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Acidification of rectal fluid in the locust Schistocerca gregaria Speight, Janet Dorothy Isabella 1967

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ACIDIFICATION OF RECTAL FLUID IN THE LOCUST SCHISTOCERCA  GREGARIA  by  JANET DOROTHY ISABELLA SPEIGHT B.Sc,  Dalhousie. U n i v e r s i t y ,  1965  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the Department o f ZOOLOGY  We accept t h i s t h e s i s as conforming r e q u i r e d standard.  t o the  THE UNIVERSITY OF BRITISH COLUMBIA August, 1967  In p r e s e n t i n g  for  thesis  an a d v a n c e d d e g r e e  that  the  Study.  thesis  Library  for  agree  at  in p a r t i a l  make i t  that  freely  or  representatives.  of  my w r i t t e n  this  thesis  may be g r a n t e d  for  permission.  Department The U n i v e r s i t y o f B r i t i s h V a n c o u v e r 8, Canada  for  Columbia  It  is  financial  of  British  available  permission  purposes  by h.i;s  fulfilment  the U n i v e r s i t y of  scholarly  publication  without  shall  I further  Department  or  this  for  the  Columbia,  I  reference  and  extensive  by  the  requirements  copying  this  Head o f my  understood  gain  of  agree  shall  that  not  be  copying  allowed  Chairman:  P r o f e s s o r John E. P h i l l i p s  i  ABSTRACT A c i d i f i c a t i o n of r e c t a l f l u i d i n Schistocerca i s due probably  gregaria  to a c t i v e s e c r e t i o n o f hydrogen i o n s ,  although bicarbonate  absorption  from H2CO3 l e a v i n g behind  hydrogen ions cannot be excluded.  This was concluded a f t e r  the f o l l o w i n g mechanisms were e l i m i n a t e d :  introduction of a c i d  from some a n t e r i o r r e g i o n of the gut; b a c t e r i a l  fermentation;  slow r e l e a s e o f hydrogen i o n s from f e c a l m a t e r i a l ; p r e f e r e n t i a l absorption  o f the b a s i c form o f the phosphate b u f f e r p a i r ;  s e c r e t i o n o f a c i d phosphate; r e l e a s e of hydrogen i o n s from the intima. Any a b s o r p t i o n o f phosphate i n the rectum must be as the monovalent i o n s i n c e the c u t i c u l a r i n t i m a was found t o l i m i t s e v e r e l y passage o f the d i v a l e n t form, as shown by s t u d i e s o f i n t i m a l p e r m e a b i l i t y a t v a r i o u s pH v a l u e s . The  r e l a t i o n s h i p o f a c i d i f i c a t i o n of r e c t a l contents t o  t o t a l acid-base  r e g u l a t i o n o f the l o c u s t i s not c l e a r , although  there i s some evidence t h a t excess a c i d i t y i s l o s t v i a the excreta.  Regulation  of the hemolymph pH i s r a t h e r slow,  with  a halfway r e t u r n to normal i n about one day when the t o l e r a n c e l e v e l has not been exceeded.  ii TABLE OF CONTENTS Page INTRODUCTION  1  MATERIALS AND METHODS  8  Animals  8  Removal o f gut contents f o r pH determination  8  Preparation  9  o f the l i g a t e d l o c u s t rectum  I n j e c t i o n and removal o f l o c u s t r e c t a l f l u i d  10  S o l u t i o n s used i n i n j e c t i o n experiments  11  D e t e c t i o n o f leakage from the rectum  12  P e r m e a b i l i t y o f the i n t i m a t o i n o r g a n i c phosphate  13  C o l l e c t i o n o f body f l u i d s o f S c h i s t o c e r c a g r e g a r i a  19  Measurement o f i n o r g a n i c phosphate by c o l o r i m e t r i c method  21  pH measurements  23  RESULTS  26  pH o f d i f f e r e n t r e g i o n s o f the gut  26  Demonstration o f a s t a t e o f dynamic e q u i l i b r i u m  27  Role o f phosphate i n a c i d i f i c a t i o n  30  Carbon d i o x i d e t e n s i o n o f a c i d i f i e d r e c t a l f l u i d  37  Changes i n the p r e p a r a t i o n ' s  capacity to a c i d i f y  buffer  39  Amount and r a t e o f a c i d s e c r e t i o n  44  P o s s i b l e r o l e o f the e x c r e t o r y system i n pH r e g u l a t i o n DISCUSSION  45 49  SUMMARY  69  LITERATURE CITED  71  APPENDIX  77  iii LIST OF TABLES TABLE I II III IV  V  VI VII  VIII  IX X  XI XII  FOLLOWING PAGE pH o f gut contents i n normal and t e t r a c y c l i n e - f e d Schistocerca gregaria.  26  Permeability pH v a l u e s .  31  o f the i n t i m a to phosphate a t three  Concentrations o f i n o r g a n i c phosphate found i n the body f l u i d s o f the l o c u s t .  34  Comparison o f amounts o f monovalent and d i v a l e n t i n o r g a n i c phosphate i n M c l l v a i n e ' s b u f f e r and l o c u s t hemolymph.  35  Phosphate s e c r e t i o n experiments. The pH and phosphate c o n c e n t r a t i o n o f r e c t a l f l u i d 20 min. f o l l o w i n g the i n j e c t i o n o f 65 >al. o f 0.002M or 0.004M M c l l v a i n e ' s b u f f e r (pH 7.2-7.4) i n t o l i g a t e d rectum.  36  Change i n pH o f a c i d i f i e d b u f f e r on exposure to a i r f o r 3 hr.  38  The pH o f r e c t a l f l u i d as measured w i t h an anaerobic e l e c t r o d e system 20 min. a f t e r i n j e c t i n g 60 JJLI. o f 0.002M M c l l v a i n e ' s b u f f e r (pH 7.4) i n t o the l i g a t e d rectum.  39  The pH o f 0.002M T r i s - H C l b u f f e r 30 min. a f t e r i n j e c t i o n o f 65 >al. i n t o the l i g a t e d rectum f o r the second or t h i r d r e p l i c a t e experiment on the same locust. 42 A c i d i f i c a t i o n o f 0.002M M c l l v a i n e ' s b u f f e r w i t h time a f t e r i n j e c t i o n o f 50 jal. i n t o l i g a t e d rectum.  43  Comparison o f hemolymph pH u s i n g two types o f g l a s s e l e c t r o d e s , as an estimate o f the e r r o r due to l o s s o f carbonic a c i d .  46  The pH o f hemolymph w i t h time a f t e r i n j e c t i o n o f 100 / i l . o f 0.1N HC1 i n t o unstarved a d u l t male locusts.46 The pH o f hemolymph o f c o n t r o l (uninjected) w i t h time.  locusts 46  XV  TABLE XIII  XIV  FOLLOWING PAGE The pH o f hemolymph w i t h time a f t e r i n j e c t i o n o f 150 ju.1. o f d i s t i l l e d water i n t o a d u l t u n s t a r v e d male l o c u s t s .  46  The pH o f f e c a l p e l l e t s o f animals i n j e c t e d w i t h 1 0 0 j u l . o f O.lN'HCl, f o l l o w e d over 72 h r .  47  V  LIST OF FIGURES FIGURE 1  2 3  FOLLOWING PAGE  Diagram o f apparatus arrangement f o r i n j e c t i o n o f experimental b u f f e r i n t o the l i g a t e d l o c u s t rectum.  10  Regions o f the gut o f S c h i s t o c e r c a ( a f t e r P h i l l i p s , 1964a).  26  gregaria  The change i n pH o f r e c t a l f l u i d w i t h time a f t e r i n j e c t i n g 0.002M M c l l v a i n e ' s b u f f e r w i t h o r i g i n a l pH values 7.2-7.4 and 3.8-3.95.  28  The change i n pH o f r e c t a l f l u i d w i t h time a f t e r i n j e c t i n g 0.002M M c l l v a i n e ' s b u f f e r with o r i g i n a l pH value 7.2-7.4 c o n t a i n i n g 4 mg./l. t e t r a c y c l i n e .  29  The pH o f 50 >ul. a l i q u o t s o f a l k a l i n e 0.002M b u f f e r w i t h added f e c a l m a t e r i a l a g a i n s t time since addition.  30  A c i d i f i c a t i o n o f 0.002M T r i s - H C l b u f f e r , pH 7.2-7.4.  33  7  A c i d i f i c a t i o n o f 0.02M M c l l v a i n e ' s b u f f e r , pH 7.2-7.4.  36  8  T i t r a t i o n o f 10 ml., o f 0.002M M c l l v a i n e ' s b u f f e r w i t h 0.1N HCl.  44  T i t r a t i o n o f 10 ml. o f 0.02M M c l l v a i n e ' s b u f f e r w i t h 0.1N H C l .  44  4  5  6  9  ACKNOWLEDGMENTS  I wish to thank my s u p e r v i s o r ,  Dr. John P h i l l i p s , f o r  h i s p a t i e n t guidance and c o n t i n u a l encouragement study.  during  this  I thank Dr. G.G.E. Scudder f o r h i s c r i t i c i s m o f the  manuscript, and a l s o Drs. A. M. Perks, D.J. R a n d a l l , and P.A. Dehnel f o r p r o f i t a b l e d i s c u s s i o n s .  I also gratefully  acknowledge the a s s i s t a n c e o f Mrs. C a r l a Beaumont w i t h the t r a c e r experiments and w i t h the diagrams.  INTRODUCTION In v e r t e b r a t e s , acid-base  r e g u l a t i o n i s accomplished  mostly by adjustment o f the b i c a r b o n a t e - c a r b o n i c system.  The c o n c e n t r a t i o n  acid buffer  o f carbonic a c i d i s f i x e d by the l i n k  between the r e s p i r a t o r y and c i r c u l a t o r y systems, and v e r t e b r a t e r e n a l mechanisms r e g u l a t e b i c a r b o n a t e Intracorpuscular  concentration.  hemoglobin i s a l s o c e n t r a l to the e f f i c i e n c y  of the system. In c o n t r a s t , there i s very mechanism o f acid-base  l i t t l e known about the  regulation i n insects.  They do not show  the same p h y s i o l o g i c a l a s s o c i a t i o n between r e s p i r a t o r y and c i r c u l a t o r y systems as do mammals.  The i n s e c t s have a t r u e  'open' c i r c u l a t o r y system r a t h e r than a c l o s e d system which i s mediated by i n t e r s t i t i a l f l u i d .  There i s o n l y one e x t r a -  c e l l u l a r f l u i d which bathes the c e l l s and c i r c u l a t e s through the body.  This i s the hemolymph.  Furthermore, the hemolymph o f  i n s e c t s i s not u s u a l l y concerned with oxygen t r a n s p o r t nor w i t h the t r a n s p o r t o f carbon d i o x i d e The  ( F l o r k i n and Jeuniaux, 1964).  t r a c h e a l system o f the i n s e c t spreads among a l l  the i n t e r n a l organs and i s i n i n t i m a t e contact with r e s p i r i n g t i s s u e s which i t s u p p l i e s with gaseous oxygen. l i q u i d phase i s minimized.  Transport  Tracheae end i n very s l e n d e r  i n the tubes  c a l l e d t r a c h e o l e s , and the f i n a l t r a n s p o r t o f oxygen takes p l a c e by d i f f u s i o n through t i s s u e f l u i d s and cytoplasm t o the mitochondria  (Miller,  1964).  Carbon d i o x i d e i s about  thirty-five  times as permeable as oxygen i n animal t i s s u e s , so t h a t i f  2 t r a c h e a l arrangement i s adequate f o r oxygen t r a n s p o r t ,  these  same c o n d i t i o n s should be more than s u f f i c i e n t f o r removal o f carbon d i o x i d e  (Miller,  1964).  There i s no c e l l u l a r element comparable t o the e r y t h r o c y t e i n the hemolymph (Jones,  1964) which c o u l d serve i n  an analogous manner to i n c r e a s e the b u f f e r i n g e f f i c i e n c y o f a bicarbonate-carbonic  a c i d system.  There are no r e s p i r a t o r y  pigments found i n i n s e c t s , except f o r the occurrence o f hemoglobin i n the hemolymph o f some chironomids ( F l o r k i n and Jeuniaux, 1964).  The green c o l o r o f the hemolymph o f the  s o l i t a r y phase o f S c h i s t o c e r c a g r e g a r i a and Locusta  migratoria  i s due t o the presence o f a y e l l o w chromoprotein, the p r o s t h e t i c groups o f which a r e 4 - c a r o t e n e and l u t e i n , and o f a b l u e chromoprotein, the p r o s t h e t i c group o f which seems to be mesobiliverdin  (Goodwin and S r i s u k h ,  absent from the gregarious  1951).  Mesobiliverdin i s  hoppers which have y e l l o w  While there i s no analog  hemolymph.  i n the i n s e c t s o f acid-base  r e g u l a t i o n v i a a l i n k between the r e s p i r a t o r y and c i r c u l a t o r y systems, i t has been suggested by P h i l l i p s  (1961) t h a t the o  rectum o f the d e s e r t l o c u s t , S c h i s t o c e r c a g r e g a r i a F o r s k a l i s f u n c t i o n a l l y analogous to the d i s t a l and c o l l e c t i n g tubes o f the mammalian kidney.  They are both able to absorb NaCl  a g a i n s t l a r g e g r a d i e n t s and can a l s o absorb water so as to produce a hypertonic of H  +  for Na  +  urine.  In the v e r t e b r a t e s ,  the exchange  i s c e n t r a l to the r e g u l a t i o n o f pH; perhaps a  s i m i l a r mechanism i s i n v o l v e d i n t h i s i n s e c t .  Before  considering  3  p r e l i m i n a r y o b s e r v a t i o n s which support of  t h i s suggestion,  a  review  the e x c r e t o r y system i n i n s e c t s and p a r t i c u l a r l y the d e s e r t  locust i s i n order. The e x c r e t o r y system of S c h i s t o c e r c a g r e g a r i a i s p r i m i t i v e and  t y p i c a l anatomically.  I t i n c l u d e s the  Malpighian  t u b u l e s which l i e f r e e i n the hemocoel and open i n t o d i g e s t i v e t r a c t between the midgut and hindgut abdominal segment. 1956;  Phillips,  Malpighian  i n the  third  There are about 2 50 tubules i n a l l (Savage,  1965), and they extend  mesothorax and back to the penultimate are blind-ended  the  and the w a l l s are one  forward  to the  abdominal segment. c e l l layer thick.  tubules s e c r e t e a f l u i d i n t o the gut and,  They The  while  this  f l u i d does not d i f f e r o s m o t i c a l l y from the hemolymph, the r e l a t i v e c o n c e n t r a t i o n of ions i s very d i f f e r e n t Shaw, 1964). times  The  concentration of K  +  (Stobbart  and  i s usually at l e a s t s i x  t h a t of the hemolymph and as much as t h i r t y times i n  some a q u a t i c forms.  The  concentration of Na  +  and C l ~ are lower  than i n the hemolymph, as found i n D i x i p p u s morosus (Ramsay, 1955b, 1956) Malpighian Dixippus  and S c h i s t o c e r c a ( P h i l l i p s ,  1964c).  The pH  of  tubule f l u i d has been shown by Ramsay (19 56) i n  to be higher than the hemolymph pH.value.  Ramsay a l s o  found t h a t when he v a r i e d the pH of the f l u i d b a t h i n g  the  t u b u l e s , the contents were always s l i g h t l y a l k a l i n e , so i t has been concluded  t h a t the M a l p i g h i a n  tubules do not f u n c t i o n i n  pH r e g u l a t i o n i n t h i s i n s e c t a t l e a s t .  B a s i c a l l y the  tubules appear to be s e c r e t o r y ; t h e i r product  Malpighian  i s further  m o d i f i e d by the e p i t h e l i u m of the hindgut and rectum, e s p e c i a l l y  4 the l a t t e r .  Knowledge of the p h y s i o l o g i c a l r o l e of the  i s rather limited seem normally  hindgut  (Stobbart and Shaw, 1964), but i t does not  to be concerned i n i o n i c r e g u l a t i o n .  The rectum o f the i n s e c t f u n c t i o n s p r i n c i p a l l y i n s e l e c t i v e r e a b s o r p t i o n o f ions s e c r e t e d by the M a l p i g h i a n Wigglesworth (1932) suggested t h a t the rectum reabsorbed and  s a l t s , and t h i s was  water  confirmed by the work of Ramsay (1950,  1953a, 1953b) on mosquito l a r v a e , and o f P h i l l i p s 1964b, 1964c, 1965)  tubules.  on the d e s e r t l o c u s t .  untenable the o l d e r hypothesis  (1961, 1964a,  T h e i r work made  t h a t the M a l p i g h i a n  are r e s p o n s i b l e f o r i o n i c r e g u l a t i o n .  tubules  alone  Ramsay (1956) s t a t e d :  'There i s nothing i n the response of the tubules  ... which  might suggest t h a t they are r e s p o n s i b l e f o r the maintenance o f the normal composition  o f the hemolymph; r a t h e r i t i s t h a t the  a c t i v i t y of the tubules alone would r a d i c a l l y a l t e r composition  the  of the hemolymph were i t not f o r the p a r t i c i p a t i o n  o f the other important r e c t a l glands'.  The  organs of the e x c r e t o r y system,  l o c u s t rectum has been shown by  to t r a n s p o r t a c t i v e l y sodium, potassium and  the  Phillips  c h l o r i d e from the  lumen a g a i n s t e l e c t r o c h e m i c a l g r a d i e n t s , independent of s o l v e n t flow.  I t modifies  generated by the M a l p i g h i a n  the primary e x c r e t o r y  tubules and i s r e s p o n s i b l e f o r  r e g u l a t i n g the normal composition The  fluid  o f the hemolymph.  s i x long r e c t a l pads, or r e c t a l glands,  c o n s i s t of  a s i n g l e l a y e r of very l a r g e columnar e p i t h e l i a l c e l l s ,  while  between the pads the e p i t h e l i u m i s c o n s i d e r a b l y reduced w i t h  5 c e l l s cuboidal The  to squamous i n shape ( P h i l l i p s , 1964a, 1965).  whole lumen of the rectum i s l i n e d w i t h a r a t h e r  thick  c h i t i n o u s c u t i c l e or i n t i m a unattached over the pads. b a s i s of c y t o l o g i c a l studies, P h i l l i p s o n l y the l a r g e e p i t h e l i a l to any  extent  On  (1965) concluded t h a t  c e l l s o f the r e c t a l pads are  i n i o n t r a n s p o r t across  involved  the rectum.  To r e t u r n to the analogy suggested by P h i l l i p s he found t h a t the contents of the rectum were a c i d a f t e r p e r s i s t e n t r i n s i n g s with a l k a l i n e b u f f e r . had  the  (1961),  (pH 5-6)  even  Ramsay (1956)  found p r e v i o u s l y i n D i x i p p u s morosus t h a t w h i l e the  urine  i s always a l k a l i n e to the serum, i t becomes a c i d i n the rectum. In Rhodnius the M a l p i g h i a n tubules  have a somewhat d i f f e r e n t  s t r u c t u r e than i n e i t h e r S c h i s t o c e r c a or Dixippus, two  f u n c t i o n a l l y d i s t i n c t regions,  the tubule portion  there  being  i . e . the d i s t a l p o r t i o n of  i s s e c r e t o r y and r e s o r p t i o n occurs i n the proximal  (Wigglesworth, 1931b).  I t was  shown t h a t the  o f the s e c r e t o r y p o r t i o n are weakly a l k a l i n e and  then become  d i s t i n c t l y a c i d i n the lower p o r t i o n of the t u b u l e . acidification,  according  contents  The  to the theory of Wigglesworth, i s  important i n n i t r o g e n e x c r e t i o n causing  p r e c i p i t a t i o n of  a c i d as u r a t i c spheres which are then e l i m i n a t e d i n the The  contents from a l l r e g i o n s  midgut f l u i d  i s 0.7  i s lower than  pH u n i t s lower, hindgut 1.4  does not appear to be  due  and this:  and r e c t a l  pH u n i t s lower than the hemolymph ( P h i l l i p s , 1961). acidification  feces.  pH of S c h i s t o c e r c a hemolymph i s about 7.13,  the pH of gut  uric  2.4  The  to the i n t r o d u c t i o n o f  6 a c i d i c m a t e r i a l from the M a l p i g h i a n Two  tubules  (Ramsay, 1956).  main p o s s i b i l i t i e s were suggested a t t h i s time by  P h i l l i p s to account f o r a c i d i f i c a t i o n o f the r e c t a l f l u i d : The  (1)  g r a d i e n t i n pH a r i s e s because the r e c t a l w a l l i s impermeable  to hydrogen i o n s , which enter  the rectum from the hindgut or  a r i s e '. from b a c t e r i a l fermentation.  (2) The e p i t h e l i u m  a c t i v e l y maintains the pH g r a d i e n t . The  experiments done by P h i l l i p s  (1961) i n v e s t i g a t i n g  the a c i d i f i c a t i o n o f r e c t a l f l u i d have been repeated  here, s i n c e  they were p r e l i m i n a r y i n nature and s i n c e i t was thought t h a t the process  might be a s s o c i a t e d w i t h acid-base  regulation. His  c o n c l u s i o n was t h a t the e p i t h e l i u m a c t i v e l y maintains the gradient.  Three mechanisms were suggested t o account f o r t h i s :  (1) The c e l l s o f the r e c t a l w a l l may themselves have a low pH due  t o the presence o f H2CO3 d e r i v e d from metabolic  activity.  The r e c t a l lumen c o u l d be i n e q u i l i b r i u m w i t h the low pH o f these c e l l s , and the g r a d i e n t would be i n s t e a d between the r e c t a l e p i t h e l i u m and the hemolymph.  (2) The b a s i c form o f a  b u f f e r p a i r i s taken up by the r e c t a l c e l l s e.g. than H2PO4 , o r HCO3  -  r a t h e r than H2CO3.  HPO4  rather  I t i s also possible  t h a t carbon d i o x i d e c o u l d d i f f u s e i n t o the lumen from the epithelium,  hydrate and d i s s o c i a t e .  p r e f e r e n t i a l l y absorbed to leave H  +  Then HC03~ c o u l d be behind.  This l a s t scheme  might r e q u i r e the presence o f c a r b o n i c anhydrase i n the  rectal  lumen s i n c e h y d r a t i o n o f carbon d i o x i d e i s slow when n o t enzymatically  catalyzed.  Or, i t c o u l d be a slow r e a c t i o n , as  7 i n the system d e s c r i b e d by P i t t s proximal  (1963) i n the lumen o f  tubules of the v e r t e b r a t e kidney.  (3)  a c t i v e s e c r e t i o n o f hydrogen i o n s by the r e c t a l  the  There i s epithelium,  e i t h e r as f r e e ions or as an a c i d which d i s s o c i a t e s . The purposes of t h i s study have been to t e s t the above hypotheses as to the source and mechanism o f a c i d i f i c a t i o n , and  to determine whether the a c i d i f i c a t i o n process  p l a y s some  r o l e i n pH r e g u l a t i o n o f the l o c u s t by f o l l o w i n g the pH e x c r e t a i n a c i d loaded  animals.  of  8 MATERIALS AND METHODS Animals o  The colony o f S c h i s t o c e r c a g r e g a r i a F o r s k a l was r e a r e d i n a c o n t r o l l e d environment  o f 2 8 ° C and 50% r e l a t i v e  under continuous l i g h t , and f e d on a d i e t o f bran, l e t t u c e , grass and tap water.  humidity  fresh  Immature a d u l t male animals  thatwere t h r e e t o four weeks p a s t the f i n a l moult and s t i l l a p i n k i s h c o l o r were taken from the colony as r e q u i r e d .  Animals  used f o r a g i v e n experiment were taken from the same egg b a t c h reared, i n a s i n g l e cage so as t o reduce v a r i a t i o n due t o age, environmental h i s t o r y and other v a r i a b l e s . were used i n a l l experiments  Unstarved animals  i n case s t a r v a t i o n had any e f f e c t  on the normal processes o f i o n i c and osmotic r e g u l a t i o n .  Young  a d u l t s were used as more body f l u i d s c o u l d f r e q u e n t l y be o b t a i n e d from these animals than from o l d e r  ones.  Removal o f gut contents f o r pH d e t e r m i n a t i o n L o c u s t s were a n a e s t h e t i z e d w i t h a mixture o f ether and carbon d i o x i d e and a l o n g i t u d i n a l c u t made along the d o r s a l surface.  Hoskins and H a r r i s o n (1934) showed t h a t ether has no  e f f e c t on the pH o f g u t contents o f the honeybee.  Gut contents  were removed by s l i t t i n g the gut a n t e r i o r l y t o p o s t e r i o r l y , care b e i n g taken t o a v o i d c o n t a c t w i t h the hemolymph.  In most  cases the contents were s u f f i c i e n t l y moist t o measure pH. However, sometimes r e c t a l contents were d r y due t o water a b s o r p t i o n , and i n these cases d i s t i l l e d water was added f o r moisture.  A d d i t i o n o f a s m a l l amount o f d i s t i l l e d water t o  a l r e a d y m o i s t m a t e r i a l d i d n o t s i g n i f i c a n t l y change the pH.  9 P r e p a r a t i o n o f the l i g a t e d l o c u s t rectum The method used i n p r e p a r a t i o n o f the l i g a t e d rectum was developed and d e s c r i b e d by P h i l l i p s w i t h the f o l l o w i n g s l i g h t m o d i f i c a t i o n s .  locust  (1961, 1964a)  The animal was p l a c e d  on i t s r i g h t s i d e i n a s h o r t l e n g t h o f g l a s s t u b i n g , diameter 1.5 cm., w i t h c o t t o n wool l o o s e l y packed a t one end and the l a s t f i v e or s i x abdominal segments p r o t r u d i n g from the end o f the tube.  To a n a e s t h e t i z e the animal, a mixture o f carbon d i o x i d e  and e t h y l ether was blown g e n t l y through the c o t t o n wool.  The  stream was c o n t i n u e d f o r about 15 to 20 min. w h i l e the l i g a t i o n was b e i n g performed;  t h i s i s a longer time than mentioned by  P h i l l i p s who used 3-5 min. o f ether vapour a f t e r minutes o f carbon d i o x i d e .  several  A l s o , i n making the i n c i s i o n , i t  was found convenient to p r i c k f i r s t a s m a l l hole i n the c u t i c l e , and t o use t h i s hole as a s t a r t i n g p o i n t f o r the s c i s s o r s . O r i g i n a l l y human h a i r was used t o t i e o f f the gut, b u t i n l a t e r experiments c o t t o n t h r e a d was used w i t h equal s u c c e s s . A c c o r d i n g to P h i l l i p s  (1961), the b l o o d osmotic p r e s s u r e  a f t e r the o p e r a t i o n i s s i m i l a r to t h a t r e p o r t e d f o r normal l o c u s t s by Treherne (1959).  Animals were i n v a r i a b l y a c t i v e  u n t i l the end o f an experiment, and f o r s e v e r a l days Three to seven days was r e p o r t e d by P h i l l i p s  (1964a).  thereafter. In  g e n e r a l , experiments were r u n immediately a f t e r the o p e r a t i o n , and an experiment would take no more than t h r e e to four hours. Measurements o f P h i l l i p s  (1961) i n d i c a t e d t h a t the r e c t a l  e p i t h e l i u m was a b l e t o move water and s a l t a c t i v e l y f o r 24 t o 48 hr. a f t e r  ligation.  10 I n j e c t i o n and removal o f l o c u s t r e c t a l  fluid  The l i g a t e d rectum was r i n s e d thoroughly u s i n g 1.2M NaCl i n j e c t e d through the anus w i t h a s y r i n g e and f i n e p o l y e t h y e l e n e t u b i n g (PE 10), so t h a t a l l f e c a l m a t e r i a l was removed b e f o r e i n j e c t i o n o f experimental b u f f e r .  Salt  was  used, although a l k a l i n e b u f f e r made up w i t h s a l t  was  used by P h i l l i p s  subsequent experiment,  solution  solution  (1961) f o r r i n s i n g w i t h no d i f f e r e n c e s i n  experimental r e s u l t s .  A f t e r completion o f an  the animal was d i s e c t e d and r e s u l t s d i s c a r d e d i f  any v i s i b l e m a t e r i a l was found i n the rectum,  or i f the  l i g a t u r e had loosened. The  t e r m i n a l segment o f the l o c u s t was waxed around a l l  the p r o t r u b e r a n c e s t o prevent l e a k i n g , and the animal p l a c e d i n a wire cage padded w i t h c o t t o n wool on the stage o f a b i n o c u l a r microscope.  The abdomen o f the animal extended  the cage and was clamped to a p i e c e o f p l a s t i c e n e . i n j e c t i o n apparatus by P h i l l i p s  from  The  ( F i g . 1) used was s i m i l a r t o t h a t designed  (1964a).  I t c o n s i s t s o f an i n j e c t i o n  needle  approximately 2 mm.in diameter w i t h the t i p p u l l e d to about 0.5 mm. and the edges w e l l annealed by h e a t i n g i n a Bunsen burner t o prevent puncture o f the c u t i c u l a r l i n i n g o f the rectum,  the i n t i m a .  The needle was s e a l e d i n one arm (1) o f a  s m a l l perspex chamber w i t h t h r e e o u t l e t s and a t t a c h e d to a micromanipulator.  The second sidearm o f the chamber  (2) l e a d  by s u r g i c a l rubber t u b i n g t o a s y r i n g e f i l l e d w i t h water. remaining opening  The  (3) was f i t t e d w i t h an 0 - r i n g through which  FIGURE 1 Diagram o f apparatus arrangement f o r i n j e c t i o n experimental b u f f e r i n t o stippling indicates solution. the  l i g a t e d l o c u s t rectum.  water; l i g h t s t i p p l i n g ,  S o l u t i o n s are  rectum by (3)  p e r i o d i c a l l y by  tube a t t a c h e d to p a r t  F l u i d i s introduced into  Samples of r e c t a l f l u i d can be  removing the  rod and  g l a s s c a p i l l a r y through p a r t s 3 and capillary.  rectum through  a d j u s t i n g the micrometer b u r e t t e w i t h the  in place.  Heavy  experimental  i n t r o d u c e d i n t o the  anus by means of a m i c r o c a p i l l a r y  1 of the perspex chamber.  rod  the  of  inserting 1 into  the  the metal  taken  a very f i n e injection  PERSPEX CHAMBER  LOCUST  MICROMETER  BURETTE  11 was pushed.a removable metal r o d .  With the r o d i n p l a c e , the  d e s i r e d amount o f experimental s o l u t i o n was drawn up i n t o the i n j e c t i o n needle.  The needle was i n s e r t e d by means o f the  micromanipulator through the anus o f the animal i n t o the r e c t a l lumen.  To stop leakage, a beeswax-resin mixture was  used to s e a l the needle i n p l a c e .  The experimental f l u i d was  then d r i v e n i n t o the rectum u s i n g the s y r i n g e t o f o r c e water down the tube and the r e s u l t i n g a i r p r e s s u r e to push the other f l u i d out o f the i n j e c t i o n needle i n t o the rectum. assured by the v e n t i l a t o r y movements o f the l o c u s t . c o u l d be withdrawn  M i x i n g was Samples  a t i n t e r v a l s by p u l l i n g o u t the metal r o d  (3) and i n s e r t i n g a long t h i n g l a s s c a p i l l a r y i n t o the injection pipette. In g e n e r a l , 65 of  15 to 20  o f f l u i d were i n j e c t e d .  One sample  was removed a t v a r i o u s time i n t e r v a l s and the  remainder c o n s t i t u t e d the f i n a l sample.  When o n l y a f i n a l  sample was r e q u i r e d , then 5 0 j a l . were i n j e c t e d i n i t i a l l y . volume changes  Thus  due to sampling were s t a n d a r d i z e d . Values f o r  a g i v e n time might c o n s i s t o f a mixture o f samples  taken i n both  these ways. S o l u t i o n s used i n i n j e c t i o n  experiments  Two b u f f e r s were used i n the i n j e c t i o n experiments.  These  were M c l l v a i n e ' s p h o s p h a t e - c i t r a t e b u f f e r and T r i s (hydroxymethyl) aminomethanewith HCl.  Both o f these were made  up w i t h 1.2M NaCl to minimize water movement i n the rectum (Phillips, to  1961).  S a l t was used i n s t e a d o f a s a c c h a r i d e s o l u t i o n  discourage b a c t e r i a l  activity.  12 To prepare 0. 02M M c l l v a i n e s b u f f e r , 0.02M Na HPC>4 was 1  2  t i t r a t e d w i t h 0.02M c i t r i c a c i d to pH 7.2-7.4. was prepared by t i t r a t i n g a c i d to pH 7.2-7.4.  A weaker b u f f e r  0.002M Na2HP04 w i t h 0.002M c i t r i c  This value o f pH was chosen s i n c e pH 7.4  i s the pH p r e d i c t e d from the Ussing equation f o r r e c t a l at  fluid  e q u i l i b r i u m i f hydrogen ions were d i s t r i b u t e d p a s s i v e l y across  the r e c t a l w a l l ( P h i l l i p s ,  1961).  An a c i d b u f f e r pH 3.8-3.95  was a l s o prepared u s i n g 0.002M c i t r i c a c i d and 0.002M Na2HPC>4 . To prepare the T r i s - H C l b u f f e r , 0.002M T r i s was t i t r a t e d w i t h 0.1N HCl to pH 7.4.  Because the a c i d was r e l a t i v e l y  concentrated  and the amount added very s m a l l , i t s a d d i t i o n was assumed not to  change the m o l a r i t y o f the o r i g i n a l NaCl s o l u t i o n  significantly. D e t e c t i o n o f leakage from the rectum Unless otherwise s t a t e d , the dye amaranth was i n c l u d e d i n a l l experimental b u f f e r s i n j e c t e d i n t o the rectum o f Schistocerca.  Two m i l l i g r a m s were added to 10 ml. o f b u f f e r  p r i o r to use. Amaranth i s n o t absorbed (Treherne, 1957, 1958).  from any p a r t o f the l o c u s t gut  Phillips  (1961, 1964a) found t h a t  amaranth behaves l i k e phenol r e d and many other dyes and i s a c t i v e l y s e c r e t e d . by the M a l p i g h i a n tubules a g a i n s t a 100- to 1000-fold c o n c e n t r a t i o n g r a d i e n t . hemolymph, amaranth was almost  When i n j e c t e d i n t o the  completely removed from the b l o o d  w i t h i n 1-2 h r . and was found concentrated i n the hindgut a n t e r i o r t o the l i g a t u r e .  Hence the leakage o f a s m a l l amount  13 of dye  c o u l d be detected by a post-experimental  the hindgut.  examination of  Leakage d i d not occur o f t e n , and any  large  punctures c o u l d be immediately detected s i n c e there was r e c o v e r y of i n j e c t e d f l u i d d u r i n g  poor  sampling.  P e r m e a b i l i t y of the i n t i m a to i n o r g a n i c phosphate (a)  P r e p a r a t i o n of the The  i n t i m a l p r e p a r a t i o n used i n s t u d i e s of p e r m e a b i l i t y  to phosphate ions was 1965)  intima  s e t up as d e s c r i b e d by P h i l l i p s  w i t h minor m o d i f i c a t i o n s .  c u t t i n g o f f the head, and  The  l o c u s t was  sacrificed  (PE 90) was  i n s e r t e d a short distance  the rectum through the anus. the tubing was  I t was  cut, and  the gut,  cut through i n the hindgut  (0.05M) was  several millimeters  leaks.  Next, the and  micromanipulator.  region several millimeters  A concentrated  s o l u t i o n of amaranth  f l u s h e d through the rectum v i a the  t u b i n g to r i n s e out any  polyethylene  s o l i d m a t e r i a l i n the rectum.  l i g a t u r e of c o t t o n thread was end of the rectum.  final  s t i l l waxed to the  p u l l e d out s l i g h t l y u s i n g the  a n t e r i o r to the rectum. dye  micromanipulator,  membrane between the penultimate  abdominal segment was was  into  waxed s e c u r e l y i n p l a c e to the l a s t segment.  of the cannula were waxed w e l l to prevent  cannula,  Polyethylene  (0.2-0.4 cm.)  While h e l d by a  The whole o f the f i n a l abdominal segment and  intersegmental  by  the animal h e l d on the stage of a  b i n o c u l a r microscope with a s l a b of p l a s t i c e n e . tubing  (1961,  A  then t i e d f i r m l y a t the a n t e r i o r  The p r e p a r a t i o n was  s o l u t i o n so t h a t leaks c o u l d be  f i l l e d w i t h the amaranth  detected.  14 To loosen the r e c t a l pads and other e x t e r n a l t i s s u e s from the c h i t i n o u s i n t i m a , the p r e p a r a t i o n was d i s t i l l e d water.  Any  soaked o v e r n i g h t i n  leaks were d e t e c t e d by the appearance of  the r e d amaranth dye i n the surrounding water.  A f t e r soaking,  the e x t e r n a l t i s s u e c o u l d be p u l l e d away from the c u t i c l e i n s t r i p s with f i n e forceps. A s m a l l hole was  cut i n the PE 9 0 tubing about 1  above the waxed j o i n t w i t h the i n t i m a . cm.  of smaller polyethylene tubing  cm.  A l e n g t h of about  (PE 10) was  i n s e r t e d through  t h i s hole down i n t o the lumen o f the sac so t h a t the end v i s i b l e w i t h i n but not rubbing a g a i n s t the s i d e . the two for  tubes was  s e a l e d w i t h wax.  The  5-10  The  was  j o i n t of  smaller tube was  used  running the p e r f u s a t e i n t o the sac, and the l a r g e r f o r  c o l l e c t i o n o f the s o l u t i o n which was  f o r c e d out.  i n t i m a was  sac whose p e r m e a b i l i t y to  l e f t then as a cannulated  The c h i t i n o u s  phosphate c o u l d be s t u d i e d i n the absence of the  cellular  material. (b)  P e r f u s i o n of the i n t i m a l Non-radioactive  sac  s o l u t i o n s were contained i n g l a s s  r e s e r v o i r s h e l d by clamps to a h o r i z o n t a l b a r . r e s e r v o i r l e d by a needle  i n t o the PE  pressure  H2O)  (range 3 5-40  cm.  was  Tubing  10 t u b i n g .  from the  Hydrosatatic  used to p e r f u s e the  The r a t e of p e r f u s i o n c o u l d thus be c o n t r o l l e d roughly  solution. by  a d j u s t i n g the h e i g h t o f the r e s e r v o i r . R a d i o a c t i v e s o l u t i o n o f i d e n t i c a l chemical to  the n o n - r a d i o a c t i v e s o l u t i o n was  composition  placed i n small p l a s t i c  15 v i a l s , and each r e c t a l sac was put i n one o f these. R a d i o a c t i v e molecules d i f f u s i n g i n t o the sac were washed out i n the p e r f u s a t e which was c o l l e c t e d f o r determination o f radioactivity.  The p l a s t i c v i a l s were kept i n a constant  temperature b a t h a t 28°C. and  A t o t a l o f 14 p r e p a r a t i o n s  was run,  f i v e s u c c e s s i v e r e p l i c a t e c o l l e c t i o n s were made f o r each  p r e p r a t i o n f o r each o f the four s o l u t i o n s r u n . (c)  S o l u t i o n s used i n study o f i n t i m a l p e r m e a b i l i t y to phosphate The p r e m e a b i l i t y o f the c u t i c u l a r i n t i m a to the v a r i o u s  i o n i c s p e c i e s o f i n o r g a n i c phosphate was s t u d i e d by o b s e r v i n g the d i f f u s i o n o f l a b e l l e d phosphate a t three d i f f e r e n t pH values. (Giese,  The d i s s o c i a t i o n o f phosphoric a c i d occurs 1962):  H3PO4 p  Using  as f o l l o w s  K  a  * H 2.12  +  + H P04~i=± H 7.20 2  +  + HP0  the Henderson-Hasselbalch equation,  z=±H 12.66  = 4  +  + P0 ^ 4  i t was c a l c u l a t e d that  a t pH 4 the phosphate i s found c h i e f l y as the monovalent i o n , and  a t pH 8 p r i m a r i l y as the d i v a l e n t form.  was chosed as an i n t e r m e d i a t e the normal r e c t a l pH v a l u e . intima before absorption  A value o f pH 6  value and s i n c e i t approximates Any molecule must penetrate the  c o n t a c t i n g the r e c t a l e p i t h e l i u m , and so f o r  to occur i t i s e s s e n t i a l that the molecule be a b l e  to pass t h i s f i r s t membrane r e l a t i v e l y u n r e s t r i c t e d .  I f the  i n t i m a were found impermeable t o phosphate a t pH 6 as some preliminary observations  suggested, involvement o f phosphate i n  a c i d i f i c a t i o n c o u l d then be e l i m i n a t e d a t the o u t s e t .  16 The b u f f e r s o l u t i o n s used c o n s i s t e d o f 20mM P 0 made up 4  w i t h the sodium and potassium KC1  s a l t s , and 5mM/l. each o f NaCl,  and MgCl / a d j u s t e d t o pH values o f 4, 6, and 8. 2  Labelled  phosphate as H3P"^C>4 was added t o the e x t e r n a l s o l u t i o n , i n such s m a l l amount as to have no e f f e c t on the b u f f e r c o n c e n t r a t i o n 32 o f phosphate o r the pH. Enough . P was added t o gxve a count c  of about 10 presence  cpm/ml. i n the o u t s i d e s o l u t i o n .  of C a  + +  To t e s t whether  had a s i g n i f i c a n t e f f e c t on the phosphate  p e r m e a b i l i t y o f the membrane, a c o n t r o l experiment was run a t pH 6 i n which the s o l u t i o n c o n t a i n e d 5mM/l. C a C l to the other ions mentioned above.  Calcium  2  i n addition  c o u l d not be added  a t extreme pH values because o f p r e c i p i t a t i o n o f c a l c i u m phosphate.  S o l u t i o n s were then a l l 35mM except f o r the  c a l c i u m s o l u t i o n which had a t o t a l s o l u t e c o n c e n t r a t i o n o f 40mM.  The wide spectrum a n t i b i o t i c t e t r a c y c l i n e - (5 mg./l.) was  i n c l u d e d t o prevent b a c t e r i a l i n c o r p o r a t i o n o f phosphate. (d)  C o l l e c t i o n and counting o f r a d i o a c t i v e samples To c o l l e c t the r a d i o a c t i v e p e r f u s a t e , a p l a n c h e t was  l e f t under the open end o f the PE 9 0 t u b i n g f o r a known l e n g t h o f time, 15 min.  f o r the f i r s t  t h e r e a f t e r f o r 3 0 min.  three p r e p a r a t i o n s and  A f t e r a change o f p e r f u s i n g s o l u t i o n ,  the new s o l u t i o n was allowed t o run H hr. b e f o r e making the f i r s t c o l l e c t i o n so as to r i n s e o u t any v e s t i g e s o f the previous s o l u t i o n .  Samples were d r i e d b e f o r e counting.  If  the volume c o l l e c t e d was i n s u f f i c i e n t t o wet the s u r f a c e o f the p l a n c h e t evenly, a few drops o f 'Tween 80*,  a detergent,  17 were used.  To measure the a c t i v i t y o f the e x t e r n a l  f o r l a t e r c a l c u l a t i o n s / 10  solutions  samples of each were counted.  Both i n i t i a l and f i n a l 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 s were checked each day.  S i g n i f i c a n t changes never o c c u r r e d i n  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 s over the course o f a day. A l l c o u n t i n g was done on a ' P h i l l i p s ' E l e c t r o n i c Counter PW403 5 and G e i g e r - M u e l l e r d e t e c t o r .  The c o u n t i n g o f samples  from a s i n g l e p r e p a r a t i o n was done on the same day t o reduce v a r i a t i o n s due t o the e f f i c i e n c y o f the instrument and amount o f r a d i o a c t i v e decay.  The t o t a l number o f counts per minute  was about four times background count f o r pH 4 where the g r e a t e s t amount o f d i f f u s i o n o c c u r r e d .  Background counts were  run f r e q u e n t l y between counts o f unknowns, and the day's average was s u b t r a c t e d from i n d i v i d u a l samples.  The number o f  counts per minute was determined from a t o t a l o f 1000counts. The range f o r the f i v e r e p l i c a t e s was about 10% o f the mean. S i n c e the m o l a r i t y o f the r a d i o a c t i v e s o l u t i o n was known, the amount o f phosphate i n 10 u l . c o u l d be c a l c u l a t e d and r e l a t e d t o the count o b t a i n e d .  From these standards, the amount  o f phosphate which had d i f f u s e d i n t o the sac c o u l d be c a l c u l a t e d . The c a l c u l a t i o n was done f o r each sample c o l l e c t e d and the f i v e values f o r a g i v e n p r e p a r a t i o n a t each of the three pH v a l u e s then averaged.  18 (e)  I n o r g a n i c phosphate p r e c i p i t a t i o n A n t i b i o t i c was i n c l u d e d i n the experimental  s o l u t i o n s to  prevent b a c t e r i a l i n c o r p o r a t i o n o f the i n o r g a n i c phosphate. MacKay and B u t l e r ' s m o d i f i c a t i o n o f Mathison's method (Peters and Van S l y k e , 1932) was used as a check f o r t h i s .  The  technique r e s u l t s i n the p r e c i p i t a t i o n o f i n o r g a n i c phosphate. I f the l a b e l l e d ^phosphate s o l u t i o n i s t r e a t e d a c c o r d i n g t o t h i s method, a l l l a b e l l e d m a t e r i a l should be contained i n the p r e c i p i t a t e i f a l l phosphate has i n f a c t remained as i n o r g a n i c phosphate.  However i f a s i g n i f i c a n t count i s o b t a i n e d from the  supernatant,  then some phosphate must be present i n dissolved,  o r g a n i c form or i n c o r p o r a t e d i n t o b a c t e r i a . , The Slyke  s o l u t i o n s were made up as g i v e n by P e t e r s and Van  (see Appendix A ) .  The q u a n t i t i e s f o r the assay were  s c a l e d down but the r e l a t i v e p r o p o r t i o n s were the same.  One  m i l l i l i t e r o f the e x t e r n a l s o l u t i o n was p i p e t t e d i n t o a s m a l l c e n t r i f u g e tube.  Then 0.2 ml. o f the magnesium c i t r a t e  o f F i s k e was added, p l u s 0.4 ml. o f s t r o n g ammonia The  mixture  water.  tube was shaken f o r a few minutes, corked and l e f t i n the  r e f r i g e r a t o r overnight.  Ten m i c r o l i t e r s o f the supernatant  o b t a i n e d a f t e r c e n t r i f u g a t i o n was p l a c e d on a p l a n c h e t f o r counting.  T h i s assay was done f o r each r a d i o a c t i v e s o l u t i o n  a t the s t a r t and a t the f i n i s h o f each day's experimental  runs.  I t was found t h a t P^2 remained 100% i n the i n o r g a n i c form, as the a c t i v i t y o f the supernatant never d i f f e r e d from background  counts.  significantly  19 C o l l e c t i o n o f body f l u i d s o f S c h i s t o c e r c a (a)  gregaria  Hemolymph Samples of hemolymph were e a s i l y obtained by c u t t i n g o f f  one  of the l a r g e jumping legs o f the l o c u s t .  Amounts o f 50-60  >ul. were c o l l e c t e d i n a c a p i l l a r y tube as the v e n t i l a t o r y movements o f the animal f o r c e d out drops of f l u i d . were used f o r measurements of pH and cases when determinations hemolymph was (b)  Samples  i n o r g a n i c phosphate.  were not made immediately  In  the  s t o r e d under p a r a f f i n o i l .  Malpighian  tubule  Malpighian  fluid  tubule f l u i d c o u l d be c o l l e c t e d by i n s e r t i n g a  f i n e g l a s s cannula with the a i d o f a micromanipulator, through the anus up  to the r e g i o n of e n t r y of the tubules  Amounts o f f l u i d from 3-5 jal. t h i s way,  though d i f f i c u l t y  gut.  c o u l d sometimes be c o l l e c t e d i n  was  m a t e r i a l i n the gut. P h i l l i p s for  i n t o the  o f t e n encountered due  to  (1961, 1964c) had used the method  c o l l e c t i o n from s t a r v e d animals.  the hindgut and p o s t e r i o r midgut was almost t o t a l l y from the M a l p i g h i a n  The  fluid collected in  assumed to be  derived  tubules as i t was  c l e a r and  watery w i t h numerous s m a l l r e d c r y s t a l s i n some cases.  I t could  be e a s i l y d i f f e r e n t i a t e d from the dark brown f l u i d found i n the midgut and  f o r e g u t , which resembles molasses i n  Consequently an a l t e r n a t e method was used, taken from Ramsay (1955a) who c o l l e c t i o n of u r i n e i n Dixippus anaesthetized  with ether and  opened a l o n g  the m i d - v e n t r a l  consistency.  more f r e q u e n t l y  used the method f o r  morosus.  The  locust  carbon d i o x i d e and  was  the abdomen  l i n e a t about the l e v e l of  entry  20 of  the M a l p i g h i a n  tubules i n t o the gut.  A l i g a t u r e of cotton  thread was t i e d around the i n t e s t i n e ; j u s t p o s t e r i o r to the j u n c t i o n o f the t u b u l e s , t a k i n g care not t o t i e o f f the tubules as w e l l .  A n t e r i o r to t h i s , a c u t was made i n t o the w a l l o f  the midgut, and any m a t e r i a l i n the area was removed u s i n g fine forceps.  F r e q u e n t l y , f l u i d could, be squeezed from t h i s  m a t e r i a l and r e c o v e r e d w i t h a c a p i l l a r y tube.  A f t e r removal o f  gut contents, a f i n e g l a s s cannula was i n s e r t e d , and e i t h e r t i e d i n t o p l a c e or merely h e l d i n p o s i t i o n .  F l u i d would  accumulate i n the l e n g t h o f gut between the l i g a t u r e s , and t h i s f l u i d had been presumably e l a b o r a t e d by the M a l p i g h i a n  tubules.  Samples u s u a l l y had t o be pooled u s i n g t h i s method: 2.5-4^1. were used i n the phosphate a n a l y s i s .  The r a t e o f c o l l e c t i o n was  not as good as t h a t g i v e n by Ramsay o f 4.0-6.5 mm?/hr. necessary, (c)  When  samples were s t o r e d under p a r a f f i n o i l .  Rectal  fluid  R e c t a l f l u i d was c o l l e c t e d by squeezing  g e n t l y the  t e r m i n a l abdominal segment o f the l o c u s t and c o l l e c t i n g any r e s u l t i n g f l u i d i n a c a p i l l a r y tube.  In some animals  a drop o f  f l u i d , could, be obtained, w h i l e i n o t h e r s a l l f l u i d had a l r e a d y been absorbed from the rectum not used s i n c e the animals  Cannuli were  were unstarved and the p i p e t t e became  clogged with s o l i d m a t e r i a l . pooled,  ( P h i l l i p s , 1964c).  Samples u s u a l l y had t o be  as volumes used i n the phosphate determination  from 2-4 jil.  ranged  The f l u i d was s t o r e d under p a r a f f i n o i l unless  measurements were made on i t  immediately.  21 Measurement of i n o r g a n i c (a)  phosphate by  c o l o r i m e t r i c method  Body f l u i d s The  method used to measure i n o r g a n i c  phosphate  c o n c e n t r a t i o n s i n the body f l u i d s o f S c h i s t o c e r c a t h a t o f Gomori small  gregaria  was  (1942) w i t h minor changes used to d e a l with  the  sample volumes.  The  Hemolymph was the  then p r e c i p i t a t e d w i t h 1 ml.  trichloroacetic acid  to 60 j u l . and 5% TCA,  the  l i s t e d i n Appendix  allowed to c l o t f o r 20 to 30 min.,  serum p r o t e i n was  micro-centrifuge  s o l u t i o n s are  (TCA)  tube.  B.  and  5%  f o r 1 hr. i n a p o l y e t h y l e n e  Hemolymph sample s i z e s ranged from 2 5  sometimes samples were pooled.  I f untreated with  sample formed a p r e c i p i t a t e when the  molybdate-sulfuric  reagent was  added, and  no b l u e c o l o r although the p r e c i p i t a t e was  a f i l t e r e d sample blue.  Fiske  had  and.  Subbarow (1925) s t a t e t h a t p r e c i p i t a t e formation means t h a t protein i s present.  They found t h a t 1 mg.  p r e c i p i t a t e 0.01  phosphorus and  mg.  of p r o t e i n  diminish  the  precipitate  was  amount.  centrifuged  a t the maximum speed i n an  E l e c t r i f u g e Model 50 c e n t r i f u g e .  TCA  the c o l o r o f  supernatant by an e q u i v a l e n t down f o r 15 min.  The  will  F i v e p e r c e n t TCA  was  also  added to samples of r e c t a l f l u i d and M a l p i g h i a n t u b u l e f l u i d although no p r e c i p i t a t e r e s u l t e d and  c e n t r i f u g a t i o n could  be  omitted. The w i t h the  amount of reagents was  m o d i f i e d from Gomori to  s m a l l q u a n t i t i e s of the unknowns but  are e s s e n t i a l l y the same. a d d i t i o n , as  follows:  The  procedure was,  the  deal  proportions  i n order o f  (1)  Blank:  0.5 ml. m o l y b d a t e - s u l f u r i c reagent 1 ml. 5% TCA 0.2 ml. 'elon' water t o 3 ml.  (2)  Standard:  0.5 ml. m o l y b d a t e - s u l f u r i c reagent 0.2 (0.4, 0.6) ml. standard 0.5mM K H P 0 2  4  1 ml. 5% TCA water to 3 ml. (3)  Unknown:  0.5 ml. m o l y b d a t e - s u l f u r i c reagent supernatant  from TCA p r e c i p i t a t i o n  0.2 ml. 'elon' water to 3 ml. The samples were i n c u b a t e d a t room temperature  f o r 45  to 90 min. and r e a d a t 675 myu on a Bausch and Lomb S p e c t r o n i c 20.  A sample was sometimes checked by the a d d i t i o n o f a known  amount o f standard i f the c o n c e n t r a t i o n was a t the lower  limit  of the curve, or by u s i n g a l a r g e r sample s i z e whenever p o s s i b l A c a l i b r a t i o n curve was prepared f o r each group o f unknowns. Sample s i z e was a d j u s t e d so t h a t p e r c e n t t r a n s m i s s i o n was not g r e a t e r than 92% and not l e s s than 32% on the c a l i b r a t i o n The experimental e r r o r i s estimated a t t ImM/l., taken  curve  from  hemolymph determinations s i n c e e r r o r due t o t r a n s f e r and c e n t r i f u g a t i o n was a t a maximum {b)  here.  Phosphate s e c r e t i o n The same method o f Gomori (1942) was used i n experiments  i n v e s t i g a t i n g p o s s i b l e phosphate s e c r e t i o n by the rectum.  In  these, amaranth dye was o m i t t e d from the i n j e c t e d b u f f e r so as  23 to a v o i d c o m p l i c a t i o n s phosphate.  The  w i t h the c o l o r i m e t r i c determination  absence o f any p r o t e i n p r e c i p i t a t e w i t h  a d d i t i o n of 5% TCA. was  the  used as an a l t e r n a t e method o f  d e t e c t i n g leaks i n t o the lumen, as the presence of any would have caused p r e c i p i t a t i o n of serum p r o t e i n . experimental  of  e r r o r was  of the order of + 0.3  hemolymph  The  mM/1.  pH measurements A l l pH measurements were made u s i n g a Radiometer Meter 27.  pH  For r o u t i n e adjustment of b u f f e r s o l u t i o n , Radiometer  Type G202B g l a s s e l e c t r o d e w i t h r e f e r e n c e e l e c t r o d e K401 used.  The  'B'  was  type o f e l e c t r o d e i s recommended by Radiometer  f o r . measurements on p o o r l y b u f f e r e d n e u t r a l s o l u t i o n s o f s t r o n g e l e c t r o l y t e s , and  hence was  made up w i t h 1.2M  B u f f e r s o l u t i o n s used i n s t a n d a r d i z i n g  NaCl.  these e l e c t r o d e s were a c c u r a t e Two  s u i t e d f o r the weak b u f f e r s  to t  0.01  pH u n i t .  types of g l a s s e l e c t r o d e were used i n measuring  the  pH o f m i c r o - q u a n t i t i e s : (1)  The Radiometer Type G2 52C w i t h r e f e r e n c e e l e c t r o d e  r e q u i r e s a t l e a s t 2>ul. f o r one 5yUl. (± 0.1  was  determination.  used as the l a r g e r sample gave b e t t e r  pH u n i t ) .  The  'C  In p r a c t i c e , accuracy  type o f e l e c t r o d e i s designed with a  s p e c i a l view to mechanical s t r e n g t h .  I t was  used f o r  pH  measurements of i n j e c t e d b u f f e r , hemolymph, gut contents, fecal material. u n t i l pH 9,  and  experimentally.  K150  S a l t e r r o r s do not occur w i t h t h i s  and  electrode  t h i s i s out of the range encountered Whenever sample s i z e permitted,  a number of  24 determinations  (4-5)  were made u s i n g e l e c t r o d e G252C.  f i r s t sample o f unknown was discarded.  Before  considered  as a r i n s e and  the next group of determinations  on another unknown, a f r e s h drop of KCl was  The readings  were made  r e l e a s e d from the  calomel e l e c t r o d e to get r i d of any m a t e r i a l which might have d i f f u s e d i n t o the KOI.  This was  not done between r e p l i c a t e s .  A l l measurements were c a r r i e d out a t room temperature. I t was  found necessary to c a l i b r a t e the  r e g u l a r l y because of v a r i a t i o n s due e l e c t r o d e s from the i d e a l and experimental  microelectrode  to d e v i a t i o n s of micro-  the r e l a t i v e weakness of  b u f f e r as compared to b u f f e r s used to  the e l e c t r o d e .  the  standardize  (This e x h i b i t s i t s e l f by a d r i f t of the meter  needle i n the case o f the weak b u f f e r w i t h a more r a p i d s t a b i l i z a t i o n f o r stronger b u f f e r . )  As an e l e c t r o d e aged, i t  a l s o sometimes r e q u i r e d c a l i b r a t i o n .  To do t h i s ,  i d e n t i c a l to t h a t used i n experiments was pH values e.g.  5.5,  glass electrode.  6.5,  7.0,  to v a r i o u s  as measured by Type G202B  These same s o l u t i o n s were then measured u s i n g  the m i c r o e l e c t r o d e values  6.0,  adjusted  0.002M b u f f e r  G252G, and a c a l i b r a t i o n curve drawn.  Mean  f o r unknowns c o u l d then be r e a d o f f the curve to o b t a i n  a corrected value.  C a l i b r a t i o n s were done o n l y i n those cases  where an a b s o l u t e value was  d e s i r e d , and not f o r those where  o n l y r e l a t i v e values were r e q u i r e d . contents,  and  Values f o r hemolymph, gut  f e c a l m a t e r i a l were not c o r r e c t e d s i n c e they have  a h i g h enough b u f f e r c a p a c i t y t h a t e x c e s s i v e does not occur.  The  time allowed  d r i f t o f the meter  f o r the needle of the meter  25 to e q u i l i b r a t e was  s t a n d a r d i z e d a t 2 min.  were a c c u r a t e to JT 0.01 (2)  pH  The b u f f e r  standards  units.  The Radiometer Type G297 e l e c t r o d e w i t h r e f e r e n c e K497  calomel e l e c t r o d e r e q u i r e d a minimum volume o f 2 0 >ul. measurements done, 50 /JLI. was  used.  In the  The electrode i s mounted  i n a water j a c k e t o f g l a s s from which o n l y the p l a s t i c o f the f i n e c a p i l l a r y t u b i n g protrude a t e i t h e r end.  tips Loss of  carbon d i o x i d e i s kept a t a minimum by t h i s design, and e l e c t r o d e w i l l h e n c e f o r t h be c o n s i d e r e d  'anaerobic'.  the  The  m i c r o e l e c t r o d e u n i t i s intended p r i m a r i l y f o r measuring the a c t u a l pH of mammalian b l o o d i n s m a l l q u a n t i t i e s a t a s p e c i f i c . temperature.  There i s no reason  to suppose the system i s l e s s  a c c u r a t e f o r measuring the pH of l o c u s t hemolymph, e.g.  there  are not l a r g e amounts of c a r b o n i c anhydrase i n the hemolymph which would cause g r e a t e r l o s s of carbon d i o x i d e . from the c i r c u l a t i o n j a c k e t keeping  thermostat  Heated water  (VTS13) flows through the  the e l e c t r o d e a t an even temperature (25°C} .  The e l e c t r o d e s were s t a n d a r d i z e d w i t h p r e c i s i o n b u f f e r s o l u t i o n , Radiometer Types S15 00 and S1510, a c c u r a t e to ± 0.005 pH  units.  Any  gas exchange w i t h the environment p r i o r to measurement  was  kept a t a minimum by s t o r i n g samples under p a r a f f i n o i l  b e f o r e drawing them i n t o the c a p i l l a r y of the e l e c t r o d e . system was  used f o r measuring pH values of l o c u s t hemolymph  and experimental dioxide.  The  b u f f e r , to check e f f e c t s due  E q u i l i b r i u m time was  s e t a t 2 min.  to l o s s of carbon  26 RESULTS pH o f d i f f e r e n t r e g i o n s o f the gut (a)  Normal v a l u e s To determine  of  i f the r e c t a l a c i d i t y i s due to i n t r o d u c t i o n  a c i d from any a n t e r i o r r e g i o n o f the gut, the pH o f i t s  contents was measured i n the d i f f e r e n t r e g i o n s  ( F i g . 2 ) . Ten  animals were used and values f o r each r e g i o n were averaged.  The  r e s u l t s are g i v e n i n Table I. The midgut f l u i d was found t o be a c i d w i t h a pH o f 5.46* 0.39, and t h i s i s s i g n i f i c a n t l y lower than a l l p o s t e r i o r v a l u e s . In  the r e g i o n o f e n t r y o f the M a l p i g h i a n tubules between midgut  and hindgut,  the contents became a l k a l i n e w i t h values o f  7.59±0.72, 7.94±0.49, and 7.13+0.47 a n t e r i o r t o p o s t e r i o r . f i n a l value of  The  o f 7.13+0.47 o c c u r r e d i n the ileum and so t h i s p a r t  the gut, w h i l e i t does not modify  found i n the rectum  the pH to the a c i d l e v e l as  (5.91*0. 44) , does lower i t somewhat.  The  v a l u e 7.13 d i f f e r s s i g n i f i c a n t l y from 7.94 but not from 7.59. M a t e r i a l was seldom found i n the colon, which appears  to a c t as  a s p h i n c t e r , although the value o f 6.17+0.14 f o r the p o s t e r i o r ileum may i n d i c a t e some forward movement o f a c i d .  The anterior'  and p o s t e r i o r r e c t a l contents were found t o have a pH o f 6.19+ 0.45 and 5.91+0.44 r e s p e c t i v e l y , which i s s i g n i f i c a n t l y more a c i d than a l l a n t e r i o r values except those f o r p o s t e r i o r ileum and midgut.  Two values a r e p r e s e n t e d s i n c e f r e q u e n t l y the f e c a l  p e l l e t c o u l d be d i v i d e d and two readings taken.  The d i f f e r e n c e ,  w h i l e not s i g n i f i c a n t a t the 0.05 l e v e l o f p r o b a b i l i t y , may be a  FIGURE 2 Regions o f the gut o f S c h i s t o c e r c a g r e g a r i a : b, oesophagus; c, crop; tubule; g, p r o c t o d a e a l sac  (after P h i l l i p s ,  a, pharynx;  d, caeca; e, v e n t r i c u l u s ; f , M a l p i g h i a n valve; h, ileum;, i ,  1964a).  colon;  j , rectal  £  TABLE I  pH o f gut contents i n normal and t e t r a c y c l i n e - f e d Schistocerca  gregaria. pH  Region o f gut  N  o  r  m  a  l  Mean - S.D. \Mid v e n t r i c u l u s •H  Tetracycline fed Mean - S.D.  5.46 - 0.39  6.12 -  0.43  Posterior ventriculus  7.59 - 0.72  7.26 -  0.75  Proctodaeal valve  7.94 - 0.49  8.07 -  0.54  Anterior  7.13 ± 0.47  7.48 ±  0.58  ileum  [ P o s t e r i o r ileum Colon  6.17 - 0.14 —  A n t e r i o r rectum  6.19 - 0.45  6.32 -  0.47  ^ P o s t e r i o r rectum  5.91 - 0.44  5.80 -  0.60  27 r e f l e c t i o n of the f a c t t h a t the more p o s t e r i o r l y o c c u r r i n g m a t e r i a l has been i n the rectum f o r a longer p e r i o d , presumably has  reached a t e r m i n a l value.  The  f e c a l p e l l e t s a l s o had a mean o f 5.95*0.55. the value  pH of  and excreted  This agrees with  f o r the most p o s t e r i o r m a t e r i a l found i n the rectum.  Some o f the v a r i a t i o n i n values p r o b a b l y to the handling l o c u s t to defecate  f o r a given r e g i o n was  due  of the animal which o f t e n caused  and probably s h i f t e d gut  the  contents p o s t e r i o r l y  generally. (b)  Values f o r animals f e d a n t i b i o t i c So t h a t the r o l e o f b a c t e r i a l fermentation  c o u l d be evaluated,  the gut  f l o r a was  in acidification  k i l l e d o f f i n ten animals.  They were f e d the normal bran and water except t h a t a l a r g e dose o f the wide spectrum a n t i b i o t i c t e t r a c y c l i n e was  included i n  the d i e t f o r four days. Values f o r t e t r a c y c l i n e t r e a t e d animals are i n c l u d e d i n Table I. gut  There was  no  s i g n i f i c a n t d i f f e r e n c e between the pH  contents o f c o n t r o l animals and  tetracycline  (p=0.05).  those t r e a t e d w i t h  I t can thus be  a c i d i f i c a t i o n occurs i n the rectum, and i n t r o d u c t i o n of a c i d from any  of  concluded t h a t i s not due  e i t h e r to  the  a n t e r i o r r e g i o n or to b a c t e r i a l  fermentation. Demonstration of a s t a t e of dynamic e q u i l i b r i u m (a)  Permeability  o f the r e c t a l e p i t h e l i u m  to hydrogen ions  Experiments were done to demonstrate t h a t the pH between the r e c t a l lumen and  hemolymph r e p r e s e n t s  gradient  a dynamic  28 equilibrium  maintained by  designed to show that  the  the r e c t a l w a l l . same a c i d pH  more a l k a l i n e or a more a c i d b u f f e r l i g a t e d rectum.  I f so,  pH  hypothesis that of the  and  because the r e c t a l w a l l buffers  a t pH  and  0.5  an  fell  reached between pH  5.95  and  pH  i n j e c t e d i n t o the  difference min.  ions.  1 to 2 min. 6.02  within  (p=0.05) between the  first  a steady  2 0 min.  10 min.  and  There was  no  When was  then, more that  solution.  same a c i d pH whether an  acid  significant  l e v e l of a c i d i t y reached a t  a f t e r i n j e c t i o n of a l k a l i n e b u f f e r same time.  as  compared to that  acid buffer  i n j e c t i o n a t the  equilibrium  value i s a r e f l e c t i o n of the b u f f e r i n g  of the experimental s o l u t i o n .  and  are  was  l o c u s t rectum, there  i n j e c t i o n of a l k a l i n e b u f f e r  i s injected.  Ten  Results  approached s i m i l a r to  system approaches the  or a l k a l i n e b u f f e r  hemolymph  When a l k a l i n e b u f f e r  units within  s l o w l y u n t i l a steady value was  C l e a r l y the  the  r i n s e d out were i n j e c t e d w i t h  i n c r e a s e i n pH most r a p i d l y i n the  observed f o l l o w i n g  of  that  3.8-3.95.  twelve w i t h the a c i d b u f f e r .  injected,  was  epithelium  e q u i l i b r a t e w i t h the  values 7.2-7.4 and  i n F i g . 3.  acidic buffer  the  rejection  i s impermeable to hydrogen  presented g r a p h i c a l l y  pH was  the  would r e q u i r e  animals w i t h the r e c t a l i g a t e d and  the pH  i s introduced into  used i n these experiments were 0.002M  Mcllvaine's buffer  alkaline buffer  whether a  the a c i d i t y a r i s e s a n t e r i o r l y and  lumen contents does not  The  experiments were  i s attained  t h i s would suggest that  i s permeable to hydrogen i o n s , the  The  The  pH  40 for  Time taken to reach an capacity  values of r e c t a l f l u i d  FIGURE 3 The  change i n pH o f r e c t a l f l u i d w i t h time a f t e r  injecting  0.002M M c l l v a i n e ' s b u f f e r w i t h o r i g i n a l pH values 7.2-7.4 and 3.8-3.95.  H o r i z o n t a l bars, mean values; v e r t i c a l b a r s ,  standard e r r o r o f the mean.  O  IO  20  30  MINUTES AFTER INJECTION OF BUFFER INTO R E C T U M  40  29 reached a t e q u i l i b r i u m i n these experiments a r e not s i g n i f i c a n t l y d i f f e r e n t from normal r e c t a l values (b)  Role o f b a c t e r i a l  (Table I ) .  fermentation  To demonstrate t h a t a c i d i f i c a t i o n o f f l u i d i n j e c t e d i n t o l i g a t e d r e c t a was due s o l e l y to the a c t i v i t y o f the r e c t a l w a l l and  not to b a c t e r i a l fermentation,  the f o l l o w i n g experiment was  performed i n which an a n t i b i o t i c was i n c l u d e d i n the b u f f e r . Ten  animals were i n j e c t e d w i t h 0.002M M c l l v a i n e ' s b u f f e r pH  7.2-7.4 to which 4 mg./l. o f t e t r a c y c l i n e had been added. A c i d i f i c a t i o n occurred  i n the presence o f the a n t i b i o t i c  i n d i c a t i n g t h a t b a c t e r i a l fermentation phenomenon.  ( F i g . 4)  i s not i n v o l v e d i n the  The pH reached a t 40 min. was 6.06-0.28 as compared to  pH 6.02^0.19 f o r the same system without t e t r a c y c l i n e .  The  d i f f e r e n c e was t e s t e d and found not to be s i g n i f i c a n t a t the 0.05 level of probability.  I t should a l s o be noted t h a t the r e c t a l  contents had been r i n s e d out,  thus d r a s t i c a l l y r e d u c i n g the  indigenous b a c t e r i a l p o p u l a t i o n . I t i s concluded from the r e s u l t s o f F i g . 3 and F i g . 4 t h a t the r e c t a l w a l l i s permeable t o hydrogen ions and t h a t the pH g r a d i e n t across  the r e c t a l w a l l r e p r e s e n t s  e q u i l i b r i u m maintained by the e p i t h e l i a l (c)  a dynamic  cells.  Possible contribution of f e c a l material Although care was taken always to r i n s e the rectum  thoroughly b e f o r e  i n j e c t i o n o f b u f f e r , and the rectum was checked  by d i s e c t i o n a f t e r the experiment, s m a l l amounts o f f e c a l m a t e r i a l c o u l d p o s s i b l y be c o n t r i b u t i n g t o the a c i d i t y  since  FIGURE 4 The change i n pH o f r e c t a l f l u i d w i t h time a f t e r  injecting  0.002M M c l l v a i n e ' s b u f f e r w i t h o r i g i n a l pH value 7.2-7.4 c o n t a i n i n g 4 mg./l. t e t r a c y c l i n e . values:  H o r i z o n t a l b a r s , mean  v e r t i c a l bars, standard e r r o r o f the mean.  IO  20  30  MINUTES AFTER INJECTION OF BUFFER INTO RECTUM  40  30 they have been shown to be a c i d themselves (pH 5.91). experiment was  An  t h e r e f o r e undertaken to check whether there i s  s i g n i f i c a n t or s u f f i c i e n t slow r e l e a s e o f hydrogen ions by m a t e r i a l to account f o r the a c i d i f i c a t i o n . f e c a l material j u s t obtained /il.  Small p i e c e s  fecal  of  from a l o c u s t were p l a c e d i n 50  o f 0.002M a l k a l i n e b u f f e r .  The  suspension was  a c a p i l l a r y tube to reduce l o s s by evaporation,  placed i n  and  measured a g a i n s t time to see i f i n f a c t there was  the pH  was  a slow r e l e a s e  of hydrogen ions o c c u r r i n g . The r e s u l t s are shown i n F i g . 5.  By i n s p e c t i o n ,  there  i s no s i g n i f i c a n t lowering of the pH a f t e r the i n i t i a l change caused by a d d i t i o n o f f e c a l m a t e r i a l so t h a t no slow r e l e a s e o f hydrogen ions took p l a c e w i t h i n a three hour p e r i o d . time i s g r e a t e r than the p e r i o d o f experimentation;  This  also i t  i s most u n l i k e l y t h a t v i s i b l e fragments such as were added here would have been c o n s i s t e n t l y undetected d u r i n g the experiments because o f the p r e c a u t i o n s  injection  a l r e a d y mentioned.  I t i s concluded t h a t there i s not a slow r e l e a s e o f hydrogen ions by f e c a l m a t e r i a l which would account f o r  the  observed l e v e l o f a c i d i f i c a t i o n i n the l i g a t e d rectum.  This i s  a f u r t h e r c o n f i r m a t i o n o f the f a c t t h a t the a c i d i t y i s produced by the r e c t a l w a l l  itself.  Role of phosphate i n a c i d i f i c a t i o n (a)  P e r m e a b i l i t y of the i n t i m a to phosphate A p o s s i b l e mechanism o f a c i d i f i c a t i o n was  v i s u a l i z e d as  the a b s o r p t i o n of the b a s i c form o f a b u f f e r p a i r , e.g. HPO4""  FIGURE 5 The  pH o f 50  a l i q u o t s o f a l k a l i n e 0.002M b u f f e r w i t h  added f e c a l m a t e r i a l  a g a i n s t time s i n c e a d d i t i o n .  are the mean o f a t l e a s t two v a l u e s . +0.04 pH u n i t s .  Points  Standard d e v i a t i o n ,  I  2  3  TIME (HOURS) AFTER ADDITION OF FECAL MATERIAL tO BUFFER  31 r a t h e r than H2P04~, or HCC>3~ r a t h e r than  these  H2CO3,  being  the two most p r e v a l e n t b u f f e r systems found i n b i o l o g i c a l material  (Phillips,  1961).  high i n the r e c t a l lumen  The c o n c e n t r a t i o n o f phosphate i s  (section c ) .  I t was decided to  i n v e s t i g a t e the r o l e o f phosphate i n the a c i d i f i c a t i o n  process  l a r g e l y because some p r e l i m i n a r y r e s u l t s had suggested t h a t the i n t i m a was impermeable to i n o r g a n i c phosphate. c o u l d be confirmed,  I f this  then any p r e f e r e n t i a l a b s o r p t i o n o f the  b a s i c form o f the phosphate b u f f e r p a i r c o u l d be e l i m i n a t e d , i . e . the phosphate must be a b l e t o penetrate  the i n t i m a  lining  the r e c t a l lumen i f a b s o r p t i o n i s to occur. The p e r m e a b i l i t y o f the i n t i m a to phosphate was determined a t three d i f f e r e n t pH values; one a t the normal r e c t a l pH o f 6 and the other  two values  chosen so t h a t the phosphate  e x i s t e d e i t h e r l a r g e l y as monovalent i o n (pH 4) or d i v a l e n t i o n (pH 8 ) .  A summary o f r e s u l t s i s given i n Table I I .  While the  i n t i m a i s almost impermeable to phosphate a t pH 8, t h i s i s not t r u e a t the other pH values t e s t e d .  The i n t i m a i s about  t h i r t e e n times more permeable a t pH 4 than a t pH 8, and about twice as permeable as a t pH 6.  D i f f e r e n c e s between a l l  p o s s i b l e p a i r s a r e s i g n i f i c a n t a t the 0.05 l e v e l o f p r o b a b i l i t y . Using  the Henderson-Hasselbalch equation and the  f o l l o w i n g pKa values f o r 25 C. (Giese, 1962): H3PO4  H  7-  pK  a  = 2.12  +  + H P0 ~7 2  4  = ± H 7.20  +  + H P 0  4  ~ = r H 12.66  i t was c a l c u l a t e d t h a t a t pH 4, phosphate e x i s t s as 76  +  + P0  S 4  TABLE I I  P e r m e a b i l i t y o f the i n t i m a to phosphate a t three pH v a l u e s . .uMPO^hr/^M pH 6 w i t h  Preparation  Ca  added  pH 8  pH 6  1  0.225  1.73  2.07  3.77  2  0.499  2.08  2.39  6.07  3  -  2.88  4.29  7.68  4  0.413  1.36  2.69  5.18  5  0.600  3.08  2.71  5.94  6  0.275  1.92  2.36  4.85  7  0.238  2.18  3.77  5.22  8  0.306  1.89  2.74  6.83  9  0.462  3.90  4.77  9.20  10  0.524  2.88  3.39  7.19  11  0.913  4.82  6.06  7.95  12  0.403  1.56  2.30  4.20  13  0.568  4.38  6.08  8.69  14  0.625  3.02  2.55  4.89  Mean  0.465  2.69  3.44  6.26  S.D.  0.093  1.08  1.37  1.70  S.E.  0.026  0.287  0.365  0.453  + +  pH 4  32 H2PO4 H2PO4  : -  1  H3PO4  and  at  pH  8  as  6.3  HPC-4-:  1  H  2  P0  4  ~.  At  pH  6,  predominates over the d i v a l e n t form i n a r a t i o o f about  16 : 1.  Thus the change i n phosphate p e r m e a b i l i t y with pH may  be due t o the i o n i c form i n which the phosphate e x i s t s a t that pH.  The d i f f e r e n c e i n s i z e between monobasic  orthophosphate  and d i b a s i c orthophosphate i s a f a c t o r o f 25% ( B r i n d l e y , 1962). The i n t i m a appears t o a c t as a molecular passage o f the l a r g e r  HPO4  f o r other m o l e c u l e s .  Absorption  -  s i e v e l i m i t i n g the  i o n , as d e s c r i b e d by P h i l l i p s  (1965)  from the rectum r e q u i r e s  molecules f i r s t pass through the c u t i c u l a r i n t i m a  that  before  c o n t a c t i n g the l a r g e e p i t h e l i a l c e l l s where mechanisms o f active': r e a b s o r p t i o n and a c i d i f i c a t i o n probably the i n t i m a probably  reside.  Hence  l i m i t s a b s o r p t i o n o f d i b a s i c orthophosphate  i n favor o f the monobasic  form.  Any p r e f e r e n t i a l a b s o r p t i o n o f  the b a s i c form o f the phosphate b u f f e r p a i r would be s e v e r e l y l i m i t e d by the low p e r m e a b i l i t y o f the i n t i m a to the d i v a l e n t form. The presence o f c a l c i u m i o n i n the medium does i n f l u e n c e the p e r m e a b i l i t y o f the i n t i m a to phosphate although the e f f e c t i s small.  This may be a charge e f f e c t ,  j u s t as i t i s p o s s i b l e  t h a t the changes i n pH i n f l i c t e d e x p e r i m e n t a l l y  on the i n t i m a  are e f f e c t i n g the charge on the membrane, and hence i t s permeability to ions.  An experiment to t e s t t h i s has been  c a r r i e d out by Chabun  (1967).  i o n s a t v a r i o u s pH v a l u e s ,  He s t u d i e d the movement o f c h l o r i d e  and showed t h a t p e n e t r a t i o n was a t  the same r a t e a t a l l pH v a l u e s .  I t can t h e r e f o r e be concluded  33  t h a t v a r i a t i o n s i n pH do not e f f e c t the charge on the membrane, and  therefore  the v a r i a t i o n i n p e n e t r a t i o n  phosphate forms must be I t can be  due  various  to d i f f e r e n c e s i n s i z e .  concluded t h a t the i n t i m a i s s l i g h t l y permeable  to phosphate a t i t s normal pH value, but the membrane s e v e r e l y b u f f e r p a i r , and reabsorption  of the  t h a t the pore s i z e of  l i m i t s e n t r y of the b a s i c form of  hence c a s t s doubt on  the  the theory o f s e l e c t i v e  o f the b a s i c form o f phosphate as a mechanism of  acidification. (b)  A c i d i f i c a t i o n of T r i s - H C l b u f f e r To i n v e s t i g a t e f u r t h e r whether phosphate b u f f e r i s  necessary f o r a c i d i f i c a t i o n , the  l i g a t e d and  occurred  i n j e c t i o n of T r i s - H C l b u f f e r  r i n s e d rectum was  performed.  I f no  into  acidification  w i t h t h i s b u f f e r , i t would suggest t h a t the phosphate  o f the p r e v i o u s l y for a c i d i f i c a t i o n .  i n j e c t e d b u f f e r s o l u t i o n might be  necessary  I f a c i d i f i c a t i o n p e r s i s t e d , i t would seem  u n l i k e l y t h a t there e x i s t s a p a r t i c u l a r mechanism f o r the uptake o f the T r i s molecule, and  t h a t more p r o b a b l y there  a c t i v e s e c r e t i o n of hydrogen ions or some a c i d i n t o the  had been an  (e.g.  carbonic)  lumen.  F i g . 6 shows the r e s u l t s o f i n j e c t i o n experiments on animals.  The  l e v e l of a c i d i t y reached a t e q u i l i b r i u m  comparable to t h a t o b t a i n e d w i t h M c l l v a i n e ' s buffer  ( i . e . 6.04  - 6.18  vs. 6.02).  w i t h both b u f f e r s i n 10 to 20 min. i d e n t i c a l l y and  judged to have no  five  was  phosphate-citrate  E q u i l i b r i u m was  reached  F i v e other animals t r e a t e d t r a c h e a l damage or  leaks  FIGURE 6 A c i d i f i c a t i o n o f 0.002M T r i s - H C l b u f f e r , pH 7.2-7.4. graph r e p r e s e n t s r e s u l t s from f i v e animals. l i n e s , mean values;  The  Horizontal  v e r t i c a l b a r s , standard e r r o r o f the mean.  7.5  I O  i  i  I  IO  2 0  3 0  MINUTES AFTER INJECTION OF BUFFER INTO RECTUM  34 f a i l e d to a c i d i f y the b u f f e r to a pH value of a t l e a s t 6.5 the f i r s t run.  This i s p o s s i b l y the r e s u l t of v a r y i n g  c u t i c u l a r p e r m e a b i l i t y to T r i s : have allowed  on  a more permeable i n t i m a might  i t s p e n e t r a t i o n to the c e l l l a y e r where i t would  be expected to cause damage of t r a n s p o r t a c t i v i t y .  The  c u t i c u l a r i n t i m a o f i n d i v i d u a l l o c u s t s i s known to e x h i b i t widely varying permeability properties ( P h i l l i p s ,  1967).  The r e s u l t s show t h a t a c i d i f i c a t i o n occurs i n the absence of phosphate b u f f e r .  They s u b s t a n t i a t e the c o n c l u s i o n drawn  from i n t i m a l p e r m e a b i l i t y s t u d i e s t h a t a b s o r p t i o n o f the b a s i c form of the phosphate b u f f e r p a i r i s not the o n l y mechanism of a c i d i f i c a t i o n , i f t h i s b u f f e r p a i r p l a y s any r o l e a t a l l . (c)  P o s s i b l e involvement of a c i d phosphate While the b a s i c form of the phosphate b u f f e r p a i r i s not  s e l e c t i v e l y taken up,  the p o s s i b i l i t y s t i l l remains t h a t the  a c i d form of the b u f f e r p a i r i s b e i n g s e c r e t e d i n t o the lumen. Before  i n v e s t i g a t i n g t h i s hypothesis  d i r e c t l y , i t was  necessary  to e s t a b l i s h where the g r a d i e n t s i n phosphate c o n c e n t r a t i o n o c c u r r r e d i n the animal,  and  so the phosphate  concentrations  o f the body f l u i d s were measured. R e s u l t s are given i n Table I I I . phosphate i n the M a l p i g h i a n the hemolymph.  The  The  concentration  t u b u l e f l u i d i s about twice t h a t of  c o n c e n t r a t i o n of phosphate i n r e c t a l  i s c o n s i d e r a b l y higher  of  than i n e i t h e r hemolymph or  fluid  Malpighian  tubule f l u i d ,  these d i f f e r e n c e s b e i n g h i g h l y s i g n i f i c a n t  ( p<0.001).  From a c o n s i d e r a t i o n o f these f i g u r e s alone, i t  TABLE I I I  Concentrations o f i n o r g a n i c phosphate found i n the body f l u i d s o f the l o c u s t .  Fluid  Hemolymph-serum  mM P O 4  6.24  S.D.  No. Obs.  Range  1.273  10  4.69-8.24  Malpighian tubule  14.6  5.65  5  9.5-23.9  Rectal f l u i d  47.9  5.89  9  14.0-81.4  35 c o u l d be supposed t h a t the high c o n c e n t r a t i o n o f phosphate i n the r e c t a l f l u i d i s due e i t h e r to s e c r e t i o n o f phosphate or to a c o n c e n t r a t i n g o f phosphate r e s u l t i n g from a b s o r p t i o n o f water i n the rectum. First, investigated.  the p a s s i v e movement o f a c i d phosphate was In p r e v i o u s  experiments i n v o l v i n g i n j e c t i o n o f  0.002M b u f f e r , the g r a d i e n t f o r d i f f u s i o n o f the a c i d form o f phosphate was  from the hemolymph i n t o the r e c t a l lumen.  i f a stronger b u f f e r  However,  (0.02M) were used, then the g r a d i e n t would  favour p a s s i v e movement o f H2P04~out of the rectum r a t h e r than inward.  This f o l l o w s from the f o l l o w i n g c o n s i d e r a t i o n s :  The phosphate c o n c e n t r a t i o n o f the hemolymph was found to be 6.24 mM/1. 7.13  (Table I I I ) .  A t the normal pH o f the hemolymph,  (Table X ) , i t can be c a l c u l a t e d from the Henderson-  H a s s e l b a l c h equation  t h a t the r a t i o o f d i v a l e n t to monovalent  phosphate i o n s i s 1 : 1.18.  Doing the same c a l c u l a t i o n  for  M c l l v a i n e ' s b u f f e r a t pH 7, the r a t i o o f d i v a l e n t to monovalent i o n s i s 1 : 1.6.  Then s i n c e the t o t a l amount o f phosphate i s  known, the amounts o f monovalent and d i v a l e n t phosphate can be calculated. IV.  A summary o f these c a l c u l a t i o n s i s g i v e n i n Table  T o t a l phosphate c o n c e n t r a t i o n f o r the b u f f e r was equated  to m o l a r i t y , s i n c e o n l y a few drops of c i t r a t e had been added to reach  t h i s pH.  (Phillips,  -  mv.  1965) with the r e c t a l lumen p o s i t i v e to the hemocoel  would support H2P04  The e l e c t r o p o t e n t i a l g r a d i e n t o f 20  a t h r e e - f o l d c o n c e n t r a t i o n g r a d i e n t , e.g. lOmM  i n the lumen would e q u i l i b r a t e with 3.38mM R^PO^" i n the  TABLE IV  Comparison o f amounts o f monovalent and d i v a l e n t i n o r g a n i c phosphate i n M c l l v a i n e ' s b u f f e r and locust  hemolymph.  m M PO, H P0  4  H P0 2  4  Hemolymph (pH 7.13)  2.86  3.38  0.002 M M c l l v a i n e ' s b u f f e r (pH 7.0)  0.77  1.23  7.7  12.3  0.02 M M c l l v a i n e ' s b u f f e r (pH 7.0)  36 hemolymph; t h i s value i s exceeded by 12.3mM the  H2PC>4~  in  0.02M b u f f e r . Seven l o c u s t s w i t h r e c t a l i g a t e d were i n j e c t e d w i t h 0.02M  Mcllvaine's  buffer.  The pH o f the i n j e c t e d b u f f e r dropped  u n i t s i n the f i r s t 10 min. and t h e r e a f t e r more s l o w l y A t 90 min. the b u f f e r had a pH value o f 6.39+0.14. a c i d i f i c a t i o n f o r the stronger t h a t f o r 0.002M b u f f e r  0.5  (Fig. 7).  The r a t e o f  b u f f e r was slow as compared t o  ( F i g . 3 ) , as expected.  E q u i l i b r i u m was  o n l y approached i n about 90 min. as compared t o 15-20 min. f o r 0.002M b u f f e r . due  to p a s s i v e  The r e s u l t s suggest t h a t a c i d i f i c a t i o n i s not d i f f u s i o n o f the a c i d i c form o f the phosphate  b u f f e r p a i r i n t o the lumen. While the p o s s i b i l i t y t h a t the a c i d form o f the phosphate b u f f e r p a i r i s moving i n t o the r e c t a l lumen p a s s i v e l y was t e s t e d by  experiments w i t h 0.02M b u f f e r , and i t was found t h a t  a c i d i f i c a t i o n occurred  even when the g r a d i e n t  movement o u t o f the rectum, there i s s t i l l  favoured  H2PO4  -  the p o s s i b i l i t y o f  a c t i v e s e c r e t i o n o f a c i d phosphate i n t o the lumen.  This was  i n v e s t i g a t e d by the f o l l o w i n g experimental procedure: 65 jal. of 0.002M M c l l v a i n e ' s pH  buffer  7.2-7.4 were i n j e c t e d i n t o t h e l i g a t e d l o c u s t rectum and  l e f t f o r 20 min.  The pH o f the a c i d i f i e d b u f f e r was measured  as w e l l as the c o n c e n t r a t i o n 1942). and  (0.0045;for two other cases) a t  o f i n o r g a n i c phosphate  Table V shows the r e s u l t s .  (Gomori,  F i v e animals were used,  three experiments were c a r r i e d out on each.  The volume  of f l u i d i n the rectum was n o t r i g o r o u s l y c o n t r o l l e d b u t  FIGURE 7  A c i d i f i c a t i o n o f 0.02M M c l l v a i n e ' s b u f f e r , pH 7.2-7.4. The graph r e p r e s e n t s r e s u l t s from seven animals.  H o r i z o n t a l bars,  mean values; v e r t i c a l bars, standard e r r o r o f the mean.  70 LU  6.5  u. u.  CD u_  6.0|  I  o.  5.5  IO  20  30  40  50  60  70  MINUTES AFTER INJECTION OF BUFFER INTO RECTUM  80  90  TABLE V  Phosphate  secretion  experiments.  The pH and phosphate  c o n c e n t r a t i o n o f r e c t a l f l u i d 20 min. f o l l o w i n g the i n j e c t i o n o f 65 > i l . o f 0.002 or 0.004 M M c l l v a i n e ' s buffer  Locust  1  2  3  4  5  (pH 7.2-7.4) i n t o l i g a t e d  Experiment  pH  rectum.  mM P O 4 Initial  Final  1  6.94  1.90  2  6.86  3  6.85  1.70  1  6.53  1.83  2  6.75  3  7.05  1.83  1  6.64  2.00  2  7.02  3  7.04  1.86  1  6.18  4.83  2  6.32  3  6.60  4.67  1  6.60  4.33  2  6.81  3  6.79  2.00  2.00  2.00  4.63  4.36  1.70  1.67  2.00  4.50  4.33 4.80  37 the 1.2M NaCl content o f the b u f f e r prevented  any s i z e a b l e water  a b s o r p t i o n over the time p e r i o d i n v o l v e d and consequent i n c r e a s e o f phosphate c o n c e n t r a t i o n from t h i s e f f e c t .  However,  some o f the v a r i a t i o n i n r e s u l t s can be a t t r i b u t e d to s l i g h t volume changes caused by water movement. I t was c a l c u l a t e d from the Henderson-Hasselbalch  equation  t h a t i f the pH was lowered from 7.2 to 6.5 o n l y by s e c r e t i o n o f a c i d phosphate then the o r i g i n a l have to r i s e to 6mM. to the extent  c o n c e n t r a t i o n o f 2mM would  The c o n c e n t r a t i o n o f phosphate never  t h a t was c a l c u l a t e d as necessary  given l e v e l of a c i d i f i c a t i o n  rose  t o reach a  d u r i n g any o f the f i v e  experiments.  For example, animal 2 lowered the b u f f e r pH to 6.53 with no i n c r e a s e i n b u f f e r phosphate c o n c e n t r a t i o n .  Furthermore, the  same phosphate c o n c e n t r a t i o n o f 1.83 mM was found even when the b u f f e r pH had a value o f o n l y 7.05 pH u n i t s . phosphate c o n c e n t r a t i o n o f 4.5 mM, to r i s e to 13.5 mM i n order  F o r an i n i t i a l  the value would be expected  to r e a c h pH 6.5, whereas a c t u a l l y  the pH went down to 6.18 and 6.32 w i t h no s i g n i f i c a n t phosphate c o n c e n t r a t i o n .  rise i n  Thus there does not appear t o be any  s e c r e t i o n o f phosphate which c o r r e l a t e s w i t h the hydrogen i o n concentration. The  carbon d i o x i d e t e n s i o n o f a c i d i f i e d r e c t a l I t i s p o s s i b l e t h a t the m e t a b o l i c  fluid  a c t i v i t y o f the r e c t a l  e p i t h e l i u m produces a high p a r t i a l pressure o f carbon d i o x i d e , and  hence c a r b o n i c a c i d , i n the e p i t h e l i u m and lumen o f the  rectum.  The lumen might then be i n e q u i l i b r i u m with a low pH  i n the e p i t h e l i u m  ( P h i l l i p s , 1961).  38 This hypothesis Mcllvaine's buffer  was t e s t e d by i n j e c t i n g a l k a l i n e  (.002M, pH 7.4) i n t o the l i g a t e d l o c u s t  rectum, and removing the a c i d i f i e d f l u i d 20-3 0 min. pH was measured a t  Q..5-1 min.  later.  The  and the r e s t o f the f l u i d kept  i n c a p i l l a r y t u b i n g f o r 3 h r . to a l l o w the carbon d i o x i d e i n the a c i d i f i e d f l u i d  to e q u i l i b r a t e with t h a t i n the a i r .  R e s u l t s a r e shown i n Table VI. b o t h a p o s i t i v e and negative was On  Small pH changes o c c u r r e d i n  direction.  The average i n c r e a s e  0.19 pH u n i t s and the average decrease was 0.16 pH u n i t s . t h i s b a s i s , l i t t l e o f the a c i d i t y o f r e c t a l f l u i d can be  a t t r i b u t e d to a high carbon d i o x i d e t e n s i o n i n the e p i t h e l i u m or lumen; the pH would have i n c r e a s e d with time i f carbon d i o x i d e were b e i n g given o f f from the b u f f e r . Readings o f pH were taken i n a l l experiments up to t h i s p o i n t u s i n g the m i c r o e l e c t r o d e  G2 52C,  i t s c h i e f advantage being  the s m a l l sample s i z e r e q u i r e d  (5>ul.).  The anaerobic  e l e c t r o d e G297 r e q u i r e d too l a r g e a volume to be used r o u t i n e l y for  a l l measurements  (50 >ul.)  an a d d i t i o n a l way o f checking  b u t i t was used a t t h i s stage as whether a c i d i f i e d b u f f e r had a  high p a r t i a l p r e s s u r e o f carbon d i o x i d e . obtained  with the anaerobic  I f the readings  e l e c t r o d e were s i g n i f i c a n t l y more  a c i d as compared t o those w i t h the m i c r o e l e c t r o d e ,  then t h i s  would i n d i c a t e t h a t s i g n i f i c a n t amounts o f carbon d i o x i d e were being  l o s t to the atmosphere i n u s i n g the open system. Mcllvaine's buffer  for  2 0 min.  (pH 7.4, 0.002M) was i n j e c t e d and l e f t  i n the l i g a t e d rectum.  On removal from the animal.  TABLE VI  Change i n pH o f a c i d i f i e d b u f f e r on exposure to a i r f o r 3 hr.  Experiment  pH o f b u f f e r  Difference  1 min.  3 hr.  1  5.98  6.21  + 0.23  2  5.81  5.65  - 0.16  3  6.29  6.51  + 0.22  4  6.12  5.94  - 0.18  5  6.00  5.87  - 0.13  6  6.22  6.33  + 0.11  Mean  +0.02  39 the sample was  kept under p a r a f f i n o i l u n t i l pH was  measured.  The sample c o u l d be drawn d i r e c t l y i n t o the c a p i l l a r y o f the e l e c t r o d e from beneath the p a r a f f i n o i l i n many cases; maximum exposure to the a i r was the change i n pH,  0.5-1  min.  i n others,  Within t h i s  time,  i f carbon d i o x i d e were i n f a c t b e i n g given  o f f , would be of the order of 0.05  pH u n i t s .  (In 5 min.,  the  experimental b u f f e r bubbled w i t h carbon d i o x i d e changed from pH 6.10  to pH 6.3 0) . Table VII g i v e s the r e s u l t s o b t a i n e d w i t h the  system.  The pH values are not s i g n i f i c a n t l y lower  anaerobic  (p=0.05) than  values o b t a i n e d p r e v i o u s l y i n experiments i n which no  special  p r e c a u t i o n s were taken to prevent escape of carbon d i o x i d e (the r e a d i n g f o r 20 min. E r r o r due  i n F i g . 3, pH 5.95*0.27).  to carbon  d i o x i d e l o s s i s not s i g n i f i c a n t  then,  and once again a h i g h p a r t i a l p r e s s u r e of carbon d i o x i d e does not appear to be the immediate cause of the a c i d i f i c a t i o n of the i n j e c t e d b u f f e r . Changes i n the p r e p a r a t i o n ' s c a p a c i t y to a c i d i f y b u f f e r (a)  Adjustment to hemolymph pH In the experiments i n which M c l l v a i n e ' s b u f f e r and  T r i s - H C l were i n j e c t e d i n t o the l i g a t e d l o c u s t rectum, no more than three runs were performed on the same animal s i n c e a p r o g r e s s i v e change was  noted i n the p r e p a r a t i o n .  For example,  pH values o b t a i n e d would f r e q u e n t l y f o l l o w t h i s p a t t e r n , given here f o r 0.002M a l k a l i n e M c l l v a i n e ' s b u f f e r :  TABLE V I I  The pH o f r e c t a l f l u i d as measured w i t h an anaerobic e l e c t r o d e system 20 min. a f t e r i n j e c t i n g 6 0 ^ 1 . o f 0.002 M M c l l v a i n e ' s b u f f e r  (pH 7.4) i n t o the l i g a t e d  rectum.  Preparation 1  Experiment  pH  1  6.29  2  6.48  2  1  6.09  3  1  5.91  2  6.34  3  6.40  4  1  6.30  . 5  1  6.08  2  6.23  3  6.31  Mean  6.24  St. deviation  0.17  St. error  0.05  40 Run 2 min. 20 min.  1 2 6.60 6.84 6.11 6.40  3 7.00 6.52  F a i l u r e to a c i d i f y b u f f e r a f t e r three to four runs i n a g i v e n p r e p a r a t i o n might" c o n c e i v a b l y show some r e l a t i o n s h i p to hemolymph pH, i . e . to a change i n the c o n c e n t r a t i o n g r a d i e n t due to a l t e r e d hemolymph pH.  I n j e c t i o n o f a c i d b u f f e r i n t o the  rectum might cause the hemolymph to become more a c i d , and i n j e c t i o n o f a l k a l i n e b u f f e r might cause the hemolymph to become more a l k a l i n e .  In the case o f the l a t t e r ,  pH g r a d i e n t observed d u r i n g the experiments might r e p r e s e n t an adjustment  the reduced then  to the higher pH o f the hemolymph  r e s u l t i n g i n r e d u c t i o n o f the s e c r e t i o n o f hydrogen  ions  into  the lumen. In the experiments, 65 / a l . o f e i t h e r a c i d or a l k a l i n e Mcllvaine's buffer  (0.002M) we're i n j e c t e d i n t o the l i g a t e d  rectum  (the pH v a l u e s f o r these same experiments are g i v e n i n F i g . 3 ) . A t the end o f 3-4 experiments on a g i v e n animal, the pH o f the hemolymph was measured.  Values a r e g i v e n below f o r 15  animals: pH o f i n j e c t e d b u f f e r 7.2-7.4 3.8-3.95  F i n a l pH o f Hemolymph Mean+S.D. Range 7.07-0.15 6.84-7.26 7.03+0.13 6.83-7.17  There i s no s t a t i s t i c a l d i f f e r e n c e between the hemolymph pH o f those animals i n j e c t e d w i t h a c i d b u f f e r compared to those i n j e c t e d w i t h a l k a l i n e b u f f e r , though pH v a l u e s o f both are s i g n i f i c a n t l y lower than c o n t r o l animals d i s c u s s e d i n the next s e c t i o n .  (unoperated), as  While the data do not demonstrate  41 t h a t the animals are a d j u s t i n g a c i d s e c r e t i o n i n response to an a l t e r e d hemolymph pH,  i t cannot be  concluded t h a t the animals  do not a d j u s t s i n c e i t i s always p o s s i b l e t h a t the change o f pH r e q u i r e d to i n i t i a t e r e g u l a t i o n i n any smaller  i n d i v i d u a l may  be  than the v a r i a t i o n i n hemolymph pH of the whole  population. The  same change a f t e r three runs was  i n which T r i s - H C l b u f f e r was  noted i n experiments  i n j e c t e d i n t o the l i g a t e d rectum  as had been observed w i t h M c l l v a i n e ' s b u f f e r , although f a i l u r e i n the case of the T r i s b u f f e r cannot be e n t i r e l y from probable c e l l u l a r damage i f the T r i s molecule to the e p i t h e l i u m (b)  A l k a l i n e pH  separated penetrated  (see e a r l i e r s e c t i o n ) . values  When f a i l u r e o f the l i g a t e d rectum to a c i d i f y i n j e c t e d f l u i d was due  f i r s t encountered, i t was  to damage to the p r e p a r a t i o n  a l s o a l t e r the pH q u e s t i o n was  to h i g h values  not c l e a r whether t h i s  was  or whether the rectum c o u l d as w e l l as a c i d v a l u e s .  asked whether, w i t h s u c c e s s i v e  The  i n j e c t i o n s , the  pH of the b u f f e r ever f l u c t u a t e d s i g n i f i c a n t l y i n the a l k a l i n e d i r e c t i o n to a value  higher  than the pH expected i f hydrogen  ions were d i s t r i b u t e d p a s s i v e l y across i s , pH  7.4  the r e c t a l w a l l .  i s the pH p r e d i c t e d from the Ussing  That  equation f o r  r e c t a l f l u i d a t e q u i l i b r i u m i f hydrogen ions were d i s t r i b u t e d p a s s i v e l y across  the r e c t a l w a l l  of the rectum i s 2 0 mv.  ( P h i l l i p s , 1961).  The  p o s i t i v e to the hemocoel so t h a t  pH of r e c t a l f l u i d should be  0.3  pH u n i t s above t h a t of  lumen the the  42 hemolymph (7.13) a t e q u i l i b r i u m , p r o v i d e d t h a t hydrogen ions move a c r o s s the r e c t a l w a l l by d i f f u s i o n o n l y . q u e s t i o n , pH values  taken 30 min.  To answer t h i s  a f t e r i n j e c t i o n of 0.002M  T r i s - H C l b u f f e r (pH 6.65-7.4) d u r i n g the second or  third  experiment on the same animal were compared to the  original  pH of the b u f f e r i n j e c t e d i n t o the rectum. presented may  i n Table V I I I .  I t can be  These values  seen t h a t w h i l e the b u f f e r  not have been a c i d i f i e d by the rectum to the u s u a l  (pH 5.19-6.1), i t has a l k a l i n e than pH  7.4.  are  extent  i n no case become s i g n i f i c a n t l y more Thus any v a r i a t i o n i n pH i s i n the  a c i d d i r e c t i o n , although  f o r animals 6 and  to t h a t value expected p a s s i v e l y .  7 the pH went up  F a i l u r e to a c i d i f y i n these  cases appears to be caused by damage to the p r e p a r a t i o n . (c)  D e t e r i o r a t i o n of p r e p a r a t i o n The o p e r a t i o n and repeated b u f f e r i n j e c t i o n s i n t o the  l i g a t e d rectum may  cause i n j u r y to the l o c u s t and r e s u l t i n a  f a i l u r e to r e g u l a t e hemolymph pH w i t h i n i t s u s u a l l i m i t s . way  One  t h i s c o u l d be t e s t e d would be to compare the pH o f hemolymph  of animals a t the end of experiments to the pH i n animals t h a t had not been s u b j e c t e d to the o p e r a t i o n .  The pH values f o r  hemolymph f o r animals which had notundergone the were drawn from Tables X and X I I .  operation  These values were compared  to the pH values f o r l o c u s t s i n which the l i g a t e d rectum  was  i n j e c t e d w i t h e i t h e r a c i d or a l k a l i n e M c l l v a i n e ' s b u f f e r (section a).  Animals s u b j e c t e d to the l i g a t i o n and  injection  procedures d i d i n f a c t have a hemolymph pH s i g n i f i c a n t l y  lower  TABLE V I I I  The pH o f 0.002M T r i s - H C l  b u f f e r 30 min. a f t e r  i n j e c t i o n o f 65 yul. i n t o the l i g a t e d rectum f o r the same  second or t h i r d r e p l i c a t e  experiment on the  locust.  B u f f e r pH Animal  Original  Final  (30 min.)  1  7.28  7.25  2  7.43  7.51  3  6.90  7.07  4  7.08  7.21  5  6.98  6.92  6  6.65  7.38  7  6.65  7.67  TABLE IX  A c i d i f i c a t i o n o f 0.002 M M c l l v a i n e ' s b u f f e r w i t h time after  Animal  i n j e c t i o n o f 50  Time b u f f e r l e f t i n rectum  into ligated  rectum.  B u f f e r pH Original  Final  1  70 min.  7.2  5.76  2  70 min.  7.2  6.38  3  2 hr.  7.10-7.15  6.36  4  2 hr.  7.10-7.15  5.91  5  2.5 h r .  7.25  5.91  6  2.5 h r .  7.25  6.83*  7  2.5 h r .  7.21  6.19  Mean  6.09  S.D.  0.261  S.E.  0.107  43 than the c o n t r o l animals  (p=0.05).  T h i s may  r e f l e c t a general  d e t e r i o r a t i o n o f the p r e p a r a t i o n as a whole, and a f a i l u r e to r e g u l a t e hemolymph pH w i t h i n i t s normal l i m i t s . animal may  A l s o the  have been unable to e x c r e t e a c i d due to l i g a t i o n .  P o s s i b l e d e t e r i o r a t i o n o f the p r e p a r a t i o n was by another method, i . e . by t e s t i n g how c o u l d m a i n t a i n the pH g r a d i e n t .  checked  long the l i g a t e d  rectum  To check whether f a i l u r e to  a c i d i f y a f t e r three runs i s due to the death o f the p r e p a r a t i o n w i t h i n the time o f the experiment, b u f f e r was  the f i r s t i n j e c t i o n o f  l e f t i n the rectum f o r a l e n g t h o f time equal to or  g r e a t e r than the time n o r m a l l y taken to perform t h r e e i n j e c t i o n s . I f the p r e p a r a t i o n were i n f a c t dying, then the pH g r a d i e n t a c r o s s the membrane would no longer be maintained. t h a t the membrane i s f r e e l y permeable  T h i s assumes  t o the a c i d i f y i n g  entity  i n both d i r e c t i o n s , and t h a t there i s not s e c r e t i o n o f a c i d which cannot b a c k - d i f f u s e . s i n c e i t was  The assumption appears  justified  shown p r e v i o u s l y t h a t the system approaches  the  same a c i d pH whether an a c i d or an a l k a l i n e b u f f e r i s i n j e c t e d ( F i g . 3), and hence i s f r e e l y permeable Seven animals were s t u d i e d ;  to hydrogen  ions.  50>ul. o f 0.002  M c l l v a i n e ' s b u f f e r , pH 7.10-7.25,were i n j e c t e d i n t o the l i g a t e d rectum f o r times v a r y i n g from 70 min. to 2.5 h r . g i v e n i n Table IX.  Only one out o f the seven animals t e s t e d  f a i l e d to a c i d i f y the experimental b u f f e r . pH value was 0.19  R e s u l t s are  The  equilibrium  6.09-0.26; t h a t f o r o r i g i n a l experiments was  (Fig. 3).  6.02 +  44 The p r e p a r a t i o n across  i s able t o m a i n t a i n  the pH g r a d i e n t  the rectum f o r a t l e a s t 2.5 h r . so t h a t f a i l u r e t o  a c i d i f y a f t e r three runs i s n o t due to the death o f the e p i t h e l i u m even though the pH o f the hemolymph o f t r e a t e d animals does tend to drop w i t h i n t h i s l e n g t h o f time. Amount and r a t e o f a c i d s e c r e t i o n In order  to estimate  the q u a n t i t y o f a c i d s e c r e t e d by  the r e c t a l e p i t h e l i u m , both 0.02M and 0.002M M c l l v a i n e ' s b u f f e r were t i t r a t e d w i t h 0.1N HCl. which was normally  Amaranth dye (2mg./10 ml.),  added t o i n j e c t e d b u f f e r s , was i n c l u d e d .  The  pH was measured w i t h a Radiometer Type G202B g l a s s e l e c t r o d e .  The  values  i n F i g . 8 and F i g . 9 r e p r e s e n t  titrations.  From the t i t r a t i o n curves,  the average from  three  i t was c a l c u l a t e d  t h a t 3.9 jil. o f 0.1N HCl a r e needed to a c i d i f y 65 j u l . o f 0.02M b u f f e r from pH 7.2-7.4 t o pH 6.39; and 0.68 >ul. o f 0.1N HCl i n the case o f 0.002M b u f f e r to reach a l e v e l o f 6.02. . Over the pH range 7.4 to 6.0, the b u f f e r i n g c a p a c i t y i s approximately constant,  so t h a t 0.007 >uequiv. H  +  were needed to lower 1  o f 0.02M b u f f e r 1 pH u n i t ; and 0.0007 >uequiv. f o r 0.002M b u f f e r . The  c a l c u l a t i o n f o r 0.02M b u f f e r g i v e s i d e n t i c a l r e s u l t s to  Phillips  (1961).  absorption  Then, assuming t h a t there was no s i g n i f i c a n t  o f b u f f e r from the rectum w i t h i n 5 min., there must  have been a t o t a l n e t i n f l u x o f a t l e a s t 0.11 ;uequiv. H , or +  l.j4yuequiv. H / h r . t o account f o r the observed a c i d i f i c a t i o n +  o f 0.02M b u f f e r . >uequiv. H / h r . +  P h i l l i p s gave a minimal estimate  o f 0.4  Both these estimates, c o u l d be much lower than  FIGURE 8 T i t r a t i o n o f 10 ml. o f 0.002M M c l l v a i n e s b u f f e r 1  0.IN H C l .  Each p o i n t i s the average from three  Amaranth dye was  included.  with titrations.  pH OF BUFFER  FIGURE 9 T i t r a t i o n o f 10 ml. o f 0.02M M c l l v a i n e ' s b u f f e r  with  0.1N  titrations.  H C l . . Each p o i n t i s the average from three  Amaranth dye was  included.  pH OF BUFFER G*  O  sj  >j  O  In  O  . «i-'  45 the a c t u a l s e c r e t i o n r a t e s i n c e the value c a l c u l a t e d depends on determining  a c c u r a t e l y the i n i t i a l  s l o p e of the  curve.  Moreover, the r a t e o f back d i f f u s i o n , which i n c r e a s e s as a c i d i f i c a t i o n proceeds, done f o r the f i r s t reasons:  i s not known.  5 min.  The  calculation  of the stronger b u f f e r f o r  was  two  the c o n c e n t r a t i o n g r a d i e n t i s lowest a t the  b e g i n n i n g of the curve and hence g i v e s the c l o s e s t e s t i m a t i o n of  r a t e i n the absence of a c o n c e n t r a t i o n g r a d i e n t ; times were  c o n s i d e r e d more accurate f o r the stronger b u f f e r . P o s s i b l e r o l e of the e x c r e t o r y system i n pH r e g u l a t i o n (a)  Tolerance  level  In the course of t h i s i n v e s t i g a t i o n , some p r e l i m i n a r y o b s e r v a t i o n s were made concerning pH r e g u l a t i o n and  the  p o s s i b l e r o l e of the e x c r e t o r y system, and more p a r t i c u l a r l y rectal acidification,  i n t h i s process.  i n j e c t e d with v a r i o u s amounts of 0.1N  Unstarved  l o c u s t s were  HCl to determine the  amount of a c i d t h a t S c h i s t o c e r c a can t o l e r a t e and whether i t can r e g u l a t e hemolymph pH when i t has been loaded w i t h The animals  were a n a e s t h e t i z e d and i n j e c t e d beneath the  c u t i c l e o f the t h i r d abdominal segment. for  acid.  One  hour was  d i s t r i b u t i o n of the a c i d throughout the animal.  allowed Hemolymph  samples were taken from the j o i n t o f a jumping l e g over a 72 hr. p e r i o d .  Animals were not f e d d u r i n g the  experimental  period. A l l measurements o f hemolymph pH made d u r i n g  these  experiments were done w i t h the m i c r o e l e c t r o d e , s i n c e o n l y  46 10-15 jil. c o u l d be taken a t each time o f sampling.  To  check  the e r r o r i n v o l v e d due t o l o s s o f c a r b o n i c a c i d due to sampling and time o f measurement  (Buck/  1953;  Phillips,  1961),  the pH  values o f l o c u s t hemolymph were compared u s i n g the two types of glass electrodes: Radiometer  Radiometer  Type G2 52C,  exposed to a i r , and  Type G297, which i s a n a e r o b i c .  About 60 >ul. o f hemolymph c o u l d be taken from the l e g j o i n t o f the l o c u s t i n t o a c a p i l l a r y tube.  This was  done as  q u i c k l y as p o s s i b l e , and the sample then kept under p a r a f f i n o i l u n t i l measured, f o r the a n a e r o b i c system.  For the o t h e r type,  hemolymph was k e p t i n 5 JOLI. c a p i l l a r y tubes and measured w i t h i n 2-5  min. The r e s u l t s f o r e i g h t animals are p r e s e n t e d i n Table X.  e r r o r was Phillips  found to be about 0.03 (1961) who  pH u n i t s .  This agrees w i t h  e s t i m a t e d t h a t the e r r o r i n measured pH value  f o r l o c u s t hemolymph i s l e s s than 0.1 pH The t o l e r a n c e l e v e l was 0.1N  HCl, s i n c e t h e r e was  unit.  judged to be about 100  of  some evidence a t l e a s t o f r e c o v e r y to  normal pH v a l u e s i n t h i s group, i . e . animals 2 and 3 i n Table XI a t 72 h r . had hemolymph pH v a l u e s o f 7.40 respectively.  and  7.12  Animals g i v e n e i t h e r 100 or 150>ul. o f 0.1N  as w e l l as the c o n t r o l animals f o r more than 72 h r .  The  (Tables X I I and X I I I ) ,  HCl,  survived  Table XII shows t h a t l o s s o f hemolymph  due to sampling does not e f f e c t pH, and Table X I I I shows t h a t there i s not a s i g n i f i c a n t change i n pH caused by the use o f d i s t i l l e d water r a t h e r than s a l i n e f o r i n j e c t i n g  acid.  TABLE X  Comparison o f hemolymph pH using two types o f g l a s s e l e c t r o d e s , as an estimate  o f the e r r o r due to l o s s  of carbonic a c i d .  Animal  Micro-electrode reading  Anaerobic reading  TV-F-F  Dxtterence  1  7.37  7.274  0.096  2  7.17  7.070  0.100  3  7.13  7.122  0.008  4  7.08  7.036  0.044  5  7.08  7.098  -0.018  6  7.13  7.121  0.009  7  7.23  7.178  0.052  8  7.08  7.110  -0.030  Mean  7.17  7.13  S.D.  0.10  0.0727  S.E.  0.0354  0.0257  0.033  TABLE XI  The pH o f hemolymphwith time a f t e r i n j e c t i o n o f 100 yul. o f 0.IN HCl i n t o unstarved a d u l t male locusts.  PH  Animal  Final Condition o f animal  1-2 hr  6-7 h r .  24 h r .  1  7.08  6.98  6.95  2  7.12  7.20  7.31  7.40  strong  3  6.68  6.97  7.09  7. 14  strong  4  6.90  6.72  6.78  6. 87  weak  Mean  TABLE X I I  •  72 hr.  -  weak  6.95  The pH o f hemolymph of  c o n t r o l (uninjected)  l o c u s t s w i t h time.  pH Animal  0 hr.  6 hr.  24 h r .  72 hr  1  7.43  7.38  7.35  7.36  2  7.22  7.42  7.56  7.30  3  7.28  7.34  7.46  7.32  4  7.33  7.37  7.40  7.44  TABLE X I I I  The pH o f hemolymph w i t h time a f t e r i n j e c t i o n of 150  o f d i s t i l l e d water i n t o a d u l t  unstarved  male l o c u s t s  Animal  p  H  1-2 h r .  6 nr.  24 h r .  72 h r .  1  7.53  7.54  7.54  7.64  2  7.44  7.33  7.45,  7.53  3  7.48  7.22  7.40  7.57  4  7.48  7.38  7.47  7.52  47 (b) pH o f f e c a l p e l l e t s o f animals i n j e c t e d w i t h a c i d The pH o f f e c a l p e l l e t s o f animals i n j e c t e d w i t h a c i d was  measured to i n v e s t i g a t e whether the degree o f  a c i d i f i c a t i o n o f e x c r e t a (and hence perhaps b u f f e r ) the a c i d l o a d o f an animal. v i a the e x c r e t a , i n which  reflects  That i s , excess a c i d might be  lost  case the r e c t a l a c i d i f i c a t i o n c o u l d  w e l l be important i n the pH r e g u l a t i o n o f the animal as a whole An a l t e r n a t i v e i s t h a t the a c i d i f i c a t i o n may to water,  Na+  and K  +  t r a n s p o r t i n the  To determine i f excess a c i d was  simply be coupled  rectum. i n f a c t l o s t i n the  e x c r e t a , s i x animals were i n j e c t e d w i t h 1 0 0 j a l . o f 0.1N  HCl,  t h i s b e i n g an amount t h a t the animal was known to t o l e r a t e . F e c a l p e l l e t s were c o l l e c t e d b e f o r e the i n j e c t i o n and f o r 72 hr thereafter.  N e i t h e r food nor water was  d u r i n g the c o l l e c t i o n time.  g i v e n to the animals  Hemolymph samples were taken from  the l e g j o i n t o f u n a n a e s t h e t i z e d animals a t the end of the experiment,  and pH measured to determine whether any r e c o v e r y  had o c c u r r e d .  The r e s u l t s are g i v e n i n Table XIV.  In a l l animals examined except animal 2, the e x c r e t a became an average o f 0.3  u n i t s more a c i d a f t e r the i n j e c t i o n  of HCl; moveover the e x c r e t a o f t h a t e x c e p t i o n was a c i d than the o t h e r s .  a l r e a d y more  The two animals from which the  number o f f e c a l p e l l e t s were c o l l e c t e d the lowest f i n a l hemolymph v a l u e s :  least  (no. 1 and no. 6) had  pH 6.88  and 6.66^  However,  the other four animals seem to have been a b l e to e x c r e t e the a c i d i n f e c a l p e l l e t s and showed a r e c o v e r y to normal hemolymph v a l u e s .  The i n i t i a l  hemolymph pH was  estimated  TABLE XIV  The pH o f f e c a l p e l l e t s o f animals i n j e c t e d 100 Jul. o f 0.1N HCl, f o l l o w e d over 72 h r .  with Distilled  water added f o r moisture except f o r u n d e r l i n e d v a l u e s .  Hours a f t e r injection  Animal 1  2  3  4  5  6  0  6.23  5.20  7.70  5.98  6.40  5.99  1-5  5.55  -  6.65  5.75  6.18  5.50  5.68  4.72  5.02  5.50  4.53  20-30  -  4.79  6. 00 5.48  40-50  5.51  60-72  5.19  5.41  5.08  5.48  6.40  5.50  5.32  5.28  5.82  5.38 5.48 5. 52  Hemolympht e r m i n a l value  6.88  7.02  7.22  7.26  7.27  6.66  48 p r e v i o u s l y a t pH 7.17  (Table X ) , and the amount o f a c i d  lowers the pH to about 6.95 (Table X I ) .  injected  The values o f hemolymph  pH measured a t the end o f 72 h r . (6.66-7.27) were comparable to those i n Table XI (6.87-7.40),  as expected s i n c e the amount o f  a c i d i n j e c t e d was the same f o r both groups o f animals.  That  the two animals which Showed the l e a s t r e c o v e r y t o normal hemolymph pH v a l u e s a l s o e x c r e t e d the l e a s t number o f f e c a l p e l l e t s may i n d i c a t e t h a t the amount o f m a t e r i a l i n the gut a t the time o f a c i d i n j e c t i o n l i m i t s the amount o f a c i d i n the e x c r e t a .  A t l e a s t two animals  eliminated  (no. 3 and no. 5) show a  t r e n d o f p r o g r e s s i v e l y more a c i d p e l l e t s and then a r e t u r n t o l e s s a c i d v a l u e s , presumably  as the a c i d l o a d i s reduced. A l l  animals were very a c t i v e a t the end o f the experiment,  regardless  o f the degree o f r e c o v e r y o f hemolymph pH values to a normal level. These p r e l i m i n a r y o b s e r v a t i o n s do suggest t h a t the l e v e l of a c i d i f i c a t i o n o f the e x c r e t a r e f l e c t s the a c i d l o a d o f the animal.  The e x c r e t o r y system appears to be one way a v a i l a b l e  to the animal f o r e l i m i n a t i n g excess a c i d i t y .  49 DISCUSSION In i n v e s t i g a t i n g the a c i d i f i c a t i o n o f r e c t a l f l u i d , the pH o f d i f f e r e n t r e g i o n s o f the gut was measured t o see i f the normal a c i d i t y o f r e c t a l contents a c i d from some a n t e r i o r r e g i o n .  was due to i n t r o d u c t i o n o f The values  found agree  roughly  w i t h those found i n the l i t e r a t u r e , many o f which have been determined c o l o r i m e t r i c a l l y r a t h e r than e l e c t r o m e t r i c a l l y . F o r example, Hastings seven orthopterans  and Pepper  (1943) s t u d i e d the body f l u i d s o f  and found t h a t the r e g u r g i t a t e d d i g e s t i v e  j u i c e s from s i x s p e c i e s o f grasshoppers f e l l w i t h i n the pH range o f 5.2 to 5.8.  This i s comparable t o the value o f 5.46  found f o r the mid v e n t r i c u l u s o f the l o c u s t where the brown f l u i d occurs.  Swingle (1931) found t h a t the m a j o r i t y o f  i n s e c t s s t u d i e d , r e p r e s e n t i n g seven orders, had a s l i g h t l y a c i d i c d i g e s t i v e t r a c t , and t h a t the pH u s u a l l y i n c r e a s e d from the mouth to the f o r e g u t and a n t e r i o r r e g i o n o f the midgut, and then decreased from the p o s t e r i o r r e g i o n o f the midgut through the hindgut.  The l o c u s t f o l l o w s t h i s p a t t e r n , a t l e a s t f o r  the areas s t u d i e d from midgut to rectum. Uvarov, 1966) s t u d i e d pH i n the a l i m e n t a r y  Payne (1961, c i t e d by canal of Schistocerca  g r e g a r i a ; he gave values o f 5.4 f o r f o r e g u t , 6.2-711 f o r midgut and  7.0 f o r hindgut.  Hindgut i n c l u d e s the ileum,  c o l o n and  rectum, so i t i s d i f f i c u l t t o compare the one f i g u r e f o r hindgut pH  to those f o r ileum and rectum as given i n Table  1.  Values  of 6.0 to 6.2 a r e n o t uncommon i n the hindgut o f grasshoppers and  l o c u s t s (Uvarov, 1966).  50 The a l k a l i n i t y i n the r e g i o n o f M a l p i g h i a n tubule e n t r y may be due to t h e i r f l u i d c o n t r i b u t i o n to the gut here.  It is  known t h a t the M a l p i g h i a n t u b u l e s e l a b o r a t e the primary excretory f l u i d i n Schistocerca ( P h i l l i p s , t h i s f l u i d i s m o d i f i e d by the rectum.  1965), and t h a t  One f e a t u r e o f the  a l t e r a t i o n i s a change i n the hydrogen-ion  concentration.  Ramsay (1956) working on e x c r e t i o n by the M a l p i g h i a n tubules o f the s t i c k i n s e c t , Dixippus morosus  (Orthoptera) found t h a t the  d i f f e r e n c e i n pH between u r i n e and serum i s somewhat v a r i a b l e but i n a l l cases the output o f the M a l p i g h i a n t u b u l e s was a l k a l i n e to serum b u t became a c i d i n the rectum.  Measurements  made o f pH o f r e c t a l f l u i d expressed through the anus were between 3.5 and 4.5 i n the s t i c k i n s e c t .  R e c t a l pH values  found  here were higher than both those g i v e n by Ramsay above and by Phillips  (1961) who found the pH o f r e c t a l contents to be  4.73±0.32 and the pH o f e x c r e t e d f e c e s to be 5.3 0 0.36 f o r the +  locust.  These a r e to be compared to pH values o f 5.91JT0.44 and  5.95-0.55 r e s p e c t i v e l y i n the p r e s e n t study.  The d i f f e r e n c e s  f o r the same experimental animal may be a t t r i b u t a b l e to d i f f e r e n c e s i n the e l e c t r o d e system used f o r d e t e r m i n a t i o n , or to d i f f e r e n c e s i n d i e t o f the animals.  In the case o f Dixippus,  there i s a s p e c i e s d i f f e r e n c e to c o n s i d e r as w e l l . There was no s i g n i f i c a n t d i f f e r e n c e between the pH of gut contents of c o n t r o l animals and those f e d t e t r a c y c l i n e i n the d i e t to k i l l o f f the gut f l o r a .  A change i n e i t h e r the  p o s i t i v e or n e g a t i v e d i r e c t i o n was c o n c e i v a b l e s i n c e F l e t c h e r and Haub (1933, c i t e d by House, 1965) d i d f i n d t h a t the pH  51 o f the a l i m e n t a r y c a n a l o f Phormia r e g i n a (Diptera) l a r v a e i s s l i g h t l y higher i n those r e a r e d n o n a s e p t i c a l l y than i n those reared a s e p t i c a l l y .  In B l a t e l l a germanica (Orthoptera),  Wigglesworth (1927) e x p l a i n e d a c i d i t y i n the crop as due t o the a c t i v i t i e s o f microorganisms carbohydrate  a c t i n g on sugar:  with a  food the pH was 4.8, w i t h p r o t e i n 6.3.  No  d i f f e r e n c e s o f t h i s magnitude were found i n S c h i s t o c e r c a by treatment w i t h a n t i b i o t i c , and so i t has been concluded t h a t b a c t e r i a l f e r m e n t a t i o n does n o t account  f o r the observed  a c i d i f i c a t i o n i n the rectum, nor f o r v a r i a t i o n i n pH along the l e n g t h o f the a l i m e n t a r y c a n a l . appears  Rather  the pH o f gut contents  to r e f l e c t the pH o f s e c r e t i o n s i n t o the gut, the  product o f a b s o r p t i v e processes by the gut l i n i n g p l u s the products o f d i g e s t i o n . The a c i d i t y i n the rectum  i s n o t due to the i n t r o d u c t i o n  o f a c i d from any a n t e r i o r r e g i o n o f the gut, s i n c e the rectum was always a c i d to a l l other r e g i o n s p o s t e r i o r to the M a l p i g h i a n t u b u l e s and s i n c e the same degree o f a c i d i f i c a t i o n was achieved by the l i g a t e d rectum.  There was some i n d i c a t i o n t h a t the  hindgut i n g e n e r a l may a c i d i f y the a l k a l i n e m a t e r i a l which enters i t ,  as evidenced by a change i n pH from 7.94 to 7.13,  the l a t t e r value taken from the a n t e r i o r p a r t o f the hindgut ( a n t e r i o r ileum) o f c o n t r o l animals.  A l t e r n a t i v e l y there may  be some forward movement o f a c i d from the rectum; or the decrease from pH 7.94 to 7.13 c o u l d a l s o mean t h a t the area under c o n s i d e r a t i o n f a i l s  to m a i n t a i n the o r i g i n a l g r a d i e n t , the  change r e s u l t i n g from n e t d i f f u s i o n o f hydrogen ions a c r o s s the gut w a l l w i t h the hemocoel.  52 The  same a c i d pH i s a t t a i n e d i f a more a l k a l i n e or more  a c i d buffer i s introduced the e p i t h e l i u m  i n t o the rectum.  T h i s suggests t h a t  i s permeable t o hydrogen i o n s , and agrees w i t h  the r e s u l t s o f P h i l l i p s p o s s i b i l i t y that  (1961).  He, too, r e j e c t e d the  ' e q u i l i b r i u m i s not a t t a i n e d because the  r e c t a l w a l l i s r e l a t i v e l y impermeable t o hydrogen ions seems u n l i k e l y i n view o f the r a p i d a b s o r p t i o n s a l t s from the r e c t u m ) .  o f water and  A c i d i f i c a t i o n occurred  1  (which  even when an  a n t i b i o t i c was i n c l u d e d i n the i n j e c t e d b u f f e r which agrees w i t h the r e s u l t s o f P h i l l i p s rather  (1961) although he used  than t e t r a c y c l i n e .  terramycin  Both are wide-spectrum a n t i b i o t i c s .  Furthermore, there was found to be n e i t h e r s i g n i f i c a n t nor s u f f i c i e n t slow r e l e a s e o f hydrogen ions by f e c a l m a t e r i a l over the experimental p e r i o d to a c i d i f y i n j e c t e d b u f f e r s o l u t i o n s . P h i l l i p s had found p r e v i o u s l y t h a t the pH o f r i n s i n g s d i d not change more than 0.1 u n i t over a 3 h r . p e r i o d a f t e r t h e i r removal from the rectum.  Evidence supports the hypothesis t h a t the  observed a c i d i f i c a t i o n i s due s o l e l y to the a c t i v i t y o f the rectal wall. F a i l u r e o f the p r e p a r a t i o n  to a c i d i f y a f t e r repeated  i n j e c t i o n was not c o r r e l a t e d w i t h a l t e r e d hemolymph pH; nor d i d the p r e p a r a t i o n  d i e w i t h i n the experimental p e r i o d as  evidenced by i t s a b i l i t y to m a i n t a i n the pH g r a d i e n t over an extended p e r i o d o f time.  Phillips  (1964b)found t h a t the  t r a n s r e c t a l p o t e n t i a l i n the same i n v i v o p r e p a r a t i o n maintained undiminished over a 3 h r . p e r i o d .  was  This i n d i c a t e s t h a t  53 f a i l u r e was  not due  versus time, and preparation  to changes i n p o t e n t i a l across  the membrane  i s i n agreement w i t h the f i n d i n g t h a t  the  s u r v i v e s f o r a t l e a s t t h i s l e n g t h of time.  It is  p o s s i b l e a l s o t h a t the.adjustment of a c i d i f i c a t i o n i s l o c a t e d i n the t i s s u e o f the rectum i t s e l f and  t h a t t h i s has  somehow  been e f f e c t e d by the experimental procedure. Another p o s s i b i l i t y i s t h a t the i n t i m a i s a c t i n g as exchange r e s i n and  a f t e r a number of runs the supply  ions f o r exchange on the c u t i c u l a r membrane may  be  an  of hydrogen  exhausted.  Some doubt i s thrown on the i d e a by c a l c u l a t i o n s of the amount o f a c i d r e q u i r e d to a c i d i f y the i n j e c t e d b u f f e r s o l u t i o n . With the stronger b u f f e r , the p r e p a r a t i o n for  two  HCl.  or three runs, i . e . e q u i v a l e n t  c o u l d be used  to the r e l e a s e o f 1.2  However i n three runs of the weaker b u f f e r , t h i s  would not have been exceeded, i . e . 0.21  still  pM. HCl.  >uM  capacity  A l s o , even i f  the i n t i m a were to a c t as an exchange r e s i n , the maintenance o f the g r a d i e n t would s t i l l epithelium,  depend on the a c t i v i t y o f  the  s i n c e the system i s i n a s t a t e o f dynamic e q u i l i b r i u m .  In view of t h i s ,  the o n l y other  p o s s i b i l i t y t h a t suggests i t s e l f  i s t h a t the chances o f heat damage as the p r e p a r a t i o n w i t h hot wax  i n c r e a s e w i t h each s u c c e s s i v e  i s sealed  injection.  P h i l l i p s suggested i n h i s study, though he d i d not the p o s s i b i l i t y e x p e r i m e n t a l l y , p r e f e r e n t i a l absorption  study  t h a t the a c i d i t y r e s u l t e d from  o f the b a s i c form of a b u f f e r p a i r .  I t has been suggested t h a t the a c i d i t y of the gut contents might be  due  to phosphoric a c i d (Hobson, 1931); and  Hoskins and  Harrison  i n the  bee,  (1934) showed t h a t the c o n c e n t r a t i o n  of  54 phosphate i n the midgut (0.046M) i s n e a r l y f i v e times t h a t o f the hemolymph (0.01M) whereas the e x c r e t a c o n t a i n o n l y 0.005M. Some work has been done by L i n d s a y and  Craig  (1942) on  the  uptake of l a b e l l e d phosphate by the gut e p i t h e l i u m of i n s e c t s . They found t h a t radiophosphate became concentrated o f a l l i n s e c t s s t u d i e d , but was hindgut  of most.  cockroach, and  absent from the f o r e g u t  and  They looked a t the waxmoth, mealworm,  firebrat.  The  c o n c e n t r a t i o n r e f e r s to t i s s u e  content r a t h e r than body f l u i d s , and was authors  i n the midgut  i n t e r p r e t e d by  as i n d i c a t i n g t h a t the midgut e p i t h e l i u m was  t i s s u e concerned w i t h f a t m o b i l i z a t i o n .  No  the  the main  information  was  g i v e n by these workers, however, as to the s i t e of phosphate r e a b s o r p t i o n i n the The  gut.  c o n c e n t r a t i o n s of phosphate i n the body f l u i d s of  S c h i s t o c e r a f o l l o w the p a t t e r n as found i n Dixippus by Ramsay (19 56). i n the M a l p i g h i a n  He  morosus  found t h a t the c o n c e n t r a t i o n of phosphorus  tubule f l u i d was  140 mg.-atoms/1., and  that  the c o n c e n t r a t i o n of phosphate was  greater i n t h i s f l u i d  than  i n the hemolymph or serum.  Other f i g u r e s f o r Dixippus  (Ramsay, 1955a) are, serum 39 meq./l. and M a l p i g h i a n fluid  51 meq./l.  S i m i l a r i l y i n the l o c u s t , the  tubule f l u i d contained  14.5*5.65 mM/1.  a hemolymph value of 6.24-0.40 mM/1. phosphate i n the r e c t a l f l u i d was  tubule  Malpighian  phosphate as compared to The  c o n c e n t r a t i o n of  c o n s i d e r a b l y higher  e i t h e r of these a t 47.9-5.89 mM/l..for the l o c u s t . found i n the l i t e r a t u r e f o r hemolymph phosphate i n Exopterygotes are:  morosus  than  Other f i g u r e s concentrations  55 Sutcliffe Wood  (1962) Odonata: (1957) Phasmida:  Aeschna grandis l a r v a e . . . 4 meq./l. Carausius(Dixippus)morosus adults...16  Phosphate values f o r Endopterygotes range from 2.8 10.3  high c o n c e n t r a t i o n of phosphate i n the r e c t a l  be a r e s u l t e i t h e r  of phosphate s e c r e t i o n i n t o  or o f r e c t a l water a b s o r p t i o n .  The  e l i m i n a t e d by t h i s i n v e s t i g a t i o n .  concluded  resulting  The  was  Although there i s a high i t cannot be  from t h i s f a c t t h a t l i t t l e phosphate i s  i n t h i s r e g i o n of the gut.  fluid  the lumen  first possibility  c o n c e n t r a t i o n o f phosphate i n the r e c t a l f l u i d ,  reabsorbed  large concentration  gradient  from water r e a b s o r p t i o n favours movement of phosphate  out o f the rectum i n t o the hemolymph. t h a t i s reabsorbed into  meq./l. to  meq./l. ( F l o r k i n and Jeuniaux, 1964). The  may  meq./l.  actual quantity  would depend on the r a t e of e n t r y o f m a t e r i a l  the rectum as w e l l as how  long the m a t e r i a l remained t h e r e .  A l s o , i n c o l l e c t i o n of r e c t a l f l u i d , only obtained  The  samples were presumably  from animals i n which a l l water had not y e t been  reabsorbed, and nothing i s known about r e l a t i v e r a t e s of a b s o r p t i o n o f water and phosphate.  T h i s f a c t may  partly  f o r the v a r i a t i o n  f o r t h i s body  fluid.  i n determinations  Phosphate i s p r e s e n t  i n the r e c t a l f l u i d i n s u f f i c i e n t  concentration that p r e f e r e n t i a l i o n c o u l d a l t e r the pH.  account  a b s o r p t i o n of one  form of  this  A t the normal r e c t a l pH of about 6,  the phosphate would be found predominantly as the a c i d form, H  2  P 0  4~-  This c o u l d be the r e s u l t o f s e l e c t i v e  o f the a l k a l i n e  form l e a d i n g to a c i d i f i c a t i o n .  reabsorption Transport  56 systems f o r b o t h the monovalent and d i v a l e n t forms a r e known to occur b i o l o g i c a l l y : penetrating  i n the f e r t i l i z e d sea u r c h i n egg the  form i s the  HPO4  -  i o n (Chambers and Whiteley, 1966);  and i n y e a s t and b a c t e r i a l c e l l s there i s t r a n s p o r t o f phosphate as H2PC>4  _  i o n (Goodman and R o t h s t e i n ,  1957; M i t c h e l l ,  1954) . Doubt i s c a s t on the theory o f s e l e c t i v e r e a b s o r p t i o n o f the b a s i c form o f the phosphate b u f f e r p a i r by experiments i n which a T r i s - H C l b u f f e r was used r a t h e r than M c l l v a i n e ' s buffer  (phosphate-citrate).  Acidification persisted i n five  out o f ten animals w i t h the T r i s b u f f e r , i . e . i n the absence o f the phosphate b u f f e r system. the same r e g a r d l e s s  The pH values  a t e q u i l i b r i u m were  o f the b u f f e r components.  Other evidence  comes from measurements o f phosphate c o n c e n t r a t i o n s  of a c i d i f i e d  b u f f e r , done to study the p o s s i b i l i t y o f phosphate s e c r e t i o n . I t can be c a l c u l a t e d t h a t i f the pH o f 2mM phosphate b u f f e r was lowered from pH 7.2 to 6.5 by removal o f HP04 f i n a l phosphate c o n c e n t r a t i o n phosphate c o n c e n t r a t i o n  would be 1.2mM.  =  only,  then the  Changes i n  d u r i n g pH change a r e not o f t h i s  magnitude. A f u r t h e r l i m i t a t i o n i s imposed upon r e a b s o r p t i o n b a s i c form o f phosphate by the c u t i c u l a r i n t i m a .  o f the  Tracer  s t u d i e s i n d i c a t e t h a t the i n t i m a i s p r o b a b l y a c t i n g to r e s t r i c t the movement o f the l a r g e r d i v a l e n t  HPO4  -  i o n and any uptake  of phosphate would be i n favor o f the monovalent form. a c i d i t y o f the rectum would f a c i l i t a t e any phosphate  The  reabsorption  57 which does occur  in* t h i s organ, s i n c e the i n t i m a i s v i r t u a l l y -  impermeable a t higher pH values  to the b a s i c form.  a c i d i t y i s a l s o important i n n i t r o g e n e x c r e t i o n  The  being  r e s p o n s i b l e f o r p r e c i p i t a t i o n o f u r i c a c i d (Wigglesworth, 1931b). The  low pH o f the r e c t a l contents  a l s o means t h a t weak a c i d s  which d i s s o c i a t e i n the hemolymph can be e x c r e t e d a t l e a s t i n p a r t i n the u n d i s s o c i a t e d form  ( i . e . c a r r y i n g out H ) , as +  d e s c r i b e d f o r the v e r t e b r a t e kidney(White, Handler and Smith, (1964). Movement o f a c i d phosphate i n t o the lumen o f the rectum, e i t h e r a c t i v e l y or p a s s i v e l y was e l i m i n a t e d as a mechanism o f acidification. H2PO4  Even when the g r a d i e n t f a v o r e d movement o f  o u t o f the rectum r a t h e r than inwards (0.02M b u f f e r ) ,  a c i d i f i c a t i o n occurred.  When the phosphate c o n c e n t r a t i o n i n  the weaker b u f f e r was checked a f t e r a c i d i f i c a t i o n and compared to the pH value o f the same sample, there was no c o r r e l a t i o n . Hence any evidence f o r involvement o f the phosphate b u f f e r system appears to be l a c k i n g . The b i c a r b o n a t e  b u f f e r system i s a l s o widespread  b i o l o g i c a l l y , and i t s involvement i n the a c i d i f i c a t i o n has been considered,  though o n l y p a r t i a l l y .  o f h i s experimental  Phillips  evidence c o n s i d e r e d  (1961) on the b a s i s  i t 'highly p o s s i b l e  t h a t a c i d i f i c a t i o n i n the l o c u s t rectum i s due to a c t i v e s e c r e t i o n o f hydrogen ions i n t o the lumen, or r e a b s o r p t i o n o f bicarbonate  from c a r b o n i c a c i d i n the r e c t a l f l u i d  (which i n  e f f e c t leads to the same r e s u l t as a c t i v e s e c r e t i o n o f hydrogen) or b o t h ' .  58 I t has been shown t h a t l i t t l e of the a c i d i t y of r e c t a l f l u i d can be a t t r i b u t e d to a h i g h carbon d i o x i d e t e n s i o n i n the lumen, by experiments i n which a c i d i f i e d b u f f e r was e q u i l i b r a t e with a i r . o r i g i n a l l y by P h i l l i p s  allowed  T h i s r e f u t e s the hypothesis (1961) t h a t the metabolic  to  suggested  a c t i v i t y of  the r e c t a l e p i t h e l i u m produces a h i g h p a r t i a l p r e s s u r e  of  carbon d i o x i d e , and hence c a r b o n i c a c i d , i n the e p i t h e l i u m  and  lumen of the rectum.  a  The  lumen i s not  in e q u i l i b r i u m with  h i g h carbon d i o x i d e t e n s i o n i n the e p i t h e l i u m . a l s o r e j e c t e d t h i s hypothesis ment.  only s l i g h t e r r o r due  l o s s d u r i n g these experiments. involvement of the b i c a r b o n a t e  e l e c t r o d e system  to carbon d i o x i d e  T h i s does not however e l i m i n a t e b u f f e r system.  there were a c t i v e r e a b s o r p t i o n of b i c a r b o n a t e a c i d and  (1961)  on the b a s i s of the same e x p e r i -  In a d d i t i o n , r e s u l t s w i t h the anaerobic  showed t h a t there was  Phillips  If in fact from carbonic  i f the r e a b s o r p t i o n were s u f f i c i e n t l y r a p i d to pro-  duce a low c o n c e n t r a t i o n of c a r b o n i c a c i d , t h i s would be i n d i s t i n g u i s h a b l e from hydrogen ion s e c r e t i o n . There are many examples of involvement of the  bicarbon-  ate system i n a c i d i f i c a t i o n to be drawn from v e r t e b r a t e s . pH g r a d i e n t s across the i n t e s t i n a l e p i t h e l i u m can be able.  Wilson  (1962) reviews s t u d i e s c a r r i e d out on  changes across the w a l l of the i n t e s t i n e . the contents contents  In most  of the jejunum are a c i d to the b l o o d  of the ileum are more a l k a l i n e .  The  consideracid-base animals,  while  Evidence suggests  t h a t these g r a d i e n t s are the r e s u l t of the a c t i v e t r a n s p o r t of  HC0 ~ and H . +  3  The  r a t i n t e s t i n e i n v i t r o has been shown by Wilson  (1954) t o develop a pH g r a d i e n t  across i t s w a l l such t h a t the  lumen i s a c i d compared t o the s e r o s a l s i d e .  Parsons  (1956)  i n v e s t i g a t e d pH changes across the r a t i n t e s t i n e i n v i v o and showed t h a t when a s a l i n e s o l u t i o n c o n t a i n i n g placed  is  i n the jejunum, the b i c a r b o n a t e i o n was almost completely  removed i n 4 h r .  In the ileum and colon,  c e n t r a t i o n r i s e s under s i m i l a r Wilson and Kazyak verted  25 mM HCO^  the b i c a r b o n a t e con-  conditions.  (1957) d i d experiments i n which i n -  sacs o f hamster ileum were incubated w i t h a b i c a r b o n a t e -  s a l i n e s o l u t i o n on both sides o f the i n t e s t i n a l w a l l . found t h a t the pH and HCO^ on the mucosal s i d e .  They  f e l l on the s e r o s a l s i d e and rose  Furthermore the l o s s from the mucosal  s i d e was equal t o the gain on the s e r o s a l s i d e .  Studies o f  f l u i d and i o n movement i n d i c a t e d t h a t the ileum s e c r e t e d an +  i s o t o n i c s o l u t i o n o f Na mucosal s i d e . increased  +  —  (or K ) HCO^  from the s e r o s a l t o the  I t was noted t h a t the carbon d i o x i d e  tension  i n the- s e r o s a l s o l u t i o n w h i l e t r a n s p o r t was  This observation of t r a n s p o r t  occuring.  was taken as an i n d i c a t i o n t h a t the mechanism  involves  exchange o f a hydrogen i o n f o r some  other c a t i o n across the s e r o s a l border o f the e p i t h e l i a l The  f o l l o w i n g mechanism was p o s t u l a t e d  by them:  cell.  BLOOD  EPITHELIAL CELL  LUMEN  HCO,  /  'OH-  HCO-, Na  From a l l these  s t u d i e s , i t seems as though the more a c i d  s i d e has a h i g h carbon d i o x i d e t e n s i o n whenever a pH g r a d i e n t i s maintained for  across the i n t e s t i n a l w a l l .  T h i s holds both  the s o l u t i o n i n the lumen o f the jejunum i n a c i d s e c r e t i o n  in vivo  (Parsons,  1956) and the s o l u t i o n o f the s e r o s a l s i d e  in a l k a l i n e s e c r e t i o n by the ileum i n v i t r o 1957) .  Wilson  (1962) c o n s i d e r s i t reasonable  (Wilson and Kazyak, t o suppose t h a t  a s i m i l a r mechanism i s i n v o l v e d i n both p a r t s o f the small i n t e s t i n e , one which i n v o l v e s the exchange or t r a n s f e r o f hydrogen ions across one border o f the e p i t h e l i a l  cell.  A second example i s the r e a b s o r p t i o n o f b i c a r b o n a t e i n the v e r t e b r a t e kidney. i n the proximal  Pitts  (1963) d e s c r i b e s the mechanism  tubule which i s s p e c i a l i z e d f o r r e a b s o r p t i o n  of most o f the b i c a r b o n a t e which enters the glomerular rate.  Sodium and b i c a r b o n a t e  i n the glomerular  filtrate.  ions enter the proximal Na  +  Na  i n t o the p e r i t u b u l a r  The r e s u l t i n g low i n t r a c e l l u l a r c o n c e n t r a t i o n o f N a  means t h a t N a H  tubule  i s c o n s t a n t l y pumped out of  the t u b u l a r c e l l s and a c t i v e l y extruded fluid.  filt-  +  +  ions o f the f i l t r a t e d i f f u s e i n t o the c e l l .  ions from the i n t e r i o r o f the c e l l a r e exchanged f o r .  +  ions i n the t u b u l a r lumen.  The H  +  -  a s s o c i a t e s w i t h HCO,.  61 of the f i l t r a t e and forms c a r b o n i c a c i d which i n t u r n decomposes i n t o CO^ The  C0  and water  (slowly, s i n c e not e n z y m a t i c a l l y c a t a l y z e d ) .  d i f f u s e s i n t o the c e l l and  2  i n f l u e n c e of c a r b o n i c anhydrase. f u r n i s h e s HCO^  a b s o r p t i o n of b i c a r b o n a t e 2  Subsequent d i s s o c i a t i o n  According  gradient  to t h i s scheme the r e -  i s i n d i r e c t i . e . v i a conversion  i n the t u b u l a r lumen.  process  under the  which d i f f u s e s down a c o n c e n t r a t i o n  i n t o the p e r i t u b u l a r f l u i d .  C0  i s hydrated  The e s s e n t i a l process  to  or c e n t r a l  i s the exchange of i n t r a c e l l u l a r hydrogen ions f o r  sodium ions i n the t u b u l a r f l u i d .  Whether the movement of  ions i s p a s s i v e or r e q u i r e s a pump i s not c e r t a i n ( P i t t s , About 90% of f i l t e r e d b i c a r b o n a t e imal t u b u l e of the kidney.  i s reabsorbed  s p e c i a l i z e d to against a  A c i d i f i c a t i o n of u r i n e to a pH below 6 r e q u i r e s  almost t o t a l removal of Phillips  1963).  i n the prox-  the remainder of the f i l t e r e d b i c a r b o n a t e  high gradient.  +  There i s a f u r t h e r mechanism i n  the c o l l e c t i n g duct of the v e r t e b r a t e kidney reabsorb  H  bicarbonate.  (1961) found t h a t there would have to be  t o t a l net i n f l u x of at l e a s t 0.07  /uequiv. H  +  or 0.4  a  ^uequiv.  H /hr. to account f o r the observed a c i d i f i c a t i o n o f b u f f e r i n +  the l i g a t e d rectum of the l o c u s t . + 1.4  /aequiv.  H / h r . was  estimated  l e a s t f o r the former estimate,  A somewhat higher value of in this investigation.  At  the net i n f l u x of hydrogen ions  i s o f the same order of magnitude as net sodium c h l o r i d e a b s o r p t i o n from the rectum i n the absence of a c o n c e n t r a t i o n gradient,  so t h a t there may  be an exchange of hydrogen ions  f o r sodium i o n s as i n the v e r t e b r a t e kidney.  Or there c o u l d be  a potassium-hydrogen i o n exchange; potassium r e a b s o r p t i o n i n the rectum i s 10 times  t h a t f o r sodium ( P h i l l i p s ,  Potassium t r a n s p o r t may  be l i n k e d to the movement o f hydrogen  i o n s i n the midgut of Hyalophora c e c r o p i a  1964b, 1965).  ( H a s k e l l , demons,  and Harvey, 1965), as suggested by i n h i b i t i o n of t r a n s p o r t a t moderately a l k a l i n e pH values, as w e l l as the e f f e c t s of carbon d i o x i d e and  inhibitory  c a r b o n i c anhydrase i n h i b i t o r s .  In the v e r t e b r a t e system i f c a r b o n i c anhydrase i s i n h i b i t e d then there i s not a c i d i f i c a t i o n , and a l s o more N a excreted. and  This supports  the i d e a the H  +  i s exchanged f o r Na  t h a t c e l l u l a r carbon d i o x i d e i s the source o f the  protons  is  +  (White, Handler, and Smith, 1964).  Carbonic  +  secreted anhydrase  has been r e p o r t e d i n the t i s s u e s of s e v e r a l i n s e c t s (Sobotka and Kann, 1941; 1958). cells  Anderson and March, 1956;  I t i s s a i d to occur (Gilmour,  i n t h i s study  1961).  No  was  He  the  i n h i b i t o r s t u d i e s have been attempted  s i n c e t h e i r value i s q u e s t i o n a b l e  conducted some s t u d i e s with  Friedman,  i n the s o l u b l e f r a c t i o n s o f  i n v e s t i g a t i o n of c e l l u l a r mechanism.  inhibitor.  Buck and  i n an i n t r o d u c t o r y  For example, Parsons  (1956)  'Diamox', a c a r b o n i c anhydrase  found t h a t i n the ileum of the r a t i t s e f f e c t  to cause the d i r e c t i o n of net movement of carbon d i o x i d e  to change to a b s o r p t i o n so t h a t the ileum became more a c i d (pH 7.2)  than i n the u n t r e a t e d animals  jejunum i t s e f f e c t was  (pH 7.5).  In  to i n h i b i t the a b s o r p t i o n of  carbon d i o x i d e so t h a t the contents  the total  of the jejunum became more  o  63 alkaline  (pH 7.0)  than  i n the untreated animals  ever he c a u t i o n s t h a t the e f f e c t s of  (pH 6.5).  How-  'Diamox' l e a d to a g e n e r a l +  d e p r e s s i o n of the c a p a c i t y to absorb water and Na t h a t the e f f e c t s of due  —  and C I ,  and  'Diamox' r e p o r t e d above are not n e c e s s a r i l y  to i t s a c t i o n as an i n h i b i t o r of the enzyme c a r b o n i c  anhydrase s i n c e c a r b o n i c anhydrase i s not even present mucosa of the s m a l l i n t e s t i n e of the r a t .  i n the  :  Hydrogen ion s e c r e t i o n has been demonstrated a l s o f o r yeast c e l l s  (e.g. Conway, 1954;  Conway and O'Malley, 1955),  i n t o the lumen of the stomach of v e r t e b r a t e s 1957,  1967;  Fleming,  Davies,  1957), from f r o g s k i n  (e.g. Davenport,  (Huf et  a l . 1951,  1957) .  In t h i s study no measurements of b i c a r b o n a t e  concen-  t r a t i o n s were made because of t e c h n i c a l problems encountered w i t h the technique 195.7) .  of m i c r o d i f f u s i o n ( Shaw,. 195 5; Conway,  Hence no d i f f e r e n t i a t i o n can be drawn a t t h i s  between hydrogen ion s e c r e t i o n as such,  stage  and b i c a r b o n a t e  re-  a b s o r p t i o n from c a r b o n i c a c i d l e a v i n g behind hydrogen i o n s . The  c o n c e n t r a t i o n of HCO^  Exopterygote,  i n the hemolymph of  Odonata: Aeschna qrandis l a r v a e , i s given  15 meq./l. by S u t c l i f f e  (1962).  Endopterygotes i s the same: S i a l i s l u t a r i a larvae  The estimate  for  as  two  15 meq./l. f o r Megaloptera:  (Shaw, 1955;  Sutcliffe,  for Diptera: Gasterophilus i n t e s t i n a l i s larvae 1950a). A c c o r d i n g to Levenbook bicarbonate  one  1962); 17 meq./l. (Levenbook,  (1950b) the c o n c e n t r a t i o n of  i s u s u a l l y low f o r t e r r e s t r i a l i n s e c t s ;  the  64 f i g u r e for Gasterophilus hemolymph of many other  blood  i s much higher  i n s e c t s e.g.  than i n the  e i g h t times t h a t of  Proden i a er idan i a . Representative  values  r e c t a l concentration 91 mM/1. adult,  of HCO^  (Shaw, 1955; 75 mM/1.  found i n the l i t e r a t u r e f o r were:  Sialis lutaria  Staddon, 1955);  Notonecta  (Wigglesworth, 1965).  hemolymph pH of 7.13,  The  l o c u s t was  slightly  found to have a  i n agreement w i t h P h i l l i p s  The b u f f e r c a p a c i t y ,  (1961).  or r e s i s t a n c e to a l t e r a t i o n i n pH,  the hemolymph i s s a i d to be g r e a t e r neutrality,  qlauca  (Staddon, 1963).  The hemolymph of i n s e c t s i s u s u a l l y very acid  and probably due  (Wigglesworth, 1965).  to b i c a r b o n a t e s ,  inorganic  P r o t e i n and b i c a r b o n a t e  important q u a n t i t a t i v e l y a c c o r d i n g  few,  i f any,  (1950b). is usually  i n f l e x i o n p o i n t s has been taken  by workers i n c l u d i n g Levenbook as an  i n d i c a t i o n of the i n -  volvement of a whole s e r i e s of o v e r l a p p i n g which no one  urates  are the most  to Levenbook  f a c t t h a t the b u f f e r curve f o r i n s e c t b l o o d  smooth and has  of  on the a c i d s i d e o f  phosphates, p r o t e i n s , amino a c i d s , and perhaps a l s o  The  larvae,  b u f f e r systems i n  o f these dominates.  The b u f f e r c a p a c i t y o f the hemolymph of i n s e c t s shows c e r t a i n c h a r a c t e r i s t i c features,  as based on s t u d i e s of  l a r v a e P i e r i s rapae and H e l i o t h i s armigera  (Craig and  1938), the a d u l t Mormon c r i c k e t Anabrus simplex Donaldson, and Hastings,  1941), and v a r i o u s  other  the  Clark,  (Pepper, Orthoptera  65 (Hastings and Pepper, 1943) and. reviewed by Levenbook  (1950b) .  The main d i f f e r e n c e from the s i t u a t i o n found i n v e r t e b r a t e s i s t h a t the b u f f e r c a p a c i t y i s always lowest  i n the r e g i o n o f the  normal pH o f the blood r a t h e r than b e i n g o p t i m a l l y b u f f e r e d at t h i s pH as i n most other  animals.  T h i s r e s u l t s i n a U-  shaped curve when b u f f e r i n g c a p a c i t y i s p l o t t e d a g a i n s t pH (Levenbook, 1950b).  Vertebrate  blood b u f f e r s l e s s e f f i c i e n t l y  on e i t h e r s i d e o f i t s normal pH and the b u f f e r value 0^shaped. withstand  I t does appear c o r r e s p o n d i n g l y  t h a t i n s e c t s can  a wider v a r i a t i o n i n pH than mammals.  The l o c u s t  does show a remarkable t o l e r a n c e o f excess a c i d i t y , denced by o b s e r v a t i o n s acid.  curve i s  as e v i -  on animals i n j e c t e d w i t h h y d r o c h l o r i c  The range o f pH i n which the animal i s s t i l l  a c t i v e i s wider than f o r example the b l o o d o f man.  relatively The l a t t e r  i s r e g u l a t e d a t pH 7.4, and i f the pH f a l l s t o 7.0, a c i d o t i c coma and death ensue, w h i l e a t a pH o f 7.8 l i f e tetany  ends i n  (Giese, 1962) . Levenbook  (1950b) has suggested a p o s s i b l e advantage o f  the U-shaped b u f f e r curve t o the i n s e c t .  The l a c t i c a c i d  r e l e a s e d i n t o the b l o o d as a r e s u l t o f muscular e x e r t i o n by the a c t i v e i n s e c t would i n c r e a s e the f r e e CO^ content b l o o d a t the expense o f b i c a r b o n a t e .  Activity  o f the  i s also associ-  ated w i t h an i n c r e a s e i n c e l l u l a r r e s p i r a t o r y r a t e and t h i s , too, would i n c r e a s e the carbon d i o x i d e t e n s i o n i n the blood. Wigglesworth  (1935) and others  l a c t i c a c i d i n the blood,  showed t h a t an i n c r e a s e i n the  or a lowered pH due t o an excess o f  66 carbon d i o x i d e causes the animal to i n c r e a s e v e n t i l a t i o n of the t r a c h e a l system.  This i s a response a l s o found i n  v e r t e b r a t e s and i s done i n an e f f o r t to remove the excess carbon d i o x i d e by i n c r e a s i n g i t s r a t e of d i f f u s i o n through the r e s p i r a t o r y system.  In the o p i n i o n o f Levenbook, the i n s e c t  t r a c h e a l system i s not as e f f i c i e n t i n t h i s r e s p e c t as i s the v e r t e b r a t e lung.  Hence the carbon d i o x i d e content of  the  hemolymph might reach a c o n c e n t r a t i o n t h a t would i n j u r e the t i s s u e s , but f o r the f a c t that the hemolymph shows an b u f f e r i n g c a p a c i t y i n these r e g i o n s .  Miller  increased  (1964) a l s o  suggests t h a t the U-shaped curve has a r e l a t i o n s h i p to water conservation, The  allowing discontinuous  r e l e a s e of carbon d i o x i d e .  enzyme c a r b o n i c anhydrase i n the e r y t h r o c y t e o f  the mammal p l a y s a c e n t r a l r o l e i n the b u f f e r i n g e f f i c i e n c y o f the b l o o d .  Carbonic  hemolymph of o n l y one (Brinkman e t . a l . ,  anhydrase has been found i n the  i n s e c t s p e c i e s , the l a r v a of Chironomus  1932).  Levenbook and C l a r k  (1950)  confirmed  the absence of c a r b o n i c anhydrase from i n s e c t b l o o d i n experiments on G a s t e r o p h i l u s  and Locusta m i g r a t o r i a .  Ramsay (1955a) i n j e c t e d s o l u t i o n s o f NaCl and KCl i n t o the hemolymph of Dixippus f o l l o w e d the process  to a l t e r the c o n c e n t r a t i o n s  of r e t u r n to normal.  i n about one  on the part, o f  day.  then  He found t h a t f o r  potassium r e g u l a t i o n , the r e t u r n t o normal had h a l f way  and  progressed  The adaptive response f o r  the e x c r e t o r y systeitKof Dixippus  seemed  sodium  f e e b l e , e s p e c i a l l y when compared to Rhodnius.  In the l a t t e r  animal excess sodium taken i n w i t h a meal i s gotten r i d of i n 3 hr. 1931a).  ( c a l c u l a t e d by Ramsay, 1955a, from  Wigglesworth,  Hence the response of an animal t o e l e v a t e d sodium  c o n c e n t r a t i o n s i n the hemolymph v a r i e s , and t h i s may h o l d f o r hydrogen-ion r e g u l a t i o n .  also  However i n the l o c u s t  -5 injected with  10  ^aM HCl, the r a t e of r e t u r n of hydro-  gen-ion c o n c e n t r a t i o n t o normal had progressed about h a l f way  i n one day,  one-  and compares to Ramsay's r e s u l t s f o r  potassium i n Dixippus  (1955a) .  For animals w i t h h y d r o c h l o r i c a c i d i n j e c t e d i n t o the hemolymph, the e x c r e t a c o l l e c t e d subsequently were more a c i d than p r i o r to i n j e c t i o n .  I t appears probable t h a t r e c t a l  a c i d i f i c a t i o n might p l a y a r o l e i n e l i m i n a t i n g excess i t y of the animal.  The normal pH o f e x c r e t a i s s i m i l a r t o  the e q u i l i b r i u m v a l u e s a t t a i n e d on i n j e c t i n g s o l u t i o n the l i g a t e d rectum.  These lower v a l u e s a f t e r a c i d  suggest the rectum has a d j u s t e d to a new brium.  more a c i d i c  equili-  In the case of v e r t e b r a t e s , to d e a l w i t h s t r o n g a c i d s (Pitts,  The a c i d s are e x c r e t e d f u l l y n e u t r a l i z e d , and t h i s i s  accomplished without l o s s of sodium by combining w i t h ammonium ions i n s t e a d . Pitts  into  injection  such as h y d r o c h l o r i c or s u l f u r i c the f o l l o w i n g occurs 1963).  acid-  the a c i d  The net r e a c t i o n as given by  (1963) i s Na S0 2  4  + 2H C0 2  3  + 2NH  3  —»  ( N H ) S 0 + 2NaHC0 excreted reabsorbed 4  2  4  3  68 There i s an enzyme, glutaminase, present  i n the kidney which  c a t a l y z e s the h y d r o l y s i s of glutamine to form glutamic and NH^  -  a I m T l o n ;  i-  a  d i f f u s e s across  the glomerular f i l t r a t e .  Here i t combines w i t h H  from i n t e r c e l l u l a r H^CO^, and e x c r e t i o n thus serves returns Na  +  lining into +  derived  i s exchanged f o r Na .  Ammonia  +  to e l e v a t e e x t r a c e l l u l a r HCO^  and  also  to the plasma which otherwise would have been  a s s o c i a t e d w i t h an anion i n the u r i n e . be  the t u b u l a r  acid  There seems then to  a b a s i c d i f f e r e n c e between the v e r t e b r a t e  treatment of  s t r o n g a c i d s i n e x c r e t i n g them n e u t r a l i z e d , and  the  observa-  t i o n t h a t i n the l o c u s t the e x c r e t a become n o t i c e a b l y more a c i d when strong a c i d i s i n j e c t e d i n t o the hemolymph.  This,  is  rectal  the o n l y evidence gathered to support the i d e a t h a t  a c i d i f i c a t i o n might be animal as a whole.  important i n the pH r e g u l a t i o n of  the  SUMMARY 1. the  A c i d i t y i n the rectum of the l o c u s t i s not due to  i n t r o d u c t i o n o f a c i d from any a n t e r i o r r e g i o n o f the gut. 2.  B a c t e r i a l a c t i v i t y does not account f o r pH  changes  along the l e n g t h o f the gut. 3. Malpighian  Average pH v a l u e s found were:  midgut 5.46-0.3 9;  tubule r e g i o n 7.5 9-0.72 and 7.94-0.49;  ileum  7.13-0.47; rectum 6.19-0.45 and 5.91-0.44. 4.  The same a c i d pH i s a t t a i n e d whether  or more a c i d b u f f e r i s i n t r o d u c e d  i n t o the l i g a t e d rectum.  A c i d i f i c a t i o n of i n j e c t e d b u f f e r occurs of a n t i b i o t i c ,  a more a l k a l i n  even i n the presence  and hence the phenomenon i s not due to  bacterial activity.  T h i s suggests t h a t the e p i t h e l i u m i s  permeable to hydrogen  ions and t h a t the pH g r a d i e n t  t a i n e d by the r e c t a l wall, i . e . a case of dynamic 5.  i s main-  equilibrium.  There i s not s u f f i c i e n t slow r e l e a s e o f hydrogen  ions by f e c a l m a t e r i a l t o account f o r the a c i d i f i c a t i o n . 6.  Phosphate c o n c e n t r a t i o n  tubule r e g i o n to rectum. both of  i n c r e a s e s from  Serum c o n c e n t r a t i o n  Malpighian  i s lower than  these. 7.  R e c t a l pH does not favor a b s o r p t i o n  of the b a s i c  form o f phosphate b u f f e r ; and s i n c e a c i d i f i c a t i o n occurs with a T r i s - H C l buffer, absorption  also  of the b a s i c form o f a  phosphate b u f f e r p a i r seems u n l i k e l y to be the only mechanism of  acidification. 8.  The intima of the rectum i s most permeable to  1  70 phosphate a t pH 4 and l e a s t permeable a t pH 8. a c t i n g as a molecular s i e v e  I t may be  t o exclude the l a r g e r  divalent  HPO4 i o n i n f a v o r o f H2PO4 . -  -  9. buffer  A c i d i f i c a t i o n takes p l a c e w i t h 0.02M phosphate  when the g r a d i e n t i s a g a i n s t p a s s i v e movement o f the  a c i d i c component o f the phosphate b u f f e r 10.  There i s no a c t i v e  of the phosphate b u f f e r  secretion  i n t o the rectum. o f the a c i d component  p a i r which c o u l d account f o r  a c i d i f i c a t i o n o f r e c t a l contents. 11.  A c i d i t y i s n o t due t o a high p a r t i a l p r e s s u r e o f  carbon d i o x i d e i n the a c i . d i f i e d b u f f e r . not  the b i c a r b o n a t e b u f f e r  However, whether o r  system i s otherwise i n v o l v e d has  not been demonstrated. 12. jal.  Schistocerca  can t o l e r a t e an i n j e c t i o n o f about 100  0.1N HCl i n t o the hemolymph, and shows some a b i l i t y to  r e g u l a t e t h i s dose.  Excess a c i d may be l o s t i n the e x c r e t a and  a i d recovery. 13. not  F a i l u r e o f a c i d i f i c a t i o n a f t e r a number o f runs i s  correlated  buffer  w i t h measurable hemolymph pH changes due to  i n j e c t i o n i n t o the rectum, nor i s i t due to death o f  the p r e p a r a t i o n . 14.  F a i l u r e i s probably due t o h e a t i n g damage.  Calculations  o f the amount o f a c i d s e c r e t e d  cast  doubt on the theory o f the c u t i c u l a r i n t i m a a c t i n g as an i o n exchange  resin.  15. dissociates  Either  hydrogen ions or some a c i d molecule which  are a c t i v e l y s e c r e t e d i n t o the rectum, o r  b i c a r b o n a t e i s absorbed from the lumen.  71 LITERATURE CITED Anderson, A.B. .. and R.B. March, 1956. I n h i b i t o r s o f carbonic anhydrase i n the American cockroach P e r i p l a n e t a americana (L.). Can. J . Z o o l . , 34:68-74. B r i n d l e y , G.S., 1962. Beats produced by simultaneous s t i m u l a t i o n of the human eye with i n t e r m i t t e n t l i g h t and i n t e r m i t t e n t or a l t e r n a t i n g e l e c t i c c u r r e n t . J . Physiol., 164:157-167. Brinkman, R., R. Margaria, N.U. Meldrum, and F.J.W. Roughton, 1932. The C02 c a t a l y s t present i n b l o o d . J . 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J . , 56:521-527.  Wilson,  T.H., 1962. I n t e s t i n a l Absorption. P h i lade l p h i a v i 263 p.  Wilson,  T.H. and L. Kazyak, 1957. Acid-base changes across the w a l l o f hamster and r a t i n t e s t i n e . Biochim. e t biophys. a c t a , 24:124-132.  Saunders,  Wood, D.W., 1957. The e f f e c t o f i o n s upon neuromuscular t r a n s m i s s i o n i n a herbivorous i n s e c t . J. Physiol., 138:119-139.  APPENDIX A.  Inorganic  phosphate p r e c i p i t a t i o n  That s i g n i f i c a n t b a c t e r i a l i n c o r p o r a t i o n of l a b e l l e d phosphate had not o c c u r r e d  during  the t r a c e r experiments  was  checked u s i n g MacKay and B u t l e r ' s m o d i f i c a t i o n o f Mathison's method (Peters and Van were as  Slyke,  1932).  The  solutions  required  follows:  (1)  Concentrated  HCl.  (2)  Magnesium c i t r a t e mixture o f F i s k e ,  To 265  a c i d d i s s o l v e d i n 3 50 cc. o f hot water, 13 gm.  gm.  cooled and  0.9 0) added.  33 0 c c . o f s t r o n g ammonia water This was  c o o l e d and  (3)  Concentrated ammonia water  (4)  D i l u t e ammonia s o l u t i o n .  The  solution  (specific gravity  d i l u t e d to one  liter.  ( s p e c i f i c g r a v i t y 0.90). One  volume of strong ammonia water  was  d i l u t e d to 15 volumes w i t h water.  B.  Measurement o f i n o r g a n i c phosphate by  c o l o r i m e t r i e method  The method used to measure i n o r g a n i c phosphate c o l o r i m e t r i c a l l y was  citric  of magnesium  oxide f r e e from carbonate were added w i t h s t i r r i n g . was  of  t h a t o f Gomori  (1942).  Solutions  concentrations required  were: (1) up (2)  10N  H2SO4-  to one  282  Molybdate-sulfuric 7j  reagent - two  p a r t 10N  Reducing agent - one  bisulfite.  H2S04into 600  cc. water made  liter.  (Na2Mo04 2H20) one (3)  c c . concentrated  The  p a r t s 5% sodium molybdate  s u l f u r i c a c i d , one gram 'elon'  l a t t e r s o l u t i o n was  p a r t water.  (metol) i n 100  cc. 3%  made f r e s h l y every three  sodium days.  78  (4)  5% t r i c h l o r o a c e t i c a c i d  (TCA.) .  (5)  Standard - 0.5mM KH2PO4 .  

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