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The function of the anal papillae of saline-water mosquito larvae Scherer, Nancy Mae 1977

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THE FUNCTION OF THE ANAL PAPILLAE OF SALINE-WATER MOSQUITO LARVAE by Nancy Mae Scherer B.S., Western Washington State University, 1973 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES Department of Zoology We accept this thesis as conforming to the requiired standard THE UNIVERSITY OF BRITISH COLUMBIA September, 1977 (c) Nancy Mae Scherer, 1977 In p r e s e n t i n g t h i s t h e s i s in p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an advanced d e g r e e at the U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t ha 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 s t u d y . I f u r t h e r a g r e e 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 p u r p o s e s may be g r a n t e d by the Head o f my Depar tment o r by h i s r e p r e s e n t a t i v e s . - I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Depar tment o f The U n i v e r s i t y o f B r i t i s h C o l u m b i a 2075 Wesbrook Place Vancouver, Canada V6T 1W5 i i ABSTRACT The r o l e o f the ana l p a p i l l a e o f s a l i n e - w a t e r m o s q u i t o l a r v a e i s u n c e r t a i n . Sodium i o n and C l ~ uptake by the p a p i l l a e o f l a r v a e i n d i l u t e media has been d e m o n s t r a t e d ( P h i l l i p s and M e r e d i t h , 1 9 6 9 a ) . I n d i r e c t u l t r a s t r u c t u r a l and p h y s i o l o g i c a l e v i d e n c e s u g g e s t s t h a t t h e s e organs might a l s o be a c t i v e i n i o n t r a n s p o r t under s a l i n e c o n d i t i o n s ( P h i l l i p s and r M e r e d i t h , 1 9 6 9 a ; M e r e d i t h and P h i l l i p s 1 9 7 3 a , b ; B r a d l e y and P h i l l i p s , 1 9 7 5 ; P h i l l i p s and B r a d l e y , 1 9 7 7 ; P h i l 1 i p s e t al_. , 1 977) . Two p o s s i b l e f u n c t i o n s of the ana l p a p i l l a e i n s a l i n e media have been s u g g e s t e d : (1) t h a t the p a p i l l a e e x c r e t e M a + and C l ~ , and (2) t h a t the p a p i l l a e t a k e up i o n s p r e s e n t i n low c o n c e n t r a t i o n i n s a l i n e w a t e r s . Uptake of K + by the a n a l p a p i l l a e was measured f o r the i n v e s t i g a t i o n o f the second h y p o t h e s i s . Com-p l e t i o n of t h i s s tudy i n v o l v e d m e a s u r i n g the t o t a l K + b a l a n c e of the l a r v a e . These h y p o t h e s e s were t e s t e d by m e a s u r i n g hemolymph o s m o l a r i t y and i o n c o n c e n t r a -t i o n s of normal and p a p i l l a e l e s s l a r v a e i n a range o f 42 + ' s a l i n i t i e s , m e a s u r i n g K i n f l u x , t o t a l body K c o n t e n t , and 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 p a p i l l a e . i i i The a n a l p a p i l a e o f the s a l i n e m o s q u i t o l a r v a , Aedes  t a e n i o r h y n c h u s , l i v i n g i n s a l i n e w a t e r s d i d not a c t i v e l y t a k e up K + . I n d i r e c t e v i d e n c e i n d i c a t e d t h a t the ana l p a p i l l a e were a l s o not i n v o l v e d i n N a + o r C l " s e c r e t i o n i n c o n c e n t r a t e d m e d i a . When A. t a e n i o r h y n c h u s l a r v a e were r e a r e d i n d i s t i l l e d w a t e r , the ana l p a p i l l a e were r e s p o n s i b l e f o r n e a r l y a l l the K + u p t a k e , as i s the case f o r s t r i c t l y f r e s h w a t e r s p e c i e s o f m o s q u i t o l a r v a e . The o b s e r v e d i n f l u x o f K + - 2 - 1 a c r o s s the p a p i l l a e of 46 nmol cm h o c c u r r e d a g a i n s t both c h e m i c a l and e l e c t r i c a l p o t e n t i a l g r a d i e n t s , i n d i c a -t i n g t h a t t r a n s p o r t i s a c t i v e . P o t a s s i u m uptake by the a n a l p a p i l l a e was a l s o o b s e r v e d i n the s a l i n e mosqu i to l a r v a , Aedes campes t r i s , f rom both d i l u t e (about 10 m.Osm) and c o n c e n t r a t e d (about 300 m;Q§m) s o l u t i o n s a l t h o u g h uptake was much reduced i n the l a t t e r . Uptake was i n c r e a s e d by a c c l i m a t i o n o f the l a r v a e to d i l u t e m e d i a . P o t a s s i u m i n f l u x o c c u r r e d a g a i n s t the c o n c e n t r a t i o n g r a d i e n t but a l o n g the e l e c t r i c a l p o -. t e n t i a l g r a d i e n t . Uptake f o l l o w e d M i c h a e l i s - M e n t e n k i n e t i c s w i t h an a p p r o x i m a t e K n c o f 3 . 2 mM and V m , v o f u . 3 max 1.1 nmo1 mg" ^ h ^ . i v TABLE OF CONTENTS Page I. . I n t r o d u c t i on I' I I . M a t e r i a l s and Methods 9.,-A. E x p e r i m e n t a l A n i m a l s 9> B. S o l u t i o n s 10 C. I n f l u x Measurements 11 D. I s o t o p e C o u n t i n g P r o c e d u r e s 13 E. T o t a l Body K + Uptake 15 F. P o t e n t i a l D i f f e r e n c e Measurements 16 G. C o l l e c t i o n of R e c t a l F l u i d 20 H. D e t e r m i n a t i o n o f Ion C o n c e n t r a t i o n s , O s m o l a r i t y , Volume 23 I. B l o c k i n g the Anus 24 J . Removal of Anal P a p i l l a e 25 K: Measurement of Damage to the C u t i c l e 25; L. Hemolymph Sample C o l l e c t i o n 26 M. R e s p i r a t i o n Rate D e t e r m i n a t i o n 27 N. C a l c u l a t i o n s 28 I I I . R e s u l t s A. F u n c t i o n of the Anal P a p i l l a e i n S a l i n e M e d i a . . . 31 31 1. S e p a r a t i o n of the Anal P a p i l l a e of A. t a e n i o r h y n c h u s L a r v a e f rom the R e c t a l F l u i d : 2 . E f f e c t o f E x p e r i m e n t a l P r o c e d u r e s on the Body Wal l o f A. t a e n i o r h y n c h u s Larvae 34 3 . Hemolymph Ion C o n c e n t r a t i o n s and O s m o l a r i t y 34 B. Uptake of K + by the Anal P a p i l l a e 42 1 . L o c a l i z a t i o n of 4 21< I n f l u x i n A^ . t a e n i o r h y n c h u s L a r v a e + 42 2. The C o n c e n t r a t i o n of K and Volume of the R e c t a l F l u i d of A. t a e n i o r h y n c h u s Larvae . . . . 50 3 . D r i n k i n g Rate o f L a r v a e i n S a l i n e Media 54 4. Uptake of 4 2 K by the Anal P a i l l a e of A^ campes t r i s La rvae 54 C. The Mechanism of K + Uptake by the Ana l P a p i l l a e . 57 1 . Net Uptake o f K + by A. t a e n i o r h y n c h u s La rvae i n FW 60 V Page 42 2. F a c t o r s I n f l u e n c i n g K I n f l u x 66 42 a . I n f l u e n c e of E x t e r n a l Ions on K Uptake i n A. c a m p e s t r i s La rvae 66 b. E f f e c t o f I n h i b i t o r s on 42« Uptake i n A. c a m p e s t r i s L a r v a e 67 c . K i n e t i c s o f 4 2 K I n f l u x i n A. c a m p e s t r i s La rvae 74 3. The 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 Anal P a p i l l a e 78 a . The P . D . A c r o s s the Ana l P a p i l l a e of A. t a e n i o rhynchus L a r v a e 78 b. The P . D . A c r o s s the Anal P a p i l l a e of A.  c a m p e s t r i s La rvae .- 91 D. R e s p i r a t i o n Rate of A. t a e n i o r h y n c h u s La rvae i n S a l i n e Media 100 IV ; D i s c u s s i o n 103 vi LIST OF TABLES Page I. Change i n the p . d . A c r o s s the Anal P a p i l l a e as a R e s u l t o f V a r y i n g the N a + o r K + Con -c e n t r a t i o n o r I o n i c C o m p o s i t i o n o r the A d d i t i o n o f M e t a b o l i c I n h i b i t o r s 90 I I . Average p . d . A c r o s s the Anal P a p i l l a e i n V a r i o u s Media 95 I I I . E q u i l i b r i u m p . d . of N a + , K + and C l " 112 IV. P ^ f o r Water Movement A c r o s s the C u t i c l e o f some A q u a t i c I n v e r t e b r a t e s 119 v i i LIST OF FIGURES Page 1. Anatomy of a M o s q u i t o L a r v a 2 . A p p a r a t u s f o r p . d . Measurement 3. A p p a r a t u s f o r C o l l e c t i o n of R e c t a l F l u i d 3 4. H2O I n f l u x i n t o Normal and P lugged A. t a e n i o r h y n c h u s La rvae 3 5. H ? 0 I n f l u x i n t o Normal and P a p i l l a e l e s s A. t a e n i o r h y n c h u s La rvae 3 6. H 9 0 I n f l u x i n t o S i n g l e and Double L i g a t u r e d A^ ^ t a e n i o r h y n c h u s La rvae 38 7. Hemolymph C h a r a c t e r i s t i c s of Normal and P a p i l l a e l e s s A. t a e n i o r h y n c h u s L a r v a e 40 42 8 . K i n f l u x i n t o A. t a e n i o r h y n c h u s l a r v a e i n V a r i o u s Sa l i n i t i e s 45 a) 200% SW b) 100% SW c) 2 5% SW / d) d i s t i 11 ed wate r . 42 9 . K I n f l u x i n t o A. t a e n i o r h y n c h u s P a p i l l a e l e s s L a r v a e i n 100% SW 47 A 0 10 . K I n f l u x A c r o s s the Anal P a p i l l a e o f A^ _ t a e n i o r h y n c h u s L a r v a e 49 1 1 . P o t a s s i u m c o n c e n t r a t i o n and Volume i n the R e c t a l F l u i d o f A. t a e n i o r h y n c h u s L a r v a e 53 12 . C a l c u l a t i o n of the D r i n k i n g Rate of La rvae i n S a l i n e Med i a 56 42 1 3 . I n d u c t i o n o f K T r a n s p o r t by the Anal P a p i l l a e of A. c a m p e s t r i s 59 14. T o t a l Body K Content o f A. t a e n i o r h y n c h u s L a r v a e . . . 62 a) net uptake 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 -fa) the e f f e c t of e x t e r n a l N a + c) the e f f e c t of externa*! pH vi i i Page 86 15 . I n f l u x o f Rb i n A. t a e n i o r h y n c h u s L a r v a e ove r 25 h 64 + 42 16 . E f f e c t o f E x t e r n a l Na on K I n f l u x i n A_;_ c a m p e s t r i s L a r v a e 69 42 17. E f f e c t o f E x t e r n a l pH on K I n f l u x i n A. c a m p e s t r i s L a r v a e 71 42 18. E f f e c t of Ions and M e t a b o l i c I n h i b i t o r s on K I n f l u x i n A. c a m p e s t r i s 73 42 19. D i f f u s i o n of K a c r o s s the C u t i c l e of A. t a e n i o rhynchus La rvae 77 20. a) K I n f l u x i n A. c a m p e s t r i s La rvae as a F u n c t i o n a l o f the E x t e r n a l K + C o n c e n t r a t i o n . . 80 42 + b) K I n f l u x at Low K C o n c e n t r a t i o n s 4 ? 2 1 . S c a t c h a r d P l o t of Net K I n f l u x i n A_^_ c a m p e s t r i s L a r v a e 82 2 2 . P . d . A c r o s s the P a p i l l a e of A. t a e n i o r h y n c h u s La rvae Reared i n D i s t i l l e d Water and 100% SW . . . 85 2 3 . P . d . A c r o s s the P a p i l l a e o f A. t a e n i o r h y n c h u s L a r v a e D u r i n g P e r f u s i o n w i t h S o l u t i o n s of V a r i o u s Ion C o n c e n t r a t i o n s and C o m p o s i t i o n s . . . . 87 24. P . d . A c r o s s the Anal P a p i l l a e as a F u n c t i o n of E x t e r n a l Na* C o n c e n t r a t i o n i n A. t a e n i o rhynch us La rvae 93 2 5 . P . d . A c r o s s the Anal P a p i l l a e of A. c a m p e s t r i s L a r v a e D u r i n g P e r f u s i o n w i t h S o l u t i o n s of V a r i o u s Ion C o n c e n t r a t i o n s 98 26 . R e s p i r a t i o n Rate of A. t a e n i o r h y n c h u s La rvae i n D i f f e r e n t S a l i n i t i e s 102 42 2 7 . D i f f u s i o n of K A c r o s s the C u t i c l e i n S a l i n e Media 122 i x LIST OF SYMBOLS Area i n t e r n a l c o n c e n t r a t i o n o f s u b s t r a t e e x t e r n a l c o n c e n t r a t i o n of s u b s t r a t e i n t e r n a l a c t i v i t y o f i s o t o p e e x t e r n a l a c t i v i t y o f i s o t o p e e q u i l i b r i u m p o t e n t i a l r e c o r d e d p . d . a c r o s s the p a p i l l a e Faraday c o n s t a n t u n i d i r e c t i o n a l f l u x d i f f u s i o n a l p e r m e a b i l i t y c o e f f i c i e n t gas c o n s t a n t ( 1 . 3 1 X 10^ e rg m o l " ^ °K t ime c o n s t a n t f o r i s o t o p e exchange a b s o l u t e t e m p e r a t u r e t i me volume o f t o t a l body water X ACKNOWLEDGEMENTS I wish to thank Dr. John E. P h i l l i p s for his support and advice. I am grateful to Dr. Wil l iam Prince for his invaluable assistance and good humor. Drs. Simon Maddrell arid Barry Irvine provided ins ights into the wi les of micro-technique. Dr. Vladamir Pa laty 's enthusiasm was as valuable as his inspired and r e a l i s t i c knowledge of transport mechanisms. I appreciate Dr. Bandoni's a id in hunting for microbial pathogens. I am thankful to Dr. Terry Crawford for numerous loans of equipment. I would l i k e to thank Drs. John Gosel ine, Dave Randal l , and G. G. E. Scudder for the i r advice in the preparation of th i s manuscript. I enjoyed pertinent and impertinent conversations with Tim Bradley, Mary Chamberloin, John Hanrahanrahan, Joan Martin and Doug Wi l l iams. Special thanks to Joan for sharing her expert ise , but most of a l l , for her f r iendship . F i n a l l y I wish to thank Don Mansfield for his lovey f r iendship and invaluable l i t e r a r y advice. 1 INTRODUCTION The p h y s i o l o g y of i n s e c t s l i v i n g in s a l i n e h a b i t a t s (some 308 s p e c i e s in 9 o r d e r s ; F o s t e r and T r e h e r n e , 1976) has been l a r g e l y u n s t u d i e d . Yet the prob lems o f i o n i c and o s m o t i c r e g u l a t i o n under t h e s e c o n d i t i o n s are e x c e p t i o n a l . D i f f e r e n t i a l p r e c i p i t a t i o n of s a l t s by e v a p o r a t i o n causes a p e r p e t u a l l y c h a n g i n g i o n i c e n v i r o n m e n t . These w a t e r s may be h i g h l y c o n c e n t r a t e d , y e t q u i c k l y d i l u t e d by r a i n or f r e s h w a t e r r u n - o f f . Thus the o s m o r e g u l a t o r y a b i l i t y r e q u i r e d to s u r v i v e t h e s e changes i s even g r e a t e r .than t h a t n e c e s s i t a t e d by A c o n s t a n t s a l i n e e n v i r o n m e n t ( B e a d l e , 1 9 3 9 ) . V e r t e b r a t e s l i v i n g in s a l i n e h a b i t a t s r e l y on both r e n a l and e x t r a - r e n a l o rgans f o r i o n i c and o s m o t i c r e g u l a -t i o n ( g i l l , t e l e o s t s ; l a c h r y m a l g l a n d , r e p t i l e s ; n a s a l g l a n d , b i r d s ; r e c t a l g l a n d , e l a s m o b r a n c h s ) . Among the s a l i n e i n s e c t s the o n l y d e m o n s t r a t i o n of r e g u l a t i o n u t i l i z i n g a s i t e o t h e r than the g u t , M a l p i g h i a n t u b u l e s or rec tum i s the ana l p a p i l l a e o f mosqu i to l a r v a e . ( P h i l l i p s and M e r e d i t h , 1 9 6 9 a , b; P h i l l i p s and B r a d ! ey , 1977') . Ion ic . ,ahd.osmo.t i c i • r e g u l a t i o n - i s a c c o m p l i s h e d by 4 o r g a n s : the g u t , M a l p i g h i a n t u b u l e s , rec tum and the a n a l p a p i l l a e ( F i g u r e 1 ) . The gut has been d e m o n s t r a t e d to a b s o r b s 95% of the i n g e s t e d i o n s and s o l u t e s ( K i c e n i u k and P h i l l i p s , 1 9 7 4 ) . The M a l p i g h i a n t u b u l e s t r a n s p o r t s o l u t e s and w a t e r out of the hemolymph. S o l u t e s e n t e r the t u b u l e s e i t h e r by a c t i v e t r a n s p o r t o r p a s s i v e l y a l o n g rectum F i g u r e 1 The anatomy of a m o s q u i t o l a r v a ? the s c a l e i s about 2 0 : 1 . The a n a l p a p i l l a e are the s i z e found i n A. t a e n i o r h y n c h u s l a r v a e r e a r e d in d i s t i l l e d w a t e r . (D iagram i s m o d i f i e d f rom W i g g l e s w o r t h , 1933a) 3 t h e i r c o n c e n t r a t i o n g r a d i e n t s and a l o c a l o s m o t i c g r a d i e n t c r e a t e d by a c t i v e K + t r a n s p o r t . The s e c r e t e d f l u i d i s i s o m o t i c w i t h the hemolymph, a l t h o u g h d i f f e r i n g i n i o n i c c o m p o s i t i o n . Thus t h e s e o rgans a r e i n v o l v e d i n i o n . e x c r e t i o n r a t h e r than o s m o t i c r e g u l a t i o n ( r e v i e w e d by M a d d r e l l , 1 9 7 1 ) . Osmot ic r e g u l a t i o n , as w e l l as s e l e c t i v e ion r e a b s o r p t i o n , i s a c c o m p l i s h e d by the r e c t u m . The rec tum of FW m o s q u i t o l a r v a e p roduces a u r i n e which i s h y p o s m o t i c to the hemolymph (Ramsay, 1 9 5 0 ) . In s a l i n e s p e c i e s the u r i n e may be e i t h e r h y p o - o r h y p e r o s m o t i c (Ramsay, 1 9 5 0 ; B r a d l e y and P h i l l i p s , 1 9 7 5 ) . The h y p e r o s m o t i c u r i n e i n s a l i n e m o s q u i t o s p e c i e s i s formed th rough ion s e c r e t i o n ( B r a d l e y and P h i l l i p s , 1 9 7 5 ) , r a t h e r than by water r e a b s o r p t i o n as o c c u r s in t e r r e s t r i a l i n s e c t s ( M a d d r e l l , 1 9 7 1 ; S t o b b a r t . and Shaw, 1 9 7 4 ) . At the base of the anus t h e r e a re 4 e x t e r n a l e p i d e r m a l e vag i nati;oris>.s c a l l e d the ana l p a p i l l a e . These o rgans are not p e c u l i a r to m o s q u i t o e s , but are found among o t h e r a q u a t i c d i p t e r i a n l a r v a e ( W i c h a r d and K o m n i c k , 1 9 7 4 ; C r e d -l a n d , 1 9 7 6 ) . The p a p i l l a e c o n s i s t of a s i m p l e e p i t h e l i u m composed of a s i n g l e c e l l t ype s e p a r a t e d o n l y by a basement membrane f rom a c e n t r a l c a v i t y wh ich i s c o n t i n u o u s w i t h the h e m o c o e l . The u l t r a s t r u c t u r e o f the a n a l p a p i l l a e has been d e s c r i b e d in both FW and s a l i n e s p e c i e s of m o s q u i t o l a r v a e . There i s a h i g h degree o f . i n f o l d i n g o f the a p i c a l and b a s a l membranes of the p a p i l l a e , e x t e n s i v e t r a c h e a t i o n and a l a r g e 4 number of m i t o c h o n d r i a ( P h i l l i p s and M e r e d i t h , 1 9 6 9 ; C o p e l a n d , 1 9 6 4 ; Sohal and C o p e l a n d , 1 9 6 6 ; M e r e d i t h and P h i l l i p s , 1 9 7 3 a , b ) . T h i s morpho logy i s c h a r a c t e r i s t i c o f t r a n s p o r t i n g e p i t h e l i a ( r e v i e w e d by B e r r i d g e and Osetinran, 1 9 7 2 ) . The f u n c t i o n of the a n a l p a p i l l a e of FW m o s q u i t o l a r v a e has been the s u b j e c t o f numerous s t u d i e s . In 1933 W i g g l e s w o r t h n o t e d t h a t the p a p i l l a e were a c t i v e in w a t e r b a l a n c e . I n d e e d , the s i z e of the p a p i l l a e was i n v e r s e l y c o r r e l a t e d w i t h the e x t e r n a l s a l i n i t y ( W i g g l e s w o r t h , 193;3a) . Subsequent s t u d i e s have shown t h a t n e a r l y a l l N a + , K + (Ramsay, 1 9 5 3 ; T r e h e r n e , 1 9 5 4 ; S t o b b a r t , 1 9 5 9 , 1 9 6 5 , 1 9 6 7 ) , C l " ( W i g g l e s w o r t h , 1 9 3 8 ; K o c h , 1 9 3 8 ; S t o b b a r t , 1967) and phosphate ( H a s s e t and J e n k i n s , 1951) uptake in FW s p e c i e s i s by the ana l p a p i l l a e . The p a p i l l a e are ve ry e f f e c t i v e i n u p t a k e , c a p a b l e of a c c u m u l a t i n g N a + f rom a 5JJM s o l u t i o n ( S t o b b a r t , 1 9 6 5 ) . Hemolymph i o n c o n c e n t r a t i o n s and o s m o l a r i t y a re h e l d w i t h i n nar row l i m i t s over the t o l e r a t e d range of s a l i n i t i e s ( S t o b b a r t and Shaw, 1 9 7 4 ) . FW l a r v a e can s u r v i v e in s o l u t i o n s as c o n c e n t r a t e d as 1% N a C l , but a re unab le to w i t h s t a n d much h i g h e r s a l i n i t i e s due to the i n a b i l i t y to produce u r i n e h y p e r o s m o t i c to the hemolymph (Ramsay, 1 9 5 0 ) . In c o n t r a s t , r e l a t i v e l y l i t t l e i s known of the f u n c t i o n of the a n a l p a p i l l a e i n s a l i n e water m o s q u i t o e s . E a r l y s t u d i e s p r o v i d e d no e v i d e n c e of any t r a n s p o r t f u n c t i o n f o r the ana l p a p i l l a e ( B e a d l e , 1 9 3 9 ; Ramsay, 1 9 5 0 ) ; r a t h e r , the 5 rec tum was p o s t u l a t e d to be the s i t e of i o n i c and o s m o t i c r e g u l a t i o n (Ramsay, 1 9 5 0 ) . In s p i t e of the p roposed v e s t i g u a l n a t u r e of the p a p i l l a e , a l l the s a l i n e s p e c i e s of m o s q u i t o l a r v a which have been s t u d i e d are a b l e to t o l e r a t e v e r y d i l u t e e x t e r n a l s o l u t i o n s (Aedes d e t r i t u s , Ramsay, 1 9 5 0 ; O p i f e x f u s c u s , M c G r e g o r , 1 9 6 5 ; Aedes  campes t r i s , P h i l l i p s and M e r e d i t h , 1 969a;, Aedes t a e n i o r - hynchus , Nayer and Sauerman, 1 9 7 4 ) . As i n FW l a r v a e , the hemolymph o s m o l a r i t y and ion c o n c e n t r a t i o n s a re r e g u l a t e d w i t h i n nar row l i m i t s i n s a l i n e m o s q u i t o s p e c i e s (Bradl .ey and P h i l l i p s , 1 9 7 7 b ) . The q u e s t i o n of the f u n c t i o n of the a n a l p a p i l l a e i n s a l i n e media was reopened i n 1969 when P h i l l i p s and M e r e d i t h d e m o n s t r a t e d t h a t the p a p i l l a e of a s a l i n e m o s q u i t o s p e c i e s c o u l d t a k e up N a + and C I " a f t e r a c c l i m a t i o n to d i l u t e s o l u t i o n s . U n l i k e the case f o r a FW s p e c i e s (Soha l and C o p e l a n d , 1 9 6 6 ) , the u l t r a s t r u c t u r e of the p a p i l l a e of s a l i n e m o s q u i t o l a r v a e in h y p e r o s m o t i c media and l a r v a e a c c l i m a t e d to h y p o s m o t i c media was s . i m i l a r . T h i s s u g g e s t s t h a t the p a p i l l a e are a l s o f u n c t i o n a l in s a l i n e m e d i a . F u r t h e r m o r e , p a p i l l a e l e s s l a r v a e in s a l i n e media had a C I " . l e v e l in the hemolymph 25% above t h a t in c o n t r o l a n i m a l s ( P h i l l i p s and M e r e d i t h , 1 9 6 9 a ) . P h i l l i p s and M e r e d i t h ( 1 969 a , l 9 7 3 a , b) p r o p o s e d t h a t the d i r e c t i o n of i o n t r a n s p o r t a c r o s s the p a p i l l a e c o u l d be r e v e r s e d under s a l i n e c o n d i t i o n s to p e r m i t N a + and CI e x c r e t i o n . T h i s 6 h y p o t h e s i s was supporteld by the work o f Nayer and Sauerman (1974) who showed t h a t p a p i l l a e l e s s A . t a e n i o r h y c h u s l a r v a e i n 25% SW were l e s s e f f e c t i v e i n c o n t r o l l i n g hemolymph o s m o l a r i t y and c h l o r i d e l e v e l s than normal l a r v a e . P h i l l i p s and Mered i th ((I969a,b)al so s u g g e s t e d t h a t the p a p i l l a e might t a k e up i o n s wh ich a re p r e s e n t i n low c o n c e n t r a t i o n s i n s a l i n e ' w a t e r s . In p a r t i c u l a r , K + might be taken up by the p a p i l l a e under s a l i n e c o n d i t i o n s to compensate f o r the h igh r a t e of K + l o s t i n the r e c t a l s e c r e t i o n ( B r a d l e y and P h i l l i p s , 1 9 7 5 ) . The p r o p o s e d f u n c t i o n s of the ana l p a p i l l a e in s a l i n e media a re c u r r e n t l y based on i n d i r e c t e v i d e n c e . A l t e r e d hemolymph c o n c e n t r a t i o n s of p a p i l l a e l e s s l a r v a e c o u l d have r e s u l t e d f rom damage to the c u t i c l e by AgNOg. The e x t e r n a l s a l i n i t y p r e s e n t d u r i n g the e a r l y deve lopment of l a r v a e used i n the h i s t o l o g i c a l s t u d i e s was not c o n t r o l l e d . T h e r e -f o r e , a r e - e x a m i n a t i o n of the r o l e of the ana l p a p i l l a e in s a l i n e water m o s q u i t o l a r v a e i s w a r r a n t e d . T h i s s tudy was u n d e r t a k e n to d e t e r m i n e the f u n c t i o n s of the ana l p a p i l l a e of m o s q u i t o l a r v a e i n d i g e n o u s to the s a l i n e h a b i t a t . Two s p e c i e s of s a l i n e wate r m o s q u i t o l a r v a e were used i n t h i s s t u d y : Aedes t a e n i o r h y n c h u s (Wiedemann) which i n h a b i t s c o a s t a l s a l t marshes a l o n g the e a s t e r n c o a s t of N o r t h , C e n t r a l and South A m e r i c a f rom B r a z i l to New Eng land and the P a c i f i c c o a s t f rom Peru to C a l i f o r n i a ; and Aedes c a m p e s t r i s (Dayer and Knub) wh ich i s 7 found i n b r a c k i s h i n l a n d l a k e s i n s e m i - a r i d r e g i o n s of Nor th A m e r i c a ( C a r p e n t e r and L a C a s s e , 1955). I examined two a s p e c t s of p a p i l l a e f u n c t i o n : (1) i f the p a p i l l a e o f l a r v a e i n s a l i n e media s e c r e t e N a + and C l " to the e x t e r n a l medium, and (2) i f the p a p i l l a e t a k e up i o n s which a r e i n low c o n c e n t r a t i o n s i n s a l i n e w a t e r s . A d i r e c t t e s t of the e x c r e t o r y f u n c t i o n was not f e a s -i b l e because I was not a b l e to s e p a r a t e the a n a l p a p i l l a e f rom the r e c t a l f l u i d . However , i n d i r e c t e v i d e n c e was o b t a i n e d by compar ing hemolymph o s m o l a r i t y and ion l e v e l s i n normal and p a p i l l a e l e s s l a r v a e . The r e s u l t s do not p r o v i d e e v i d e n c e t h a t the ana l p a p i l l a e o f l a r v a e l i v i n g i n c o n c e n t r a t e d w a t e r s e x c r e t e N a + or sC1 — . T r a n s p o r t of K + was chosen f o r the second p a r t o f t h i s s tudy s i n c e t h i s ion i s o f t e n p r e s e n t in s a l i n e w a t e r s a t lower c o n c e n t r a t i o n s than found i n the hemolymph. The p o t a s s i u m ion i s known to be t r a n s p o r t e d by the p a p i l l a e of FW s p e c i e s , and uptake c o u l d compensate f o r the h i g h l o s s of K + in the r e c t a l f l u i d of A. t a e n i o r h y n c h u s . N o t h i n g i s c u r r e n t l y known about the p r o p e r t i e s of K + t r a n s p o r t by the a n a l p a p i l l a e of s a l i n e s p e c i e s of m o s q u i t o l a r v a e , nor has the mechanism of uptake been c h a r a c t e r i z e d i n FW m o s q u i t o e s . The f i r s t s t e p i n the s t u d y o f K + uptake by the a n a l 42 p a p i l l a e was to l o c a l i z e the r o u t e of K e n t r y i n t o l a r v a e i n d i f f e r e n t s a l i n i t i e s to d e t e r m i n e the r e l a t i v e i m p o r t a n c e •/ > 8 of the p a p i l l a e in K T u p t a k e . These e x p e r i m e n t s d e m o n s t r a -t e d t h a t K + uptake o c c u r s v i a the ana l p a p i l l a e o n l y under FW c o n d i t i o n s . Subsequent e x p e r i m e n t s were u n d e r t a k e n to d e t e r m i n e i f t h e r e was' a net uptake o f K + a g a i n s t both c h e m i c a l and e l e c t r i c a l g r a d i e n t s ; t h a t i s , i f uptake was a c t i v e . The p r o p e r t i e s of the uptake mechanism were i n -d i c a t e d by the e f f e c t s of v a r i o u s i o n s , m e t a b o l i c i n h i b i t o r s and pH on K + u p t a k e . These e f f e c t s were gauged by m e a s u r i n g + 42 t o t a l body K l e v e l s , K i n f l u x and changes in 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 a n a l p a p i l l a e . I a l s o measured the amount o f K + l o s t in the r e c t a l f l u i d and g a i n e d th rough d r i n k i n g and d i f f u s i o n t h r o u g h the g e n e r a l body w a l l to d e t e r m i n e the t o t a l K + b a l a n c e of the l a r v a e . 9 MATERIALS AND METHODS A. EXPERIMENTAL ANIMALS Aedes t a e n i o r h y n c h u s eggs were o b t a i n e d f rom the E n t o m o l o g i c a l R e s e a r c h C e n t e r , Vero B e a c h , F l o r i d a and the l a r v a e were r e a r e d a c c o r d i n g , to Nayer ( 1 9 6 7 ) . F o u r t h i n -s t a r l a r v a e were used in a l l e x p e r i m e n t s . They were s t a r v e d i n f r e s h r e a r i n g media f o r 1 to 2 days b e f o r e e x p e r i m e n t a t i o n to reduce the v a r i a b i l i t y due to the n u t r i t i o n a l s t a t e of the l a r v a e . L a r v a e were not s t a r v e d p r i o r to measurement o f the e 1 e c t r o p o t e n t i a 1 d i f f e r e n c e ( p . d . ) a c r o s s the ana l p a p i l l a e . The w e i g h t of f o u r t h i n s t a r l a r v a e v a r i e d between 1 . 5 and 5 mg, w i t h a mean of 3 mg. A l l w e i g h t s p r e s e n t e d in t h i s t h e s i s a r e wet w e i g h t s . Aedes c a m p e s t r i s l a r v a e were c o l l e c t e d on 3 A p r i l 1977 f rom C t e n o c l a d u s p o n d , a Na-MgS.O^ pond near K a m l o o p s , B . C . ( B T i n n , 1 9 6 9 ) . A t the t ime o f c o l l e c t i o n most o f the l a r v a e were in the second i n s t a r ; however , t h e r e were a few f i r s t and t h i r d i n s t a r s . L a r v a e d e v e l o p e d to the f o u r t h i n s t a r a t e i t h e r room t e m p e r a t u r e (23+2 ° C ) , o r 20 °C, or 10°?C s i n c e i t was not known which t e m p e r a t u r e would r e s u l t i n the b e s t s u r v i v a l . La rvae were a b l e to comple te deve lopment a t a l l t e m p e r a t u r e s . A l l l a r v a e were kept a t room t e m p e r a -t u r e f o r a t l e a s t 4 days b e f o r e e x p e r i m e n t a t i o n . Water i n most r e a r i n g pans was a e r a t e d to encourage the growth of b a c t e r i a and p r o t o z o a upon which the l a r v a e f e e d . The d i e t was s u p p l e m e n t e d w i t h b r e w e r ' s y e a s t . The w e i g h t of 10 i f o u r t h i n s t a r l a r v a e was between 2 and 8 mg, a v e r a g i n g 6 mg. Four days a f t e r c o l l e c t i o n , h a l f the l a r v a e were t r a n s -f e r r e d f rom C t e n o c l a d u s water to a s o l u t i o n c o n t a i n i n g 1 mM KC1 and 5 mM N a C l . A l a r g e amount of o r g a n i c m a t e r i a l ( m a i n l y dead b r i n e shr imp and m o s q u i t o e s ) i n the bottom of the pans c o n t r i b u t e d to the ion c o n c e n t r a t i o n . The l a r v a e remained i n t h i s s o l u t i o n f o r 10 days by wh ich t i m e most l a r v a e were i n the f o u r t h i n s t a r . These l a r v a e were s t a r v e d in a f r e s h s o l u t i o n of 5 mM NaCl f o r 2 to 3 days p r i o r to e x p e r i m e n t a t i o n to d e p l e t e the l a r v a e o f K + . L a r v a e wh ich remained i n C t e n o c l a d u s water t h r o u g h o u t t h e i r deve lopment were s t a r v e d i n f r e s h pond water f o r 2 to 3 days b e f o r e use in e x p e r i m e n t s . B. SOLUTIONS E x p e r i m e n t s used A. t a e n i o r h y n c h u s l a r v a e r e a r e d i n a number o f d i f f e r e n t m e d i a : Vancouver a rea sea water ( r e -f e r r e d to as 100% SW, c o n t a i n i n g 347 mM N a + , 405 mM C l " , 8 mM K + , 725 mOsm at pH 7 . 8 to 8 . 3 ) ; 25% SW p r e p a r e d by d i l u t i o n w i t h d i s t i l l e d w a t e r , 200% SW o b t a i n e d by e vapor a--' t i o n of SW a t room t e m p e r a t u r e ; a s o l u t i o n c o n t a i n i n g 1 mM KC1 and 5 mM N a C l ; and d i s t i l l e d w a t e r . The r e a r i n g s o l u t i o n c a l l e d " d i s t i l l e d w a t e r " a c t u a l l y c o n t a i n s low s a l t l e v e l s due to added y e a s t , e x c r e t o r y p r o d u c t s of the l a r v a e and dead l a r v a e . Ion l e v e l s r e a c h a maximum of about 1 imM N a + and 0 . 5 mM K; + . 11 C t e n o c l a d u s pond water wh ich had been f i l t e r e d t h r o u g h l a y e r s of c h e e s e c l o t h to remove l a r g e p a r t i c l e s was found to c o n t a i n 225 mM N a + , 8 . 4 mM C I " , 3 . 6 mM K + , 308 mOsm a t pH 8 . 7 . From the d a t a of B r a d l e y and P h i l l i p s (1977) and K i c i n i u k and P h i l l i p s ( 1 9 7 4 ) , the S O ^ - c o n c e n t r a t i o n i s e s t i m a t e d a t 190 mM and H C 0 3 " a t l l : m M . S o l u t i o n s used f o r f l u x and e l e c t r o p o t e n t i a l s t u d i e s were p r e p a r e d from a n a l y t i c a l grade c h e m i c a l s in g l a s s d i s t i l l e d wate r and when n e c e s s a r y the pH was a d j u s t e d to 7 . 0 to 7 . 5 w i t h c o n c e n t r a t e d HC1, NaOH, H 2 S 0 4 , or KOH. S o l u t i o n s were e q u i l i b r a t e d to room t e m p e r a t u r e (23+2 °C) b e f o r e use and a l l e x p e r i m e n t s were p e r f o r m e d at t h a t 42 t e m p e r a t u r e . S o l u t i o n s used in -K s t u d i e s were f i l t e r e d t h r o u g h 1 .2 jJmm mi 11 i pore f i l t e r s . The s o l u t i o n c o n t a i n i n g 42 1 mM KC1 and 5 mM NaCl used f o r the K f l u x s t u d i e s w i t h a c c l i m a t e d A. c a m p e s t r i s l a r v a e a l s o c o n t a i n e d 0 . 5 mM C a C l 2 . C. INFLUX MEASUREMENTS The s i t e s of K + uptake i n the l a r v a e were d e t e r m i n e d 42 by m e a s u r i n g the K i n f l u x i n t o 3 groups o f l a r v a e : (1) n o r m a l , (2) l a r v a e l i g a t u r e d a t the neck w i t h f i n e s i l k t h r e a d , and (3) l a r v a e l i g a t u r e d at the neck and a n a l segment (segment 9 , these l a r v a e h e r e a f t e r are c a l l e d d o u b l e l i g a t u r e d ) . A l l i n f l u x e x p e r i m e n t s were c a r r i e d out in the same medium i n which the l a r v a e were r e a r e d , e x c e p t 12 t h a t a s o l u t i o n of 1 mM KC1 and 5 mM NaCl was used in the p l a c e o f d i s t i l l e d w a t e r . T o t a l K + uptake was a s s e s s e d 42 by m e a s u r i n g K i n f l u x i n t o normal l a r v a e . I n f l u x t h r o u g h the ana l p a p i l l a e and the g e n e r a l body s u r f a c e was e s t i m a t e d u s i n g l a r v a e l i g a t u r e d a t the n e c k . The K + i n f l u x a c r o s s the body w a l l was measured u s i n g d o u b l e l i g a t u r e d l a r v a e . I n f l u x t h r o u g h the a n a l p a p i l l a e was c a l c u l a t e d from the d i f f e r e n c e between the i n f l u x e s i n t o neck l i g a t u r e d and double l i g a t u r e d l a r v a e . The p h y s i o l o g i c a l i m p o r t a n c e of the K + f l u x t h r o u g h the a n a l p a p i l l a e to the l a r v a was a s s e s s e d by compar ing t h i s d i f f e r e n c e w i t h the t o t a l K + u p t a k e . To s t a r t an i n f l u x e x p e r i m e n t , groups of 15 l a r v a e were p l a c e d in p l a n c h e t s c o n t a i n i n g 1 ml of s o l u t i o n l a b e l e d 42 w i t h K ( o b t a i n e d as KC1 in water f rom New Eng land N u c l e a r ; L a c h i n e , Quebec) of the a p p r o p r i a t e s p e c i f i c a c t i v i t y wh ich v a r i e d between 0 . 0 3 to 0 . 4 mCi mg \ In a l l c a s e s the i n c r e a s e i n the c o n c e n t r a t i o n of K + due to the a d d i t i o n of i s o t o p e was n e g l i g i b l e . The p l a n c h e t s were p l a c e d in s m a l l g l a s s p e t r i d i s h e s c o n t a i n i n g d i s t i l l e d water to 42 m a i n t a i n a c o n s t a n t h u m i d i t y . To d e t e r m i n e K i n f l u x , l a r v a e were s a m p l e d . a f t e r 5 m i n J 9 and 1 , 3 , and 5 h. P r e l i m i n a r y e x p e r i m e n t s i n d i c a t e d t h a t t h i s p e r i o d , f e l l 42 w i t h i n the i n i t i a l l i n e a r phase of "K u p t a k e . Samples taken a t 5 m i n , were used to e s t i m a t e the c o n t r i b u t i o n o f i s o t o p e a d h e r i n g to the c u t i c l e . T h i s amount was n e g l i g i b l e . 13 L a r v a e (n=10 f o r each t r e a t m e n t a t each s a m p l i n g t ime f o r A. t a e n i o r h y n c h u s , and n;=k6 f o r A. c a m p e s t r i s ) were r i n s e d i n r u n n i n g tap wate r f o r 5 min to remove i s o t o p e a d h e r i n g to the c u t i c l e . The l a r v a e were b l o t t e d dry on K l e e n e x and i n d i v i d u a l l y weighed on an August S a u t e r 50 mg b a l a n c e , a c c u r a t e to + 0 . 5 nig. They were t h e n - t r a n s f e r r e d to i n -d i v i d u a l p l a n c h e t s and the h e a d , ana l segment and l i g a -t u r e s removed to a v o i d c o n t r i b u t i o n of i s o t o p e a c c u m u l a t e d beyond the l i g a t u r e s . The head c a p s u l e was a l s o removed f rom normal l a r v a e . The w e i g h t l o s s due to t h i s amputa -t i o n was c o r r e c t e d by s u b t r a c t i o n of a mean head and l i g a t u r e w e i g h t of 10 l a r v a e f rom the same r e a r i n g pan-. The a n a l segment p l u s l i g a t u r e w e i g h t was i n s i g n i f i c a n t . R a d i o a c t i v i t y was measured as d e s c r i b e d in S e c t i o n D. P r e l i m i n a r y e x p e r i m e n t s used ^ R b , r a t h e r than ^ K . Rub id ium i o n i s commonly t r a n s p o r t e d by K + c a r r i e r mechanisms in i n s e c t s ( C e r c r o p i a m i d g u t , Harvey ^ a1_^:"1968';> I ' . -C a 1 1 i p h o r a s a l i v a ry g l a n d , B e r r i d g e e t aj_. , 1 9 7 6 ) . The 86 l o n g e r h a l f l i f e o f Rb of 18 d a y s , compared to 1 2 . 4 h 42 86 f o r :K , f a c i l i t a t e d e x p e r i m e n t a l d e s i g n . I n f l u x of Rb 42 was measured u s i n g s i m i l a r p r o c e d u r e s to t h o s e in the K e x p e r i m e n t e x c e p t t h a t the head and a n a l segment p l u s l i g a t u r e s were not removed p r i o r to c o u n t i n g . D. ISOTOPE COUNTING PROCEDURES Samples c o n t a i n i n g t r i t i u m were p l a c e d in l o w - a l k a l i g l a s s s c i n t i l l a t i o n v i a l s (Kent L a b o r a t o r i e s , L t d . ; V a n c o u v e r , 14 B . C . ) c o n t a i n i n g 10 ml of K e n t f l u o r (Kent L a b o r a t o r i e s ) and c o u n t e d f o r 10 min in a N u c l e a r Ch icago I socap 300 l i q u i d s c i n t i l l a t i o n s y s t e m . A c t i v i t y of the i s o t o p e in the e x t e r n a l s o l u t i o n was c a l c u l a t e d as the average o f at l e a s t 4 d e t e r m i n a t i o n s . A l l data were n o r m a l i z e d w i t h r e s p e c t to the s p e c i f i c a c t i v i t y o f the e x t e r n a l s o l u t i o n . Samples were c o r r e c t e d f o r background and quench ing by the c h a n n e l s r a t i o method (Wang and W i l l i s , 1 9 6 5 , p. 1 3 2 - 1 3 4 ) . To d e t e r m i n e the c o n t e n t o f t r i t i u m in the whole body , l a r v a e were d i g e s t e d f o r 12 h in 0 . 4 ml of a 10% (w/v) s o l u t i o n of K0H at 27°C. The samples were n e u t r a l i z e d w i t h 0 . 8 ml o f 10 N H 2 S 0 4 , 10 ml o f K e n t f l u o r added and the v i a l s c o u n t e d as d e s c r i b e d p r e v i o u s l y . One ,ul hemolymph samples or i n d i v i d u a l l a r v a e l a b e l e d QC AO w i t h Rb o r K were p l a c e d i n 0 . 5 ml o f d i s t i l l e d wate r on s t e e l p l a n c h e t s ( A m e r s h a m - S e a r l e , A r l i n g t o n H e i g h t s , 1 1 1 . ) , e v a p o r a t e d to d r y n e s s under an i n f r a r e d l a m p , and counted 86 42 f o r 10 min ( Rb) or 1 min ( ... K) in a N u c l e a r Ch icago Model 470 Gas Flow D e t e c t o r . Whole body samples were f i n e l y m a c e r a t e d w i t h f o r c e p s and e v e n l y d i s t r i b u t e d ove r the bot tom of the p l a n c h e t b e f o r e d r y i n g . The s h o r t h a l f l i f e ( 1 2 . 4 2 h) and the l a r g e number of samples n e c e s s i t a t e d 42 the s h o r t c o u n t i n g t ime f o r samples l a b e l e d w i t h K. B a c k -ground a c t i v i t y was e s t i m a t e d by i n c l u d i n g p l a n c h e t s c o n -t a i n i n g " c o l d " l a r v a e as b l a n k s in eve ry r u n . " C o l d " l a r v a e were a l s o i n c l u d e d in p l a n c h e t s used to d e t e r m i n e 15 the a c t i v i t y o f the e x t e r n a l s o l u t i o n to compensate f o r s e l f a b s o r p t i o n o f p p a r t i c l e s . C o r r e c t i o n f o r decay 42 of K was made as d e s c r i b e d i n The Handbook of R a d i o -a c t i v e N u c l i d e s (Wang 9,69; : p. 74-75) . • , E. TOTAL BODY K + UPTAKE 42 W h i l e s t u d i e s u t i l i z i n g K i n d i c a t e d the r o u t e and magni tude of K + i n f l u x , they d i d not a l o n e d e m o n s t r a t e net uptake of t h i s c a t i o n . To d e t e r m i n e i f t h e r e was net uptake 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 , t o t a l body K was measured i n A. t a e n i o r h y n c h u s l a r v a e as a f u n c t i o n of e x t e r n a l K + and N a + c o n c e n t r a t i o n s and pH. S o l u t i o n s used to t e s t the e f f e c t of e x t e r n a l K + c o n c e n t r a t i o n s c o n t a i n e d 0 . 0 5 , 0 . 5 , 2 . 5 , 5 or 25 mM KC1 and a l l s o l u t i o n s c o n t a i n e d 5 mM N a C l . S o l u t i o n s used to t e s t the e f f e c t of e x t e r n a l + 4-Na c o n c e n t r a t i o n on K c o n t e n t of the l a r v a e c o n t a i n e d 0 . 1 , i l , 10 and 100 mM NaCl and a l l s o l u t i o n s c o n t a i n e d . 1 mM KC1. Ten mM sodium phosphate b u f f e r s o l u t i o n s c o n -t a i n i n g 1 mM KC1 were a d j u s t e d to pH 5 , 6 , 7 , 8 , or 9 w i t h c o n c e n t r a t e d HC1 or NaOH to s tudy the e f f e c t s of pH on K upta k e . Groups of 10 to 20 l a r v a e were p l a c e d in 35 X 10 cm p l a s t i c p e t r i d i s h e s c o n t a i n i n g 5 ml of the e x p e r i m e n t a l s o l u t i o n and were i n c u b a t e d a t room t e m p e r a t u r e f o r 12 h. F o r s a m p l i n g , the l a r v a e were t r a n s f e r r e d to a tea s t r a i n e r and r i n s e d w i t h d i s t i l l e d wate r to remove s u p e r f i c i a l K + . 16 The r i n s e d l a r v a e were b l o t t e d dry on K l e e n e x , weighed in t a r e d p l a t i n u m boats and. ashed o v e r n i g h t a t 425 °C. The b o a t s were p l a c e d in p l a s t i c v i a l s and the ash e l u t e d w i t h 1 ml of 0 . 1 M HC1. T w e n t y - f i v e jul samples of the e l u a n t were c o l l e c t e d in c a l i b r a t e d p i p e t t e s ( C o r n i n g ) and the K + c o n c e n t r a t i o n d e t e r m i n e d as d e s c r i b e d in S e c t i o n H. T o t a l body p o t a s s i u m i s i n d i c a t e d as " K + " , however , because o f the a s h i n g p r o c e d u r e both bound and i o n i c p o t a s s i u m a r e i n -c l u d e d i n t h i s v a l u e . There was no c o n t a m i n a t i o n by K + f rom s o l u t i o n s or the boats as v e r i f i e d by t e s t i n g boats c o n t a i n i n g d i s t i l l e d water or 0 .1 M HC1 as a c o n t r o l . F. POTENTIAL DIFFERENCE ACROSS THE ANAL PAPILLAE To d e t e r m i n e i f net a b s o r p t i o n of K + o c c u r r e d a g a i n s t an 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 ( p . d . ) , the p . d . a c r o s s the a n a l p a p i l l a e was measured between the hemocoel and the e x t e r n a l s o l u t i o n . L a r v a e were l i g a t u r e d a t the head and s i p h o n and suspended a c r o s s a n o t c h e d p a r t i t i o n in a f P e r s p e x ( m e t h y l m e t h a c r y l a t e po l ymer ) chamber ( F i g u r e 2 ) . 1 The head and t h o r a x were submerged in heavy p a r a f f i n and the c a u d a l end was c o n t i n u o u s l y p e r f u s e d w i t h the e x p e r i -menta l s o l u t i o n s . The d i m e n s i o n s of the p e r f u s i o n chamber were 0 . 7 X 1 . 3 X 0 . 3 cm. The chamber was p e r f u s e d w i t h s o l u t i o n s by g r a v i t y feed a t a r a t e of 2 ml min ^ and the s o l u t i o n removed by s u c t i o n . E q u i l i b r a t i o n w i t h new s o l u t i o n s , as checked w i t h a s o l u t i o n c o n t a i n i n g the dye a m a r a n t h , o c c u r r e d w i t h i n 10 s e c . I t was assumed t h a t 17 agar bridge between reference electrode and chamber Figure 2 Apparatus used to measure el 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 anal p a p i l l a e . The r e c o r d i n g m i c r o e l e c t r o d e i s placed in a drop of hemolymph under l i q u i d p a r a f f i n . The referenc e e l e c t r o d e i s connected to the chamber c o n t a i n i n g perfusate v i a an agar bridge. 18 hemolymph c o n c e n t r a t i o n s were c o n s t a n t d u r i n g the s h o r t p e r i o d s ( g e n e r a l l y 1 to 2 min) when l a r v a e were n o t . p e r f u s e d w i t h the r e f e r e n c e s o l u t i o n . A Ag/AgCl r e f e r e n c e e l e c t r o d e , type RC-1 (W-P I n s t r u -m e n t s ; I n c . ; Hamden, Conn. ) was c o n n e c t e d to the p e r f u s i o n chamber by a s a l t b r i d g e made f rom p o l y e t h y l e n e t u b i n g (PE 90) f i l l e d w i t h 3 M KC1 in 3% a g a r . The r e c o r d i n g e l e c t r o d e was p u l l e d f rom g l a s s c a p i l l a r y tubes ( 1 . 2 mm d i a m e t e r , a l k a l i - f r e e g l a s s ; F i s h e r , P i t t s b u r g , P a . ) on a David Kopf Model 700C v e r t i c a l .. m.r .cropipette p u l l e r (Dav id Kopf I n s t r u m e n t s , T u j u n g a , Ca . ) and f i l l e d w i t h 3 M KC1 i n 3% 5 a g a r . These e l e c t r o d e s had l a r g e t i p s of about 5 X 1 0 ohm r e s i s t a n c e . The m i c r o e 1 e c t r o d e was mounted i n a W-P model EH-1S e l e c t r o d e h o l d e r s e c u r e d on a N a r i s h i g e (Model 479) m i c r o m a n i p u l a t o r . The e l e c t r o d e s were c o n n e c t e d . t o a K e i t h l e y Model 602 e l e c t r o m e t e r ( K e i t h l e y I n s t r u m e n t s , I n c . ; C l e v e l a n d , Ohio) and the p . d . r e c o r d e d on a F i s h e r R e c o r d a l l S e r i e s 500 c h a r t r e c o r d e r (Houston I n s t r u m e n t s ; A u s t i n , T e x a s ) . The asymmetry v o l t a g e of the r e c o r d i n g e l e c t r o d e was measured w i t h r e s p e c t to the r e f e r e n c e e l e c t r o d e w h i l e both e l e c t r o d e s were immersed in the r e f e r e n c e s o l u t i o n . The asymmetry v o l t a g e was l e s s than 5 mV and s t a b l e ; f o r most r e c o r d i n g s i t was l e s s than 1 mV. I n i t i a l l y the r e -c o r d i n g e l e c t r o d e was p l a c e d in the r e f e r e n c e s o l u t i o n at 1 min i n t e r v a l s to check the asymmetry v o l t a g e . The v o l t a g e 19 change was never g r e a t e r than 2 mV, and o n l y r a r e l y more than 1 mV so the p r a c t i c e was d i s c o n t i n u e d . D e f e c t i v e e l e c t r o d e s were e a s i l y r e c o g n i z e d by i n s t a b i l i t y i n the v o l t m e t e r r e a d i n g . The p . d . was measured between a drop of hemolymph e x t r u d e d f rom the - thorax of a l a r v a and the s o l u t i o n around the a n a l p a p i l l a e . C o n t a c t was made on the hemocoel s i d e of the a n a l p a p i l l a e by c a r e f u l l y p u n c t u r i n g the t h o r a x w i t h f i n e d i s s e c t i n g s c i s s o r s (John Weiss & S o n s , L t d . ; L o n d o n , Eng land ) and p l a c i n g the r e c o r d i n g e l e c t r o d e i n the drop of hemolymph which formed under the p a r a f f i n . T h i s p r o c e d u r e had no a p p a r e n t e f f e c t on the l a r v a ' s a c t i v i t i e s . The a o r t a c o n t i n u e d to pump, m o u t h p a r t s to move, and the rec tum to e x c r e t e f o r s e v e r a l h o u r s . A c r i t i c a l a s s u m p t i o n of t h i s method of m e a s u r i n g the p . d . a c r o s s the p a p i l l a e was t h a t ; t h e a n a l p a p i l l a e fo rm the path of l e a s t e l e c t r i c a l r e s i s t a n c e a c r o s s the body w a l l . The a n a l p a p i l l a e o f FW l a r v a e ( W i g g l e s w o r t h , 1 9 3 3 a , Ramsay, 1950) and s a l i n e l a r v a e ( P h i l l i p s and B r a d l e y , 1 977) . . ;. ) a re w e l l known to be the a r e a of the c u t i c l e most permeable to both i o n s and w a t e r , so t h i s a s s u m p t i o n appears j u s t i f i e d . The a n a l p a p i l l a e were p e r f u s e d w i t h v a r i o u s s o l u t i o n s f o r p e r i o d s of up to 3 . 5 h. P e r f u s i o n of a g i v e n s o l u t i o n was c o n t i n u e d u n t i l the p . d . s t a b i l i z e d a t a new v a l u e , u s u a l l y w i t h i n 1 m i n . R e c o r d i n g s were t e r m i n a t e d i f the 20 p . d . d e c l i n e d m a r k e d l y or the r e s p o n s e became e r r a t i c . P e r f u s i o n w i t h the e x p e r i m e n t a l s o l u t i o n was b r a c k e t t e d between p e r f u s i o n s o f the r e f e r e n c e s o l u t i o n to check f o r any d r i f t of the r e f e r e n c e p o t e n t i a l . Return of the p . d . to the r e f e r e n c e v a l u e b e f o r e and a f t e r p e r f u s i o n w i t h the e x p e r i m e n t a l s o l u t i o n was the c r i t e r i a f o r a c c e p t i n g the e x p e r i m e n t a l p o t e n t i a l measurement . The d a t a a r e p r e s e n t e d as the ave rage d i f f e r e n c e i n the e x p e r i m e n t a l p o t e n t i a l measurement f rom the r e f e r e n c e p o t e n t i a l ( i .e . the p . d . r e c o r d e d when the p a p i l l a e were p e r f u s e d w i t h the r e a r i n g m e d i a , o r a s o l u t i o n c o n t a i n i n g 1 mM KC1 and 5 mM NaCl in the case o f l a r v a e r e a r e d i n d i s t i l l e d w a t e r ) . T h i s compensated f o r d r i f t i n g of the r e f e r e n c e p o t e n t i a l d u r i n g the c o u r s e of the e x p e r i m e n t . G. COLLECTION OF RECTAL FLUID To e s t i m a t e the b a l a n c e of K + l o s s and uptake by v a r i o u s r o u t e s in A. t a e n i o r h y n c h u s l a r v a e , i t was n e c e s -s a r y to measure the l o s s of K + i n the r e c t a l f l u i d . A m o d i f i c a t i o n o f the t e c h n i q u e d e v e l o p e d by Ramsay (1953) was used to c o l l e c t r e c t a l f l u i d ( F i g u r e 3 ) . A l a r v a was s n a r e d l o o s e l y b e h i n d the head and around the s i p h o n w i t h f i n e s i l k t h r e a d . G e n e r a l l y the l i g a t u r e a round the s i p h o n was t i g h t e n e d to a v o i d f l o o d i n g the t r a c h e a l sys tem w i t h p a r a f f i n f rom the c o l l e c t i o n b a t h . L a r v a e can e a s i l y s u r v i v e f o r e x t e n d e d p e r i o d s w i t h o u t a c c e s s to a i r th rough the s i p h o n due to c u t i c u l a r r e s p i r a t i o n ( f o r 36 h , 21 Fi gure 3 Apparatus used f o r the c o l l e c t i o n of r e c t a l f l u i d from A. taeniorhynchus l a r v a e . 22 B e a d l e , 1 9 3 9 ; f o r 17 h, S t o b b a r t , 1 9 7 1 ) . Samples f rom l a r v a e w i t h open or b l o c k e d s i p h o n s had a s i m i l a r K + c o n c e n t r a t i o n . The l i g a t u r e d l a r v a was s e c u r e d in a p a r t i t i o n e d P e r s p e x chamber (2 X 1 . 3 X 0 . 3 cm) by s e a l i n g the abdomen i n t o a n o t c h i n the p a r t i t i o n w i t h V a s e l i n e and t e t h e r i n g the ends o f the l i g a t u r e s w i t h p l a s t i c i n e to the s i d e s of the chamber. In t h i s manner the h e a d , f u l l y c a p a b l e of d r i n k i n g , was submerged i n the r e a r i n g medium w h i l e the ana l segment was c o v e r e d w i t h heavy p a r a f f i n ( w h i t e , heavy d o m e s t i c ; F i s h e r ) . R e c t a l f l u i d was c o l l e c t e d as i t was e x c r e t e d I ' in ./ : mi c rop i p e t t e s ( l o w - a l k a l i c o a g u l a t i o n t u b e s ; F i s h e r ) h e l d n e x t to the anus . The p i p e t t e f i l l e d by c a p i l l a r y a c t i o n and c o n t a i n e d p a r a f f i n s t a i n e d w i t h Sudan B l a c k to p r e v e n t e v a p o r a t i o n o f the s a m p l e . P o t a s -s ium ion c o n c e n t r a t i o n and volume were d e t e r m i n e d on samples c o l l e c t e d over a 1 to 2 . 5 h p e r i o d . O c c a s i o n a l l y some f l u i d e n t e r e d the c a p i l l a r y tube w i t h i n a few m i n u t e s of the tube b e i n g p l a c e d a t the a n u s . T h i s f l u i d was d i s c a r d e d s i n c e B r a d l e y and P h i l l i p s (1975) found i t was i s o s m o t i c w i t h the hemolymph and t h e r e f o r e p r o b a b l y d e r i v e d d i r e c t l y f rom the Ma 1pigta'an t u b u l e s . a n d the m i d g u t . The volumes g i v e n u n d e r e s t i m a t e the t o t a l volume s e c r e t e d s i n c e t h e r e i s some l o s s of f e c a l m a t e r i a l d u r i n g s a m p l i n g . Feces would f r e q u e n t l y adhere to the w a l l of the c o l l e c t i n g p i p e t t e or would have to be s e p a r a t e d from the u r i n e w i t h 23 a g l a s s rod to p e r m i t volume d e t e r m i n a t i o n of the drop of u r i n e . The p r e s e n c e of f e c e s i n the sample d i d not a l t e r the K + c o n c e n t r a t i o n s i n c e samples composed a l m o s t e n t i r e l y of f e c e s had K + c o n c e n t r a t i o n s e q u i v a l e n t to samples composed a l m o s t e n t i r e l y of u r i n e . H. DETERMINATION OF ION CONCENTRATIONS, OSMOLARITY AND VOLUME Sodium ion and K + c o n c e n t r a t i o n s i n l a r v a e and s o l u t i o n s were d e t e r m i n e d w i t h a T e c h t r o n AA 120 ( V a r i a n T e c h t r o n P t y . L t d . ; M e l b o u r n e , V i c t o r i a ) a t o m i c a b s o r p t i o n s p e c t r o -photometer in the a b s o r p t i o n mode. One p i samples were p l a c e d i n p l a s t i c v i a l s c o n t a i n i n g 3 ml of d i s t i l l e d water f o r N a + d e t e r m i n a t i o n s and r e a d a t 5 8 9 . 4 nm, or 1 ml of a swamp s o l u t i o n c o n s i s t i n g o f 500 juM NaCl and read a t 7 6 6 . 9 nm f o r K + d e t e r m i n a t i o n s . C h l o r i d e ion c o n c e n t r a -t i o n s in 2 jul samples were measured w i t h a CMT 10 C h l o r i d e t i t r a t o r ( R a d i o m e t e r , Copenhagen) . Osmot ic c o n c e n t r a t i o n s were d e t e r m i n e d u s i n g a B i o l o g i c a l C r y o s t a t / N a n o l i t e r Osmometer ( C l i f t o n T e c h n i c a l P h y s i c s , New Y o r k ) . At l e a s t t h r e e d e t e r m i n a t i o n s of the o s m o l a r i t y were made on each f l u i d sample and a v e r a g e d . The volume o f n a n o l i t e r - s i z e samples was e s t i m a t e d f rom the d i a m e t e r of the sample drop under l i q u i d p a r a f f i n ( l i g h t , d o m e s t i c ; F i s h e r ) . The c o n v e r s i o n between d i a m e t e r and volume was c a l c u l a t e d by m e a s u r i n g the r a d i o a c t i v i t y o f drops f rom a s t a n d a r d 24 " I^C- inul i n s o l u t i o n ( B r a d l e y and P h i l l i p s , 1 9 7 5 ) . I. BLOCKING THE ANUS To d.ert.e.rmi ne if-; t''h>.ecan:a"n p a p i l l a e were r e s p o n s i b l e f o r i on s e c r e t i o n i t was n e c e s s a r y to p r e v e n t e x c r e t i o n of r e c t a l f l u i d . The ana l c a n a l was p lugged by i n j e c t i n g d e n t a l " p i t and f i s s u r e s e a l a n t " (a g i f t o f Dr . R. H. Roydhouse , D e n t i s t r y , U n i v e r s i t y of B r i t i s h C o l u m b i a ) . The 2 g l u e r e s i n s were t h o r o u g h l y mixed on a s h e e t of " P a r a f i l m " i m m e d i a t e l y b e f o r e i n j e c t i o n . The m i x t u r e was taken up i n a m i c r o p i p e t t e and the t i p was m a n u a l l y i n t r o d u c e d i n t o the a n a l c a n a l w h i l e the l a r v a was l o o s e l y r e s t r a i n e d w i t h f o r c e p s . Glue was i n j e c t e d by b l o w i n g t h r o u g h r u b b e r t u b i n g a t t a c h e d to the c a p i l l a r y t u b e . P l a c e m e n t o f the p l u g was d e t e r m i n e d v i s u a l l y s i n c e the p i n k g l u e was e a s i l y v i s i b l e t h r o u g h the t r a n s l u c e n t c u t i c l e , The p l u g o f t e n f i l l e d the ana l c a n a l , rec tum and ex tended i n t o the i l e u m . S u c c e s s i n s e a l i n g the rec tum was d e t e r -mined by n o t i n g the absence o f f e c e s i n the e x t e r n a l medium, d i s s e c t i o n of the l a r v a and v i s u a l i n s p e c t i o n of the p l u g s i z e and h a r d n e s s , and f i n a l l y by i s o t o p e f l u x e x p e r i m e n t s . In a n o t h e r e x p e r i m e n t the anus was g l u e d . b y a p p l i c a t i o n of Eastman 910 t i s s u e a d h e s i v e (Eastman Chemica l P r o d u c t s ; Ki n g s t o n , T e n n . ) . 25 J . REMOVAL OF THE ANAL PAPILLAE Hemolymph ion l e v e l s and s u r v i v a l o f p a p i l l a e l e s s l a r v a e were compared w i t h those of normal l a r v a e to d e t e r -mine i n d i r e c t l y the ' f u n c t i o n of the ana l p a p i l l a e . The p a p i l l a e were removed by i n c u b a t i n g f i r s t i n s t a r l a r v a e i n a Mo s o l u t i o n of AgNO^ f o r 30 s e c . T h i s was the s h o r t e s t p e r i o d wh ich s u c c e s s f u l l y removed the ana l p a p i l l a e . An i n a p p r o p r i a t e AgNO^ c o n c e n t r a t i o n or immers ion t ime e i t h e r k i l l e d the l a r v a e o r d i d not remove the p a p i l l a e . L a r v a e were t r e a t e d w i t h AgNO^ as e a r l y in deve lopment as p o s s i b l e to a l l o w the maximum r e c o v e r y t i m e . S u r v i v i n g f o u r t h i n s t a r l a r v a e had m o l t e d 3 t i m e s , wh ich e n s u r e d c o m p l e t e h e a l i n g of the wound. La rvae t r e a t e d w i t h AgNO^ i m m e d i a t e l y a f t e r h a t c h i n g d i e d . S u r v i v a l was h i g h when t r e a t m e n t o c c u r r e d 24 h a f t e r h a t c h i n g . A f t e r b e i n g immersed i n AgNO^j l a r v a e were t h o r o u g h l y r i n s e d in tap water and r e t u r n e d to the r e a r i n g m e d i a . The e f f e c t of AgNO^ t r e a t m e n t on the body w a l l p e r m e a b i l i t y was checked by 3 m e a s u r i n g Wfl i n f l u x i n t o t r e a t e d and u n t r e a t e d l a r v a e as d e s c r i b e d in S e c t i o n D. These i n f l u x s t u d i e s were c a r r i e d out u s i n g l a r v a e t h a t had been i n c u b a t e d in 1% A g N 0 ^ f o r 1 m i n . L a r v a e used i n l a t e r e x p e r i m e n t s were exposed to AgN0 3 f o r 30 s e c . K. MEASUREMENT OF DAMAGE TO THE CUTICLE P r e l i m i n a r y e x p e r i m e n t s s u g g e s t e d t h a t h a n d l i n g p r o -cedures might be a l t e r i n g the p e r m e a b i l i t y of the body 26 w a l l . T h e r e f o r e , "^ H^O f l u x a c r o s s the c u i t i c l e was measured to a s s e s s the e f f e c t o f l i g a t u r i n g and AgNO^ t r e a t m e n t on c u t i c u l a r p e r m e a b i l i t y . In the f i r s t e x p e r i m e n t i n f l u x a i n t o l a r v a e w i t h the neck l i g a t u r e d was compared, to i n f l u x i n t o doub le l i g a t u r e d l a r v a e ; i n the second s t u d y normal and p a p i l l a e l e s s l a r v a e were compared . S o l u t i o n does not e n t e r th rough the anus as d e t e r m i n e d by immers ing l a r v a e l i g a t u r e d a t the head in a c o n c e n t r a t e d amaranth s o l u t i o n . Koch (1938) a l s o f o u n d t h a t f l u i d d i d not e n t e r v i a the a n u s . Groups of 15 to 20 S W - r e a r e d l a r v a e were p l a c e d in capped p l a s t i c v i a l s c o n t a i n i n g 2 . 5 ml o f SW and 0 .1 mCi 3 - 1 of H^O h a v i n g a s p e c i f i c a c t i v i t y of 1 mCi g . In the f i r s t e x p e r i m e n t samples were taken a t 0 . 5 , 1 . 5 , 3 and 6 h. S a m p l i n g t i m e s f o r the AgMO^ e x p e r i m e n t were 2 , 4 and 7 h. In both e x p e r i m e n t s samples were a l s o taken w i t h i n one minute o f b e i n g p l a c e d in the t r i t i a t e d s o l u t i o n to e s t i m a t e n o n - s p e c i f i c i s o t o p e b i n d i n g to the c u t i c l e . T h i s amount was i n s i g n i f i c a n t . At the a p p r o p r i a t e t i m e s l a r v a e were removed f rom the s o l u t i o n , r i n s e d f o r 3 min in r u n n i n g t a p wate r and b l o t t e d dry on K l e e n e x , and a hemolymph sample was c o l l e c t e d f rom i n d i v i d u a l l a r v a e f o r s c i n t i l l a -t i o n c o u n t i n g as d e s c r i b e d in S e c t i o n L. L. COLLECTION OF HEMOLYMPH SAMPLES Hemolymph samples were c o l l e c t e d f rom i n d i v i d u a l l a r v a e or p o o l e d f rom 2 to 4 l a r v a e . Larvae were b r i e f l y 27 r i n s e d w i t h d i s t i l l e d w a t e r , b l o t t e d dry on K l e e n e x and p l a c e d on a s h e e t of " P a r a f i l m " . The c u t i c l e was t o r n w i t h c a r e to a v o i d r u p t u r i n g the g u t , and the hemolymph was i m m e d i a t e l y c o l l e c t e d w i t h a c a l i b r a t e d (1 or 2 jul) Drummond m i c r o c a p or a g l a s s m i c r o p i p e t t e ( l o w - a l k a l i g l a s s , F i s h e r ) f i l l e d w i t h p a r a f f i n to p r e v e n t e v a p o r a t i o n . The amount of hemolymph was e s t i m a t e d by compar ing the l e n g t h o f the f l u i d column to the t o t a l volume of the m i c r o c a p (Kaufman and P h i l l i p s , 1 9 7 3 ) . Samples c o l l e c t e d by t h i s method had a c o m p o s i t i o n e q u i v a l e n t to samples c o l l e c t e d w h i l e the l a r v a e was h e l d under l i q u i d p a r a f f i n ( B r a d l e y and P h i l l i p s , 1 9 7 5 ) , so e v a p o r a t i o n of the hemolymph + + d u r i n g c o l l e c t i o n was n e g l i g i b l e . Samples f o r Na and K a n a l y s i s were i m m e d i a t e l y p l a c e d i n p l a s t i c v i a l s and measured as d e s c r i b e d in S e c t i o n H. Samples to be a n a l y s e d f o r C l " or o s m o t i c p r e s s u r e were i m m e d i a t e l y p l a c e d under l i q u i d p a r a f f i n and d e t e r m i n a t i o n s c o m p l e t e d w i t h i n 2 h; M. DETERMINATION OF RESPIRATION RATE The 0^ consumpt ion of A. t a e n i o r h y n c h u s l a r v a e r e a r e d and t e s t e d i n 25, 1 00 , and 200%.1-.SW was measured to d e t e r m i n e i f the m e t a b o l i c r a t e was l i n k e d to the o s m o t i c and i o n i c w o r k l o a d . F i v e groups of 15 l a r v a e each were p l a c e d i n 5 ml o f r e a r i n g medium in Gi1 son m e t a b o l i c f l a s k s c o n t a i n i n g 0 . 2 ml o f 6 M KOH i n the c e n t r a l w e l l to t r a p C 0 2 - F l a s k s 28 were mounted on a Warburg manometer and submerged in a water bath a t 23°C. A f t e r 1 h e q u i l i b r a t i o n , d e t e r m i n a t i o n s o f gas volume were made a t 0 . 5 h i n t e r v a l s over the nex t 4 h. R e s u l t s were e x p r e s s e d as jul 0^ mg"^ , based on an average wet w e i g h t f o r a l a r v a of 3 mg. N. CALCULATIONS 42 The K i n f l u x a c r o s s the a n a l p a p i l l a e was c a l c u l a t e d as the d i f f e r e n c e between the mean i n f l u x e s i n t o i n d i v i d u a l neck l i g a t u r e d and d o u b l e l i g a t u r e d l a r v a e . The d r i n k i n g r a t e of l a r v a e i n s a l i n e media was 4 2 c a l c u l a t e d as the d i f f e r e n c e of the mean K i n f l u x i n t o normal and neck l i g a t u r e d l a r v a e . 42 The p a s s i v e d i f f u s i o n of K a c r o s s the body w a l l was 42 measured by the K i n f l u x i n t o doub le l i g a t u r e d l a r v a e . The e n t r y o f a s m a l l m o l e c u l e by d i f f u s i o n f o l l o w s an e x p o n e n t i a l curve h a v i n g the f o r m : Cj = C Q ( 1 - e x p ( - t / T ) ) , where C. and C a re the i n t e r n a l and e x t e r n a l a c t i v i t i e s 1 0 ( a p p r o x i m a t e d by u s i n g the hemolymph a n d . e x t e r n a l c o n -42 ' c e n t r a t i o n s ) o f K, t i s t i m e , and T i s the t ime c o n s t a n t AO * * of K exchange (Shaw, 1 9 5 5 ) . A p l o t o f l n ( l - C . / C ) v e r s u s t s h o u l d r e s u l t in a s t r a i g h t l i n e w i t h a s l o p e of - 1 / T . The d i f f u s i o n a l p e r m e a b i l i t y c o e f f i c i e n t (P^) of the c u t i c l e to t r i t i a t e d water was c a l c u l a t e d from the f o r m u l a : P d = V/TA, where V i s the volume o f t o t a l body w a t e r , T i s the t ime c o n s t a n t f o r H o 0 i n f l u x and A i s the a r e a of the 29 c u t i c l e (Shaw, 1 9 5 5 ) . The volume of t o t a l body water was c a l c u l a t e d assuming t h a t water c o n s t i t u t e d 85% of the body v o l u m e , a v a l u e found f o r a n o t h e r s a l i n e m o s q u i t o l a r v a , O p i f e x f u s c u s ( N i c h o l s o n and L e a d e r , 1 9 7 4 ) . The t o t a l body volume was c a l c u l a t e d f rom the e q u a t i o n : y = - 0 . 1 9 + 3 1 . 0 7 x , where y i s the volume i n mm and x i s the w e i g h t of the l a r v a in mg. T h i s e q u a t i o n was found by B r a d l e y (1976) to d e s c r i b e the r e l a t i o n s h i p of volume to w e i g h t in A.  t a e n i o r h y n c h u s . The s u r f a c e a r e a of the c u t i c l e was e s t i -mated f rom d a t a of B r a d l e y (1976) f o r A. t a e n i o r h y n c h u s . The e q u i l i b r i u m p o t e n t i a l (E^) of v a r i o u s i o n s was c a l c u l a t e d a c c o r d i n g to the N e r n s t e q u a t i o n : l o g C-/C = ( z F / 2 . 3 0 3 RT) ( E r ) , where C. and C Q r e p r e s e n t the hemolymph and e x t e r n a l i on a c t i v i t i e s ( a p p r o x i m a t e d by the use of c o n c e n t r a t i o n r a t h e r than a c t i v i t y ) , z i s the v a l e n c e , R the gas c o n s t a n t , T a b s o l u t e t e m p e r a t u r e , F the Faraday c o n s t a n t , and E i s the p . d . measured a c r o s s the a n a l r r pa p i 11ae . 42 The u n i d i r e c t i o n a l K f l u x , J , was c a l c u l a t e d as d e s c r i b e d by P r u s c h ( 1 9 7 6 ) . The s p e c i f i c a c t i v i t y e q u a l s the i s o t o p e c o n c e n t r a t i o n (cpm m l - ' ' ) d i v i d e d by the ion c o n c e n t r a t i o n (nmol m l " " ' ) . The t r a c e r f l u x e q u a l s the s l o p e of the i n i t i a l l i n e a r i n f l u x g raphed v e r s u s t ime (cpm h ^) d i v i d e d by the s u r f a c e a r e a of the ana l p a p i l l a e (.cm"). The f l u x , J (nmol cm h ) , i s then c a l c u l a t e d as the - 2 - 1 t r a c e r f l u x (cpm cm h ) d i v i d e d by the s p e c i f i c a c t i v i t y (cpm nmo1~^) . 30 S t a t i s t i c a l s i g n i f i c a n c e of the d a t a was a n a l y s e d u s i n g S t u d e n t ' s t t e s t i f a p p l i c a b l e . When the v a r i a n c e s were not homogeneous the Welch p r o c e d u r e f o r a 2 sample t e s t w i t h unequal v a r i a n c e s was used to t e s t the s i g n i f i c a n c e l e v e l ( B r o w n l e e , 1 9 6 5 , p. 2 9 9 - 3 0 3 ) . The v a r i a b i l i t y of the data i s e x p r e s s e d as the s t a n d a r d e r r o r of the mean ( S . E . M . ) P r o b a b i l i t i e s a re g i v e n e i t h e r to the P< 0 .01 or P< 0 . 0 5 (2 t a i l e d t e s t ) l e v e l . 31 RESULTS A. THE FUNCTION OF THE ANAL PAPILLAE IN SALINE MEDIA 1. SEPARATION OF THE ANAL PAPILLAE OF A. TAENIORH.YNCHUS LARVAE FROM THE RECTAL FLUID The o r i g i n a l o b j e c t i v e o f t h i s s tudy was to e x p l o r e the p o s s i b i l i t y t h a t the ana l p a p i l l a e are i n v o l v e d i n i o n e x c r e t i o n i n s a l i n e m e d i a . I t was f i r s t n e c e s s a r y to s e p a r a t e the ana l p a p i l l a e f rom the r e c t a l f l u i d s i n c e B r a d l e y and P h i l l i p s (1975) d e m o n s t r a t e d t h a t i t c o n t a i n s i o n s . S e v e r a l methods were a t t e m p t e d to s top the r e c t a l e x c r e t i o n i n c l u d i n g l i g a t i n g the abdomen ( t h e r e b y c u t t i n g o f f i n n e r v a t i o n by the abdomina l g l a g l i a ) , g l u i n g the a n u s , and p l a c i n g a p l u g i n the ana l c a n a l . These approaches were u n s u c c e s s f u l . L i g a t e d l a r v a e c o n t i n u e d to e j e c t 8 6 f e c e s f o r s e v e r a l h o u r s , Rb e f f l u x f rom " h o t " l a r v a e w i t h the ana l segment g l u e d was up to 4 t imes h i g h e r than Q C the e f f l u x f rom normal l a r v a e , and Rb e f f l u x f rom p lugged l a r v a e was c o n s i s t a n t l y h i g h e r than the e f f l u x f rom doub le l i g a t u r e d l a r v a e . The p l u g was p a r t i a l l y e f f e c t i v e i n Q C b l o c k i n g the r e c t a l f l u i d s i n c e Rb e f f l u x f rom p lugged l a r v a e was l e s s than h a l f the e f f l u x f rom normal l a r v a e . However , p l u g g i n g the anus damaged the c u t i c l e as i n d i c a t e d 3 by the g r e a t e r i n f l u x of H,,0 th rough the body w a l l o f p l u g g e d v e r s u s normal l a r v a e ( F i g u r e 4 ) . 32 F i g u r e 4 3 ttr,0 i n f l u x i n t o A. t a e n i o r h y n c h u s l a r v a e r e a r e d i n 100% SW: u n t r e a t e d l a r v a e (e) and l a r v a e w i t h the ana l c a n a l o c c l u d e d w i t h d e n t a l p i t and f i s s u r e s e a l a n t (•). A l l l a r v a e were l i g a t u r e d a t the neck to p r e v e n t d r i n k i n g . Each p o i n t r e p r e s e n t s the mean i n f l u x i n t o 5 l a r v a e e x c e p t at 0 . 5 hr when 4 l a r v a e were u s e d . Means are s i g n i f i c a n t l y d i f f e r e n t (P< 0 . 0 5 ) a t 0 . 5 and 1 . 5 h r . Bars i n d i c a t e + .. S . E . M . 33 34 2 . EFFECT OF EXPERIMENTAL PROCEDURES ON THE BODY WALL OF A. TAENIORHYNCHUS LARVAE S i n c e i t was not f e a s i b l e to s e p a r a t e the p a p i l l a e f rom the r e c t a l f l u i d , I t r i e d an a l t e r n a t e approach of d e s t r o y i n g the ana l p a p i l l a e w i t h AgNO^. T h i s t r e a t m e n t d i d not a l t e r the p a s s i v e p e r m e a b i l i t y of the c u t i c l e s i n c e 3 the i n f l u x of H^O i n t o p a p i l l a e l e s s l a r v a e e q u a l l e d the i n f l u x i n t o normal l a r v a e a t the 3 and 7 h s a m p l i n g t imes ( F i g u r e 5 ) . I n f l u x was s i g n i f i c a n t l y d i f f e r e n t (P<0 .05) a t 2 h. The e f f e c t of doub le l i g a t u r i n g on the p e r m e a b i l i t y 3 of the c u t i c l e was a l s o m e a s u r e d . I n f l u x o f H2O i n t o l a r v a e l i g a t u r e d a t the ana l segment was not s i g n i f i c a n t l y d i f f e r e n t (P<0.05) f rom the i n f l u x i n t o normal l a r v a e ( F i g u r e * 6 ) . 3 . HEMOLYMPH ION CONCENTRATIONS AND OSMOLARITY To d e t e r m i n e the s i g n i f i c a n c e of the anal p a p i l l a e i n i o n i c and o s m o t i c r e g u l a t i o n , hemolymph c o m p o s i t i o n of normal and p a p i l l a e l e s s A. t a e n i o r h y n c h u s l a r v a e r e a r e d i n v a r i o u s s a l i n i t i e s were compared . Ion l e v e l s and o s m o l a r i t y of the hemolymph were found to be r e m a r k a b l y c o n s t a n t f o r normal l a r v a e r e a r e d i n ! d i s t i l l e d w a t e r , 25 , 1 00 , and 200% SW . ( F i g u r e 7 ) . Over t h i s range the e x t e r n a l o s m o l a r i t y changes by 1440 mOsm, N a + by 700 mM, C l " by 800 mM, and K + by 16 mM. the c o r r e s p o n d i n g 35 F i g u r e 5 3 H 20 i n f l u x f rom 100% SW i n t o A. t a e n i o r h y n c h u s l a r v a e r e a r e d i h 100% SW. Normal (•) and p a p i l l a e l e s s l a r v a e (•) are compared . P a p i l l a e were removed by i n c u b a t i o n of l a r v a e in a 1% s o l u t i o n of AgN03 f o r 1 m i n . A l l l a r v a e were l i g a t u r e d a t the n e c k . The p o i n t s are means c a l c u l a t e d f rom the number of l a r v a e i n d i c a t e d nex t to each p o i n t . Bars i n d i c a t e + S . E . M . hours 37 F i g u r e 6 3 i n f l u x f rom 100% SW i n t o A. t a e n i o r h y n c h u s l a r v a e r e a r e d i n 100% SW. La rvae l i g a t u r e d at the neck (•) and doub le l i g a t u r e d l a r v a e (•) a re compared . Each p o i n t r e p r e s e n t s the mean i n f l u x i n t o 5 l a r v a e e x c e p t at 0 . 5 h when 4 l a r v a e were u s e d . Bars i n d i c a t e + S . E . M . 38 C O I o C L o X Z3 o CVJ C O 1 6 8 hours 39 F i g u r e 7 Hemolymph ion l e v e l s and o s m o l a r i t y i n A. taen i o rhynchus r e a r e d i n d i s t i l l e d w a t e r , 2 5 , 1 00 , and 200% SW. (CI was measured i n l a r v a e r e a r e d i n a s o l u t i o n c o n t a i n i n g 1 mM KC1 and 5 mM NaCl r a t h e r than d i s t i l l e d w a t e r ) . The number of d e t e r m i n a t i o n s used to c a l c u l a t e the mean o f each p o i n t i s i n d i c a t e d n e x t to the p o i n t . Most samples f o r hemolymph ion d e t e r m i n a t i o n s were p o o l e d f rom 2 to 4 l a r v a e . V a l u e s a re g i v e n f o r normal (•) and p a p i l l a e l e s s l a r v a e (•). Bars r e p r e s e n t + S . E . M . hemolymph osmolarity (mOsm) 41 1 a changes i n hemolymph o s m o l a r i t y and ion c o n c e n t r a t i o n w e r e : + - + o s m o l a r i t y , 14 mOsm; Na , 33 mM; Cl , 16 mM; and K , 2 mM.. P a p i l l a e l e s s l a r v a e c o u l d not be r e a r e d i n d i s t i l l e d w a t e r , i n agreement w i t h p r e v i o u s o b s e r v a t i o n s t h a t the ana l p a p i l l a e are r e s p o n s i b l e f o r i o n uptake f rom d i l u t e s o l u t i o n s ( W i g g l e s w o r t h , 1 9 3 3 ; K o c h , 1 9 3 9 ; Ramsay, 1 9 5 3 ; T reherne , 1 954 ; P h i l l i p s and M e r e d i t h , 1 9 6 9 a ) . P a p i l l a e l e s s l a r v a e r e a r e d i n s a l i n e s o l u t i o n s tended to be s l i g h t l y s m a l l e r i n s i z e and have a s l o w e r r a t e o f deve lopment than normal l a r v a e , but appeared normal i n a l l o t h e r r e s p e c t s . P u p a t i o n and e c l o s i o n were not a f f e c t e d . M o r t a l i t y of p a p i l l a e l e s s l a r v a e i n 25 and 100% SW was l o w , so the f o l l o w i n g data are not the r e s u l t o f s e l e c t i o n of a few g e n e t i c a l 1 y p re -adapxed ; 1 a r v a e . S u r v i v a l o f p a p i l l a e l e s s l a r v a e i n 200% SW was not h i g h ; however i n many groups of l a r v a e , few normal l a r v a e s u r v i v e d so the low s u r v i v a l r a t e can not n e c e s s a r i l y be a t t r i b u t e d to l o s s of p a p i l l a e . P a p i l l a e l e s s l a r v a e r e a r e d i n 100% SW had the same hemolymph o s m o l a r i t y and i o n l e v e l s as normal l a r v a e ( F i g u r e 7 ) . Hemolymph from p a p i l l a e l e s s l a r v a e r e a r e d i n 25% SW was s i g n i f i c a n t l y (P<0.01) l ower i n o s m o l a r i t y (297 + 7 mOsm v e r s u s 325 + 5 mOsm) and h i g h e r i n C l " (75 + 2 mM v e r s u s 56 + 1 mM) than the hemolymph of normal l a r v a e . T h i s i n d i c a t e d some l o s s of o s m o r e g u l a t o r y a b i l i t y . Sodium i o n (125 + 6 and 118 + 2 mM) and K + (11 + 1 and 10 + 1 mM) l e v e l s were not d i f f e r e n t f rom those i n normal l a r v a e . 42 These o b s e r v a t i o n s d i d not s u p p o r t the s u g g e s t i o n t h a t the a n a l p a p i l l a e of l a r v a e l i v i n g i n h y p e r o s m o t i c media e x c r e t e Na + and C l ~ to the e x t e r n a l . s o l u t i o n ( P h i l l i p s and M e r e d i t h , 1 969a;; Nayer and Sauerman, 1 974 ; P h i l l i p s and B r a d l e y , 1 9 7 7 ) . However , the approach used i n my e x p e r i m e n t of t e s t i n g l a r v a e l o n g a f t e r removal o f the p a p i l l a e does not e x c l u d e the p o s s i b i l i t y t h a t normal hemolymph l e v e l s i n p a p i l l a e l e s s l a r v a e r e s u l t e d from compensatory a d j u s t -ment by the e x c r e t o r y s y s t e m . The hemolymph ion c o n c e n t r a t i o n s i n A. c a m p e s t r i s l a r v a e were s i m i l a r to those i n A. t a e n i o r h y n c h u s . L a r v a e l i v i n g i n C t e n o c l a d u s pond w a t e r had hemolymph ion c o n c e n t r a -t i o n s o f 133 + 7 mM N a + (n=6) and 7 + 1 mM K + (n=12) . A f t e r a c c l i m a t i o n to a s o l u t i on- c o n t a i n i ng 1 mM KC1 and 5 mM NaCl f o r 13 d a y s , the hemolymph N a + f e l l to 114 + 2 mM (n=6) and K + c o n c e n t r a t i o n rose to 1 2 + 1 mM (n=6) . B. UPTAKE OF K + BY THE ANAL PAPILLAE 1 . LOCALIZATION OF 4 2 K INFLUX INTO A. TAENIORHYNCHUS LARVAE A l t h o u g h o b s e r v a t i o n s w i t h p a p i l l a e l e s s l a r v a e d i d not i n d i c a t e t h a t N a + or CI were s e c r e t e d by the ana l p a p i l l a e , the u l t r a s t r u c t u r a l morphology of the p a p i l l a e from l a r v a e l i v i n g i n s a l i n e media s u g g e s t e d t h a t some t r a n s -p o r t a c t i v i t i e s o c c u r r e d under, t h e s e c o n d i t i o n s . A second h y p o t h e s i s was t e s t e d ; t h a t i o n s wh ich a re i n s h o r t s u p p l y i n v a r i o u s s a l i n e w a t e r s would be a c t i v e l y taken up by the 43 ana l p a p i l l a e . P o t a s s i u m ion uptake i s d e s c r i b e d in t h i s s t u d y . 42 The i n f l u x of K was measured i n n o r m a l , neck l i g a t u r e d and double l i g a t u r e d A. t a e n i o r h y n c h u s l a r v a e . F i g u r e 8 42 i l l u s t r a t e s the p a t t e r n of K i n f l u x f o r l a r v a e r e a r e d i n d i f f e r e n t s a l i n i t i e s . In l a r v a e r e a r e d i n d i s t i l l e d water 42 n e a r l y a l l K i n f l u x was th rough the a n a l p a p i l l a e . No i n f l u x o c c u r r e d th rough the p a p i l l a e of l a r v a e r e a r e d i n 25% SW e x c e p t a t the 5 h s a m p l i n g t ime when the i n f l u x was s i g n i f i c a n t l y g r e a t e r (P<0.05) than i n f l u x th rough the 42 c u t i c l e a l o n e . Uptake of K d i d not o c c u r a c r o s s the p a p i l l a e of l a r v a e r e a r e d in 100 or 200% SW. T h i s was 42 v e r i f i e d i n a s e p a r a t e e x p e r i m e n t i n which K i n f l u x was measured i n n o r m a l , neck l i g a t u r e d , doub le l i g a t u r e d and p a p i l l a e l e s s l a r v a e r e a r e d in 100% SW ( F i g u r e 9 ) . The i n f l u x i n p a p i l l a e l e s s , as w e l l as head l i g a t u r e d l a r v a e , was n e g l i g i b l e compared to uptake by i n g e s t i o n . 42 The r a t e of K i n f l u x th rough the ana l p a p i l l a e of l a r v a e r e a r e d i n d i s t i l l e d water i s shown i n F i g u r e 1 0 . The l a c k o f uptake ove r the f i r s t hour was o b s e r v e d because i n f l u x a c r o s s the p a p i l l a e was n e g l i g i b l e compared to i n -f l u x a c r o s s the body w a l l . I n f l u x was l i n e a r between 1 and 42 5 h. U n i d i r e c t i o n a l K i n f l u x " , J , was c a l c u l a t e d as d e s c r i b e d by P r u s c h ( 1 9 7 6 ) . T h i s c a l c u l a t i o n was based on 2 a s u r f a c e a rea o f the ana l p a p i l l a e of 0 . 0 0 7 2 7 cm per l a r v a . The a r e a was c a l c u l a t e d f rom the mean d i m e n s i o n s of the 44 F i g u r e 8 42 K i n f l u x i n t o A. t a e n i o r h y n c h u s l a r v a e r e a r e d i n d i s t i l l e d water ( d ) , 25% SW ( c ) , 100% SW (b) and 200% SW ( a ) . A l l l a r v a e were t e s t e d in t h e i r r e s p e c t i v e r e a r i n g s o l u t i o n s e x c e p t a s o l u t i o n of 1 mM KC1 and 5 mM NaCl was s u b s t i t u t e d f o r d i s t i l l e d water to p r o v i d e a known ion c o m p o s i t i o n . Normal (•) , neck l i g a t u r e d (•) and double l i g a t u r e d (A ) l a r v a e a r e compared . P o i n t s r e p r e s e n t the mean of 10 l a r v a e . Bars denote + S . E . M . 46 Figure 9 42 K inf lux into normal (•), neck l igatured (•) double l igatured (A) and pap i l l ae less A.  taen iorhynchus larvae (•) reared in 100% SW. Points are the mean'.of 5 determinations. Bars denote + S.E hours 48 F i g u r e 10 42 K i n f l u x t h r o u g h the a n a l p a p i l l a e o f A.  t a e n i o r h y n c h u s l a r v a e r e a r e d i n d i s t i 1 1 e d ' w a t e r . The r e g r e s s i o n c o e f f i c i e n t of the l i n e c o n n e c t i n g the e x p e r i m e n t a l p o i n t s i s 0 . 9 9 8 8 , so i n f l u x can be c o n -s i d e r e d l i n e a r o v e r t h i s p e r i o d . Each p o i n t i s the mean o f the d i f f e r e n c e o f the mean i n f l u x i n t o 10 normal and 10 doub le l i g a t u r e d l a r v a e . Bars r e p r e s e n t + S . E . M . 49 hours 50 ana l p a p i l l a e o f A. t a e n i o r h y n c h u s l a r v a e r e a r e d i n d i s t i l l e d wate r (NayaV and Sauerman, 1 9 7 4 ) . Each p a p i l l a was c o n s i d e r e d to be a c y l i n d e r w i t h one end open . S i n c e i n f l u x v a l u e s were r e c o r d e d per mg, t h i s a r e a was c o r r e c t e d - 1 2 f o r an average w e i g h t o f 3 mg l a r v a , or 0 . 0 0 2 4 2 cm . 42 The s l o p e of the r e g r e s s i o n l i n e d e s c r i b i n g K uptake in - 1 - 2 - 1 cpm mg v e r s u s t ime was 3 7 2 . 7 5 J was 46 nmol cm h 2 . THE CONCENTRATION OF K + AND VOLUME OF THE RECTAL FLUID OF A. TAENIORHYNCHUS LARVAE 42 S i n c e the a n a l p a p i l l a e d i d not t a k e up K i n s a l i n e m e d i a , i t was n e c e s s a r y to c o n f i r m the l o s s of K + i n the r e c t a l f l u i d r e p o r t e d by B r a d l e y and P h i l l i p s ( 1 9 7 7 b ) . These a u t h o r s found t h a t A. t a e n i o r h y n c h u s l a r v a e l i v i n g i n 100% SW, which c o n t a i n s 10 mM K + , m a i n t a i n e d hemolymph c o n c e n t r a t i o n of 10 mM K + y e t the r e c t a l f l u i d c o n t a i n e d 158 mM K + . I f t h i s 1 oss wene. o v e r - e s t i m a t e d , the f i n d i n g 42 t h a t the p a p i l l a e were, not a c t i v e i n K uptake i n s a l i n e media would be e x p l a i n e d . O t h e r w i s e , a n o t h e r uptake s i t e must e x i s t to compensate f o r t h i s l o s s i f the l a r v a e are to be i n s t e a d y - s t a t e . I t i s p o s s i b l e t h a t the h i g h l e v e l o f K + i n the r e c t a l f l u i d r e p o r t e d by B r a d l e y and P h i l l i p s (1977b) was an a r t i f a c t of t h e i r e x p e r i m e n t a l p r e p a r a t i o n owing to d i s -t e n s i o n o f the r e c t a l w a l l . T h e i r method r e l i e d on s e a l -i n g o f f the rectum and a l l o w i n g f l u i d to a c c u m u l a t e f o r up to 2 h p r i o r to s a m p l i n g . To a v o i d t h i s p rob lem I c o l l e c t e d 51 r e c t a l f l u i d as i t was n o r m a l l y e x c r e t e d f rom the a n u s . The mean K + c o n c e n t r a t i o n and f l u i d s e c r e t i o n r a t e o f the r e c t a l f l u i d were not 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 (P<0.05) between l a r v a e r e a r e d i n d i s t i l l e d w a t e r , 25 or 100% SW ( F i g u r e 1 1 ) . However f l u i d f rom l a r v a e r e a r e d i n 200% SW was s i gn i f i can t l y ='(?< 0.05) Tower in K + c o n c e n t r a t i o n '..than f l u i d from larvae which had'been-reared in 100% .SW. The average volume e x c r e t e d was 41 + 8 n l h~^ i l a r v a " ^ f o r l a r v a e r e a r e d i n d i s t i l l e d wate r and 43 +_ 1 1 , 54 + 1 0 , and 31 +1:1 n l h"^ l a r v a " ^ f o r l a r v a e r e a r e d i n 2 5 , 100 and 200% SW, r e s p e c t i v e l y . K + c o n c e n t r a t i o n s i n the r e c t a l f l u i d were 64 + 15 mM i n l a r v a e i n d i s t i l l e d w a t e r , and 57 + 1 1 , 56 + 8 , and 34 + 51 mM f o r l a r v a e i n 2 5 , 100 and 200% SW, r e s p e c t i v e l y . A l t h o u g h r e c t a l f l u i d c o l l e c t e d f rom l a r v a e e x c r e t i n g n o r m a l l y had lower K + l e v e l s than d i d f l u i d f rom b l o c k e d or l i g a t e d r e c t a (158 mM), the volume o f the s e c r e t i o n was g r e a t e r than t h a t found by B r a d l e y and P h i l l i p s (38 + 8 n l 2 h ~ ^ ) . T h e r e f o r e , the t o t a l K + l o s s i s s i m i l a r i n both s t u d i e s . The q u a n t i t y of K + l o s t in the r e c t a l f l u i d i s much h i g h e r than the K + c o n c e n t r a t i o n i n the e x t e r n a l media (1 to 16 mM) and hemolymph (10 mM). The q u e s t i o n s t i l l remains as to the s i t e a t wh ich the l a r v a e t a k e up K + to m a i n t a i n body l e v e l s . T h i s m a t t e r i s c o n s i d e r e d f u r t h e r i n t h e d i s c u s s i o n . 52 F i g u r e 11 P o t a s s i u m ion c o n c e n t r a t i o n (A) and f l u i d s e c r e t i o n r a t e (•) of the r e c t a l f l u i d e x c r e t e d by A. t a e n i o r h y n c h u s l a r v a e r e a r e d i n d i s t i l l e d w a t e r , 2 5 , 100 and 200% SW. Samples were c o l l e c t e d over an i n t e r v a l o f 1 to 2 . 5 h. The c o n c e n t r a t i o n of K + over t h i s p e r i o d was found to be i n d e p e n d e n t of the t ime of s a m p l i n g . P o i n t s a r e the mean f o r the number of d e t e r m i n a t i o n s i n d i c a t e d n e x t to each p o i n t . Bars denote + S . E . M . f l u i d secretion rate (nl mg"1 h"1) K concentration of secretion (mM) es 54 3 . DRINKING RATE OF LARVAE IN SALINE MEDIA 42 I n f l u x o f K i s r e p r e s e n t a t i v e of the d r i n k i n g r a t e i f a l l i n f l u x o c c u r s th rough i n g e s t i o n , as was the case f o r l a r v a e i n s a l i n e media ( F i g u r e 8 , F i g u r e 2 1 ) . However i n A. t a e n i o r h y n c h u s l a r v a e i n 25% SW and A. c a m p e s t r i s l a r v a e i n C t e n o c l a d u s w a t e r , t h e r e was a s m a l l i n f l u x of 42 K a c r o s s the a n a l p a p i l l a e so the d r i n k i n g r a t e s of t h e s e l a r v a e are a p p r o x i m a t e . The average d r i n k i n g r a t e over 5 h was 3 0 , 27 and 14 nl mg~^ h"^ f o r A. t a e n i o r h y n c h u s l a r v a e r e a r e d and t e s t e d i n 2 5 , 100 and 200% SW ( F i g u r e 1 2 ) . The r a t e ave raged 37 n l mg~^ h~^ i n A. c a m p e s t r i s l a r v a e i n C t e n o c l a d u s w a t e r . 4 . UPTAKE OF K+ BY THE ANAL PAPILLAE OF A . CAMPESTRIS LARVAE The p r e v i o u s e x p e r i m e n t s have shown t h a t K + i s t r a n s -p o r t e d by the a n a l p a p i l l a e of A. t a e n i o r h y n c h u s under FW c o n d i t i o n s but not i n s a l i n e m e d i a . S i n c e l a t e r e x p e r i -ments were c o n d u c t e d on A. c a m p e s t r i s , i t was n e c e s s a r y to c o n f i r m a s i m i l a r r o l e of the p a p i l l a e i n t h i s s p e c i e s . I t was a l s o of i n t e r e s t to see i f l a r v a e which had u n d e r -gone i n i t i a l deve lopment i n s a l i n e w a t e r s c o u l d t u r n on K + uptake by the p a p i l l a e a f t e r a c c l i m a t i o n to a d i l u t e s o l u t i o n . A . t a e n i o r h y n c h u s l a r v a e were not s u i t a b l e f o r t h i s e x p e r i m e n t s i n c e the l a r v a l s t a g e l a s t s o n l y 5 d a y s . 55 F i g u r e 12 The d r i n k i n g r a t e o f l a r v a e i n t h e i r r e s p e c t i v e r e a r i n g media c a l c u l a t e d as the d i f f e r e n c e of the 42 mean i n f l u x of K i n t o 10 normal and 10 doub le l i g a t u r e d l a r v a e in the case of A. t a e n i o r h y n c h u s or 6 l a r v a e o f each t r e a t m e n t f o r A. c a m p e s t r i s . A.  taen i orhyn ch us l a r v a e r e a r e d and t e s t e d i n 200% SW ( o ) , 100% SW (•) and 25% SW ( A ) , and A. c a m p e s t r i s l a r v a e r e a r e d and t e s t e d i n C t e n o c l a d u s water (•) are compared . Bars denote + S . E . M . 56 2 3 4 5 hours 57 S i t e s o f K uptake i n A. c a m p e s t r i s were d e t e r m i n e d 42 as d e s c r i b e d p r e v i o u s l y . Uptake o f K by l a r v a e l i v i n g i n C t e n o c l a d u s wate r was compared to uptake by l a r v a e wh ich had been t r a n s f e r r e d to a s o l u t i o n c o n t a i n i n g 1 mM KC1 and 5 mM NaCl i n the second i n s t a r and a c c l i m a t e d f o r 13 d a y s . La rvae wh ich were r e a r e d i n C t e n o c l a d u s pond wate r 42 e x h i b i t e d a s m a l l but s i g n i f i c a n t K i n f l u x a c r o s s the p a p i l l a e compared to i n f l u x by d r i n k i n g and d i f f u s i o n t h r o u g h the body w a l l ( F i g u r e 1 3 a ) . A. c a m p e s t r i s l a r v a e a c c l i m a t e d 42 ta - ia d i l u t e s o l u t i o n e x h i b i t e d an i n c r e a s e d K uptake by the p a p i l l a e ( F i g u r e 1 3 b ) . At the 3 and 5 h s a m p l i n g t i m e s the i n f l u x a c r o s s the p a p i l l a e a c c o u n t e d f o r most 42 of the t o t a l body u p t a k e . The K i n f l u x th rough the ana l p a p i l l a e o f A . c a m p e s t r i s l a r v a e i n the d i l u t e medium a v e r a g e d 0 . 0 8 3 nmol mg~^ h~^ v e r s u s 0./038 nmol mg~^ h~^ i n l a r v a e i n C l e n o c l a d u s w a t e r . C. THE MECHANISM OF K+ UPTAKE BY THE ANAL PAPILLAE IN F.W. Once i t was known t h a t the a n a l p a p i l l a e were r e s p o n s i b l e 42 f o r most of the K i n f l u x i n A. t a e n i o r h y n c h u s l a r v a e r e a r e d i n d i s t i l l e d w a t e r , i t was of i n t e r e s t to d e t e r m i n e : (1) i f t h i s i n d i c a t e d a net uptake o f K + r a t h e r than exchange d i f f u s i o n , (2) i f uptake .was c o u p l e d to the movement of o ther , , i o n s , and (3) i f any net movement o c c u r r e d a g a i n s t 58 Figure 13 42 Influx of K into A. campestris larvae (a) living and tested in Ctenocladus water and (b) larvae acclimated to a solution containing 1 mM KC1 and 5 mM NaCl for 13 days. Normal (t), neck ligatured (•) and double ligatured (*) larvae are compared. Points are the mean of 6 determinations. Bars represent +_ S.E.M. hours b) 0. 60 an e l e c t r o c h e m i c a l g r a d i e n t , thus i n d i c a t i n g an a c t i v e t r a n s p o r t mechanism. 1 . NET UPTAKE OF K+ BY THE A. TAENIORHYNCHUS LARVAE Net uptake o f K + 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 was d e t e r m i n e d by c h e m i c a l a n a l y s i s of K + c o n t e n t i n A.  t a e n i o r h y n c h u s l a r v a e . L a r v a e were r e a r e d i n d i s t i l l e d wate r and then t r a n s f e r r e d to s o l u t i o n s of v a r i o u s K + or N a + c o n c e n t r a t i o n s o r a t d i f f e r e n t pH l e v e l s to a s s e s s the e f f e c t of N a + and H + c o n c e n t r a t i o n on K + u p t a k e . A f t e r 12 h i n c u b a t i o n i n KC1 s o l u t i o n s the t o t a l K + c o n t e n t of l a r v a e i n c r e a s e d ove r the c o n t e n t of c o n t r o l l a r v a e i n d i s t i l l e d wate r ( F i g u r e 1 4 a ) . S i n c e the hemolymph K + c o n c e n t r a t i o n i s about 10 mM, net uptake against the chemical gradient occurred -. f rom the 0 . 5 , 2 . 5 and 5 mM KC1 s o l u t i o n s . Maximum body K + l e v e l s were a t t a i n e d when the e x t e r n a l K + c o n c e n t r a t i o n was 2 . 5 mM i n one t r i a l and 5 mM i n the o t h e r t r i a l . These two t r i a l s were done at d i f f e r e n t t i m e s u s i n g d i f f e r e n t groups of l a r v a e . Under such c o n d i t i o n s t h i s o l e v e l o f v a r i a b i l i t y i s not unusual ( S t o b b a r t , 1 9 6 7 ) . Net uptake 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 was a l s o 8 6 d e m o n s t r a t e d i n a s e p a r a t e e x p e r i m e n t i n which Rb i n f l u x i n A. t a e n i o r h y o c h u s l a r v a e was measured ove r a 25 h 86 p e r i o d . The Rb a c t i v i t y i n the l a r v a e was more than ten t i m e s g r e a t e r than the a c t i v i t y of the e x t e r n a l s o l u t i o n ( F i g u r e 1 5 ) . Over t h i s p e r i o d the same i s o t o p e l e v e l s were 61 F i g u r e 14a , b , c T o t a l body K c o n t e n t i s measured as a f u n c t i o n of e x t e r n a l K + c o n c e n t r a t i o n ( a ) , N a + c o n c e n t r a t i o n (b) and pH (c ) i n A. t a e n i o r h y n c h u s l a r v a e r e a r e d i n d i s t i l l e d w a t e r . Each p o i n t i s the mean K c o n t e n t of 15 to 20 l a r v a e a f t e r 12 h-n i n c u b a t i o n i n the s p e c i f i e d s o l u t i o n s . Each graph i n c l u d e s the r e s u l t s of two e x p e r i m e n t s (••,•) u s i n g l a r v a e r e a r e d a t d i f f e r e n t t i m e s . 5 6 7 8 9 external r>H 63 F i g u r e 15 86 Rb c o n t e n t of A. t a e n i o r h y n c h u s l a r v a e r e a r e d i n d i s t i l l e d wate r a f t e r t h i s i s o t o p e was added to the e x t e r n a l medium. Normal (•) , neck l i g a t u r e d (•) and d o u b l e d l i g a t u r e d l a r v a e [A) a re compared. E q u i l i b r i u m was reached by 8 h fo r - the f i r s t 2 groups of l a r v a e . P o i n t s r e p r e s e n t the mean f o r 5 l a r v a e . Bars denote 8 6 +_ S . E . M . The d o t t e d l i n e i n d i c a t e d the l e v e l of Rb i n the e x t e r n a l s o l u t i o n . 64 hours 65 m a i n t a i n e d in normal and neck l i g a t u r e d l a r v a e ; w h i l e the much lower a c t i v i t y in doub le l i g a t u r e d l a r v a e i n d i c a t e d t h a t the ana l p a p i l l a e were r e s p o n s i b l e f o r n e a r l y a l l up-t a k e . The t o t a l body K + c o n t e n t was a l s o measured a t a c o n -s t a n t e x t e r n a l K + c o n c e n t r a t i o n of 1 mM and v a r i a b l e N a + c o n c e n t r a t i o n s o f 0 , 0 . 1 , 1 , 10 or 100 mM. A l t h o u g h the a b s o l u t e l e v e l o f t o t a l body K + d i f f e r e d between t r i a l s , as i n the p r e v i o u s e x p e r i m e n t , the p a t t e r n of r e s p o n s e was s i m i l a r in both groups o f l a r v a e ( F i g u r e 1 4 b ) . A peak K + c o n t e n t o c c u r r e d when the e x t e r n a l N a + c o n c e n t r a -t i o n was 1 and 5 mM i n d i f f e r e n t t r i a l s . P o t a s s i u m c o n -t e n t d e c l i n e d on e i t h e r s i d e of t h i s maximum. The d i f f e r -ence between the K + c o n t e n t of c o n t r o l l a r v a e i n c u b a t e d i n d i s t i l l e d w a t e r and t h a t of l a r v a e i n the NaCl s a l t s o l u t i o n s was 5 nmol mg~^ i n both t r i a l s . T h i s was s i m i l a r to the d i f f e r e n c e o f 6 nmol mg ^ o b s e r v e d when the KC1 c o n c e n t r a t i o n was a l t e r e d ( F i g u r e 1 4 a ) . A l t h o u g h the d a t a f o r i n d i v i d u a l e x t e r n a l c o n c e n t r a t i o n s were not s u i t a b l e f o r s t a t i s t i c a l a n a l y s i s , an i n c r e a s e i n t o t a l body K c o n t e n t o f s i m i l a r magni tude was o b s e r v e d i n both N a + and K + s a l t s o l u t i o n s , i n d i c a t i n g the change was not random. The e f f e c t o f the e x t e r n a l pH on K + uptake was a s s e s s e d by i n c u b a t i n g groups of 15 l a r v a e i n a 10 mM sodium p h o s -phate b u f f e r s o l u t i o n c o n t a i n i n g 1 mM KC1 at pH 5 , 6 , 7 , 8 , o r 9 . I f a K + / H + exchange were o c c u r r i n g , a h i g h e r pH 66 would be e x p e c t e d to f a v o r K T u p t a k e . In both t r i a l s the K + c o n t e n t was g r e a t e s t a t pH 7 , and d e c r e a s e d a t h i g h e r and lower pH v a l u e s ( F i g u r e 1 4 c ) . The peak may r e f l e c t the pH optimum o f a c a r r i e r f o r K + . L a r v a e appeared norma1 a t a l l pH l e v e l s . 2 . FACTORS INFLUENCING 4 2 K INFLUX A n o t h e r approach to s t u d y i n g the p r o p e r t i e s of the K + uptake mechanism i n the ana l p a p i l l a e was to o b s e r v e the e f f e c t o f s o l u t i o n s v a r y i n g i n i o n i c c o m p o s i t i o n or C O I I T 42 t a i n m g m e t a b o l i c i n h i b i t o r s on the i n i t i a l l i n e a r K i n f l u x . A c a m p e s t r i s l a r v a e wh ich had been a c c l i m a t e d to a s o l u t i o n c o n t a i n i n g 1 mM KC1 and 5 mM' NaCl f o r 13 days were used i n t h i s e x p e r i m e n t . S i n c e n e a r l y a l l o f the 42 K i n f l u x o c c u r r e d a c r o s s the a n a l p a p i l l a e ( F i g u r e 1 3 ) , u n l i g a t u r e d l a r v a e c o u l d be used i n t h e s e e x p e r i m e n t s . 42 a . The I n f l u e n c e of E x t e r n a l Ions on !< Uptake i n A. c a m p e s t r i s La rvae A. c a m p e s t r i s l a r v a e a c c l i m a t e d to a s o l u t i o n c o n t a i n i n g 1 mM KC1 and 5 mM NaCl were p l a c e d i n v a r i o u s s o l u t i o n s v a r y i n g i n i o n i c c o m p o s i t i o n and 42 l a b e l e d w i t h K f o r 3 h to a s s e s s the e f f e c t o f d i f -42 f e r e n t e x t e r n a l i o n c o n c e n t r a t i o n s on K i n f l u x . I n f l u x ove r t h i s p e r i o d was l i n e a r ( F i g u r e 1 3 ) . I n -42 f l u x of K f rom a s o l u t i o n c o n t a i n i n g 1 mM KC1 and 67 10 mM NaCl was used as a r e f e r e n c e v a l u e . 42 + Uptake of : K was i n d e p e n d e n t o f e x t e r n a l Na c o n c e n t r a t i o n ove r the range of 0 to 100 mM ( F i g u r e 1 6 ) . T h i s i ndi cated] t h a t N a + d id/ not compete f o r the K + uptake mechanism. I n f l u x was s i g n i f i c a n t l y g r e a t e r (P<0.01) a t pH 6 than at o t h e r pH l e v e l s ( F i g u r e 1 7 ) , a l t h o u g h the i n c r e a s e was s m a l l . 4 2 To d e t e r m i n e i f the K i n f l u x was c o u p l e d to movement o f o t h e r i o n s , SO^" or H C 0 3 " were s u b s t i t u t e d f o r C I " and NH^ + f o r N a + . The pH of a l l s o l u t i o n s was between 7 . 0 and 7 . 5 . S u b s t i t u t i o n of S O ^ - had no 42 e f f e c t on the ' K i n f l u x , however HCO^ caused a s i g n i f i c a n t (P^O .05) i n c r e a s e of 13% above the r e f e r e n c e v a l u e ( F i g u r e 1 8 ) . S u b s t i t u t i o n of NH^+ f o r N a + caused a s i g n i f i c a n t (P<0.05) d e c r e a s e of 11% 42 i n the K i n f l u x . 42 The E f f e c t o f I n h i b i t o r s on K Uptake i n A. c a m p e s t r i s L a r v a e . 4? The e f f e c t of i n h i b i t o r s on "K i n f l u x was a l s o d e t e r m i n e d . Ouabain ( s t r o p h a n t h i n - G ) , a s p e c i f i c i n h i b i t o r of N a + - K + A T P a s e , a t a c o n c e n t r a t i o n o f 10 M caused a s i g n i f i c a n t (P<0.05) d e c r e a s e i n i n f l u x ( F i g u r e 1 8 ) . The d e c r e a s e was of the same magni tude as o c c u r r e d when 'NH^* was s u b s t i t u t e d f o r N a + ( F i g u r e 1 8 ) . A c e t a z o l a m i d e ( D i a m o x ) , an i n h i b i t o r 68 F i g u r e 16 42 K i n f l u x as a f u n c t i o n o f the e x t e r n a l NaCl c o n c e n t r a t i o n i n t o A. c a m p e s t r i s l a r v a e a c c l i m a t e d to a s o l u t i o n of 1 mM KC1 and 5 mM NaCl f o r 13 d a y s . A l l s o l u t i o n s a l s o c o n t a i n e d 1 mM KC1. P o i n t s are the mean i n f l u x i n t o 6 groups of 4 l a r v a e e a c h . Bars denote + S . E . M . 69 0-1 1.0 10 e x t e r n a l N a + c o n c e n t r a t i o n (itiM)-70 F i gure 17 42 K i n f l u x a t d i f f e r e n t pH l e v e l s i n t o A.  c a m p e s t r i s l a r v a e a c c l i m a t e d to a s o l u t i o n c o n t a i n i n g 1 mM KC1 and 5 mM NaCl f o r 13 d a y s . Tes t s o l u t i o n s c o n t a i n e d a 10 mM sodium phosphate b u f f e r and 1 mM KC1. P o i n t s are the mean i n f l u x i n t o 6 groups of 4 l a r v a e e a c h . The r e f e r e n c e v a l u e + S . E . M . f o r i n f l u x f rom a s o l u t i o n c o n t a i n i n g 1 mM KC1 and 10 mM NaCl i s i n d i c a t e d by the d o t t e d l i n e s . Bars denote + S . E . M . 71 0.48H external pH i 9 72 F i g u r e 18 The e f f e c t o f i on s u b s t i t u t i o n s and i n h i b i t o r s on 42 K i n f l u x i n t o A. c a m p e s t r i s l a r v a e a c c l i m a t e d to a s o l u t i o n c o n t a i n i n g 1 mM KC1 and 5 mM N a C l . C h l o r i d e -f r e e s o l u t i o n s c o n t a i n e d 1 mM KHCO^ and 5 mM NaHCO^ or 0 . 5 mM K 2 S 0 4 and 2 . 5 m M N a ^ O ^ . The N a — f r e e s o l u t i o n c o n t a i n e d 1 mM KC1 and 5 mM N H ^ C l . A s o l u t i o n c o n t a i n i n g 1 mM KC1 and 5 mM NaCl w i t h e i t h e r Diamox at a c o n -c e n t r a t i o n of 1 0 " 3 M, o u a b a i n a t 1 0 ~ 3 M, or DNP at 1 0 _ 5 M . Each p o i n t r e p r e s e n t s the mean i n f l u x i n t o 6 groups of 4 l a r v a e e a c h . The r e f e r e n c e v a l u e + S . E . M . i s f o r i n f l u x f rom a s o l u t i o n c o n t a i n i n g 1 mM KC1 and 10 mM NaCl and i s i n d i c a t e d by the d o t t e d l i n e s . Bars denote + S . E . M . 0.48H 74 of c a r b o n i c a n h y d r a s e , would be e x p e c t e d to cause a r e d u c t i o n i n the movement o f an ion i f H + o r HCO^ were c o u p l e d w i t h i t s t r a n s p o r t . N e i t h e r Diamox a t a c o n c e n t r a t i o n o f 10 nor 2 , 4 - d i n i t r o p h e n o 1 (DNP) - 5 42 a t 10 M d e c r e a s e d the K i n f l u x . The p o s s i b i l i t y t h a t K + t r a n s p o r t i s a c t i v e i s •' 42 not e x c l u d e d because t h e s e i n h i b i t o r s d i d not a f fec t K i n f l u x . The i n h i b i t o r s may not have r e a c h e d the t r a n s p o r t s i t e due to the i m p e r m e a b i l i t y o f the c u t i c l e , p e r f u s i o n on the "wrong" s i d e of the membrane, or an i n s u f f i c i e n t e x p o s u r e to the i n h i b i t o r . 42 c . The K i n e t i c s of K I n f l u x in A. c a m p e s t r i s L a r v a e The n e x t s t e p i n the c h a r a c t e r i z a t i o n of K + t r a n s p o r t was to d e t e r m i n e the k i n e t i c a s p e c t s o f 42 K i n f l u x . P r e v i o u s e x p e r i m e n t s w i t h A. c a m p e s t r i s l a r v a e have shown t h a t K + uptake by the ana l p a p i l l a e can be t u r n e d on i n FW ( F i g u r e 13) and uptake i s i n -f l u e n c e d by the i o n i c c o m p o s i t i o n o f the e x t e r n a l s o l u t i o n . These d a t a s u g g e s t the p r e s e n c e o f a c a r r i e r - m e d i a t e d K + t r a n s p o r t s y s t e m . I f such a 42 sys tem e x i s t s , then i n i t i a l K i n f l u x might be e x p e c t e d to e x h i b i t e n z y m e - l i k e k i n e t i c s , such as s a t u r a t i o n . The k i n e t i c s of K + t r a n s p o r t i n A. c a m p e s t r i s a c c l i m a t e d to a s o l u t i o n c o n t a i n i n g 1 mM KC1 and 5 mM NaCl l a r v a e were i n v e s t i g a t e d by t r a n s f e r r i n g 75 l a r v a e to s o l u t i o n s o f v a r i o u s K + c o n c e n t r a t i o n s 42 l a b e l e d w i t h K and m e a s u r i n g the i n i t i a l i n f l u x over 42 the f i r s t 3 h. Uptake of K. was l i n e a r d u r i n g t h i s p e r i o d ( F i g u r e 1 3 ) . 42 Data were c o r r e c t e d f o r K i n f l u x by d r i n k i n g and d i f f u s i o n th rough the body w a l l a t d i f f e r e n t 42 e x t e r n a l c o n c e n t r a t i o n s . E s t i m a t e s of uptake of K by d r i n k i n g the e x t e r n a l s o l u t i o n d u r i n g the 3 h t ime c o u r s e of t h i s e x p e r i m e n t were c a l c u l a t e d u s i n g a d r i n k i n g r a t e of 37 n l mg~^ h~^. T h i s was the average d r i n k i n g r a t e f o r A. c a m p e s t r i s o v e r a 5 h p e r i o d ( F i g u r e 1 2 ) . The i n f l u x by d i f f u s i o n a c r o s s the body w a l l was c a l c u l a t e d i n the f o l l o w i n g manner. The 42 K i n f l u x ove r 3 h i n t o d o u b l e l i g a t u r e d A. t a e n i o r h y n -chus l a r v a e was p l o t t e d a g a i n s t the e x t e r n a l K + c o n -c e n t r a t i o n . The r e l a t i o n s h i p was l i n e a r ( r = 0 . 9 2 8 4 ) as p r e d i c t e d f o r d i f f u s i o n ( F i g u r e 1 9 ) . An i n f l u x 42 - 1 of K o v e r a 3 h p e r i o d o f 0 . 0 8 5 nmol mg was measured in doub le l i g a t u r e d A. c a m p e s t r i s l a r v a e i n C t e n o c l a d u s w a t e r . T h i s was c o n s i d e r e d to r e p r e s e n t 4 2 the r a t e of p a s s i v e K i n f l u x a c r o s s the body w a l l o f A. c a m p e s t r i s l a r v a e . T h i s v a l u e f e l l on the r e g r e s s i o n l i n e d e s c r i b i n g the i n f l u x i n t o A.  t a e n i o rhynchus l a r v a e was c a l c u l a t e d based on the s l o p e of t h i s r e g r e s s i o n l i n e . 42 A f t e r c o r r e c t i n g f o r K i n f l u x by d r i n k i n g and 76 F i g u r e 19 42 K i n f l u x ove r a 3 h p e r i o d i n t o doub le l i g a t u r e A. t a e n i o r h y n c h u s l a r v a e (n=10) (A ) as a f u n c t i o n of + 42 the e x t e r n a l K c o n c e n t r a t i o n . I n f l u x o f K i n t o doub le l i g a t u r e d A. c a m p e s t r i s l a r v a e (n=6) i n C t e n o c l a d u s wate r (A) was 0 . 0 8 5 nM mg~^ 3 h ~ ^ . The s l o p e o f the r e g r e s s i o n l i n e ( r = 0 . 9 2 8 4 ) i s 0 . 0 3 . Bars denote + S . E . M . 77 78 d i f f u s i o n t h r o u g h the body w a l l , the r e m a i n i n g i n f l u x e x h i b i t e d s a t u r a t i o n a t h igh e x t e r n a l K + c o n c e n t r a t i o n s ( F i g u r e 2 0 a ) . Uptake of K conforms to M i c h a e l i s -Menten k i n e t i c s ( F i g u r e 2 0 b ) . A S c a t c h a r d p l o t o f the d a t a shown i n F i g u r e 20b y i e l d s a Kg 5 o f , 3 . 2 mM and a V m a x o f 1.1 nmol mg~^ h"^ ( F i g u r e 2 1 ) . 3 . THE POTENTIAL DIFFERENCE ACROSS THE ANAL PAPILLAE W h i l e n e t i n f l u x o f K + a c r o s s the a n a l p a p i l l a e a g a i n s t a c o n c e n t r a t i o n d i f f e r e n c e has been d e m o n s t r a t e d , i n f l u x c o u l d be e x p l a i n e d by a f a v o r a b l e e l e c t r i c a l g r a d i e n t w i t h o u t the need to p o s t u l a t e an a c t i v e t r a n s p o r t mechan ism. T h e r e f o r e , the p o t e n t i a l d i f f e r e n c e ( p . d . ) a c r o s s the ana l p a p i l l a e was measured under a v a r i e t y of c o n d i t i o n s i n o r d e r to d e s c r i b e the p r o p e r t i e s of the K + t r a n s p o r t mechanism. a . The p . d . A c r o s s the Anal P a p i l l a e o f A. t a e n i o r h y n c h u s L a r v a e In the f i r s t s e t of e x p e r i m e n t s . , the p . d . a c r o s s the ana l p a p i l l a e of A. t a e n i o r h y n c h u s l a r v a e r e a r e d i n d i s t i l l e d wate r was r e c o r d e d a t 1 min i n t e r v a l s to d e t e r m i n e the magni tude and s t a b i l i t y of the normal p . d . A f t e r p l a c i n g the e l e c t r o d e i n the hemolymph, the p . d . i m m e d i a t e l y rose to w i t h i n a few mV of the f i n a l v a l u e . T h i s l e v e l was g e n e r a l l y m a i n t a i n e d w i t h i n 5 mV f o r 2 or 3 h o u r s . The p . d . a c r o s s the 79 F i g u r e 20 '4 2 a) K i n f l u x i n t o A. c a m p e s t r i s l a r v a e as a f u n c t i o n of the e x t e r n a l K + c o n c e n t r a t i o n . Po ' in ts (•) r e p r e s e n t the mean i n f l u x i n t o 6 groups of 4 l a r v a e e a c h . The e x p e r i m e n t a l da ta a re c o r r e c t e d f o r d i f -42 f u s i o n of K th rough the g e n e r a l body s u r f a c e a l o n e 42 ( A ) and t h i s c o r r e c t i o n p l u s the amount of :K i n g e s t e d by d r i n k i n g ( A ) . These c o r r e c t e d v a l u e s (A) r e p r e s e n t the i n f l u x th rough the ana l p a p i l l a e a l o n e . Bars denote + S . E . M . 4 2 + b) i n f l u x o f K a t low e x t e r n a l K c o n c e n t r a -t i o n s as shown i n (a) on an expanded s c a l e . 81 F i g u r e 21 42 A S c a t c h a r d p l o t o f the i n i t i a l K i n f l u x c a l c u l a t e d f rom the d a t a shown i n F i g u r e 20b. Each p o i n t r e p r e s e n t s the mean net i n f l u x i n t o 6 groups of 4 l a r v a e e a c h . The c a l c u l a t e d K n c i s 3 .2 mM and V 0 . 5 ma isi".IhmoT'! mg~^ h~^. These v a l u e s were o b t a i n e d f rom t h e . r e g r e s s i o n l i n e ( r = 0 . 9 9 2 6 ) . 82 83 p a p i l l a e of l a r v a e i n the r e f e r e n c e s o l u t i o n (of 1 mM KC1 and 5 mM NaCl ) ave raged .4-36 + 1 mV, hemocoel p o s i t i v e , over a 10 min p e r i o d ( F i g u r e 2 2 ) . The p . d . was s t a b l e f o r i n d i v i d u a l l a r v a e . The p . d . was a l s o measured a c r o s s the p a p i l l a e of l a r v a e r e a r e d i n 100% SW to d e t e r m i n e i f the p . d . i n d i l u t e media r e f l e c t e d the t r a n s p o r t o f i o n s . The p a p i l l a e of these l a r v a e do not t a k e up K + ( F i g u r e 8 b ) . The i n i t i a l p . d . was+6.3 mV and averaged+8 .5 mV over 10 min ( F i g u r e 2 2 ) . The low p . d . a c r o s s the p a p i l l a e of l a r v a e r e a r e d i n 100% SW and the r a p i d change o f the p . d . upon e x p o s u r e of the ana l p a p i l l a e of l a r v a e r e a r e d i n d i s t i l l e d w a t e r to d i f f e r e n t i o n i c c o n -c e n t r a t i o n s i n d i c a t e d t h a t the p . d . a c r o s s the p a p i l l a e was a s s o c i a t e d w i t h ion uptake i n FW. The r e m a i n i n g e x p e r i m e n t s were aimed at d e f i n i n g the b a s i s of the p . d . a c r o s s the p a p i l l a e by r e c o r d i n g * the change i n p . d . i n r e s p o n s e to p e r f u s i o n of the a n a l p a p i l l a e w i t h v a r i o u s s o l u t i o n s . The p . d . was r e l a t i v e l y i n s e n s i t i v e to changes in K + c o n c e n t r a t i on ,.i n the e x t e r n a l s o l u t i o n , i n c r e a s i n g by o n l y 9 mV when e x t e r n a l KC1 c o n c e n t r a t i o n s were i n c r e a s e d f rom 0 to 25 mM ( F i g u r e 2 3 ) . T h i s s u g g e s t s t h a t e i t h e r K + i s not t r a n s p o r t e d by an e l e c t r o g e n i c mechanismoor t h a t t r a n s p o r t i s dominated by the more r a p i d t r a n s p o r t o f some o t h e r i o n , such as N a + . 84 F i g u r e 2 2 The p . d . a c r o s s the ana l p a p i l l a e of .„ t a e n i o rhynchus l a r v a e r e a r e d i n d i s t i l l e d wate r w h i l e bathed i n a s o l u t i o n c o n t a i n i n g 1 mM KC1 and 5 mM NaCl (•); and l a r v a e r e a r e d i n 100% SW w h i l e bathed i n 100% SW ( A ) . Hemolymph s i d e i s p o s i t i v e . Each p o i n t i s the mean p . d . o f a t l e a s t 8 l a r v a e . Bars r e p r e s e n t + S . E . M . p.d. (mV) hemolymph si 86 F i g u r e 2 3 The p . d . a c r o s s the ana l p a p i l l a e of A. t a e n i o r h y n c h u s l a r v a e i n r e s p o n s e to v a r i o u s e x t e r n a l KC1 and I^SO^ c o n -c e n t r a t i o n s . A l l s o l u t i o n s a l s o c o n t a i n e d 5 mM N a C l . The v a l u e o f the r e f e r e n c e p o t e n t i a l ( i e . the p . d . r e -c o r d e d when the p a p i l l a e were p e r f u s e d w i t h a s o l u t i o n c o n t a i n i n g 1 mM KC1 and 5 mM NaCl ) i s i n d i c a t e d by the d o t t e d l i n e . P o i n t s r e p r e s e n t the mean of the number of d e t e r m i n a t i o n s r e c o r d e d in Tab le I. Bars denote +_ S . E . M . 88 I f CI were t r a n s p o r t e d w i t h K' to m a i n t a i n e l e c t r o n e u t r a l i t y , then p e r f u s i o n w i t h C l ~ - f r e e s o l u t i o n s c o n t a i n i n g l e s s permeable a n i o n s s h o u l d i n c r e a s e the change i n p . d . caused by the K + c o n c e n t r a t i o n . F i g u r e 23 and Tab le I show t h a t SO^ ; s u b s t i t u t i o n had no e f f e c t on the magni tude o f the p . d . c h a n g e , however , the o v e r a l l l e v e l o f the p . d . i n c r e a s e d by an average of 8 mV. The a n a l p a p i l l a e were p e r f u s e d w i t h Diamox to d e t e r m i n e i f a r e d u c t i o n i n c a r b o n i c a n h y d r a s s e a c t i v i t y wou ld r e s u l t i n a d e c r e a s e i n K + t r a n s p o r t wh ich would be i n d i c a t i v e o f c o u n t e r t r a n s p o r t o f H + f o r K + . No change i n the p . d . o c c u r r e d a f t e r p e r f u s i o n w i t h the r e f e r e n c e s o l u t i o n c o n t a i n i n g ' - 3 Diamox a t a c o n c e n t r a t i o n o f 10 'M ( T a b l e I):,";. Ouabain was p e r f u s e d to i n v e s t i g a t e the p o s s i b i l i t y + ' + - 3 o f Na /K c o u n t e r - t r a n s p o r t . A 10 M s o l u t i o n o f o u a b a i n had no e f f e c t on the p . d . DNP a t a concentration _ 5 of 10 M a l s o had no e f f e c t . As p r e v i o u s l y m e n t i o n e d , t h i s l a c k o f r e s p o n s e does not n e c e s s a r i l y mean t h a t the t r a n s p o r t mechanism i s i n s e n s i t i v e to the i n -h i b i t o r . F a c t o r s a f f e c t i n g the p . d . a c r o s s the ana l p a p i l l a e were s t u d i e d f u r t h e r to i n v e s t i g a t e the g e n e r a l mechanism of ion t r a n s p o r t . T r a n s p o r t of N a + was the major s o u r c e o f the p . d . a c r o s s the a n a l p a p i l l a e . Removal of e x t e r n a l N a + 89 T a b l e I The p . d . r e c o r d e d a c r o s s the ana l p a p i l l a e of A.  t a e n i o r h y n c h u s and A. c a m p e s t r i s l a r v a e w h i l e the p a p i l l a e were p e r f u s e d w i t h v a r i o u s s o l u t i o n s . The p . d . i s e x p r e s s e d as the average change in the p . d . i n r e s p o n s e to the e x p e r i m e n t a l s o l u t i o n f rom the p . d . r e -c o r d e d i n the r e f e r e n c e s o l u t i o n . The r e f e r e n c e s o l u t i o n was the r e a r i n g or a c c l i m a t i o n media i n a l l c a s e s , e x c e p t a s o l u t i o n c o n t a i n i n g 1 mM KC1 and 5 mM NaCl was used r a t h e r than d i s t i l l e d w a t e r ) . P e r f u s i o n w i t h the r e f e r e n c e s o l u t i o n was b r a c k e t t e d , , between p e r f u s i o n s of the e x p e r i m e n t a l s o l u t i o n s . A l l p . d . v a l u e s are g i v e n as the p o l a r i t y of the hemolymph w i t h r e s p e c t to the p e r f u s e d s o l u t i o n . TABLE I Changes in p.d. Across the Anal Papil lae Species Rearing Condition Perfused Sol uti on A. taeniorhynchus 1 mM KC1 5 mM NaCl Average (n) p.d.(mV) D i s t i l l e d Water Average (n) p.d.(mV) A. campestris Ctenocladus Pond Water Average (n) p.d.(mV) 1 mM KC1 5 mM NaCl Average (n) p.d.(mV) 1 mM KC1-0 mM NaCl -15 + 3 4 - 4 1 + 3 8 + 8 + 4 8 0.1 mM NaCl -15 + 4 3 -37 + 6 8 + 9 + 4 7 1 mM NaCl - 8 + 3 3 -19'+ 3 8 + 6 + 3 7 10 mM NaCl + 3 + 1 3 + 5 + 1 7 0 + 1 7 100 mM NaCl + 6 + 7 3 + 4 + 5 6 +18 + 5 6 0 mM KC1-1 mM NaCl - 7 + 4 3 - 4 + 1 8 -22 + 4 5 - 1 + 0 8 0.05 mM KC1- - 5 + 7 7 - 5 + 0 9 -20 + 4 5 - 1 + 1 8 0.5 mM KC1- - 4 + 1 7 - 1 + 7 . 8 -19 + 4 5 + 1 + 1 9 5 mM KC1- + 3 + 1 6 + 1 + 1 8 -19 + 3 5 + 1 + 1 8 25 mM KC1- + 5 + 2 6 + 5 + 3 8 -12 + 3 4 + 9 + 2 8 u.u^o mn ^2 : > u/j"' 2.5 mM K 2 S0 4 -5 mM K 2 S0 4 -V u 4 + 3 + 2 + 5 + 2 +15 + 2 5 9 5 1 mM KHC03 - 5 mM Na HC03 1 mM K+ Acetate- 5 mM NaC2H302 1 mM KCL - 5 mM NH4C1 1 0 " 3 M Diamox - 1 mM KC1- 5 mM NaCl 10" 3 M Ouabain - 1 mM KC1- 5 mM NaCl 10" 5 M DNP - 1 mM KC1- 5 mM NaCl -17 + 6 -19 + 6 - 2 + 1 - 3 t 1 - 1 + 1 5 5 5 5 4 +20 + 8 + 7 + 5 +11+5 0 + 1 0 0 0 6 5 4 3 3 3 vo o 91 caused a r a p i d drop in the p . d . o f 41 mV, i n some cases the p . d . r e v e r s e d p o l a r i t y ( F i g u r e 2 4 ) . Most of the change in p . d . o c c u r r e d over the range of 0 . 1 to 10 mM N a C l . P e r f u s i o n o f the p a p i l l a e w i t h 100 mM NaCl d i d not cause a s i g n i f i c a n t i n c r e a s e (P<0.01) above the r e f e r e n c e p o t e n t i a l . The p . d . a c r o s s the a n a l p a p i l l a e was a l s o measured i n l a r v a e t h a t had been r e a r e d i n a s o l u t i o n c o n -t a i n i n g 1 mM KC1 and 5 mM NaCl to d e t e r m i n e i f e x -posure to low s a l t c o n c e n t r a t i o n s would a l t e r the f u n c t i o n of the ana l p a p i l l a e . In A. t a e n i o r h y n c h u s l a r v a e , the p . d . was g r e a t l y reduced compared to the p . d . i n l a r v a e i n d i s t i l l e d w a t e r , a v e r a g i n g o n l y 1 1 + 9 mV. The l a r g e s t a n d a r d e r r o r r e s u l t e d f rom the r e v e r s e d p o l a r i t y of the p . d . i n some l a r v a e ( T a b l e I I ) . A 12 mV change i n the p . d . o c c u r r e d i n r e s p o n s e to the e x t e r n a l K + c o n c e n t r a t i o n which was e q u i v a l e n t to t h a t o b s e r v e d in l a r v a e r e a r e d i n d i s t i l l e d w a t e r . However , a much s m a l l e r change of 21 mV o c c u r r e d when the e x t e r n a l N a + c o n c e n t r a t i o n was v a r i e d (Tab! e I ) . b. The p . d . A c r o s s the Ana l P a p i l l a e o f A. c a m p e s t r i s L a r v a e The mechanism of i o n t r a n s p o r t by the a n a l p a p i l l a e o f A. c a m p e s t r i s was i n v e s t i g a t e d by m e a s u r i n g the p . d . a c r o s s the a n a l p a p i l l a e . 92 F i g u r e 24 The p . d . a c r o s s the a n a l p a p i l l a e of A. t a e n i o r h y n c h u s l a r v a e r e a r e d i n d i s t i l l e d wate r as a f u n c t i o n of the e x t e r n a l N a + c o n c e n t r a t i o n . The p o i n t s r e p r e s e n t the mean of the number of d e t e r m i n a t i o n s l i s t e d in T a b l e I. The r e f e r e n c e p . d . i s i n d i c a t e d by the dashed l i n e . B a r s denote + S . E . M . 94 T a b l e II P . d . r e c o r d e d a c r o s s the ana l p a p i l l a e of i n d i v i d u a l A. t a e n o r h y n c h u s and A. c a m p e s t r i s l a r v a e w h i l e the a n a l p a p i l l a e were bathed i n the r e f e r e n c e s o l u t i o n . The r e f e r e n c e s o l u t i o n i s the r e a r i n g o r a c c l i m a t i o n s o l u t i o n e x c e p t a s o l u t i o n c o n t a i n i n g 1 mM KC1 and 5 mM NaCl was used r a t h e r than d i s t i l l e d w a t e r . The r e c o r d i n g e l e c t r o d e was p l a c e d in a drop of hemolymph e x t r u d e d f rom the t h o r a x and the r e f e r e n c e e l e c t r o d e was in the chamber c o n t a i n i n g p a p i l l a e . These v a l u e s are the i n i t i a l s t a b l e , p . d . r e c o r d e d w i t h i n the f i r s t minute a f t e r the l a r v a e was i m p a l e d w i t h the r e c o r d i n g e l e c t r o d e . The p o l a r i t y i n d i c a t e d i s f o r the hemolymph s i d e . TABLE II P . d . A c r o s s the Anal P a p i l l a e S p e c i es R e a r i n g C o n d i t i o n A. t a e n i o rhynchus A. c a m p e s t r i s D i s t i l l e d Water + 40 + 50 + 55 + 20 + 44 + 60 +• 8 + 25 + 35 x SEM + 37 + 6 1 mM KC1 5 mM NaCl + 38 + 12 + 25 + 18 + 10 - 25 - 15 + 11 + 9 100% SW + 3 + 9 + 5 + 4 + 15 + 6 + 10 - 2 + 6 + 2 C t e n o c l a d u s Pond Water - .5 0 - 6 + 10 + 8 - 6 0 + 3 1 mM KC1 5 mM NaCl - 34 - 54 - 43 - 48 - 51 - 31 - 52 - 41 - 46 - 52 - 46 - 45 + 2 96 To d e t e r m i n e i f the p . d . was a r e s u l t of ion t r a n s p o r t , l a r v a e which had been a c c l i m a t e d to a s o l u t i o n c o n t a i n i n g 1 mM KC1 and 5 mM NaCl f o r 13 days were compared to l a r v a e r e a r e d i n C t e n o c l a d u s w a t e r . The average p . d . a c r o s s the a n a l p a p i l l a e o f l a r v a e i n C t e n o c l a d u s water was 0 + 3 mV compared to - 4 5 +_ 2 mV i n l a r v a e a c c l i m a t e d to d i l u t e media . T h i s r e d u c t i o n i n p . d . i n c o n c e n t r a t e d media was a l s o o b s e r v e d i n A. t a e n i o r h y n c h u s l a r v a e ( T a b l e I I ) . P e r f u s i o n e x p e r i m e n t s , as d e s c r i b e d f o r A.  t a e n i o r h y n c h u s l a r v a e , were c a r r i e d out to i n v e s t i g a t e the mechanism of ion t r a n s p o r t . In A. c a m p e s t r i s l a r v a e the r e s p o n s e to changes o f the e x t e r n a l s o l u t i o n was s l o w e r and more e r r a t i c and the r e f e r e n c e p o t e n t i a l was more v a r i a b l e than i n A. t a e n i o r h y n c h u s l a r v a e . T h e r e f o r e , the f o l l o w i n g v a l u e s s h o u l d be c o n s i d e r e d as a s e m i - q u a l i t a t i v e , r a t h e r than q u a n t i -t a t i v e , measure o f the p . d . In A. c a m p e s t r i s l a r v a e a c c l i m a t e d to a s o l u t i o n c o n t a i n i n g 1 mM KC1 and 5 mM NaCl the p . d . i n c r e a s e d . , by about 10 mV between 0 and 25 mM K + or.O ahdcT.00mMJNa (^;Figure •2.:5).. A l l the i n c r e a s e in the p . d . o c c u r r e d at h igh c a t i o n c o n c e n t r a t i o n s . T h i s change was i n the d i r e c t i o n of the d i f f u s i o n p o t e n t i a l . S i n c e o n l y s m a l l changes o f the p . d . r e s u l t e d f rom v a r y i n g the Na+ or K + c o n c e n t r a t i o n s , Cl t r a n s -a 97 F i g u r e 2 5 The change i n p . d . a c r o s s the ana l p a p i l l a e of A.  c a m p e s t r i s i n r e s p o n s e to p e r f u s i o n of the p a p i l l a e w i t h s o l u t i o n s of d i f f e r e n t i o n c o m p o s i t i o n and c o n c e n t r a t i o n . Each p o i n t r e p r e s e n t s the mean of the number of d e t e r -m i n a t i o n s l i s t e d i n T a b l e I. Bars denote + S . E . M . 98 99 p o r t remained as the l i k e l y s o u r c e o f the p . d . Sub -s t i t u t i o n of HCO^ or a c e t a t e caused a s m a l l i n c r e a s e (•of 7 and 11 mV) i n the p . d . ( T a b l e I) wh ich c o u l d be e x p l a i n e d i f the CI t r a n s p o r t mechanism were not s p e c i f i c , but had a somewhat lower a f f i n i t y f o r these i o n s . S u b s t i t u t i o n of SO^ r e s u l t e d in a 43% r e -d u c t i o n i n the magni tude of the p . d . from the r e -f e r e n c e p o t e n t i a l , i n d i c a t i n g t h a t C I " was a major s o u r c e of the p . d . _ 3 When the r e f e r e n c e s o l u t i o n c o n t a i n e d 10 M - 3 - 5 Diamox, 10 M o u a b a i n or 10 M DNP, t h e r e was no e f f e c t on the p . d . ( T a b l e I ) . Changes i n the p . d . in r e s p o n s e to the K + c o n -c e n t r a t i o n were a l s o measured in A. c a m p e s t r i s i n C t e n o c l a d u s wate r ( T a b l e I ) . P e r f u s i o n of the p a p i l l a e w i t h s o l u t i o n s c o n t a i n i n g 0 to 25 mM KC1 and 5 mM NaCl caused a l a r g e r e d u c t i o n i n the p . d . The r e -d u c t i o n p r o b a b l y r e s u l t e d f rom the much l o w e r ion c o n c e n t r a t i o n s i n the p e r f u s e d s o l u t i o n s compared to the r e f e r e n c e s o l u t i o n of C l e n o c l a d u s w a t e r . Over t h i s range of K + c o n c e n t r a t i o n s , the p . d . i n c r e a s e d by 10 mV, a s i m i l a r change to t h a t o b s e r v e d f o r A . c a m p e s t r i s l a r v a e ( T a b l e I) wh ich had been a c c l i m a t e d * to d i 1 u t e me d i a . 100 V D. RESPIRATION RATE OF A. TAENIORHYNCHUS LARVAE IN SALINE MEDIA. A wide range o f o s m o t i c g r a d i e n t s e x i s t s between the hemolymph and e x t e r n a l media i n t h e s e e x p e r i m e n t s and c o r r e s p o n d i n g l y l a r g e d i f f e r e n c e s i n the m e t a b o l i c r e -q u i r e m e n t s of i o n i c and o s m o t i c r e g u l a t i o n . These d i f f e r e n t energy r e q u i r e m e n t s f o r r e g u l a t i o n might be e x p e c t e d to i n f l u e n c e the t o t a l m e t a b o l i c r a t e as i n d i c a t e d by changes i n the r e s p i r a t i o n r a t e . The 0^ consumpt ion of A. t a e n i o r h y n c h u s l a r v a e r e a r e d and t e s t e d i n 2 0 0 , 100 and .25% SW was measured a t 23 °C ( F i g u r e 2 6 ) . There, was no d i f f e r e n c e i n the i n i t i a l r e s p i r a t i o n r a t e o f l a r v a e r e a r e d i n 2 5 , 100 and 200% SW, the r a t e a v e r a g i n g about 0 . 2 AJ 1 0^ mg" 1 h \ The r a t e i n l a r v a e r e a r e d i n 200% SW, a l t h o u g h i n i t i a l l y the same as i n o t h e r l a r v a e , f e l l to s i g n i f i c a n t l y l ower v a l u e s P < 0 . 0 1 ) of about 0 . 1 ^ 1 0 2 m g - 1 h " 1 between 1 . 5 and 4 h ( F i g u r e 2 6 ) . T h i s d e c l i n e may r e s u l t f rom the l o w e r amount of d i s s o l v e d 0 o i n 200% SW. 101 F i g u r e 26 0 2 consumpt ion of 200 ( A ) , 100 (•), and 25% SW (o) r e a r e d A. t a e n i o r h y n c h u s l a r v a e i n t h e i r r e s p e c t i v e rear-^ i n g m e d i a . Each p o i n t i s the mean of 5 groups of 15 l a r v a e e a c h . La rvae were e q u i l i b r a t e d i n the m e t a b o l i c f l a s k s at 2 3 C C f o r 1 hr b e f o r e 0 2 consumpt ion was m e a s u r e d . V a l u e s a re based on a mean weight for l a r v a e of 3 mg. Bars r e p r e s e n t + S . E . M . 103 DISCUSSION The s i m i l a r hemolymph o s m o l a r i t i e s and N a + , K + , and CI c o n c e n t r a t i o n s of normal and p a p i l l a e l e s s A.  t a e n i o r h y n c h u s : l a r v a e r e a r e d i n 100% SW ( F i g u r e 7) i n -d i c a t e t h a t t h e r e was no d i f f e r e n c e i n the i o n i c or o s m o t i c r e g u l a t o r y a b i l i t i e s of t h e s e l a r v a e . T h i s s i m i l a r i t y a l s o d e m o n s t r a t e s t h a t AgNO^ t r e a t m e n t of p a p i l l a e l e s s l a r v a e d i d not damage the c u t i c l e . T h i s 3 c o n c l u s i o n i s s u p p o r t e d by the e q u i v a l e n t H^O f l u x i n t o normal and p a p i l l a e l e s s l a r v a e ( F i g u r e 4 ) . P h i l l i p s and M e r e d i t h (1969a) found i n c r e a s e d C I " l e v e l s i n the hemolymph of p a p i l l a e l e s s A. c a m p e s t r i s l a r v a e i n s a l i n e m e d i a . On t h i s b a s i s , as w e l l as the u l t r a - s t r u c t u r a 1 morphology of the p a p i l l a e , they p r o -posed t h a t the a n a l p a p i l l a e e x c r e t e N a + and CI in s a l i n e m e d i a . I have found t h a t CI l e v e l s i n the hemolymph of p a p i l l a e l e s s A. t a e n i o r h y n c h u s l a r v a e i n 25% SW were h i g h e r than i n normal l a r v a e ( F i g u r e 7 ) . However , the equa l C I " c o n c e n t r a t i o n s o b s e r v e d i n the hemolymph of l a r v a e r e a r e d i n 100% SW does not s u p p o r t the hypothe s i s >.. o f P h i l l i p s and M e r e d i t h . These i n v e s t i g a t i o n s d i d not e x c l u d e the p o s s i b i l i t y t h a t t r e a t m e n t of the l a r v a e w i t h AgNO^ had damaged the c u t i c l e . In t h e i r s tudy the change in C l ~ c o n c e n t r a t i o n c o u l d have been e x p l a i n e d by i n c r e a s e d p a s s i v e e n t r y of C l ~ . T h i s would not have been 104 a f a c t o r i n my s t u d y . However , t h e r e i s a d i f f e r e n c e i n t h e e l a p s e d t i m e between the removal o f the p a p i l l a e and use o f the l a r v a e i n my e x p e r i m e n t and t h a t of P h i l l i p s and M e r e d i t h . In t h e i r e x p e r i m e n t , the p a p i l l a e were removed s h o r t l y b e f o r e e x p e r i m e n t a t i o n , but i n my s t u d y the l a r v a e had m o l t e d 3 t i m e s b e f o r e b e i n g t e s t e d . The ex tended p e r i o d d e c r e a s e s the p r o b a b i 1 i t y of damage to the c u t i c l e , but d u r i n g t h i s t ime c o m p e n s a t i o n by the excretory system for the losscof the anal papi l lae may have ' occurred.-The K + l e v e l in the hemolymph of A. t a e n i o r h y n c h u s was c o n s t a n t i n a l l s a l i n i t i e s , i n agreement w i t h the f i n d i n g s o f B r a d l e y and P h i l l i p s ( 1 9 7 7 b ) . However , t h i s c o n s t a n t l e v e l may not be s o l e l y r e l a t e d to the c a p a c i t y f o r r e g u l a t i o n of K + over a wide range of e x t e r n a l c o n -c e n t r a t i o n s . The hemolymph c o n t a i n s o n l y 6.4% o f the t o t a l body K + ( S t o b b a r t , 1 9 6 7 ) , so changes i n the hemolymph K + l e v e l may be d e t e r m i n e d by an exchange of K + between the t i s s u e s and the hemolymph wh ich might be governed by the n u t r i t i o n a l s t a t e of the l a r v a e . T h i s s u g g e s t i o n i s s u p -p o r t e d by the f o l l o w i n g c o m p a r i s o n s . The K + l e v e l i n the hemolymph o f u n s t a r v e d t h i r d i n s t a r A. c a m p e s t r i s l a r v a e was measured s h o r t l y a f t e r c o l l e c t i o n o f the l a r v a e f rom C t e n o c l a d u s pond and found to be 20 + 1 mM (n=6) . A f t e r the same l a r v a e had been a c c l i m a t e d to a s o l u t i o n c o n t a i n i n g 1 mM KC1 and 5 mM NaCl f o r 13 days and s t a r v e d i n the same 105 medium f o r 1 day , the K + f e l l to 1 2 + 1 mM (n=6). However when t h e s e l a r v a e were s t a r v e d f o r 2 days in a s o l u t i o n c o n t a i n i n g 5 mM NaCl the K + d e c l i n e d f u r t h e r to 5 . 4 + 0 . 5 mM, 27% of the " n o r m a l " l e v e l . T h e r e f o r e , the K + c o n c e n t r a -t i o n of the hemolymph appeared to be dependent on the n u t r i -t i o n a l s t a t e of the l a r v a e , a l t h o u g h the K + c o n c e n t r a t i o n of the e x t e r n a l s o l u t i o n a l s o had an e f f e c t . 42 E x p e r i m e n t s m e a s u r i n g K i n f l u x i n t o normal l a r v a e , l a r v a e wh ich were p r o h i b i t e d f rom d r i n k i n g and l a r v a e which 42 c o u l d n e i t h e r d r i n k ' n o r e x c r e t e d e m o n s t r a t e d t h a t K i n f l u x t h r o u g h the ana l p a p i l l a e , was not s i g n i f i c a n t i n A. t a e n i o r h y n c h u s l a r v a e r e a r e d i n 2 0 0 , 100 or 25% SW ( F i g u r e ' 8 ) . In f a c t i n l a r v a e r e a r e d i n 100% SW t h e r e was 42 no d i f f e r e n c e i n K uptake between p a p i l l a e l e s s and double l i g a t u r e d and neck l i g a t u r e d 1 a r v a e ' ( F i g u r e 9 ) . Under s a l i n e c o n d i t i o n s most uptake of K + o c c u r r e d by i n g e s t i o n . 42 Uptake o f K by the a n a l p a p i l l a e of A. c a m p e s t r i s can be i n d u c e d by a c c l i m a t i o n o f the l a r v a e to d i l u t e s o l u t i o n s ( F i g u r e 1 3 ) . M o r e o v e r , i n t h i s s p e c i e s t h e r e was 42 a s l i g h t , but s i g n i f i c a n t , K uptake by the ana l p a p i l l a e of l a r v a e i n C t e n o c l a d u s w a t e r . C t e n o c l a d u s water has a c o n c e n t r a t i o n of 308 mOsm, but has a r e l a t i v e l y low K + c o n c e n t r a t i o n of 3 . 6 mM. T h e r e f o r e , the a n a l p a p i l l a e are not n e c e s s a r i l y i n a c t i v a t e d by c o n c e n t r a t e d s o l u t i o n s , but may t r a n s p o r t n e c e s s a r y i o n s wh ich a re p r e s e n t i n low c o n c e n t r a t i o n , as s u g g e s t e d by M e r e d i t h and P h i l l i p s ( 1 9 6 9 , 106 1 9 7 3 b ) . C h l o r i d e ion has a l s o been d e m o n s t r a t e d to be taken up by the p a p i l l a e o f A. c a m p e s t r i s l a r v a e f rom NaHCO^ s a l i n e water ( P h i l l i p s and B r a d l e y , 1 977?).' ' . However , the t r a n s p o r t o f both K + and C l ~ i n s a l i n e media was v e r y reduced compared to the r a t e under FW c o n d i t i o n s . In A. . t a e n i orhyn chus l a r v a e r e a r e d i n d i s t i l l e d w a t e r 42 n e a r l y a l l the K i n f l u x was t h r o u g h the ana l p a p i l l a e ( F i g u r e 8). Thus s a l i n e water s p e c i e s of m o s q u i t o l a r v a e r e a r e d i n FW take up K + i n the same manner as FW s p e c i e s , by the a n a l p a p i 1 1 a e . A c c o r d i n g to B r a d l e y and P h i l l i p s (1975) ' t h e r e was a l a r g e l o s s of K + in the r e c t a l f l u i d , o f A. t a e n i o r h y n c h u s i n s a l i n e media which i s not b a l a n c e d by the K + g a i n e d t h r o u g h d r i n k i n g the e x t e r n a l s o l u t i o n . The q u e s t i o n then a r i s e s as to why the ana l p a p i l l a e do not t a k e up K + i n s a l i n e m e d i a . B r a d l e y and P h i l l i p s ' e s t i m a t e o f K + b a l a n c e was based on measurements o f K + l e v e l s i n the u r i n e o b t a i n e d f rom r e c t a whch had been b l o c k e d by l i g a t u r i n g or s e a l i n g the anus w i t h t i s s u e a d h e s i v e . They s u g g e s t e d t h a t t h e i r v a l u e might be i n c o r r e c t due to a l t e r e d i o n r a t i o s i n the u r i n e caused by the h igh h y d r o s t a t i c p r e s s u r e d e v e l o p e d i n the s e a l e d r e c t a by the s e c r e t i o n of f l u i d . I f t h e i r v a l u e s a r e c o r r e c t then a s o u r c e of K + o t h e r than the a n a l p a p i l l a e must e x i s t . T h i s q u e s t i o n was r e i n v e s t i g a t e d by m e a s u r i n g the K + l e v e l s i n the r e c t a l f l u i d of A. t a e n i o r h y n c h u s l a r v a e which 107 had been e x c r e t e d n o r m a l l y ( F i g u r e 1 1 ) . The K c o n t e n t of t h i s u r i n e a v e r a g e d a b o u t 59 mM r a t h e r than 158 mM as found by B r a d l e y and P h i l l i p s . However , the r a t e of s e c r e t i o n was a l m o s t t w i c e as g r e a t , so the magni tude of K + l o s s i n the r e c t a l f l u i d was a l m o s t as h igh as the v a l u e of B r a d l e y and P h i l l i p s . The q u e s t i o n i s answered by c a l c u l a t i n g the o v e r a l l K + b a l a n c e of l a r v a e i n s a l i n e media to d e t e r m i n e whether the l a r v a e are in s t e a d y - s t a t e w i t h r e s p e c t to K w i t h o u t a d d i t i o n a l uptake by the a n a l p a p i l l a e . In 25% SW, A. t a e n i o r h y n c h u s l a r v a e i n g e s t e d about 0.06 nmol K + mg ^ h \ a n o t h e r O.OZnmol K + mg ^ h ^ e n t e r e d by d i f f u s i o n t h r o u g h the g e n e r a l body w a l l ( F i g u r e 8) and 2 . 4 5 . ' nmol K + mg~^ h~^ i s e x c r e t e d in the u r i n e ( F i g u r e 1 1 ) . T h i s r e s u l t s i n a K + d e f i c i t o f i i *"'. 2.3s7 nmol K + mg~^ h~^. S i m i l a r c a l c u l a t i o n s f o r l a r v a e i n 100 and 200% SW y i e l d d e f i c i t s o f 2 . 7 6j a , , - . a n d 0.45 nmol K + mg -"' h~^, r e s p e c t i v e l y . Food i n the gut i s a p o s s i b l e s o u r c e o f K + wh ich c o u l d compensate f o r t h i s l o s s of K + . The l a r v a e used i n t h e s e e x p e r i m e n t s were u n f e d , however the gut remains f u l l o f m a t e r i a l f o r s e v e r a l d a y s . B r e w e r ' s y e a s t , wh ich i s the main food f o r these l a r v a e , has a K + c o n t e n t of 29 nmol K + per mg y e a s t (by c h e m i c a l a n a l y s i s of ashed y e a s t ) . T h e r e f o r e ,1arvae in 25% SW would have to e x t r a c t K f rom 0 . 0 8 mg y e a s t h - 1 to compensate f o r the K + d e f i c i t . La rvae 108 in 100 and 200% SW would r e q u i r e 0 . 1 0 and 0 .02 mg y e a s t h \ r e s p e c t i v e l y . The midgut of A. t a e n i o r h y n c h u s has a volume o f 0 . 3 1 ^ 1 ( M a r t i n , p e r s o n a l c o m m u n i c a t i o n ) , so i f the y e a s t i s assumed to have a h y d r a t e d d e n s i t y of 1.21 g ml \ the midgut can c o n t a i n 0 . 3 7 5 mg of y e a s t . T h i s amount o f y e a s t would compensate f o r the K + l o s s f o r 5 , 4 , and 23 h f o r l a r v a e i n 2 5 , 1 0 0 , and 200% SW, r e s p e c t i v e l y . T h e r e f o r e , under normal c o n d i t i o n s when the l a r v a e are f e e d i n g , the K + l o s s i n s a l i n e media may be compensated by a b s o r p t i o n of K+ f rom food i n the g u t . To c o n f i r m t h a t a l l o f the K + i n f l u x i n t o l a r v a e i n s a l i n e media o c c u r s by i n g e s t i o n , as shown i n F i g u r e 8 , 42 the d r i n k i n g r a t e was c a l c u l a t e d based on K i n f l u x . The 42 i n f l u x of * K s h o u l d be a good measure of the d r i n k i n g r a t e i f a l l uptake o c c u r s by d r i n k i n g . 42 D r i n k i n g r a t e s based on K i n f l u x were 30 and 27 n l mg 1 h - 1 f o r A. t a e n i o r h y n c h u s l a r v a e r e a r e d i n 25 and 100% SW, r e s p e c t i v e l y ( F i g u r e 1 2 ) . B r a d l e y and P h i l l i p s (1975) r e p o r t e d a d r i n k i n g r a t e of 3 3 . 5 n l m g - 1 h ^for- A. t a e n i o rhynchus l a r v a e i n 100% SW based on the i n f l u x of 14 C - i n u l i n . These e s t i m a t e s are i n good a g r e e m e n t , s u g -42 g e s t i n g t h a t K i n f l u x o c c u r s by i n g e s t i o n i n t h e s e m e d i a . The average d r i n k i n g r a t e of l a r v a e i n 200% SW over a 5 h p e r i o d was o n l y 14 n l m g - 1 h~\ T h i s r a t e was a lways s i g n i f i c a n t l y (P<0.01) l o w e r than the d r i n k i n g r a t e i n l a r v a e i n 25% SW and s i g n i f i c a n t l y (P<0.05) l ower than 109 t h a t of l a r v a e in 100% SW e x c e p t a t the end of the 5 h p e r i od ( F i gure 1 2 ) . T h i s method f o r c a l c u l a t i o n of the d r i n k i n g r a t e i s 42 not v a J i d f o r l a r v a e r e a r e d in d i s t i l l e d water s i n c e K uptake a l s o o c c u r r e d v i a the a n a l p a p i l l a e ( F i g u r e 8) and t h i s i s not i n d i c a t i v e of water movement. The n e x t phase of t h i s s tudy was to e l a b o r a t e the mechanisms by wh ich the ana l p a p i l l a e t r a n s p o r t K + under FW c o n d i t i o n s . T r a n s - e p i t h e 1 i a 1 t r a n s p o r t of K + i s w i d e -s p r e a d among i n s e c t s and has been d e m o n s t r a t e d in a v a r i e t y o f s y s t e m s , most n o t a b l y by the M a i p i g h i a n t u b u l e s ( r e -v iewed by M a d d r e T l , 1 9 7 1 ) . P o t a s s i u m uptake in A. t a e n i o r h y n c h u s o c c u r r e d a g a i n s t an e l e c t r o c h e m i c a l g r a d i e n t ( T a b l e I, F i g u r e 1 4 a ) , t h e r e was an optimum pH f o r i t s uptake ( F i g u r e 1 4 c ) , and uptake was i n f l u e n c e d by the e x t e r n a l N a + c o n c e n t r a -t i o n ( F i g u r e 1 4 b ) . These o b s e r v a t i o n s i n d i c a t e t h a t K + uptake i s a c c o m p l i s h e d by. an a c t i v e ' t r a n s p o r t mechanism. T o t a l body K + c o n t e n t was i n f l u e n c e d by the e x t e r n a l N a + c o n c e n t r a t i o n wh ich s u g g e s t s t h a t K + t r a n s p o r t , i s l i n k e d to N a + movement i n A. t a e n i o r h y n c h u s . The n a t u r e of t h i s i n t e r a c t i o n can not be d e f i n e d based on the data o b t a i n e d i n t h i s s t u d y . The e f f e c t c o u l d r e s u l t i n d i r e c t l y f rom changes i n the p e r m e a b i l i t y o f the p a p i l l a e to K + , changes i n the p . d . a c r o s s the p a p i l l a e , or r e s u l t f rom the no p r e s e n c e o f a common c a r r i e r f o r the c a t i o n s . I t i s not p o s s i b l e to d e t e r m i n e i f K + t r a n s p o r t a c r o s s the a n a l p a p i l l a e o f A . t a e n i o r h y n c h u s i s e l e c t r o g e n i c s i n c e the p . d . - r e s u l t e d a l m o s t e n t i r e l y f rom N a + t r a n s p o r t ( F i g u r e 2 4 ) . E l e c t r o g e n i c K + t r a n s p o r t o c c u r s i n the midgut of Hyalophora c e r c r o p i a (Harvey e_t aj_, 1 9 6 8 ) , C a l l i p h o r a e r y t h r o c e p h a 1 a s a l i v a r y g l a n d s (Oschman and B e r r i d g e , 1970) and Sarcophaga b u l l a t a h i n d g u t ( P r u s c h , 1 9 7 6 ) . Measurements o f the p . d . a c r o s s the p a p i l l a e i n d i c a t e t h a t K + t h a t K + uptake wa.r>e i n d e p e n d e n t o f Cl movement s i n c e p e r f u s i o n of the p a p i l l a e w i t h SO^ - b a s e d s o l u t i o n s had no e f f e c t on the magni tude of the change i n the p . d . r e s u l t i n g from v a r y i n g the e x t e r n a l K + c o n c e n t r a t i o n 4 2 ( F i g u r e 2 3 , Tab le I ) . However , K i n f l u x must be measured under t h e s e c o n d i t i o n s to v e r i f y t h i s s u g g e s t i o n . The f a c t o r s a f f e c t i n g the p . d . a c r o s s the ana l p a p i l l a e of A. t a e n i o r h y n c h u s i n d i l u t e media were s t u d i e d f u r t h e r to i n v e s t i g a t e g e n e r a l i on t r a n s p o r t mechan isms . The p a p i l l a e d i d not behave as a N a + e l e c t r o d e s i n c e the N e r n s t s l o p e ( i e . the chance i n p . d . f o r a 10 f o l d change i n the e x t e r n a l c o n c e n t r a t i o n o f the i o n ) was o n l y 21 mV (between 0 .1 and 10 mM N a + ) . Some of the N a + may e n t e r a s s o c i a t e d w i t h Cl s i n c e the p . d . i n c r e a s e d by about 8 mV when SO^ was s u b s t i t u t e d f o r C l ~ ( F i g u r e 2 3 ) . The r e m a i n d e r might be exchanged w i t h H + o r N H ^ + . The l a t t e r would be advantageous s i n c e N H d + i s the major I l l e x c r e t o r y p r o d u c t i n a q u a t i c a r t h r o p o d s ( P r o s s e r , 1 9 7 3 , p . 2 7 9 ) . Exchange of N H ^ + f o r N a + has been d e m o n s t r a t e d i n the FW c r a y f i s h , A s t a c u s , (Shaw, 1960) and i s common among FW t e l e o s t s (Maetz and G a r c i a - R o m e a u , 1 9 6 4 ; E v a n s , 1 9 7 5 ) . Evans n o t e s t h a t N a + / N H ^ + exchange o c c u r s in some t e l e o s t s even i n s a l i n e media s i n c e the e x c r e t o r y f u n c t i o n of the exchange t a k e s p recedence over the d i s -a d v a n t a g e o u s N a + u p t a k e . The p . d . was c o n s t a n t above an e x t e r n a l c o n c e n t r a t i o n of 10 mM N a + , wh ich i s c o n s i s t e n t w i t h s a t u r a t i o n o f a N a + t r a n s p o r t mechanism or d e c r e a s e d p e r m e a b i l i t y of the p a p i l l a e to N a + a t h i g h e x t e r n a l N a + c o n c e n t r a t i o n s . The e q u i l i b r i u m p o t e n t i a l f o r CI "was +60 mV (Table III).. Th is i s equa l to the maximum r e c o r d e d p . d . a c r o s s the p a p i l l a e ( T a b l e I I ) , so C l ~ d i s t r i b u t i o n w a s p r o b a b l y p a s s i v e . When HCO^ or a c e t a t e were s u b s t i t u t e d f o r C l ~ , the p . d . d e c r e a s e d by about 50% from the r e f e r e n c e p o t e n t i a l ( T a b l e I) c o u l d i n d i c a t e e i t h e r the p r e s e n c e of an a c t i v e uptake mechanism f o r these i o n s , or g r e a t e r a p e r m e a b i l i t y compared to C l " . The p . d . a c r o s s the p a p i l l a e of A. t a e n i o r h y n c h u s appeared to be d i r e c t l y c o r r e l a t e d w i t h the amount of c a t i o n t r a n s p o r t s i n c e the p . d . d e c l i n e d by 87% a c r o s s the p a p i l l a e of l a r v a e r e a r e d i n 100% SW compared to the p . d . i n l a r v a e i n d i s t i l l e d water ( F i g u r e 2 2 ) . The p a p i l l a e of l a r v a e 42 r e a r e d i n 100% SW d i d not t r a n s p o r t K ( F i g u r e 8b) and TABLE I I I . The e q u i l i b r i u m p o t e n t i a l s ( E . ) f o r Na , K , and C l " c a l c u l a t e d f rom t h e N e r n s t e q u a t i o n . A l l hemolymph c o n c e n t r a t i o n s ( C ) a re based on a t l e a s t 6 d e t e r m i n a t i o n s e x c e p t f o r the C l ~ l e v e l s i n A. c a m p e s t r i s , in C t e n o c l a d u s wate r where the v a l u e was t a k e n f rom B r a d l e y (1975)^ T h i s was assumed to be the C l " c o n c e n t r a t i o n i n a c c l i m a t e d l a r v a e . C i s the e x t e r n a l K + c o n c e n t r a t i o n , and E the r e c o r d e d p . d . a c r o s s the ana l p a p i l l a e . r S p e c i es A. t a e n i o r h y n c h u s A. c a m p e s t r i s R e a r i ng C o n d i t i o n D i s t i l 1ed Water 1 mM KC1 , 5mM NaCl Ion C. i C E. 0 1 E r Ion C . i C 0 E . i E r (mM) (mM) (mV) (m'V) (mM) (mM) (mV) (mV) Na + 110 5 - 7 8 Na + 114 5 - 7 9 K + 10 1 - 5 8 + 37 K + 12 1 - 6 3 - 4 7 C l " 6'5 6 +60 Cl " 53 6 + 55 R e a r i n g C o n d i t i o n 100% SW C t e n o c l a d u s Water Na + 114 347 + 22 Na + 1 49 225 + 10 K + 10 8 - 6 +9 K + 7 4 - 1 4 0 C l " 68 405 +45 C l " 53 8 . + 48 113 t h e i r s i z e i s g r e a t l y reduced as compared to l a r v a e r e a r e d i n d i s t i l l e d wate r (Nayer and Sauerman, 1 9 7 4 ) . Even minor i n c r e a s e s in the e x t e r n a l s a l t c o n c e n t r a t i o n caused a marked change in the p a p i l l a e . The p . d . a c r o s s the p a p i l l a e of l a r v a e r e a r e d i n a s o l u t i o n c o n t a i n i n g 1 mM KC1 and 5 mM NaCl d e c r e a s e d by 70% compared to the p . d . i n l a r v a e r e a r e d i n d i s t i l l e d water (Tab l e. IT) ; and the r e s p o n s e to the e x t e r n a l N a + c o n c e n t r a t i o n was reduced ( T a b l e I ) . The s i z e o f the p a p i 1 1 a e o f A. t a e n i o r h y n c h u s i s a l s o g r e a t l y reduced i n l a r v a e r e a r e d i n even d i l u t e s a l t c o n c e n t r a t i o n s ( i e . tap w a t e r ; Nayer and Sauerman, 1 9 7 4 ) . T h i s r e d u c t i o n i n s i z e i n ve ry d i l u t e s a l t s o l u t i o n s a l s o o c c u r s i n A. a e g y p t i ( W i g g l e s w o r t h , 1 9 3 3 a ) . The same q u e s t i o n s r e g a r d i n g the mechanism of K + uptake by the ana l p a p i l l a e of A. t a e n i o r h y n c h u s were a l s o c o n s i d e r e d f o r A. c a m p e s t r i s l a r v a e which had been a c c l i -mated to a s o l u t i o n c o n t a i n i n g 1 mM KC1 and 5 mM N a C l . In t h i s s p e c i e s s e v e r a l o b s e r v a t i o n s i n d i c a t e t h a t K + uptake was by a c a r r i e r mechanism. Uptake f o l l o w e d M i c h a e l i s - M e n t e n k i n e t i c s ( F i g u r e 2 1 ) , was i n f l u e n c e d by the i o n i c c o m p o s i t i o n ( F i g u r e 18) and e x h i b i t e d a pH optimum ( F i g u r e 1 7 ) . " A l a r g e e l e c t r i c a l g r a d i e n t (o f - 4 5 mV) f a v o r e d p a s s i v e K + e n t r y so t h a t a c t i v e t r a n s p o r t i s not n e c e s s a r i l y i n d i c a t e d e x c e p t a t low e x t e r n a l K + c o n c e n t r a t i o n s ( i e . below 3 mM K, based on the K + c o n -c e n t r a t i o n of the hemolymph of an u n s t a r v e d l a r v a e and 114 c a l c u l a t e d u s i n g the N e r n s t e q u a t i o n ) . 42 Uptake of K f o l l o w e d M i c h a e 1 i s - M e n t e n k i n e t i c s w i t h a K n , o f 3 .2 mM and V • of 1.1 nmol rng"1 h " 1 ( F i g u r e 2 1 ) . u . o max These v a l u e s a re s i m i l a r to the K n K o f 2 mM and V m = v of 1 .5 u•y max nmol mg 1 h 1 f o r NaCl uptake by A. c a m p e s t r i s l a r v a e a c c l i m a t e d to a s o l u t i o n c o n t a i n i n g 5 mM NaCl ( P h i l l i p s 42 and M e r e d i t h , 1 9 6 9 ) . I n f l u x o f K was i n d e p e n d e n t of the e x t e r n a l N a + c o n c e n t r a t i o n ( F i g u r e 1 6 ) , i n d i c a t i n g l a c k o f c o m p e t i t i o n f o r a common t r a n s p o r t mechanism. In the FW m o s q u i t o , A. a e g y p t i uptake of t h e s e c a t i o n s a l s o o c c u r r e d i n d e p e n d e n t l y ( T r e h e r n e , 1 9 5 4 ) . Uptake was maximal a t p'H 6 , wh ich may i n d i c a t e a pH optimum f o r the K + t r a n s p o r t mechanism. I n f l u x was c o n s t a n t . a t a l l o t h e r pH l e v e l s o v e r the range o f pH 5 -9 ( F i g u r e 1 7 ) . T h i s i n s e n s i t i v i t y to pH i s h i g h l y a d a p t i v e i n l a r v a e which must f a c e ex t remes o f pH due to r a p i d d i l u t i o n by r a i n or FW r u n - o f f ( a c i d i c ) or e v a p o r a t i o n of the a l k a l i n e pond w a t e r . The pH o f the ponds f rom which A. c a m p e s t r i s was c o l l e c t e d i n c r e a s e s i n the summer to above pH 10 ( B l i n n , 1 9 6 9 , K i c i n i u c k and P h i l l i p s , 1 9 7 4 ) . T r a n s p o r t o f K + was not a s s o c i a t e d w i t h Cl move-ment s i n c e s u b s t i t u t i o n of S0^ f o r Cl does not a l t e r the 42 K i n f l u x ( F i g u r e 1 8 ) . However , s u b s t i t u t i o n of HCO^ caused a s i g n i f i c a n t (P<0.05) i n c r e a s e i n i n f l u x . T h i s i n c r e a s e was not a f u n c t i o n of a c h a n g e . i n the e l e c t r i c a l g r a d i e n t p r o m o t i n g K + e n t r y . An i n c r e a s e d i n f l u x would o c c u r i f the ana l p a p i l l a e were more permeable to HCO^ 115 + than CI . A l t e r n a t e l y , W r i g h t (1977) s u g g e s t e d t h a t H C 0 3 ' may s t i m u l a t e K + uptake i n d i r e c t l y t h r o u g h a c t i v a t i o n of N a + - K + ATPase i n f r o g c h o r o i d p l e x u s . However t h e r e i s no e v i d e n c e t h a t N a + ^ K ~ ATPase i s r e s p o n s i b l e f o r i o n t r a n s p o r t a c r o s s the p a p i l l a e . The i n c r e a s e i n i n f l u x caused by HCO^", though s i g n i f i c a n t , was not l a r g e so the i m p o r t a n c e o f HCO^" i n t o t a l K + uptake i s m i n i m a l , i : ;\'; Uptake of K vwa.s s i g n i f i c a n t l y (P<0. 01) reduced when NH^ was s u b s t i t u t e d f o r M a + in the e x t e r n a l s o l u t i o n ( F i g u r e 1 8 ) . The d e c r e a s e c o u l d have r e s u l t e d from e x t e r n a l NH'^  c o m p e t i n g f o r the K + uptake s i t e . Such a c o m p e t i t i o n has been o b s e r v e d f o r the K + b i n d i n g s i t e of N a + - K + ATPase i n the mammalian red b l o o d c e l l ( P o s t e t aj_. , 1 9 6 0 ) . I n t e r e s t -i n g l y , o u a b a i n i n the e x t e r n a l s o l u t i o n caused a d e c r e a s e 42 of the K i n f l u x of the same magni tude as t h a t caused by NH^ + s u b s t i t u t i o n . However i n v iew of the l a c k o f e f f e c t of e x t e r n a l N a + c o n c e n t r a t i o n on the i n f l u x , medat ion + + + of K uptake by a Ma-K ATPase i s u n l i k e l y . 42 A second e x p l a n a t i o n of the d e c r e a s e d K i n f l u x i n the p r e s e n c e of e x t e r n a l NH^ + i s the e x i s t e n c e of a t r a n s -p o r t mechanism which c o u p l e d K + uptake w i t h NH^ + o r NH^ e x c r e t i o n . Such a mechanism would s e r v e the dual f u n c t i o n of uptake o f a n e c e s s a r y i o n and n i t r o g e n e x c r e t i o n . The e f f e c t o f N H 4 + on K + t r a n s p o r t was not a r e s u l t o f changes i n the membrane p . d . , as r e p o r t e d f o r S . b u l l a t a h i n d g u t 116 ( P r u s c h , 1 9 7 6 ) , s i n c e the s u b s t i t u t i o n o f N H ^ + f o r N a + i n the e x t e r n a l s o l u t i o n d i d not a l t e r the p . d . a c r o s s the p a p i l l a e ( T a b l e I ) . I n t e r e s t i n g l y the p . d . a c r o s s the p a p i l l a e of A.  c a m p e s t r i s l a r v a e a c c l i m a t e d to a s o l u t i o n c o n t a i n i n g 1 mM KC1 and 5 mM NaCl was of o p p o s i t e p o l a r i t y and of even g r e a t e r magni tude than the p . d . o f A. t a e n i o r h y n c h u s l a r v a e . The n e g a t i v e p . d . might r e s u l t f rom C l " t r a n s p o r t s i n c e the C l " c o n c e n t r a t i o n i s low i n C t e n o c l a d u s water (8 mM) so uptake would be n e c e s s a r y to m a i n t a i n hemolymph l e v e l s . T r a n s p o r t of C l " by the ana l p a p i l l a e of A.  c a m p e s t r i s l a r v a e f rom both d i l u t e ( P h i l l i p s and M e r e d i t h , 1969) and c o n c e n t r a t e d ( P h i l l i p s and B r a d l e y , 1 9 7 7 ) , ' ; s o l u t i o n s has been d e m o n s t r a t e d . + + The p . d . was not a f f e c t e d by t h e e x t e r n a l Na or K c o n c e n t r a t i o n s e x c e p t a t h i g h c a t i o n c o n c e n t r a t i o n s ( F i g u r e 2 5 ) . The movement of t h e s e i o n s a l o n g t h e i r e l e c t r o c h e m i c a l g r a d i e n t would c r e a t e a l a r g e d i f f u s i o n p o t e n t i a l and reduce the p . d . At low K + c o n c e n t r a t i o n s the p . d . a c r o s s the a n a l p a p i l l a e e q u a l l e d the r e f e r e n c e p . d . ( F i g u r e 2 5 ) . However a t low N a + c o n c e n t r a t i o n s the p . d . was about 7 mV h i g h e r than the r e f e r e n c e p o t e n t i a l . T h i s i n c r e a s e was p r o b a b l y due to the d e c r e a s e d l e v e l of C l ~ i n the e x t e r n a l s o l u t i o n s i n c e the a n i o n c o n c e n t r a t i o n was not h e l d c o n s t a n t . When the ana l p a p i l l a e were p e r f u s e d w i t h 10 mM N a C l , the p . d . d ropped to the r e f e r e n c e 117 p o t e n t i a l , as would o c c u r i f the p . d . were r e s p o n d i n g to changes i n Cl r a t h e r than N a + c o n c e n t r a t i o n i n the more d i l u t e NaCl s o l u t i o n s . T h i s was v e r i f i e d by p e r f u s i o n w i t h a s o l u t i o n c o n t a i n i n g 5 mM NH^Cl r a t h e r than 5 mM N a C l . The r e s u l t i n g p . d . was equa l to the r e f e r e n c e p o t e n t i a l , so the N a + c o n c e n t r a t i o n was not a f f e c t i n g the p . d . The d i f f e r e n c e i n the m e t a b o l i c c o s t of o s m o t i c and i o n i c r e g u l a t i o n i n d i f f e r e n t s a l i n i t i e s may be c o n s i d e r -a b l e . I t i s r e m a r k a b l e t h a t t h e r e was no d i f f e r e n c e i n the ( ^ c o n s u m p t i o n of A. t a e n i o r h y n c h u s l a r v a e i n TOO and 25% SW as w e l l as the i n i t i a l 0^ consumpt ion o f l a r v a e i n 200% SW. The reduced 0^ consumpt ion o f l a r v a e i n 200% SW a f t e r 2 h may r e s u l t f rom the d e c r e a s e d 0^ c o n t e n t of 200% SW. The s o l u b i l i t y o f 0 2 in 200% SW i s about 25% l e s s than the s o l u b i l i t y i n 100% SW ( S m i t h , 1 9 2 8 , p . 2 7 2 ) . G e n e r a l l y 0 2 consumpt ion i n c r e a s e s w i t h s a l i n i t y , as o c c u r r e d i n the c o r i x i d l a r v a , S i g a r a s t a g n a l i s ( C l a u s , 1 9 3 7 ; c i t e d i n F o s t e r and T r e h e r n e , 1 9 7 6 ) . T h i s i n c r e a s e may r e s u l t f rom an i n c r e a s e i n the r a t e o f i o n t r a n s p o r t s i n c e i n g a s t r i c mucosa ( D a v i e s , 1951) and f r o g s k i n ( L e a f and Renshaw, 1 9 5 7 ; Z e r a h n , 1956) the r a t e of 0 2 c o n s u m p t i o n was p r o p o r t i o n a l to the r a t e of a c t i v e t r a n s p o r t . However , Rhodnius p r o l i x u s M a l p h i g a n t u b u l e s ( M a d d r e l l and G a r d i n e r , 118 c i t e d i n F o s t e r and T r e h e r n e , 1 976) and Hyal ophora c e r c r o p i a midgut ( H a r v e y , H a s k e l l and Z e r a h n ; 1967) showed a h i g h r a t e o f 0^ consumpt ion which was i n d e p e n d e n t of the r a t e of ion t r a n s p o r t . 3 Data from H 2 0 i n f l u x e x p e r i m e n t s p e r m i t s a compar i son of the c u t i c u l a r p e r m e a b i l i t y to wate r of A. t a e n i o r h y n c h u s w i t h t h a t o f o t h e r a q u a t i c i n v e r t e b r a t e s . When the data 3 f o r H^O i n f l u x i n t o normal l a r v a e ( F i g u r e 6) a re p l o t t e d as d e s c r i b e d i n the m a t e r i a l s and m e t h o d s , the s l o p e of the r e g r e s s i o n l i n e p a s s i n g th rough the o r i g i n ( r = 0 . 9 9 0 2 ) was 0 . 3 0 , r e s u l t i n g in a T ' v a l u e o f 3 . 3 h . Based on a t o t a l 3 body w a t e r volume of 2 . 5 8 mm and a s u r f a c e a r e a of the c u t i c l e of 22 mm , the P^ was c a l c u l a t e d to be 3 . 6 - 3 - 1 X 10 cm h T h i s v a l u e i s s l i g h t l y l e s s than the P d o f - 3 - 1 4 . 8 X 10 cm h . f o u n d f o r a n o t h e r s a l i n e m o s q u i t o , O p i f e x f u s c u s ( N i c h o l s o n and L e a d e r , 1 9 7 4 ) . Both 0 . f u s c u s and A. t a e n i o r h y n c h u s are l e s s permeable to wate r than FW a q u a t i c i n s e c t s by a f a c t o r o f 10 ( T a b l e I V ) , but 10 t i m e s more permeable than A r t e m i a s a 1 i n a . Dec reased p e r m e a b i l i t y i s a c h a r a c t e r i s t i c common to many o r g a n i s m s i n h a b i t i n g s a l i n e w a t e r s and i s advantageous in l i m i t i n g o s m o t i c wate r l o s s and s a l t e n t r y ( B e a d l e , 1 9 6 9 ; S t o b b a r t and Shaw, 1 9 74) . 119 TABLE IV A c o m p a r i s o n of d i f f u s i o n a l p e r m e a b i l i t y c o e f f i c i e n t s (P .) f o r the d i f f u s i o n of w a t e r a c r o s s the c u t i c l e of of some a q u a t i c i n v e r t e b r a t e s . S p e c i e s (cm P d hr 1 ) Source I l y c o r i s c i m i c o i d e s 1 . 7 X 10" 2 S t a d d o n , 1966 N o t o n e c t a Kjlauca 1 . 4 X 10" 2 S t a d d o n , 1966 C o r i x a d e n t i p e s 2 . 3 X 10" 2 S t a d d o n , 1966 S i a 1 i s 1 u t a r i a 1 . 8 X 10" 2 Shaw, 1955 Op i f e x f u s e us 4 . 8 X 10" 3 N i c h o l s o n & L e a d e r , 1 9 7 4 Aedes t a e n i o r h y c h u s 3 . 6 X 10" 3 P r e s e n t Study A r t e m i a s a l i n a 4 . 1 X 10" 4 S m i t h , 1969 Amoeba 1 .4 X 1 0" 3 c i t e d i n P r o s s e r . , 1 9 7 3 120 42 The c u t i c u l a r p e r m e a b i l i t y to K was a l s o c a l c u l a t e d from t h e data on doub le l i g a t u r e d l a r v a e ( F i g u r e s 8 and 1 3 ) . The t ime c o n s t a n t s f o r exchange ( T ^ v a l u e s ) f o r 4 2 K d i f -f u s i o n were 91 h f o r A. t a e n i o r h y n c h u s l a r v a e r e a r e d i n 25% SW and 114 and 67 h f o r l a r v a e i n 100 and 200% SW ( F i g u r e 2 7 ) . The c o r r e s p o n d i n g P^ v a l u e s are 1 . 3 X 1 0 ~ 4 , 1 .0 X 1 0 " 4 and 1 .8 X 1 0 ~ 4 cm h " 1 f o r the 2 5 , 100 and 200% SW c o n d i t i o n s r e s p e c t i v e l y . S i m i l a r c a l c u l a t i o n s f o r A.  c a m p e s t r i s l a r v a e i n C t e n o c l a d u s wate r r e s u l t s i n a T ' v a l u e of 104 h and a P d of 1 .7 X 1 0 " 4 cm h " 1 , based on a 3 t o t a l body w a t e r volume of 5 . 2 0 mm and s u r f a c e a r e a of the 2 c u t i c l e of 30 inm . P h i l l i p s and M e r e d i t h ( 1 9 6 9 a , b) found t h a t the u l t r a s t r u c t u r e of the ana l p a p i l l a e of A. c a m p e s t r i s l a r v a e l i v i n g i n s a l i n e media was s i m i l a r to t h a t o b s e r v e d i n l a r v a e i n . F W . T h i s s i m i l a r i t y s u g g e s t e d t h a t the p a p i l l a e were a c t i v e i n i o n t r a n s p o r t i n s a l i n e , as w e l l as f r e s h w a t e r . However uptake by the p a p i l l a e of K + ( F i g u r e 8,Figure C l ~ ( P h i l l i p s and B r a d l e y , 1 977; ) 3 : ] and N a + (as i n d i c a t e d by the reduced p . d . a c r o s s the p a p i l l a e of A. t a e n i o r h y n c h u s l a r v a e , T a b l e I I ? P h i l l i p s and M e r e d i t h , 1 969 a,) d i d not o c c u r or was g r e a t l y reduced under s a l i n e c o n d i t i o n s . The p r e s e n t s tudy a l s o p r o v i d e d i n d i r e c t e v i d e n c e t h a t the ana l p a p i l l a e of A. t a e n i o r h y n c h u s were not i n v o l v e d i n N a + o r C l " e x c r e t i o n under s a l i n e c o n d i t i o n s . There are two 121 F i gure 27 42 The d i f f u s i o n a l i n f l u x of K a c r o s s the c u t i c l e of A. t a e n i o r h y n c h u s l a r v a e r e a r e d i n 200% SW (•), 100% SW (A) and 25% SW (•) and A. c a m p e s t r i s l a r v a e i n C t e n o c l a d u s 4 ? w a t e r ( o ) . P o i n t s r e p r e s e n t the mean i n f l u x of : - K i n t o 10 (A. t a e n i o r h y n c h u s ) or 6 (A. c a m p e s t r i s ) doub le l i g a t u r e d l a r v a e . S l o p e s were c a l c u l a t e d from a r e g r e s -s i o n l i n e i n c l u d i n g the o r i g i n . The r e g r e s s i o n c o e f f i c i e n t s a re 0 . 9 9 8 2 , 0 . 9 9 6 2 , 0 . 9 9 9 7 and 0 . 9 9 8 5 per the 2 0 0 , 100 and 25% SW and C t e n o c l a d u s water c o n d i t i o n s r e s p e c t i v e l y . Bars denote + S . E . M . 122 hours 123 p o s s i b i l i t i e s which e x p l a i n the e q u i v a l e n t u l t r a s t r u c t u r e of the p a p i l l a e i n SW and FW c o n d i t i o n s . The f i r s t i s t h a t the e l a b o r a t e u l t r a s t r u c t u r e o f the p a p i l l a e i n s a l i n e c o n d i t i o n s was caused by e x p o s u r e of the l a r v a e a t one t ime d u r i n g t h e i r deve lopment to d i l u t e s o l u t i o n s ( a t l e a s t more d i l u t e than 25% SW) , or c o n c e n t r a t e d s o l u t i o n s which were h y p o t o n i c w i t h r e s p e c t to an e s s e n t i a l i o n . Once d e v e l o p e d , the u l t r a s t r u c t u r e may not a t r o p h y under s a l i n e c o n d i t i o n s . S i n c e the c o n c e n t r a t i o n of the ions i n the n a t i v e h a b i t a t may be h i g h l y v a r i a b l e , i t wou ld be advantageous to r e t a i n the c a p a c i t y to t r a n s p o r t i o n s . A l t e r n a t e l y , an e l a b o r a t e u l t r a s t r u c t u r e may be p r e s e n t under a l l c o n d i t i o n s i f the p a p i l l a e were i n v o l v e d + ++ i n r o l e s not c o n s i d e r e d i n t h i s s t u d y , such as NH^ , Mg , o r S0„ e x c r e t i o n or amino a c i d u p t a k e . 124 SUMMARY Indirect evidence indicated that the anal papillae of A. taeniorhynchus larvae were not active in Na + and Cl excretion in saline media. The anal papillae of A. taeniorhynchus larvae did not take up K+ in saline media. However the p a i l l a e were responsible for nearly a l l K+ uptake in larvae reared in d i s t i l l e d water, as occurs in FW species. Under FW conditions, net K+ uptake occurred against large chemical and e l e c t r i c a l gradients in A.  taeniorhynchus larvae, indicating that influx is active. The unidirectional flux was 46 jimo'l! mg 1 h '. In SW influx of K+ occurred through ingestion. Transport of K+ in A. taeniorhynchus larvae was in-fluenced by the external Na + concentration and hald a pH optimum of 7. Transport of Na + was the major source of the p.d. of +37 mV (hemolymph positive) across the anal papillae. The p.d. was r e l a t i v e l y unaffected by removal of external Cl . The anal papillae of A. campestris larvae take up K+ from a concentrated solution (Ctenocladus water), however uptake was much reduced compared to the rate in larvae acclimated to a dilute (6 mM) solution. Uptake of K+ was not affected by removal of Cl or Na + from the external solution and had a pH optimum 125 of 6 . T r a n s p o r t o f CI was the major source of the p . d . o f - 4 5 mV (hemolymph n e g a t i v e ) a c r o s s the p a p i l l a e . The p . d . was u n a f f e c t e d by removal of e x t e r n a l N a + . 126 BIBLIOGRAPHY B e a d l e , L. C. , 1 9 39,, R e g u l a t i o n o f the hemolymph i n the s a l i n e water m o s q u i t o l a r v a Aedes d e t r i t u s Edw. J . Exp. B i o l . 1 6 : 3 4 6 - 3 6 2 . B e r r i d g e , M . J . , B .D . L i n d l e y and W..T. P r i n c e , 1976. S t u d i e s on the mechanism of f l u i d s e c r e t i o n by i s o l a t e d s a l i v a r y g l a n d s o f Ca11i p h o r a . J . Exp. B i o l . 6 4 : 3 1 1 - 3 2 2 . B e r r i d g e , M . J . and J . L . Oschman, 1972. T r a n s p o r t i n g E p i t h e l i a . Academic P r e s s , New Y o r k . p . 9 1 . B l i n n , D .W. , 1969. A u t o e c o l o g y o f C t e n o c l a d u s (Ch1orophyceae ) i n s a l i n e e n v i r o n m e n t s . P h . D . T h e s i s , U n i v e r s i t y o f B r i t i s h C o l u m b i a . B r a d l e y , T . J . , 1976. The mechanism of h y p e r o s m o t i c u r i n e f o r m a t i o n i n the r e c t a o f s a l i n e - w a t e r m o s q u i t o l a r v a e . P h . D . t h e s i s , U n i v e r s i t y of B r i t i s h C o l u m b i a . B r a d l e y , T . J . , and J . E . P h i l l i p s , 1 9 7 5 . The s e c r e t i o n of h y p e r o s m o t i c f l u i d by the rectum o f a s a l i n e -w a t e r m o s q u i t o l a r v a , Aedes t a e n i o r h y n c h u s . J . Exp . B i o l . 6 3 : 3 3 1 - 3 4 2 . B r a d l e y , T . J . , and J . E . P h i l l i p s , 1977a . R e g u l a t i o n o f r e c t a l s e c r e t i o n i n s a l i n e - w a t e r m o s q u i t o l a r v a e l i v i n g i n w a t e r s of d i v e r s e i o n i c c o m p o s i t i o n . J . Exp . B i o l . 6 6 : 8 3 - 9 6 . B r a d l e y , T . J . , and J . E . P h i l l i p s , 1977b. The e f f e c t o f e x t e r n a l s a l i n i t y on d r i n k i n g r a t e and r e c t a l s e c r e t i o n i n the l a r v a e o f the s a l i n e - w a t e r m o s q u i t o Aedes t a e n i o rhyn ch us . J . Exp . B i o l . 6 6 : 9 7 - 1 1 0 . B r a d l e y , T . J . , and J . E . P h i l l i p s , 1 9 7 7 c . The l o c a t i o n and mechanism of h y p e r o s m o t i c f l u i d s e c r e t i o n i n the rec tum of the s a l i n e - w a t e r m o s q u i t o l a r v a e Aedes t a e n i o r h y n c h u s . J . Exp. B i o l . 6 6 : 1 1 1 - 1 2 6 . B r o w l e e , K . A . , 1 9 6 5 . S t a t i s t i c a l t h e o r y and methodo logy i n s c i e n c e and e n g i n e e r i n g . John W i l e y and Sons I n c . , New Y o r k . C a r p e n t e r , S . J . , and W . J . L a C a s s e , 1955. M o s q u i t o e s of Nor th A m e r i c a . U n i v e r s i t y of C a l i f o r n i a P r e s s , B e r k e l e y , p . 3 6 0 . C o p e l a n d , E . , 1964. A m i t o c h o n d r i a l pump i n the c e l l s of the a n a l p a p i l l a e of m o s q u i t o l a r v a e . J . C e l l B i o l . 2 3 : 2 5 3 - 2 6 3 . 127 C r e d l a n d , P . F . , 1976. A s t r u c t u r a l s t u d y of the ana l p a p i l l a e o f the midge Chironomus r i p a r i u s Meigen ( D i p t e r a : C h i r o n o m i d a e ) . C e l l and T i s s u e Res . 1 6 6 : 5 2 1 - 5 4 0 . D a v i e s , R . E . , 1 9 5 1 . The mechanism of h y d r o c h l o r i c a c i d p r o d u c t i o n by the s t o m a c h . B i o l . Rev. 2 6 : 8 7 - 1 2 0 . E v a n s , D . H . , 1975. I o n i c exchange mechanisms i n f i s h g i l l s . Comp. B i o c h e m . P h y s i o l . 5 1 ( A ) : 4 9 1 - 4 9 5 . F o s t e r , W . A . , and J . E . T r e h e r n e , 1976. I n s e c t s of mar ine s a l t m a r s h e s : p rob lems and a d a p t a t i o n s , p . 5 - 4 2 . J_n L. Cheng ( e d . ) , M a r i n e i n s e c t s . E l s e v i e r Pub. C o . , New Y o r k . H a r v e y , W . R . , and J . A . H a s k e l l and S . N e d e r g a a r d , 1968. A c t i v e t r a n s p o r t by the C e r c r o p i a m i d g u t . I I I . midgut p o t e n t i a l g e n e r a t e d d i r e c t l y by a c t i v e K-t r a n s p o r t . J . Exp . B i o l . 4 8 : 1 - 1 2 . Harvey. , W . R . , J . A . H a s k e l l , and K. Z e r a h n , 1 9 6 7 . A c t i v e t r a n s p o r t o f p o t a s s i u m and oxygen consumpt ion i n the i s o l a t e d midgut of H y a l o p h o r a c e r c r o p i a . J . Exp . B i o l . 4 6 : 2 3 5 - 2 4 8 . H a s s e t t , C C , and D.W. J e n k i n s , 1 9 5 1 . The uptake and e f f e c t o f r a d i o p h o s p h o r u s i n m o s q u i t o e s . P h y s i o l . Z o o l . 2 4 : 2 5 7 - 2 6 6 . Kaufman, W . R . , and J . E . P h i l l i p s , 1 9 7 3 . Ion and wate r b a l a n c e in the I x o d i d t i c k , Dermacentor  a n d e r s o n i . J . Exp . B i o l . 5 8 : 5 4 9 - 5 6 4 . K i c e n i u c k , J . W . , and J . E . P h i l l i p s , 1974. Magnesium r e g u l a t i o n i n m o s q u i t o l a r v a e , Aedes  c a m p e s t r i s , l i v i n g i n w a t e r s of h igh MgS0„ c o n t e n t . J . Exp . B i o l . 6 1 : 7 4 9 - 7 6 0 . 4 K o c h , H . J . , 1938. The a b s o r p t i o n of c h l o r i d e i o n s by the ana l p a p i l l a e of D i p t e r a l a r v a e . J . Exp . B i o l . 1 5 : 1 5 2 - 1 6 0 . L e a f , A . , and A. Renshaw, 1957. Ion t r a n s p o r t and r e s p i r a -t i o n of i s o l a t e d f r o g s k i n . B i o c h e m . J . 6 5 : 8 2 - 9 3 . M a d d r e l l , S . H . P . , 1 9 7 1 . The mechanisms o f i n s e c t e x c r e t o r y s y s t e m s . Adv. I n s e c t P h y s i o l . 8 : 1 9 9 - 3 3 1 . , M a e t i , J . , and E. G a r c i a - R o m e u . , 1964. The mechanism of sodium and c h l o r i d e uptake by the g i l l s o f a f r e s h w a t e r f i s h , C a r a s s i u s a u r a t u s . I I . e v i d e n c e f o r NH A/Na and HCC, and Cl e x c h a n g e . J . G e n . P h y s i o l . 47:1209-1227. J 128 M c G r e g o r , D . D . , 1 9 6 5 . A s p e c t s o f the b i o l o g y o f Op i f ex  f u s c u s Hut ton ( D i p t e r a : C u l i c i d a e ) . P r o c . R. E n t . S o c . Lond . 40 ( A ) : 9 - 1 4 . M e r e d i t h , J . , and J . E . P h i l l i p s , 1973a. U l t r a s t r u c t u r e of the ana l p a p i l l a e of a s a l t - w a t e r mosqu i to l a r v a , Aedes  c a m p e s t r i s . J . I n s e c t P h y s i o l . 1 9 : 1 1 5 7 - 1 1 7 2 M e r e d i t h , J . , and J . E . P h i l l i p s , 1973b. U l t r a s t r u c t u r e of the a n a l p a p i l l a e f rom a s e a w a t e r m o s q u i t o l a r v a (Aedes  t o g o i : T h e o b a l d ) . Canad ian J . Z o o l . 51 ( 3 ) : 3 4 9 - 353 . N a y a r , J . K . , 1967. The p u p a t i o n rhythum i n Aedes t a e n i o r h y n c h u s ( D i p t e r a : C u l i c i d a e ) . Ann E n t . S o c . Am. 5 9 ( 6 ) : 1 2 8 3 - 1 2 8 5 . N a y a r , J . K . , and D.M. Sauerman, 1974. O s m o r e g u l a t i o n i n the l a r v a e o f the s a l t m a r s h m o s q u i t o , Aedes  t a e n i o r h y n c h u s . E n t . Exp. & A p p l . 1 7 : 3 6 7 - 380. N i c h o l s o n , S . W . , and J . P . L e a d e r , 1974. The p e r m e a b i l i t y to w a t e r of the c u t i c l e o f the l a r v a o f Opi fex  f u s c u s ( H u t t o n ) ( D i p t e r a : C u l i c i d a e ) . J . Exp. B i o l . 6 0 : 5 9 3 - 6 0 4 . Oschman, J . L . , and M . J . B e r r i d g e , 1970. S t r u c t u r a l and f u n c t i o n a l a s p e c t s of s a l i v a r y f l u i d s e c r e t i o n i n C a l l i p h o r a . T i s s u e and C e l l 2 ( 2 ) : 2 8 1 - 310. P h i l l i p s , J . E . , and T . J . B r a d l e y , 1977. Osmot ic and i o n i c r e g u l a t i o n i n s a l i n e - w a t e r m o s q u i t o l a r v a e . In. B . L . G u p t a , R . B . M o r e t o n , J . L . Oschman, and B . J . Wal l ( e d . ) T r a n s p o r t o f i o n s and wate r i n an imal t i s s u e s , Academic P r e s s , London . P h i l l i p s , J . E . , T . J . B r a d l e y , and S . H . P . M a d d r e l l . In K. Schmi d t - N e i 1 sen and B o l i s ( e d . ) . Pro;c. I n t e r n a t . Congress P h y s i o l , ( i n p r e s s ) . P h i l l i p s , J . E . , and J . M e r e d i t h , 1 9 6 9 a . A c t i v e sodium and c h l o r i d e t r a n s p o r t by ana l p a p i l l a e of a s a l t -w a t e r mosqu i to l a r v a (Aedes c a m p e s t r i s ) . N a t u r e . 2 2 2 ( 5 1 8 9 ) : 1 6 8 - 1 6 9 . P h i 1 1 i p s , J . E . , and J . M e r e d i t h , 1 9 6 9 b . Osmot ic and i o n i c r e g u l a t i o n i n a s a l t - w a t e r m o s q u i t o l a r v a e Aedes  campes t r i s Amer. Z o o l . 9 : 5 8 8 P o s t , R . L . , C R . M e r r i t , C R . K i n s o l v i n g , and C D . A l b r i g h t , 1960. Membrane a d e n o s i n e t r i p h o s p h a t a s e as a p a r t i c i -pant in the a c t i v e t r a n s p o r t o f sodium and p o t a s s i u m in the human e r y t h r o c y t e . J . B i o l . Chem. 2 3 5 : 1 7 9 6 - 1 8 0 2 . 129 P r o s s e r , C L . ( e d . ) . C o m p a r a t i v e ana ima l p h y s i o l o g y . 3rd Ed . W.B. Saunders C o . , P h i l a d e l p h i a . V o l . I. P r u s c h , R . D . , 1976. U n i d i r e c t i o n a l i o n movements i n the h i n d g u t of l a r v a l Sarcophaga b u l l a t a ( D i p t e r a : S a r c o p h a g i d a e ) J . Exp. B i o l . 6 4 : 8 9 - 1 0 0 . Ramsay, J . A . , 1950 . Osmot ic r e g u l a t i o n i n mosqu i to l a r v a e . J . Exp. B i o l . 2 7 : 1 4 5 - 1 5 7 . Ramsay, J . A . , 1953. Exchanges of sodium and p o t a s s i u m i n m o s q u i t o l a r v a e . J . Exp . B i o l . 3 0 : 7 9 - 8 9 . Shaw, J . , 1 9 5 5 . The p e r m e a b i l i t y and s t r u c t u r e of the c u t i c l e o f the a q u a t i c l a r v a of S i a 1 i s 1 u t a r i a . J . Exp . B i o l . 3 2 : 3 3 0 - 3 5 2 . Shaw, J . , 1960. The a b s o r p t i o n of sodium i o n s by the c r a y f i s h A s t a c u s pa l 1 i p e s L e r e b o u l l e t . I I I . The e f f e c t o f o t h e r c a t i o n s i n the e x t e r n a l s o l u t i o n . J . Exp . B i o l . 3 7 : 5 4 8 - 5 5 6 . S m i t h , D . F . , 1928. The s o l u b i l i t y o f gasses i n s o l u t i o n s . In E.W. Washburn ( e d . ) . I n t e r n a t i o n a l C r i t i c a l T a b l e s of N u m e r i c a l D a t a , P h y s i c s , C h e m i s t r y and Techno1ogy . V o l . 3 . S m i t h , P . G . , 1969. The i o n i c r e l a t i o n s of e l e c t r i c a l p o t e n t i a l d i f f e r e n c e . I I . f l u x e s of s o d i u m , c h l o r i d e and w a t e r . J . Exp . B i o l . 5 1 : 7 3 9 - 7 5 5 . S o h a l , R . S . and E. C o p e l a n d , 1966. U l t r a s t r u c t u r a l v a r i a t i o n s i n the a n a l p a p i l l a e o f Aedes a e g y p t i ( L . ) a t d i f f e r e n t e n v i r o n m e n t a l s a l i n i t i e s . J . I n s e c t P h y s i o l . 1 2 : 4 2 9 - 4 3 9 . S t a d d o n , B . W . , 1966 . The p e r m e a b i l i t y to water of the c u t i c l e s o f some a d u l t w a t e r bugs . J . Exp. B i o l . 4 4 : 6 9 - 7 6 . S t o b b a r t , R . H . , 1959 . S t u d i e s on the exchange and r e g u l a t i o n o f sodium i n the l a r v a o f Aedes a e g y p t i ( L . ) I. s t e a d y - s t a t e e x c h a n g e . J . Exp . B i o l . 3 6 : 6 4 1 - 6 5 3 . S t o b b a r t , R . H . , 1960 . S t u d i e s on the exchange of sodium i n the l a r v a of Aedes a e g y p t i ' ( L . ) II the net t r a n s p o r t and the f l u x e s a s s o c i a t e d w i t h i t . J . Exp . B i o l . 3 7 : 5 9 4 - 6 0 8 . 130 S t o b b a r t , R . H . , 1965. The e f f e c t of some c a t i o n s and a n i o n s upon the f l u x e s and net uptake of sodium i n the l a r v a e of Aedes a e g y p t i ( L . ) . J . Exp. B i o l . 4 2 : 2 9 - 4 3 . S t o b b a r t , R.H., 1967 . The e f f e c t of some a n i o n s and c a t i o n s upon f l u x e s and net uptake of c h l o r i d e i n the l a r v a o f Aedes a e g y p t i ( L .) , and the n a t u r e of the uptake mechanisms f o r sodium and c h l o r i d e . J . Exp . B i o l . 4 7 : 3 5 - 5 7 . S t o b b a r t , R.H., 1 9 7 1 a . E v i d e n c e f o r Na/H and C1/HC0 3 exchanges d u r i n g i n d e p e n d e n t sodium and c h l o r i d e uptake by the l a r v a o f the m o s q u i t o Aedes a e g y p t i ( L . ) . J . Exp. B i o l . 5 4 : 1 9 - 2 7 . S t o b b a r t , R . H . , 1971b. The c o n t r o l o f sodium uptake by the l a r v a of the m o s q u i t o Aedes a e g y p t i ( L . ) . J . Exp . B i o l . 5 4 : 2 9 - 6 6 . S t o b b a r t , R.H., 1 9 7 1 c . F a c t o r s a f f e c t i n g the c o n t r o l o f body volume i n the l a r v a e of the m o s q u i t o e s Aedes a e g y p t i (L . ) . and Aedes d e t r i t u s . Edw. J . Exp . B i o l . 5 4 : 6 7 - 8 2 . S t o b b a r t , R.H., 1974. E l e c t r i c a l p o t e n t i a l d i f f e r e n c e s and i o n i c t r a n s p o r t i n the l a r v a of the mosqu i to A. a e g y p t i (L . ) . J . Exp . B i o l . 6 0 : 4 9 3 - 5 3 3 . S t o b b a r t , R . H . , and J . Shaw, 1974. S a l t and wate r b a l a n c e ; e x c r e t i o n , p . 3 6 2 - 4 4 6 . In M. R o c k s t e i n ( e d . ) . the P h y s i o l o g y of the I n s e c t a . V o l . 5 . Academic P r e s s , New Y o r k . T r e h e r n e , J . E . , 1954. The exchange o f l a b e l e d sodium i n the l a r v a of Aedes a e g y p t i ( L . ) . J . Exp. B i o l . 3 1 : 3 8 6 - 4 0 1 . Wang, Y. ( e d . ) 1969. Handbook of r a d i o a c t i v e n u c l i d e s . Chemica l Rubber C o . , C l e v e l a n d , p . 9 6 0 . Wang, C . H . , and D .L . W i l l i s , 1965 . R a d i o t r a c e r methodo logy i n b i o l o g i c a l s c i e n c e . P r e n t i c e - H a l l I n c . , New J e r s e y , p . 3 8 2 . W i c h a r d , W. , and H. K o m n i c k , 1 9 7 4 . F e i n s t r u k t u r and F u n k t i o n d e r A n a l p a p i 1 1 en a q u a t i s c h e r Schweb-f l i e g e n Larven ( D i p t e r a : S y r p h i d a e ) E n t o m o l . Germ. 1 (1 ) : 1 - 1 0 . 131 W i g g l e s w o r t h , V . B . , 1 9 3 3 a . The f u n c t i o n of the ana l g i l l s o f the m o s q u i t o l a r v a . J . Exp . B i o l . 9 (4) : 1 - 1 5 . W i g g l e s w o r t h , V . B . , 1933b. The a d a p t a t i o n of m o s q u i t o l a r v a e to s a l t w a t e r . J . Exp . B i o l . 9 ( 4 ) : 2 7 - 3 7 . W i g g l e s w o r t h , V . B . , 1 9 3 8 . The r e g u l a t i o n of o s m o t i c p r e s s u r e and c h l o r i d e c o n c e n t r a t i o n i n the hemolymph o f m o s q u i t o l a r v a e . J . Exp . B i o l . 1 5 : 2 3 5 - 2 4 7 . W r i g h t , E .M- . , 1 9 77 . E f f e c t of b i c a r b o n a t e and o t h e r b u f f e r s on choroid / p l e x u s N a + / K + pump. B i o c h e m . e t B i o p h y s . A c t a . 4 6 8 : 4 8 6 - 4 8 9 . Z e r a h n , K . , 1 9 5 6 . Oxygen c o n s u m p t i o n and a c t i v e t r a n s p o r t i n the i s o l a t e d and s h o r t c i r c u i t e d f r o g s k i n . A c t a . P h y s i o l . S c a n d . 3 6 : 3 0 0 - 3 1 8 . 

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