h i g h e r than i n the f l u i d s t o r e d i n the g l a n d . Therefore c h l o r i d e i o n s c o u l d be p a s s i v e l y d i s -t r i b u t e d across the r e s o r p t i o n t u b u l e of an unfed t i c k . Knowledge of the i n t r a c e l l u l a r potassium c o n c e n t r a t i o n enabled c o n s i d e r a t i o n of the movement of t h i s i o n across the i n d i v i d u a l c e l l membranes. Potassium was found t o move from a c o n c e n t r a t i o n of 16 m e q / l i t e r i n the c u l t u r e medium (Chapter I I I ) t o a c o n c e n t r a t i o n of 33 m e q / l i t e r v / i t h i n the c e l l (Chapter V ) . These measurements v/ere made i n an unfed t i c k and hence assumed to be under e o u i l i b r i u m con-d i t i o n s v/ith no net water movement. Thus I could apply the Nernst e q u a t i o n to determine whether the ions were moving p a s s i v e l y v/ith the e l e c t r o p o t e n t i a l g r a d i e n t . The observed e l e c t r o p o t e n t i a l g r a d i e n t of 10 mv could not account f o r the c o n c e n t r a t i o n g r a d i e n t . Thus potassium was p r o b a b l y a c t i v e l y t r a n s p o r t e d across the s e r o s a l membrane. Since the potassium c o n c e n t r a t i o n r e p o r t e d f o r the i n i t i a l drop of c o x a l f l u i d v/as a minimum value (Chapter I I I ) i t i s not p o s s i b l e to say f o r sure whether potassium was a c t i v e l y or -Im-p a s s i v e l y t r a n s p o r t e d across the l u m i n a l membrane. How-ever, potassium c o n c e n t r a t i o n s i n the c o x a l f l u i d were known t o be h i g h e r when t h a t f l u i d stood f o r some time i n c o n t a c t w i t h the r e s o r p t i o n c e l l s (Table X I ) . P o s s i b l y then potassium was p a s s i v e l y d i s t r i b u t e d across the l u m i n a l membrane, t h i s membrane having a low p e r m e a b i l i t y to the i o n so t h a t leakage i n t o the c o x a l f l u i d was minimal. I n summary sodium i o n s were shown to be a c t i v e l y t r a n s p o r t e d from the c o x a l f l u i d t o the hemolymph; the more l i k e l y s i t e of t h i s pump was the s e r o s a l membrane. Potassium was shown to be a c t i v e l y t r a n s p o r t e d from the hemolymph i n t o the r e s o r p t i o n c e l l s but pr o b a b l y p a s s i v e l y d i s t r i b u t e d across a l u m i n a l membrane of low p e r m e a b i l i t y . The evidence i n d i c a t e d t h a t c h l o r i d e i o n s c o u l d be p a s s i v e l y d i s t r i b u t e d across the r e s o r p t i o n c e l l s . These r e s u l t s are s i m i l a r t o those obtained from s t u d i e s on some ot h e r e p i t h e l i a l membranes; e.g. f r o g s k i n (Koefoed-Johnsen and U s s i n g , 1958) and the p r o x i m a l tubule of a v e r t e b r a t e kidney ( P i t t s , 1968). Using the f r o g - s k i n model of Koefoed-Johnsen and Us s i n g as a b a s i s , a s i m i l a r c e l l model may be proposed f o r the r e s o r p t i o n t u b u l e of the t i c k c o x a l gland ( F i g . 4 0 ). These are the c o n d i t i o n s p r e v a i l i n g i n an unfed t i c k . The model i n d i c a t e s t h a t these t r a n s p o r t mechanisms e x i s t i n the r e s o r p t i o n t u b u l e but does not n e c e s s a r i l y r e f l e c t the p r o p e r t i e s of an a c t i v e l y e x c r e t i n g g l a n d . The e l e c t r o -p o t e n t i a l g r a d i e n t between lumen and hemolymph of an a c t i v e -156-FIGURE 4-0 MODEL FOR LOCATION OF TRANSPORT MECHANISMS IN COXAL GLAND DERIVED FROM CONDITIONS EXISTING IN UNFED TICK Legend: a c t i v e t r a n s p o r t p a s s i v e d i f f u s i o n ( h i g h r a t e ) p a s s i v e d i f f u s i o n (low r a t e ) Hemolymph 10 mv Cell 44 mv Coxal f lu id 34 mv. + - + -[ C l - ] 110 meq / liter [Naf] 140 meq / liter [K"^ 16 meq / liter [ d ] <68 meq / liter Low [Nafl ? [K"^ >33 meq/ l i te r [Naf l - 112 meq / liter • [ K + ] > 20 meq / liter P > P -157-gland was v e r y s i m i l a r t o t h a t measured i n the unfed t i c k . I f the assumption i s made t h a t the same t r a n s p o r t mech-anisms are o p e r a t i v e under these two c o n d i t i o n s , the i o n c o n c e n t r a t i o n s measured i n r a p i d l y formed c o x a l f l u i d may he e x p l a i n e d as f o l l o w s . The h i g h e r c o n c e n t r a t i o n s of c h l o r i d e ( i . e . c l o s e r to those of the hemolymph) could r e s u l t i f e q u i l i b r i u m was not a t t a i n e d due t o the f a s t r a t e of f l o w through the t u b u l e . The low potassium con-c e n t r a t i o n which was eaual t o t h a t of the hemolymph probably r e f l e c t s such a low r a t e of d i f f u s i o n of potassium across the l u m i n a l membrane t h a t the c o x a l f l u i d was unchanged as i t flowed p a s t the r e s o r p t i o n c e l l s . This h y p o t h e s i s i s r e i n f o r c e d by the n e g a t i v e c o r r e l a t i o n which emerged between c h l o r i d e c o n c e n t r a t i o n s ' i n the c o x a l f l u i d and the r a t e of f l o w of t h i s f l u i d through the tub u l e (Chapter I I I ) . I should again s t r e s s t h a t the evidence f o r t h i s model was f a r from complete. I n developing the model much weight was p l a c e d on the s i m i l a r i t y of t h i s data to t h a t obtained from some ot h e r e p i t h e l i a l t i s s u e s . As such, i t should be regarded as a working h y p o t h e s i s on which to base f u r t h e r experiments. CHAPTER VI SUMMARY AND FINAL DISCUSSION -159-The i n t e n t of t h i s t h e s i s was to demonstrate the mechanism by which the t i c k Ornithodorus moubata r e g u l a t e d the volume and composition of i t s body f l u i d s d u r i n g and a f t e r f e e d i n g . A t t e n t i o n was focussed not o n l y on the c o x a l gland but a l s o on the gut w a l l . C h l o r i d e and pr o b a b l y sodium were shown t o be a c t i v e l y t r a n s p o r t e d from the gut t o the hemolymph. Potassium i o n s may be a c t i v e l y t r a n s -p o r t e d from the hemolymph i n t o the gut or they may be p a s s i v e l y d i s t r i b u t e d across a gut e p i t h e l i u m of low p e r -m e a b i l i t y . Water movement across the gut was shown to be depend-ent on the sodium and c h l o r i d e c o n c e n t r a t i o n s i n the gut f l u i d , the q u a n t i t y of sodium and c h l o r i d e i o n s t r a n s -p o r t e d across the gut w a l l and the osmotic g r a d i e n t across the gut w a l l . These parameters c o u l d be interdependent so t h a t movement of i o n s from the gut causes a f a v o r a b l e osmotic g r a d i e n t t o be maintained. However, the p o s s i b -i l i t y was not e l i m i n a t e d t h a t water movement v/as l i n k e d t o the t r a n s p o r t of i o n s across the gut e p i t h e l i u m by a process such as l o c a l osmosis. F u r t h e r s t u d i e s u s i n g meals hyperosmotic t o the hemolymph are needed t o d i s t i n g u i s h betv/een these p o s s i b i l i t i e s . However, as d i s c u s s e d e a r l i e r , c e r t a i n t e c h n i c s ! d i f f i c u l t i e s must be overcome before attempts can be made to feed t i c k s on such meals. Constant sodium and c h l o r i d e c o n c e n t r a t i o n s were -160-found to be maintained i n the hemolymph when t i c k s were f e d meals of w i d e l y d i f f e r i n g compositions p r o v i d e d these meals were i s o s m o t i c w i t h normal b l o o d . This r e g u l a t o r y a b i l i t y was l o s t when the osmotic c o n c e n t r a t i o n of the b l o o d meal was r a i s e d or when the i o n c o n c e n t r a t i o n of the gut absorbate e q u a l l e d t h a t of the hemolymph. The l a t t e r was always a s s o c i a t e d w i t h blood meals of i n c r e a s e d os-motic p r e s s u r e . Thus the e f f e c t on i o n c o n c e n t r a t i o n of the gut absorbate due t o i n c r e a s e d i o n c o n c e n t r a t i o n i n the b l o o d meal c o u l d not be d i s t i n g u i s h e d from the e f f e c t due t o i n c r e a s e d osmotic pr e s s u r e of the blood meal. S e v e r a l c r i t e r i a f o r the e x i s t e n c e of a f i l t r a t i o n membrane i n the c o x a l gland were s a t i s f i e d . The gland d i d not, under any c o n d i t i o n s produce a f l u i d which was hyper-t o n i c t o the hemolymph. Clearance s t u d i e s w i t h i n u l i n r e -v e a l e d a U/P r a t i o of u n i t y or s l i g h t l y h i g h e r over a wide range of i n u l i n c o n c e n t r a t i o n s i n the hemolymph. This i s most u n l i k e l y t o r e s u l t from a s e c r e t o r y mechanism. P r e s -sure measurements i n the hemolymph showed t h a t a p o s i t i v e h y d r o s t a t i c p r e s s u r e d i d e x i s t t o d r i v e f l u i d through the h y p o t h e t i c a l f i l t r a t i o n membrane and t h a t f o r m a t i o n of c o x a l f l u i d was p r e s s u r e s e n s i t i v e . The s i t e of primary u l t r a f i l t r a t i o n was r e v e a l e d when f l u o r e s c e i n - l a b e l l e d albumun was trapped w i t h i n the t h i n membraneous s t r u c t u r e e n v e l o p i n g the t u b u l a r p a r t of the g l a n d . F i n a l l y an u l t r a s t r u c t u r a l study d i s c l o s e d t h a t t h i s membrane was v e r y s i m i l a r i n g e n e r a l appearance to the f i l t r a t i o n membrane -161-i n the v e r t e b r a t e kidney, namely the g l o m e r u l a r c a p i l l a r y w a l l . This, evidence l e a v e s v e r y l i t t l e doubt t h a t the c o x a l gland produces f l u i d by a process of u l t r a f i l t r a t i o n . S ince i o n i c c o n c e n t r a t i o n s of c o x a l f l u i d were g e n e r a l l y lower than those of hemolymph, i t was obvious t h a t the p r i m a r y u l t r a f i l t r a t e must be m o d i f i e d by r e -a b s o r p t i o n i n the t u b u l a r p a r t of the c o x a l gland. T h i s system appeared to have some r e s o r p t i v e c a p a c i t y f o r amino a c i d s but the l a t t e r were not recovered or r e g u l a t e d to the same degree as i n o r g a n i c i o n s . A c e l l model was pro-posed f o r i o n t r a n s p o r t from the c o x a l f l u i d to hemolymph. The s i m i l a r i t y of t h i s model and the u l t r a s t r u c t u r e of the r e s o r p t i o n c e l l s t o some other e p i t h e l i a l t i s s u e s was d i s c u s s e d . I n l i g h t of s t u d i e s of comparative p h y s i o l o g y , i s a f i l t r a t i o n - r e s o r p t i o n system to be expected i n t h i s organ-ism? A c t i v e s e c r e t i o n o f water across e p i t h e l i a l c e l l s i s dependent on the r a t e of i o n t r a n s p o r t and the p e r m e a b i l i t y o f the membranes to water and i o n s . I f the membranes have h i g h p e r m e a b i l i t y to water and i o n s , a h i g h r a t e of i o n back d i f f u s i o n may r e s u l t i n a low net r a t e of water t r a n s -p o r t . This would r e q u i r e h i g h energy i n p u t p e r u n i t of water t r a n s p o r t e d . P o s s i b l y a decrease i n i o n p e r m e a b i l i t y t o l i m i t back d i f f u s i o n i s concommittant w i t h a decrease i n water p e r m e a b i l i t y . Thus i f the p e r m e a b i l i t y to i o n s v/ere d r a s t i c a l l y reduced, more i o n s would have to be t r a n s --162-p o r t e d p e r u n i t of water t r a n s p o r t e d . Again, a p o i n t would be reached where c o u p l i n g of water movement to i o n t r a n s p o r t would r e q u i r e such a high energy i n p u t as to be no l o n g e r f e a s i b l e . Consequently the r a t e c a p a c i t y o f a s e c r e t o r y mechanism can be i n c r e a s e d o n l y so f a r . I t has been c l e a r l y demonstrated t h a t i n s e c t s u t i l i z e a s e c r e t o r y mechanism f o r e x c r e t i o n . Consider volume r e g -u l a t i o n i n the blood s u c k i n g i n s e c t Rhodnius p r o l i x u s . The f i f t h i n s t a r i s about the same weight as an a d u l t female Ornithodorus moubata; i t i n g e s t s and e x c r e t e s s i m i l a r volumes o f f l u i d . However, i n Rhodnius t h i s volume i s e x c r e t e d i n about 250 minutes compared w i t h about 70 minutes f o r the t i c k . Moreover, t h a t f r a c t i o n o f u r i n e produced d u r i n g the f e e d i n g p e r i o d of Rhodnius i s o n l y s i x m i c r o l i t e r s ( M a d d r e l l , 1964-) compared t o 80 m i c r o l i t e r s i n Ornithodorus moubata. T h i s i s an important d i f f e r e n c e s i n c e i t means t h a t Rhodnius s w e l l s up to a f a r g r e a t e r extent than Ornithodorus moubata. The more r a p i d e x c r e t i o n o f f l u i d enables the t i c k t o i n g e s t a s u b s t a n t i a l b l o o d meal without an e x c e s s i v e i n c r e a s e i n t o t a l body s u r f a c e area. A d u l t Rhodnuis circumvent the problem by i n g e s t i n g a f a r s m a l l e r b l o o d meal (Wiggles-worth, 194-3). Some homopteran i n s e c t s a l s o have to manage l a r g e volumes o f f l u i d s i n c e they feed on the watery j u i c e s o f p l a n t s . T h e i r gut i s m o d i f i e d so t h a t the t e r m i n a l r e g i o n -163-of the midgut comes i n t o i n t i m a t e r e l a t i o n v/ith the lower end of the oesophagus or the beginning of the mid-gut.- This arrangement enables water to bypass the midgut and thus avoids d i l u t i o n of the hemolymph and l o a d i n g the M a l p i g h i a n t u b u l e s (Weber, 1930). The term ' f i l t r a t i o n chamber* has been a p p l i e d to t h i s complex system although the exact nature of the process by which water i s separated from n u t r i e n t s i s not known. The important p o i n t t o r e a l i z e i s t h a t t i c k s and many i n s e c t s which must handle l a r g e f l u i d volumes, e i t h e r t o l e r a t e a prolonged p e r i o d of ex-t e n s i o n or u t i l i z e an e x c r e t o r y mechanism which i s i n d e -pendent of the M a l p i g h i a n t u b u l e s . As d i s c u s s e d i n Chapter I , not a l l t i c k s r a p i d l y i n -gest a l a r g e blood meal. Rapid e x c r e t i o n of v/ater i s un-necessary i n the hard I x o d i d t i c k s s i n c e they feed over a p e r i o d of s e v e r a l days. Moreover, s i n c e the a d u l t female I x o d i d t i c k s o n l y feed once, not o n l y would i t be non-e s s e n t i a l to prevent e x c e s s i v e d i s t e n s i o n but i t might be advantageous to them s i n c e t h i s v/ould r e s u l t i n more space f o r the development of the l a r g e s i n g l e batch of eggs. Thus there i s no need f o r the r a p i d f l u i d e x c r e t i o n o f f e r e d by a f i l t r a t i o n mechanism. I n s t e a d , these t i c k s have been found to s a l i v a t e c o p i o u s l y back i n t o the host ( T a t c h e l l , 1967, 1969) by means of a s e c r e t o r y r a t h e r than f i l t r a t i o n mechanism (W. R. Kaufman, 1971, i n p r e p a r a t i o n ) . Thus t i c k s employ both s e c r e t o r y (as i n i n s e c t s ) and f i l t r a t i o n (as i n crustaceans) mechanisms i n t h e i r accessory e x c r e t o r y -164-organs. Many new avenues of r e s e a r c h were suggested by the experiments which e s t a b l i s h e d t h a t c o x a l f l u i d was formed by u l t r a f i l t r a t i o n . For example, the i n i t i a t i o n and r a t e of c o x a l f l u i d p r o d u c t i o n seemed to be, at l e a s t p a r t i a l l y , under nervous c o n t r o l ; the v a l v e at the c o x a l o r i f i c e was i m p l i c a t e d . H andling the t i c k r o u g h l y o r warming i t i s known t o e l i c i t the.sudden appearance of c o x a l f l u i d whether or not the t i c k has r e c e n t l y f e d . Robinson (194-2) showed a l s o t h a t male t i c k s r e l e a s e drops of c o x a l f l u i d w h i l e mating. However, i n the u n d i s t u r b e d t i c k which i s f e e d i n g , what i s i t t h a t i n i t i a t e s the p r o d u c t i o n of c o x a l f l u i d ? I t i s not sim p l y the g e n e r a l h y d r o s t a t i c pressure i n the t i c k s i n c e c o x a l f l u i d does not appear i f the t i c k i s squeezed (Lees, 1946). Nor i s i t an o v e r a l l d i s t e n s i o n to a c r i t i c a l volume. P o s s i b l y a sensory organ i s s t i m -u l a t e d by the i o n i c and/or osmotic c o n d i t i o n s p r e v a i l i n g i n the hemolymph or gut. A c o n s i s t e n t minimum c o n c e n t r a t i o n o f . c h l o r i d e occurred i n the hemolymph at the time when c o x a l f l u i d f i r s t appeared. This was o n l y observed i n t i c k s fed meals which were i s o s m o t i c w i t h human blood and i n which s u c c e s s f u l r e g u l a t i o n of i o n c o n c e n t r a t i o n s i n the hemolymph r e s u l t e d . I t was not so f o r t i c k s fed h.b.+ NaCl when c o x a l f l u i d p r o d u c t i o n was i n i t i a t e d at a con-s i s t e n t l y h i g h e r c h l o r i d e c o n c e n t r a t i o n . The suggestion i s t h a t c h l o r i d e i s not i t s e l f the primary or s i n g l e s t i m u l u s f o r c o x a l f l u i d p r o d u c t i o n but, i n the case of -165-i s o s m o t i c meals, i t s c o n c e n t r a t i o n r e f l e c t s some ot h e r parameter. F u r t h e r study of the i o n i c and osmotic con-c e n t r a t i o n s i n the d i f f e r e n t f l u i d compartments at the time of c o x a l f l u i d p r o d u c t i o n might c a s t f u r t h e r l i g h t on t h i s problem. In a d d i t i o n , one cannot exclude the e x i s t e n c e of a d i u r e t i c hormone whose r e l e a s e c o u l d p e r -haps be t r i g g e r e d by the act of f e e d i n g . The hemolymph c o n c e n t r a t i o n s of amino a c i d s are d i f f i c u l t t o compare w i t h those found i n other t i c k s be-cause the v a r i a t i o n was so g r e a t . I h e s i t a t e , i n viev; o f t h i s , t o p l a c e too much f a i t h i n the r e s u l t s of s i n g l e assays presented by o t h e r authors (Rehacek and B r z o s t o w s k i , 1969; T a t c h e l l , 1969). Since most of the amino a c i d s were at a h i g h e r pH than t h e i r i s o e l e c t r i c p o i n t , the general-t r e n d of r e a b s o r p t i o n i n the c o x a l gland p o s s i b l y r e s u l t s from t h e i r p a s s i v e movement down the e l e c t r o p o t e n t i a l g r a d i e n t . However a r g i n i n e , which was almost c e r t a i n l y p o s i t i v e l y charged ( i s o e l e c t r i c p o i n t 10.8) was c o n s i s t e n t l y reabsorbed a g a i n s t an e l e c t r o p o t e n t i a l and c o n c e n t r a t i o n g r a d i e n t . Very l i t t l e i s known about amino a c i d t r a n s p o r t i n arthropods ( e x c l u d i n g crustaceans) although i t has r e c e n t l y been shown t o be an a c t i v e process i n the rectum of the d e s e r t l o c u s t ( B a l s h i n and P h i l l i p s , 1971). Thus i t would be extremely i n t e r e s t i n g t o f u r t h e r i n v e s t i g a t e the r e a b s o r p t i o n of a r g i n i n e i n the c o x a l gland of the t i c k . The.development of an i n v i t r o p r e p a r a t i o n of the r e s o r p t i o n t u b u l e would g r e a t l y f a c i l i t a t e the study of t r a n s p o r t -166-i n t h i s system which i s so r e m i n i s c e n t of the p r o x i m a l c o n v o l u t e d t u b u l e i n the v e r t e b r a t e kidney. -167-REFERENCES CITED Anderson, E. and Harvey, W.R. (1966). A c t i v e t r a n s p o r t by the Cec r o p i a midgut. I I . F i n e s t r u c t u r e of the midgut e p i t h e l i u m . J . C e l l B i o l . J51: 107-134. B a l s h i n , M. and P h i l l i p s , J.E. (1971). A c t i v e a b s o r p t i o n of amino a c i d s i n the rectum of the desert l o c u s t S c h i s t o c e r c a g r e g a r i a . Nature (London) I n p r e s s . B e n t z e l , C.J., P a r s a , B. and Hare, D. (1969). 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