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Mechanism of hyperosmotic urine formation in the recta of saline-water mosquito larvae Bradley, Timothy Jud 1976

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THE MECHANISM OF HYPEROSMOTIC URINE FORMATION IN THE RECTA OF SALINE-WATER MOSQUITO LARVAE by Timothy Jud Bradley B . A . , Vanderbilt U n i v e r s i t y ,  1971  M.S., University of Oklahoma, 1973  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS OF THE DEGREE OF DOCTOR OF PHILOSOPHY  in THE FACULTY OF GRADUATE STUDIES Department of Zoology  We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA Play, 1976  ©  Timothy Jud Bradley  In  presenting  this  an a d v a n c e d d e g r e e the I  Library  further  for  agree  in p a r t i a l  fulfilment  of  at  University  of  Columbia,  the  make  that  it  freely  permission  s c h o l a r l y p u r p o s e s may  by h i s of  shall  thesis  this  w r i t ten  representatives. thesis  for  available for  financial  gain  of  The U n i v e r s i t y  of  British  2075 Wesbrook P l a c e V a n c o u v e r , Canada V6T 1W5  by  the  Columbia  shall  not  requirements  reference copying of  Head o f  i s understood that  pe rm i s s i o n .  Department  for  extensive  be g r a n t e d  It  British  the  I  agree  and  be a l l o w e d  that  study.  this  thesis  my D e p a r t m e n t  copying or  for  or  publication  without  my  i  ABSTRACT  The osmoregulatory function of the l a r v a l recta of two s a l i n e water mosquitoes, Aedes campestris and A,, taeniorhynchus, was examined. In hyperosmotic waters the rectum was shown to be the s i t e of formation of a concentrated urine by secretion of a hyperosmotic f l u i d into the r e c t a l lumen.  In s i m i l a r concentrations of sea water, both species  produced a r e c t a l secretion having an osmotic concentration and i o n i c composition s i m i l a r to that of sea water, with the exception that potassium levels are elevated 18- to 21-fold in the s e c r e t i o n . Osmoregulatory strategies in both species involve the rapid ingestion of the external medium.  In  taeniorhynchus this drinking  —1 —1 r a t e , (100  nl mg  h  ) did not vary s i g n i f i c a n t l y i n s a l i n i t i e s  10$ and 200$ sea water.  It  by a rapid rate of drinking;  i s suggested that two purposes are served 1)  dissolved nutrients can be taken up  when particulate food i s limited and medium i s large relative  between  2) when the uptake of  external  to the loss of water by osmosis across the  c u t i c l e , the concentration of urine necessary to maintain osmotic homeostasis i s close to that of the external medium. Rectal function i s A., campestris was examined in three media, a l l with an osmotic concentration of 700 mOsm but varying in ionic ratios.  their  These media contained the i o n i c r a t i o s found i n sea  water and two types of ponds in which A., campestris naturally occur. The larvae were able to survive in a l l three media, suggesting that the larvae found in one saline environment are not physiologically limited  ii  to those i o n i c conditions.  I demonstrated that changes i n the r e l a t i v e  transport rates of ions i n the rectum are very s i g n i f i c a n t in the acclimation process. Na , K , Mg  , CI  The rectum was found to be the major s i t e of  and probably HCO^ excretion, while the Malpighian  tubules are the source of some of the magnesium and almost a l l of the sulphate in the urine.  The ion r a t i o s , exclusive of K  +  found i n the  r e c t a l secretion matched those in the external medium, with the exception that an unidentified anion (probably HC0~) substituted f o r SO" i n (l\)a + Wg)S0~ medium.  A model i s proposed showing the s i t e s  of ion secretion and uptake in the Malpighian tubules, rectum and anal papillae in a l l three media. Rectal function in A,, taeniorhynchus larvae was examined in 10$,  100$  and  200$  sea water.  In • hyposmotic•  media the rectum does  not secrete a f l u i d and i t i s postulated that s a l t and water resorption occur in the rectum under these conditions as in s t r i c t l y f r e s h +  water species. Mg  ++  and E l  In hyperosmotic media the concentrations of Na , K ,  as well as the osmotic concentration of the secreted f l u i d  increase with increasing external s a l i n i t y . K  +  +  Due to the high rate of  secretion by the rectum, potassium uptake by the anal papillae i s  postulated.  Sodium and chloride may be excreted at this s i t e as w e l l .  An examination of the effect of varying hemolymph concentrations of sodium and chloride on the rate of secretion of these ions in the rectum, showed an a l l o s t e r i c relationship rather than the MichaelisMentin k i n e t i c s observed with most transport processes ( e . g . the Malpighian tubules).  iii  An i n v i t r o preparation (lacking tracheal and neural con* nections) of the l a r v a l rectum of A,, taeniorhynchus was used to examine the function of the anterior and posterior r e c t a l segments. regions, which d i f f e r functions in. v i t r o ,  These two  morphologically, were shown to have separate  the posterior segment secreting a hyperosmotic  f l u i d while the f l u i d i n the lumen of the anterior segment decreased i n osmotic concentration and showed no change i n volume. E l e c t r i c a l potential differences were measured across the basal and a p i c a l membranes as well as across the entire r e c t a l wall in vivo.  Based on these observations i n a r t i f i c i a l  hemolymphs of  various i o n i c compositions, a model i s presented of ion  transport  processes occuring i n the posterior r e c t a l segments during the s e c r e tion of a hyperosmotic f l u i d .  The model accounts f o r the ion concen-  trations and i o n i c r a t i o s observed in r e c t a l secretion from larvae reared i n d i f f e r e n t media.  iv  TABLE OF CONTENTS  Page  Chapter I  Chapter II  GENERAL INTRODUCTION The Formation of Urine i n Insects  1  Rectal Function in Insects  2  THE SECRETION OF HYPEROSMOTIC FLUID BY THE RECTUM OF A SALINE-WATER MOSQUITO LARVA AEDES TAENIORHYNCHUS (WIEDEMANN) Introduction  9  Materials and Methods  12  Results  20  Estimation of Osmotic Load  20  Osmolality of Rectal F l u i d  22  Changes in volume of the Rectal Contents  25  Composition of Rectal Secretion  29  Discussion Chapter III  8  REGULATION OF RECTAL SECRETION IN SALINEWATER MOSQUITO LARVAE IN ENVIRONMENTS OF VARYING IONIC COMPOSITION  33 37  Introduction  38  Materials and Methods  39  Results  44  Survival of A. campestris in various media  44  Survival of A,, taeniorhynchus in various media  47  V  Hemolymph Anion Concentrations in A., campestris  48  Composition of Rectal Secretion i n Different Media Sodium  49  Potassium  53  Magnesium  55  Chloride  55  Sulphate - A. campestris  57  Sulphate - A. taeniorhynchus  58  Osmolality  61  Discussion Chapter IV  49  63  THE EFFECT OF EXTERNAL SALINITIES ON DRINKING RATE AND RECTAL SECRETION IN THE LARVAE OF THE SALINE-WATER MOSQUITO AEDES TAENIORHYNCHUS  70  Introduction  71  Materials and Methods  71  Results  75  Drinking Rates  75  Hemolymph Ion Levels  81  Ionic and Osmotic Concentrations of Rectal Secretion  86  In 10$  Sea water  86  In 100$ Sea water  88  In 200$ Sea water  89  Factors Regulating Ionic and Osmotic Concentrations of Rectal Secretion Discussion  90 99  vi  Chapter V  THE USE OF AN IN VITRO RECTAL PREPARATION TO DIFFERENTIATE THE FUNCTIONS OF THE ANTERIOR AND POSTERIOR RECTAL SEGMENTS IN AEDES TAENIORHYNCHUS Introduction  108  Materials and Methods  108  Results  109  Secretion by Rectal Segments  109  Osmotic Concentrations of Rectal Secretion in, v i t r o  112  Discussion  Chapter VI  THE MECHANISM OF HYPEROSMOTIC FLUID SECRETION IN THE RECTUM OF LARVAE OF THE SALINE-WATER MOSQUITO AEDES TAENIORHYNCHUS  116  119  Introduction  120  Materials and Methods  120  Results  123  Trans-rectal E l e c t r i c a l Potential Difference  123  Influence of Hemolymph Ion Concentrations on the Trans-rectal P.D.  124  Discussion Chapter VII  107  GENERAL SUMMARY  141 150  REFERENCES  156  APPENDICES  161  vii  LIST OF TABLES  Table  Page  1  Ionic concentration (mlfl) and osmolality (mOsm) of a r t i f i c i a l and natural hemolymph.  17  2  The determination of increase in volume of rectal contents following l i g a t i o n o§ larvae using ^ C - i n u l i n volume marker.  27  Ionic concentrations of external media (NaCl, NaHC0 and (Na + Mg)S0 ) a l l of which had a t o t a l osmolality of 700 mOsm.  41  4  The i o n i c (mfll) and osmotic concentrations (mOsm) of normal and low chloride hemolymph.  43  5  Total mortality observed four days a f t e r A,. campestris larvae were transferred to three experimental media (700 mOsm) of different i o n i c composition.  46  6  A comparison of the external ionic and osmotic concentrations i n the three acclimation media with the same parameters in r e c t a l secretion from two rectal preparations.  50  7  The ionic (mfll) and osmotic (mOsm) concentretions in the a r t i f i c i a l hemolymphs used to study the effects of varying hemolymph parameters.  74  8  The osmotic concentrations of r e c t a l f l u i d removed from lumina of in, v i t r o preparations of whole recta and i s o l a t e d anterior and posterior r e c t a l segments.  113  9  The osmotic concentration (mOsm) of r e c t a l secretion produced by whole, iti v i t r o recta i n a r t i f i c i a l hemolymph with the addition of potential stimulatory agents.  115  a  3  3  s  a  4  viii  10  Paired measurements of the t r a n s - r e c t a l e l e c t r i c a l potential difference in the ant e r i o r and posterior r e c t a l segments of recta bathed in normal a r t i f i c i a l hemolymph uiithin ten minutes of d i s s e c t i o n .  124  11  Paired measurements of the t r a n s - r e c t a l e l e c t r i c a l potential difference i n the anterior and posterior rectal segments of recta bathed i n normal hemolymph, two hours a f t e r d i s s e c t i o n .  126  12  Paired measurements of the t r a n s - r e c t a l potential difference within the r e c t a l c e l l s and the r e c t a l lumen i n the anterior and posterior rectal portions.  128  ix  LIST OF FIGURES  Figure  Page  Measurements demonstrating the l i n e a r relationship between the r a d i o a c t i v i t y of drops of I^C-inulin solution and the calculated volume of those drops based on t h e i r measured diameter.  14  A schematic diagram showing the three most posterior segments of the mosquito larva with the position of ligatures used to i s o l a t e the rectum.  16  The amount of external medium consumed with time as estimated from the increase i n whole body r a d i o a c t i v i t y with time.  21  The change in osmotic concentration of r e c t a l f l u i d and hemolymph with time a f t e r the rectum was l i g a t e d .  23  The increase in osmotic concentration of r e c t a l f l u i d with time in preparations bathed in a r t i f i c i a l hemolymph.  24  A comparison of the i o n i c and osmotic concentrations of r e c t a l f l u i d with hemolymph and sea water.  31  Paired determinations of the osmotic concentration (mOsm) and sodium concentration (m ) in the r e c t a l f l u i d from whole larvae.  54  Paired determinations of the sulphate concentration i n the hemolymph and r e c t a l f l u i d of whole larvae.  59  A diagrammatic representation of the proposed locations of ion transport processes i n the Malpighian tubule, rectum and anal papillae of A_. campestris larvae, in each of three media used i n this study.  64  m  X  10  The drinking rate of larvae i n the media i n which they were reared  76  11  The relationship between the weight and v o l ume of A,, taeniorhynchus larvae*  78  12  The relationship between the weight and surface area of A,, taeniorhynchus larvae*  79  13  The relationship between the volume and drinking rate of larvae i n four s a l i n i t i e s , 10$, 50$ 100$ and 200$ sea water.  80  14  The relationship between the surface area and drinking rate of larvae i n four s a l i n i t i e s ; 10$, 50$ 100$ and 200$ sea water.  82  15  The mean concentrations of sodium, potassium magnesium and calcium in the hemolymph of larvae reared i n sea water of d i f f e r i n g salinity.  83  16  The mean c h l o r i d e , sulphate and osmotic concentrations i n the hemolymph of larvae reared in three media of d i f f e r i n g s a l i n i t y .  85  17  The i o n i c and osmotic concentrations of r e c t a l f l u i d from whole larvae in 10$j 100$, and 200$ sea water.  87  18  The effect of a r t i f i c i a l hemolymph d i f f e r i n g only i n osmotic (sucrose) concentration on the osmotic concnetration of r e c t a l s e c r e t i o n .  92  19  The e f f e c t of varying chloride concentration in a r t i f i c i a l hemolymph on the volume, chloride concentration and osmotic concentration of r e c t a l secretion c o l l e c t e d a f t e r 1.5 h.  94  20  The effect of varying sodium concentration i n a r t i f i c i a l hemolymph on the volume, sodium concentration and osmotic concentration of r e c t a l secretion c o l l e c t e d a f t e r 1.5 h.  96  21  The relationship between the concentration of chloride or sodium in a r t i f i c i a l hemolymph bathing the rectum and the rate of transport of that i o n .  98  22  The t r a n s - r e c t a l e l e c t r i c a l potential d i f ference observed i n anterior r e c t a l segments bathed i n normal a r t i f i c i a l hemolymphs or a r t i f i c i a l hemolymphs d i f f e r i n g in the concentrations of the ion i n d i c a t e d .  130  23  The t r a n s - r e c t a l e l e c t r i c a l potential d i f ference observed i n the posterior r e c t a l segment bathed i n normal a r t i f i c i a l hemolymph or a r t i f i c i a l hemolymphs d i f f e r i n g i n the concentrations of the ion i n d i c a t e d .  131  24  The effect of increasing the potassium concentration of the a r t i f i c i a l hemolymph on the t r a n s - r e c t a l e l e c t r i c a l potential difference of the posterior r e c t a l segment.  133  25  The effects of varying the sodium concentration of the a r t i f i c i a l hemolymph on the transrectal e l e c t r i c a l potential difference of the posterior r e c t a l segment.  135  26  The effect of varying the chloride concentration of the a r t i f i c i a l hemolymph on the transr e c t a l e l e c t r i c a l potential difference of the posterior r e c t a l segment.  136  27  The t r a n s - r e c t a l e l e c t r i c a l potential d i f ference observed in two posterior r e c t a l segments bathed in normal a r t i f i c i a l hemolymph or a r t i f i c i a l hemolymph containing 1.3 mlCl".  138  28  The t r a n s - r e c t a l e l e c t r i c a l potential d i f ference observed in the posterior r e c t a l segments bathed in normal a r t i f i c i a l hemolymph or a r t i f i c i a l hemolymphs d i f f e r i n g in the concentration i f the ion i n d i c a t e d .  139  29  A model of the process occurring in the c e l l s of the posterior r e c t a l segment during the secretion of hyperosmotic f l u i d .  143  xii  LIST OF PLATES  Plates  Page Photographs of in, vivo r e c t a l preparations 5 min. and 2 h after l i g a t i o n Photographs of in, v i t r o rectal preparations 10 min. and 2 h after dissection and l i g a t i o n  26  111  xiii  ACKNOWLEDGEMENTS  I would l i k e to thank Dr. John P h i l l i p s for his assistance and guidance, his f a i t h i n me and patience with me. I am indebted to Dr. Simon Maddrell for invaluable technical advice and stimulating d i s cussions. I am grateful to Drs. John G o s l i n e , Dave Randall and G.G.E. Scudder for their useful comments regarding the form of this t h e s i s . I wish to thank M.S. Haswell for technical assistance i n measuring ionic concentrations, Dr. J . K . Nayar f o r sharing his knowledge of c u l t u r i n g techniques and Dr. E.P. Marks f o r information regarding the in v i t r o preparation. I am grateful to Joan Martin and Doug Williams f o r i n t e r e s t i n g discussions, pertinent and otherwise. I wish to thank my wife, L i s a , f o r typing the f i r s t draft of the manuscript as well as contributing to the progress of this thesis by other less tangible means.  CHAPTER I GENERAL INTRODUCTION  la  1b  THE FORMATION OF URINE IN INSECTS  Urine formation i n most of those insects which have been s t u d i e d , i s a two-step process, involving non-selective secretion of a f l u i d followed by s e l e c t i v e resorption of solutes and/or water in appropriate amounts p r i o r to excretion.  The formation of the primary excretory  f l u i d occurs in the Malpighian tubules (Ramsay, 1950-1961) by a process of K  +  s e c r e t i o n , which forms an osmotic gradient leading to d i f f u s i o n of  water into the lumen of the tubule.  Ramsay suggested that other solutes  enter the lumen following t h e i r respective concentration gradients, r e s u l t i n g i n a urine which i s isosmotic to the hemolymph.  The rate of  urine secretion was found to be proportional to the hemolymph potassium concentration but phosphate ions were also necessary for s e c r e t i o n , and sodium could drive secretion when potassium concentrations were very low.  In some insects Na i s the major cation transported (reviewed by +  Maddrell, 1971).  More recent investigations have revealed the active  transport of a c i d i c dyes (Maddrell et a l , 1974), M g Maddrell, 1975)  ++  ( P h i l l i p s and  and SO* (Maddrell and P h i l l i p s , 1975).  The primary excretory f l u i d travels down the Malpighian tubules to the junction of the hind- and midgut.  In come species, resorp-  tion occurs in the lower portions of the tubule (Ramsay, 1952; Messing, 1967; I r v i n e , 1969; Maddrell and P h i l l i p s , 1975b).  Upon reaching the  alimentary t r a c t , the excretory f l u i d i s directed posteriorly into the rectum v i a the i n t e s t i n e .  Maddrell (1971) reviewed the evidence p o i n t -  ing to resorption of ions and water in the i n t e s t i n e of several i n s e c t s .  2  He suggests a s i g n i f i c a n t role f o r this region of the gut, p a r t i c u l a r l y in insect species such as the locust where molecular sieving by the r e c t a l c u t i c l e may reduce the rate at which large organic molecules are absorbed i n the rectum.  ( P h i l l i p s and D o c k r i l l , 1968).  With the exception of those cases already c i t e d , the preceding events lead to the passage into the rectum of a f l u i d approximately i s osmotic to the hemolymph.  Clearly osmotic homeostasis i s maintained by  means other than those yet described.  RECTAL FUNCTION IN INSECTS  In most i n s e c t s , "primary urine" from Malpighian tubules i s modified i n the hindgut or rectum to maintain osmotic homeostasis (Maddr e l l , 1971).  The urine i s concentrated i n the rectum in t e r r e s t r i a l and  saline-water insects and made more dilute i n freshwater i n s e c t s .  Hyper-  osmotic excreta are formed in t e r r e s t r i a l insects,by the resorption of water i n the rectum from the primary excretory f l u i d supplied from the Walpighian tubules (reviewed by P h i l l i p s , 1970).  The resorption of wat-  er i s thought to be driven by l o c a l osmotic gradients formed by ions that are a c t i v e l y resorbed from the r e c t a l lumen, and concentrated within the l a t e r a l i n t e r c e l l u l a r channels between c e l l s .  The water drawn by this  process from the lumen sweeps the ions along these channels to areas in which ion resorption back into the r e c t a l c e l l s occurs.  This results  i n a d i l u t i o n of the f l u i d moving through the r e c t a l epithelium, such that i n dehydrated i n s e c t s , the resorbate which f i n a l l y enters the hemo-  3  lymph at the mouth of the l a t e r a l channels i s not only hyposmotic to the rectal f l u i d but may be more d i l u t e than the hemolymph as w e l l . In conditions of excess ingestion of water, this mechanism would obviously be inappropriate for the maintenance of osmotic homeostasis.  In these circumstances, fewer ions are presumably resorbed  as the f l u i d passes along the i n t e r c e l l u l a r channels, r e s u l t i n g in a hyperosmotic resorbate, and thus a hyposmotic excreta.  The desert locust  Schistocerca greaaria forms hyperosmotic excreta under a l l conditions, but other i n s e c t s , such as the blowfly and cockroach, can form either hyper- or hyposmotic excreta ( P h i l l i p s , 1964a, b, c ; 1970). The rectum of freshwater aquatic insects produces a urine which i s hyposmotic to the hemolymph (reviewed by Shaw and Stobbart, 1963). The d i l u t i o n of the urine i n these insects i s believed to occur i n the rectum by means of selective ion resorption from the primary excretory fluid.  The- term fresh-water insect when applied to some species refers  more to an i n a b i l i t y to produce hyperosmotic urine than i t does to the environments in which they are found (Beadle 1943, 1969).  Often an i n -  sect group can be divided into two categories, those inhabiting fresh water very hyposmotic to the hemolymph, and those inhabiting saline water s l i g h t l y hyposmotic to the hemolymph.  This separation of niches may  occur at the l e v e l of genera, Limneohilus s p . , Anabolia sp. 1961a» b)j  (Sutcliffe  s p e c i e s , Cenocorixa b i f i d a . C. exoleta (Scudder, 1969a, b;  Scudder ejb a l , 1972); or that of physiological races of the same spec i e s , Cricotoous v i t r i o e n n i s ( S u t c l i f f e  1960).  Yet a l l of these i n s e c t s ,  and indeed a l l that are termed freshwater forms, cannot form hyperosmotic  4  urine, and must adjust to hyperosmotic environments, i f  at a l l , by allow-  ing their hemolymph concentration to r i s e . In sharp contrast to this mode of regulation i s that employed by the euryhaline saline-water insects which can form hyperosmotic urine. Ramsay (1950) measured the osmotic concentration of various body f l u i d s in the saline-water larvae of Aedes d e t r i t u s . raised i n sea water.  The f l u i d  from the Malpighian tubules was very s l i g h t l y hyperosmotic to the hemolymph and the r e c t a l f l u i d was markedly hyperosmotic, with a mean concentration near that of sea water. Nayar (1975) has shown that Aedes taeniorhynchus i s capable of maintaining hemolymph levels of sodium(173-218mM)  and  chloride(48-78mM)  within narrow l i m i t s in the face of a wide range of external concentrations(0-300$ sea water).  The fflalpighian tubules of this species produce  a f l u i d which i s very s l i g h t l y hyperosmotic to the hemolymph by a mean value of 15 mOsm (ffladdrell and Bradley, unpublished observations).  This  agrees with Ramsay's (1950) findings for A. d e t r i t u s , whereas in A,. campestris the Malpighian tubule f l u i d i s isosmotic to the hemolymph ( P h i l l i p s and Waddrell, 1975).  Therefore, the extreme concentration of  the urine to a l e v e l approximately equal to sea water must occur in the rectum. Shaw and Stobbart (1963) suggest that the concentration of the urine in the rectum of A,, detritus might occur by the same mechanism as in terrestrial  insects, i . e .  amounts of s o l u t e .  resorption of water without proportional  Meredith and P h i l l i p s (1973a) investigated the r e c t -  a l ultrastructure of the saline-water larva A. campestris in both hypo-  5  and hyperosmotic environments.  They found the ultrastructure  of the  anterior r e c t a l segment of A. campestris which contains only one c e l l type to be very s i m i l a r to that of the rectum of A,, aegypti and suggested that this portion resorbs ions from the primary excretory f l u i d when the larva i s i n hyposmotic environments.  This would mean that this a c t -  i v i t y i s associated with straight l a t e r a l membranes, moderately i n f o l d ed a p i c a l ones, extensively infolded basal membranes and mitochondria distributed evenly throughout the c e l l . d i s s i m i l a r i t y to the recta of t e r r e s t r i a l  Based on the  ultrastructural  i n s e c t s , Meredith and P h i l l i p s  also suggested that the posterior rectum forms a concentrated excreta by the secretion of a hyperosmotic f l u i d into the lumen. um of t e r r e s t r i a l  Whereas the  rect-  insects i s characterized by r e l a t i v e l y straight basal  membranes', moderately infolded a p i c a l ones, and highly infolded l a t e r a l membranes i n close association with most of the c e l l ' s mitachondria, the posterior rectum of A_. campestris showed straight  l a t e r a l membranes,  moderately infolded basal ones, and a highly infolded apical membrane associated with most of the mitochondria. having a rectum u l t r a s t r u c t u r a l l y  The only t e r r e s t r i a l  insect  s i m i l a r to this i s Thermobia domestica.  This surely represents a s u p e r f i c i a l resemblance, since the l a t t e r rectum has been implicated in the uptake of water from subsaturated a i r Nesbitt, 1973).  (Noble-  A s i m i l a r structure i s observed in the paunch of the  termite Cephalotermes which i s separate from the rectum, and which i s thought to have evolved to serve b a c t e r i a l symbionts.  (Noirot +Noirot-  Thimotee, 1967). In summary, for the three regulatory e p i t h e l i a just discussed,  6  s a l t resorption to form a hyposmotic urine i s associated with w e l l developed a p i c a l membranes and evenly d i s t r i b u t e d mitochondria.  Water  resorption to form a hyperosmotic urine i s found i n recta having highly folded l a t e r a l membranes associated with many mitochondria.  Hyper-  osmotic secretion i s hypothesized for tissues showing extensive apical i n f o l d i n g associated with many mitochondria (reviewed by Meredith and P h i l l i p s , 1973a; Wall and Oschman, 1975). The research described i n Chapter II  was undertaken to obtain  d i r e c t experimental evidence f o r or against the hypothesis of P h i l l i p s and Meredith (1969a) and Meredith and P h i l l i p s (1973a) that the r e c t a l epithelium of saline-water mosquito larvae produces a hyperosmotic excreta by s a l t secretion into the r e c t a l lumen, rather than by water r e s o r p t i o n , as occurs in t e r r e s t r i a l i n s e c t s .  Once i t had been e s t a b l i s h -  ed that hyperosmotic urine i s formed by s a l t secretion the ion concentrations of the r e c t a l secretion was measured to demonstrate that the f l u i d produced was contributing to osmoregulation.  These measurements were  made f o r larvae reared in sea water of varying concentration and also (Na+fflg)S0^ and NaHCO^ waters to investigate the a b i l i t y of the larvae to adjust secretion to varying ionic environments i n which they are naturally found (Chapters III  + IV).  Meredith and P h i l l i p s (1973a) also suggest that the anterior rectum i s the s i t e of s a l t resorption from primary excretory f l u i d in fresh and saline water and that the posterior rectum i s the s i t e of s a l t secretion in saline water.  This hypothesis was tested using an iri v i t r o  rectal preparation (Chapter V).  In addition} e l e c t r i c a l potential mea-  7  surements were c a r r i e d out in order to ascertain which ions are a c t i v e ly transported i n the l a r v a l rectum and in what region secretion may occur. A study of the kinetics of the transport mechanisms for sodium and c h l o r ide in the rectum was carried out.  F i n a l l y , I propose a model f o r the  organization of transport processes i n e p i t h e l i a l c e l l s of the posterior rectum, which i s consistent with experimental observations presented i n this thesis (Chapter  VI).  A word of explanation concerning the organization of this thesis i s i n order. P h i l l i p s , 1975).  Chapter II  i s presented as published (Bradley and  Chapters III-VI are s l i g h t l y modified from the form  i n which they were submitted as three papers to Journal of Experimental Biology.  I trust the reader w i l l excuse some overlap of introductory  material and discussion which i s appropriate i n separate p u b l i c a t i o n s .  CHAPTER  II  THE SECRETION OF HYPEROSMOTIC FLUID BY THE RECTUM OF A SALINE-WATER MOSQUITO LARVA AEDES TAENIORHYNCHUS (WIEDEMANN)  8  9  INTRODUCTION  The rectum of insects i s generally considered to be the s i t e of s e l e c t i v e reabsorption from the primary excretory f l u i d produced by the Malpighian tubules.  This a c t i v i t y has been shown i n t e r r e s t r i a l  l i p s , 1964a-cj 1970)  and freshwater insects (reviewed by Stobbart and  Shaw, 1964)  and i s thought to be ultimately  (Phil-  responsible f o r osmotic regu-  l a t i o n i n most i n s e c t s . A s i m i l a r mechanism has been postulated f o r saline-water sects.  in-  Several euryhaline insects are known to produce strongly hyper-  osmotic urine (reviewed by Stobbart and Shaw, 1964; Leader, 1972).  Ram-  say (1950) showed that the f l u i d entering the rectum of Aedes detritus larvae derives from the Malpighian tubules and i s 5-10$ hyperosmotic to the hemolymph, while the rectal f l u i d leaving the anus had a concentration s i m i l a r to sea water and up to three times the osmotic concentration of the hemolymph.  It  has since been suggested that the f l u i d became  centrated in the rectum i n the same manner as i n t e r r e s t r i a l  con-  insects  (Stobbart & Shaw, 1964), that i s by s e l e c t i v e reabsorption of water without proportional amounts of solute (reviewed by P h i l l i p s , 1970; Maddrell, 1971).  Ramsay associated the a b i l i t y of this saline-water larva to pro-  duce hyperosmotic excreta with an additional r e c t a l segment absent in freshwater species of mosquito larvae. However, P h i l l i p s and Meredith (1969a), and Meredith and P h i l l i p s (1973a) found that the u l t r a s t r u c t u r a l  features associated with the  production of hyperosmotic urine in the rectum of most t e r r e s t r i a l  in-  10  s e c t s , namely elaborate development of the l a t e r a l membranes which are associated with most of the mitochondria of the c e l l s , were absent i n the r e c t a l epithelium of another saline-water mosquito l a r v a , Aedes campestris.  Instead,  highly developed.  the a p i c a l membrane facing the lumen was most  Since these larvae were observed to drink saline  water at high rates and thereby gain water, these authors suggested that the larvae need only r i d themselves of excess ions so ingested to achieve osmotic balance.  They proposed, therefore,  that hyperosmotic  urine was produced by active secretion of these excess ions across the elaborately developed a p i c a l membranes of the posterior rectum. This proposal gained support from the observation of Prusch (1971-1974) that ammonium ions are secreted into the hindgut of Sarcophaoa bullata to produce a hyperosmotic f l u i d .  However, these larvae  have no rectum and the secretion of ammonium ions might represent an i s o l a t e d adaptation to an environment r i c h i n this i o n , and thus might be inapplicable to 3 a l i n e - w a t e r insect larvae. The work described in this Chapter was undertaken to obtain d i r e c t physiological evidence f o r or against the suggestion of  Meredith  and P h i l l i p s (1973a), that mosquito larvae l i v i n g in saline water produce hyperosmotic urine by secretion of ions into the rectum.  Aedes  taeniorhynchus» a mosquito native to coastal sea water swamps i n North America, was used as the experimental animal because of the ease with which i t can be reared year-round in the laboratory (Nayar, 1967). Nayar & Sauerman (1975) have shown that this euryhaline species i s c a pable of maintaining hemolymph levels of sodium (l73-218mffl) and c h l o r -  11  ide (48-78mM) within narrow l i m i t s i n the face of a wide range of external concentrations (0-300$ sea water).  12  MATERIALS AND METHODS  A culture of Aedes taeniorhynchus was obtained from the Entomological Research Center, Vero Beach, F l o r i d a and maintained according to the method of Nayar (1967) in sea water obtained i n the Vancouver area (832 - 8.8 mOsm).  Fourth i n s t a r larvae were starved f o r 1-2 days  before being used i n experiments.  The larvae were reared at 27°C and  a l l experiments were carried out at that  temperature.  Drinking rate was determined by measuring the i n i t i a l  linear  increase i n whole-body a c t i v i t y following transfer of larvae to sea water 14  containing  / \ C - i n u l i n (.New England Nuclear C o r p . ) .  Larvae were removed  in groups of 5 a f t e r various time i n t e r v a l s , weighed, placed i n 1 ml of 10$ KOH, macerated, incubated f o r one hour at 90°C and allowed to c o o l . The solution was then neutralized with 1 ml of appropriate  strength  H^SO^ and 1 ml aliquots were added to 10 ml of ' S c i n t i v e r s e * (Fisher S c i e n t i f i c Co.) and counted in a Nuclear Chicago *Isocap 300'  liquid  s c i n t i l l a t i o n system, Hemolymph was obtained from larvae blotted dry on f i l t e r paper and torn open so as to allow the hemolymph to flow onto a sheet of •Parafilm'.  No difference i n estimates of hemolymph composition was  observed between larvae removed d i r e c t l y rinsed i n d i s t i l l e d water.  from sea water and those f i r s t  The hemolymph was immediately taken up i n a  1 pi Drummond pipette and volume estimated from the length of the f l u i d column (Kaufman and P h i l l i p s , 1973).  Samples of r e c t a l f l u i d were ob-  tained by tearing open the integument of the larvae on f i l t e r paper and  13  thus allowing the hemolymph to be absorbed.  A sharp glass micropipette  f i l l e d with l i q u i d paraffin was used to puncture the rectum and suck up the contents.  The resultant drop was immediately expelled into mineral  o i l in a p e t r i dish coated with paraffin wax. was estimated by measuring i t s diameter.  The volume of the drop  This volumetric technique was 14  calibrated by estimating radioactivity in drops of a standard solution ( F i g .  C-inulin  1).  Ionic concentrations of hemolymph and r e c t a l f l u i d s were measured by transferring the samples to v i a l s containing 1 ml of the following:  d i s t i l l e d water for sodium, 500 uM NaC1 for potassium, and  1.5% NaEDTA for magnesium.  Concentrations were determined using a  'Techtron AA 120' atomic absorption spectrophotometer i n the transmission (Na , K ) or absorption (Mg ) +  mode.  Chloride concentrations  were measured according to the f i r s t electrometric method of Ramsay, Brown and Croghan (1955).  Osmotic concentrations were determined us-  ing a nanoliter osmometer ( C l i f t o n Technical Physics Ltd.) or the c r y oscopic method of Ramsay (1949). To measure changes in rectal f l u i d concentration with time, larvae were ligatured with fine s i l k thread behind the midgut, approximately between the s i x t h and seventh abdominal segments, thus e l i m i n ating delivery of f l u i d from the midgut and Malpighian tubules.  A  second ligature around the anal segment prevented any f l u i d from entering or leaving the rectum other than by passing through the wall of the rectum and a short portion of the i n t e s t i n e .  The larva was severed in  front of the anterior ligature to produce the experimental preparation  Figure 1.  Measurements demonstrating the l i n e a r relationship between 14  the r a d i o a c t i v i t y of drops of  C - i n u l i n solution and the calculated  volume of those drops based on t h e i r measured diameter.  The radio-  a c t i v i t y of drops of 1u1 s i z e measured by t h i s method and by 1JJ1 "Microcap" pipettes were also i n agreement.  15  shown in Figure 2.  This posterior portion of the animal was suspended  from the surface of a solution by means of the respiratory siphon,  there-  by assuring a constant supply of a i r to the rectum via the tracheae. P h i l l i p s (unpublished observation) found that chloride was a c t i v e l y absorbed by the anal papillae of Aedes campestris adapted to fresh water at comparable rates i n intact larvae and in posterior segments prepared i n the manner outlined above.  The above preparation was suspended i n  sea water, and hemolymph and r e c t a l samples were taken a f t e r varying periods of  time. I n i t i a l studies indicated that the concentration of the l i m i t -  ed volume of hemolymph i n the above preparation increased d r a s t i c a l l y with time.  To eliminate this problem in l a t e r experiments, the same  preparation was floated i n an a r t i f i c i a l tegument was torn open. in a relatively  hemolymph solution and the i n -  Under these circumstances the rectum was bathed  large volume of a r t i f i c i a l  to i t s normal tracheal supply.  medium but was s t i l l connected  The a r t i f i c i a l  hemolymph was a modified  form of that used by Berridge (1966) and contained the following i n mg/l00ml of water: NaC1 175.3, Na c i t r a t e 69.7, KC1 44.8, 29.0,  CaC1 .2H 0 2  2  ClgC1 . 6H 0 264, T . C . Yeastolate 400, P e n i c i l l i n "G" 10, Strepto2  2  mycin 20, Na succinate 200, Malic acid 200, Na glutamate 235, glucose 200, maltose 200, trehalose 200, glycine 20, proline 53, glutamine 40. The concentrations of the major inorganic ions are given in Table 1. The preparation described above was used to measure changes in the volume of r e c t a l contents in two ways.  Photographs were taken of the  recta of larvae immediately a f t e r , or two hours a f t e r , l i g a t i o n .  In  Figure 2.  A schematic diagram shouting the three most posterior  segments of the mosquito larva with the position of used to i s o l a t e the rectum. AP anal p a p i l l a e , AR anterior  ligatures  Abbreviations: A anus, AC anal c a n a l , rectum, H hemolymph, I  intestine,  M Malpighian tubules, PR posterior rectum, S siphon, T tracheae, X anterior  ligature,  Y posterior  ligature.  16b  17  Table 1.  Ionic concentrations (mfll) and osmolality (mOsm) of  and natural hemolymph (mean - S . E . ) .  Hemolymph Na K  +  +  |Ylg  ++  Cl" Osmolality  A r t i f i c i a l hemolymph  149-3  148  16-1  17  10-1  24  98-5  97  348 - 17  315  artificial  18  the second method, after the anterior ligature mas i n place but before 14 the posterior one had been a p p l i e d , a known volume of  m  C - i n u l i n i n 10%  sea water was injected into the rectum by means of a micropipette i n serted through the anus.  After 20 seconds the rectum was emptied using  the same micropipette, and the volume and r a d i o a c t i v i t y of the r e s u l t :ing drop was measured for comparison with the known values for the f l u i d injected.  Assuming uniform d i s t r i b u t i o n of i n u l i n , the percent-  age of the injected r a d i o a c t i v i t y recovered from the rectum corresponds to the percentage of the rectal f l u i d removed, and the volume recovered could be measured, an estimate of the f l u i d volume remaining in the rectum could be made.  The anal segment of the larva was then ligatured  to" i s o l a t e the rectum, the exterior c u t i c l e t o r n , and the preparation floated i n a r t i f i c i a l hemolymph f o r two hours.  At that time the rectum  was emptied as described above and the volume and osmotic concentration of the contents recovered were determined.  The difference between the  i n i t i a l and f i n a l estimates of volume gave a minimum estimate of the i n crease in the volume of the r e c t a l contents, i . e .  total secretion.  The composition of the r e c t a l f l u i d secreted by ligated post e r i o r segments was compared with that from i n t a c t animals.  Determining  the composition of the rectal contents from normal, unligated larvae presented some d i f f i c u l t i e s .  Larvae empty the rectum i n response to almost  any disturbance, making i t impossible to introduce a pipette into the rectum via the anus before the r e c t a l contents have been voided.  For  this reason the anus must be closed to enable the r e c t a l f l u i d to accumulate and remain in the rectum u n t i l sampled.  Since a ligature around the  19  anus crushes the tissue and might seriously stress the whole animal, an alternative method, which permitted retention of normal neural and tracheal connections to the rectum, was used.  This consisted of block-  ing the anus of the intact larva with Eastman 910 tissue adhesive. These larvae were placed in sea water f o r one hour, at which time the r e c t a l contents were removed as described above.  20  RESULTS  Estimation of Osmotic Load The saline-water mosquito l a r v a , Aedes campestris, ingests large quantities of external medium ( P h i l l i p s & Meredith, 1969b; Kiceniuk & P h i l l i p s , 1974).  The unavoidable intake of ions associated  with this f l u i d ingestion i s the main source of s a l t entry into  this  species ( P h i l l i p s + Bradley, in press) and hence largely establishes the rate of ion secretion required to achieve i o n i c balance in this animal.  The drinking rate of Aedes taeniorhynchus i n sea water was  therefore measured to provide a minimum estimate of ion and water turnover. 14 The i n i t i a l accumulation of  C - i n u l i n by whole larvae i n sea  water ( F i g . 3) indicated that they ingested 33.5 n l mg body weight -1 h  +  -1  , or 100 - 2 n l h  weight of 3.0 - 0.5 mg.  _1 larva  + mean - S . E . f o r larvae having a mean  Thus, £ . taeniorhynchus larvae drink  own body weight every 29.6 h.  their  This i s comparable to the rates for A..  campestris in saline media reported by Kiceniuk & P h i l l i p s (1974). the sodium concentration of sea water i s nearly 3 times that of  Since  larval  hemolymph, the hemolymph content of t h i s ion must be turned over in approximately 5.0 - 6.6 h, assuming hemolymph to body volume ratios of 1/2 arid 2/3 respectively.  Chloride must turn over even more rapidly since the  blood level of this anion i s 2/3 that of sodium.  In spite of this  rapid turnover, the larvae maintain the i o n i c and osmotic concentration of their hemolymph at about one-third that of sea water (Table  1).  Figure 3.  The amount of external medium consumed with time as  estimated from the increase in whole body r a d i o a c t i v i t y with time a f t e r placing mosquito larvae in sea water containing 14 C-inulin. each point.  V e r t i c a l bars denote S . E . of the means, N=6 for  22  Osmolality of Rectal F l u i d In order to demonstrate that the urine of A,, taeniorhynchus becomes hyperosmotic to the hemolymph while in the rectum, larvae were ligated as shown in F i g . 2 and placed in sea water.  During the manip-  ulations and p r i o r to l i g a t i o n of the rectum, defecation  invariably  occurred and the rectum r e f i l l e d with a very small volume of f l u i d from the midgut and Malpighian tubules.  Not s u r p r i s i n g l y , therefore, samples  of r e c t a l f l u i d collected within a few minutes of l i g a t i o n were isosmotic with the hemolymph ( F i g . 4),  in agreement with observations on several  other insect species (reviewed by P h i l l i p s , 1970).  The osmotic con-  centration of the rectal f l u i d measured a f t e r one and two hours showed a continual increase.  Hemolymph osmotic concentration did not change s i g -  n i f i c a n t l y over the f i r s t second.  hour, but increased s i g n i f i c a n t l y during the  Indeed, over the second hour, the osmotic concentration  ence across the r e c t a l wall did not change appreciably.  differ-  This increase  i n hemolymph concentration was attributed to passive net exchange of water and ions across the body wall of the l a r v a . I wished to separate the increase i n r e c t a l f l u i d concentration from the influence of the increasing hemolymph concentration. the same type of preparation was placed i n a large volume of hemolymph (Table 1)  To this end, artificial  and the body wall was torn open to expose the  um to this external medium.  rect-  Under these conditions the osmotic pressure  of the r e c t a l f l u i d increased within 0.5 h to a value 2.4 times that of the bathing medium, but did not change s i g n i f i c a n t l y over the next 1.5 h ( F i g . 5).  The osmotic gradients produced by the two types of  preparation  Figure 4.  The change in osmotic concentration of r e c t a l  fluid  and hemolymph 0  with time after the rectum was ligated as  shown i n F i g . 1 .  The preparation was placed i n sea water.  V e r t i c a l bars denote S . E . of the means.  (n=10)  Time (h)  Figure 5. uiith time.  The increase in osmotic concentration of r e c t a l f l u i d The rectum was ligated as shown i n F i g . 1, the i n -  tegument was t o r n , and the preparation was placed in hemolymph.  V e r t i c a l bars denote S . E . of the means.  artificial  1000  0  25  did not d i f f e r s i g n i f i c a n t l y at 1 h (P<0.1), i n d i c a t i n g that the rectum was able to function equally well in a r t i f i c i a l  and natural hemolymph.  In summary, the rectal wall of this species i s capable of developing large osmotic gradients, thereby producing urine strongly hyperosmotic to the hemolymph.  The question remains whether this i s achieved by  s e l e c t i v e water resorption from the small volume of isosmotic f l u i d i n itially  present in the lumen, or by secretion of a hyperosmotic f l u i d  into the rectum.  These p o s s i b i l i t i e s were d i f f e r e n t i a t e d  by measuring  the change in volume of the r e c t a l contents under the experimental conditions described above.  Changes in Volume of the Rectal Contents Increases in rectal volume during the production of hyperosmotic urine by ligated recta were apparent from the swelling of the rectum and the larger volume of f l u i d which could be recovered, using micropipettes, at the end of a l l experiments.  This considerable i n -  crease in r e c t a l volume was documented by comparing photographs of recta taken within 5 min or at 2 h a f t e r i s o l a t i o n of the rectum by l i g a t i o n (Plate 1).  The rectum, which i s nearly empty a f t e r being l i g a t e d , swells  considerably with f l u i d which can only have been produced by way of al secretion.  rect-  In a d d i t i o n , d i s t i n c t posterior and anterior segments  of  the rectum are c l e a r l y delineated in these photographs. A more quantitative  estimate of this volume increase was made  using ^ C - i n u l i n as a volume indicator (Table 2 ) . to be evenly distributed in the rectum, the i n i t i a l  Assuming the i n u l i n volume of  rectal  Explanation of Plate 1  Photographs of recta 5 minutes ( l e f t ) and 2 h ( r i g h t ) a f t e r  ligation.  At 2 h the recta are swollen with f l u i d secreted into the lumen by the rectum.  Exposing the rectum to view destroys the  preparation,  therefore d i f f e r e n t recta are shown in each photograph at the same magnification.  Abbreviations!  tubules, PR posterior rectum.  AR anterior  rectum, M Malpighien  Table 2.  The determination of increase i n volume of r e c t a l contents  following l i g a t i o n of larvae, as shown in F i g . 1.  Recta were injected  14 with a known amount of  C - i n u l i n by means of a micropipette inserted  through the anus before the posterior ligature was a p p l i e d .  The same  pipette was used to retrieve as much f l u i d from the rectum as possible . a f t e r 20 seconds had elapsed ( I n i t i a l ) .  The percent of the i n i t i a l  rad-  i o a c t i v i t y recovered was proportional to the percent of r e c t a l contents removed, allowing an estimate of volume of remaining r e c t a l f l u i d to be made.  The anal segment was then ligated and a f t e r 2 hours the  rectal  contents were measured for volume and osmotic concentration ( F i n a l ) . The volume of secretion i s a minimum estimate because not a l l the f l u i d secreted during the 2 hour period could be recovered by micropuncture.  Final (2 h)  Initial  Series  Volume recovered from rectum  % Radio activity recovered  Calculated volume remaining in rectum (nl)  Volume of rectal f l u i d removed (nl)  Minimum volume secreted by rectum (n1)  Osmolality of r e c t a l f l u i d (mOsm)  (nl) 1 2 3. 4 5  46 17 113 17 36  0  65  65  834  1.5  34  32.5  651  0  45  45  73  6.3  24  17.7  794  91  3.6  23  '19.4  1106  103 92 103  1039  Mean  45.8  92.4  2.3  38.2  35.9  884  S.E.  17.7  5.5  1.2  7.8  8.8  186  29  f l u i d could be estimated from the percent recovery of injected i n u l i n , and was found to be very small (2.3 - 1.2 n l ) .  C-  Within 2 h of  l i g a t i o n , the volume of r e c t a l f l u i d had increased to a mean value of 38.2 - 7.8 n l .  The largest volume increase was 65 n l a f t e r 2 h and a l l  recta showed an increase.  In every case the volume increase was asso-  ciated with a large increase i n osmotic concentration of the r e c t a l f l u i d which did not d i f f e r s i g n i f i c a n t l y (P>0.2) from previous estimates ( F i g . 5).  C l e a r l y , hyperosmotic urine i s formed by secretion of  a hyperosmotic f l u i d into the rectal lumen rather than by selective absorption of water without s o l u t e .  Composition of Rectal Secretion If  the rectum i s s o l e l y responsible f o r i o n i c regulation and  i f a l l ingested ions are absorbed i n the midgut (suggested by Kiceniuk & P h i l l i p s , 1974; Maddrell & P h i l l i p s , 1975) then the r e l a t i v e i o n i c composition of the rectal secretion should r e f l e c t that of sea water  and  the t o t a l concentration of the former f l u i d should be greater to compensate f o r osmosis across the body w a l l .  The small volume of f l u i d s e c r e t -  ed by i n d i v i d u a l recta made i t necessary to pool the f l u i d from 7-10 parations f o r a single chemical determination.  pre-  The average ion concen-  trations i n r e c t a l f l u i d 2 h after l i g a t i o n of r e c t a , which were bathed in a r t i f i c i a l 25 mM-Mg  ++  hemolymph, were!  285 mW-Na (n=1l), 158 mM-K (n = 5), +  (n=5), 425 mW-C1 (n=5), 818 mOsm (n=18). -  +  Standard errors are  recorded i n F i g . 6 , which compares the mean values f o r rectal secretion to those of sea water. Rectal secretion would seem inadequate to explain completely  30  the maintenance of i o n i c balance.  The f l u i d i s not more concentrated  than sea water and the potassium excretion i s many times higher than be accounted f o r by drinking.  can  The larvae were starved p r i o r to the  experimental period, therefore the excess potassium might have been due to autolysis of tissue but probably was not the result of ions present i n the food.  It was f e l t that the unexpected Na:K r a t i o may  have been due to decreased a c t i v i t y of the preparation, or lack of hormonal or neural stimulation of s e c r e t i o n .  With these p o s s i b i l i t i e s  i n mind, r e c t a l f l u i d was c o l l e c t e d from i n t a c t , whole larvae reared in sea water, 1 h a f t e r the anus was blocked with tissue adhesive to allow the accumulation of s u f f i c i e n t r e c t a l f l u i d f o r a n a l y s i s .  The  mean concentrations of a l l ions i n this r e c t a l f l u i d were s l i g h t l y higher ( F i g 6 ) , but not s i g n i f i c a n t l y so (P>0.05 f o r a l l ions measured), and the ion concentration ratios remained unchanged: 192 mm-K (n=5), 36 mm-Mg +  ++  435 mM-Na (n=5), +  (n=5), 468 mlV)-C1 (n=6), 920 mOsm (n=6). =  Occasionally during the sampling of the r e c t a l contents, the rectum was punctured i n such a way that no f l u i d could be seen to leak out and the rectum was observed to empty completely into the p i p e t t e .  This occurred  three times during the sampling of the recta of the larvae with the anus blocked, and yielded volumes of 21, 21 and 92 n l . In whole larvae with the anus blocked, recta are exposed to normal hemolymph and natural hormones, have i n t a c t innervations, and receive f l u i d from the midgut.  The f l u i d derived under these conditions  i s very s i m i l a r to that secreted by the i s o l a t e d , ligated r e c t a l preparation.  Both preparations demonstrate that the l a r v a l rectum of Aedes  taeniorhynchus i n sea water secretes a f l u i d which, though 16 to 19  Figure 6. water.  A comparison of r e c t a l f l u i d with hemolymph and sea  The ionic and osmotic concentrations of  hemolymph  natural  j secretion from ligated r e c t a l preparations ( F i g .  with a torn c u t i c l e , in a r t i f i c i a l  hemolymph  1)  ; r e c t a l f l u i d from  i n t a c t larvae with the anus plugged and placed i n sea water and Vancouver sea water means.  V e r t i c a l bars denote the S . E . of the  Osmolality (mOsm)  32  times higher in potassium than sea water,  i s otherwise nearly  to sea water in i t s i o n i c and osmotic c h a r a c t e r i s t i c s ( F i g .  6).  identical  33  DISCUSSION  The results c l e a r l y confirm the hypothesis ( P h i l l i p s & Meredith, 1969a) that saline-water mosquito larvae produce hyperosmotic urine by ion secretion into the lumen rather than by water reabsorption, as occurs in t e r r e s t r i a l  insects.  This appears to be  the f i r s t example of an insect rectum which functions in this way, although Prusch (1971-1974) has shown s i m i l a r a c t i v i t y in the hindgut of another dipteran l a r v a , Sarcophaga b u l l a t a .  Since the  anterior and posterior rectum were not i s o l a t e d from one another i n the present experiments i t  i s not yet possible to assign the  secretory a c t i v i t y to a s p e c i f i c segment of the rectum, although Meredith and P h i l l i p s (1973a) have presented u l t r a s t r u c t u r a l which implicates the posterior rectum in this a c t i v i t y .  evidence  They have  suggested that the anterior rectum i s involved i n s e l e c t i v e reabsorption when larvae are in hyposmotic environments. The observed concentrations of ions i n the secretion from ligated recta were not s i g n i f i c a n t l y different  from those of sea water,  except that potassium concentration was much higher i n the s e c r e t i o n . Using cryoscopic c o e f f i c i e n t s f o r pure manovalent s a l t s o l u t i o n s , the mean l e v e l s of K , Na* and C l ~ w i l l account f o r 96% of the observed +  osmotic pressure of the r e c t a l f l u i d (818 - 37 mOsm, n=18).  However,  when the magnesium concentration i s included, there i s an excess of cations over anions t o t a l l i n g 68 mEquiv. 1 \  suggesting that other  anions prevalent in sea water ( e . g . S0 ) may be present (suggested by A  34  Maddrell & P h i l l i p s , 1975).  If,  to simplify c a l c u l a t i o n s , one assumes  that the entire anion d e f i c i t i s sulphate ions associated with magnesium and sodium i o n s , the calculated osmotic concentration of the f l u i d using cryoscopic c o e f f i c i e n t s i s 856 mOsm.1 standard error of the observed osmotic pressure.  rectal  which i s within the When the same c a l c u l a -  tions are made using the i o n i c concentrations i n the r e c t a l f l u i d from i n t a c t larvae with blocked anuses, the anion d e f i c i t i s 231 mEquiv. 1 and the t o t a l calculated osmotic pressure i s 1144 mOsm 1  , which ex-  ceeds the standard error of the observed osmotic concentration equal to 137$ sea water).  It  (i.e.  i s therefore necessary to postulate some  ion binding to polyvalent anions, e . g . macromolecules of f e c a l material, as reported f o r magnesium in Ades campestris (Kiceniuk & P h i l l i p s , 1974).  In summary, the major components of the r e c t a l secretion have  been i d e n t i f i e d with the exception of a small anion d e f i c i t .  The s e c -  retion of a l l of these ions (Na , K , fflg , C1 ) occurs against concentration differences of 2 to 1 0 - f o l d .  large  E l e c t r i c a l potentials across  the r e c t a l wall must be measured to determine which of these transport processes are a c t i v e . Aedes taeniorhynchus larvae, i n 100$ sea water, drink and probably assimilate 100 nl of external f l u i d per larva per hour. Some of this water i s presumably lost by osmosis across the body w a l l . Nicholson and Leader (1974) have estimated this loss f o r another s a l i n e water mosquito (Opifex fuscus) of s i m i l a r s i z e , ligatured at the neck and -2 -1 anus, at 0.0026 jul mm h .  Assuming a s i m i l a r osmotic permeability  for  2 Aedes taeniorhynchus, and an approximation of body surface area of 14.6mm ,  35  the calculated net loss of water across the body wall i s 38 n l . h  .  Subtracting this value from the drinking rate gives a value of 62 n l h  for water loss through the excretory system.  The largest  _1 volume of r e c t a l secretion observed was 96 n l h  but the mean was  _1 19 n l h  .  While secretion of Malpighian tubule f l u i d might account  f o r the balance of excreted f l u i d , such f l u i d i s isosmotic to the hemolymph and would not contribute to osmotic regulation ( P h i l l i p s + Maddrell, 1975; Maddrell +, P h i l l i p s , 1975).  The same i s true f o r  any f l u i d which might pass down the i n t e s t i n e from the midgut (Ramsay, 1950; Kiceniuk + P h i l l i p s , 1974). It  would be premature to conclude that r e c t a l secretion i s  inadequate to account f o r osmotic regulation, since the calculations described above f o r larvae with the anus blocked suggest that the effective concentration of the secretion can reach 137$ sea water.  In  order to c o l l e c t enough f l u i d f o r a n a l y s i s , the anus was blocked i n a l l experiments.  The accumulation of f l u i d in the rectum might lead to  under-estimation of both ion concentrations and volume of the r e c t a l secretion for various reasonst 1)  Stretching of the r e c t a l wall might  lead either d i r e c t l y , or i n d i r e c t l y through stretch receptors, to i n creased passive permeability of the r e c t a l w a l l .  2)  The buildup of  hydrostatic pressure in the rectum might oppose further f l u i d s e c r e t i o n . 3) Reabsorption of ions or water might occur in the anterior rectum when distension leads to direct continuity between the two rectal segments. Ramsay (1950), observed that rectal f l u i d of A, detritus was only isosmotic or s l i g h t l y hyposmotic to the sea water medium.  A.  36  taeniorhynchus can survive i n 300$ sea water.  If  the r e c t a l f l u i d i s  isosmotic to the external environment in this s i t u a t i o n , then the rectum i s c l e a r l y capable of creating larger gradients than have been observed.  If  Ramsay's observations with i n t a c t larvae and the present  values f o r larvae with the anus ligated indicate the true upper l i m i t f o r hyperosmosity of r e c t a l secretion then other s i t e s of s a l t s e c r e t i o n , such as the anal p a p i l l a e , must be invoked. P h i l l i p s & Meredith (1969b) have presented preliminary evidence that the anal papillae of A,, campestris larvae l i v i n g in hyperosmotic media might a c t i v e l y secrete chloride to the external medium.  If sodium  i s also secreted by anal papillae in exchange f o r an inward movement of potassium, ( e . g . as in the g i l l s of saltwater t e l e o s t s ; Maetz, 1971), this might balance the loss of K  +  ions by r e c t a l secretion ( F i g .  6).  Leadem (unpublished observation) i n our laboratory has obtained some preliminary evidence for such a mechanism. A,, campestris larvae which l i v e in waters of high NaHCO^ or high MgSO^ content are also known to produce hyperosmotic urine although f l u i d leaving their Malpighian tubules i s isosmotic to the hemolymph ( P h i l l i p s & Meredith, 1969aj Kiceniuk & P h i l l i p s , 1974; P h i l l i p s & Maddrell, 1975; Maddrell & P h i l l i p s , 1975).  Presumably the dominant  ions i n the r e c t a l secretion of this species, which can also l i v e i n sea water, can be varied as dictated by the environment i n which the larvae develop.  Otherwise, i t i s necessary to postulate d i s t i n c t p h y s i o l o g i c a l  races of this species in saline waters having different  dominant i o n s .  •CHAPTER  III  REGULATION OF RECTAL SECRETION IN SALINE -WATER MOSQUITO LARVAE IN ENVIRONMENTS OF VARYING IONIC COMPOSITION  37  38  INTRODUCTION  The osmoregulatory capacities of saline-water were f i r s t  investigated by Beadle (1939).  mosquito larvae  He demonstrated that ion  regulation in Aedes detritus was dependent on tissues in the posterior end of the animal.  Ramsay (1950) extended this work by sampling f l u i d  from various body compartments in  detritus  larvae adapted to sea  water and showed that hyperosmotic urine was formed largely during passage through the rectum. 1975; and Chapter Ii)  In a previous paper (Bradley and P h i l l i p s ,  we presented evidence that the urine was concen-  trated in another saline-water  mosquito l a r v a , Aedes taeniorhynchus. by  secretion of a hyperosmotic f l u i d into the r e c t a l lumen.  It  was of i n -  terest to extend these observations to another euryhaline species of mosquito l a r v a , Aedes campestris, which l i v e s in a l k a l i having a wide range of ionic compositions.  salt-lakes  I wished to determine  wheth-  er the i o n i c composition of rectal secretion r e f l e c t s that of the natural water to which larvae are adapted; i . e .  can the ion ratios of  the secretion be r a d i c a l l y altered from those observed i n A. taeniorhynchus.  Such changes would suggest elaborate control of ion transport  mechanisms and possibly even induction of new protein c a r r i e r s , a process rare amongst metazoans. Scudder (1969a) examined the d i s t r i b u t i o n of several insect species, including A., campestris, found in saline-water ish Columbia.  ponds i n  Brit-  His analyses of the water from these ponds indicates that  A., campestris could survive not only in waters of extremely  variable  39  osmotic concentrations, but d i f f e r i n g i o n i c composition as w e l l .  Larvae  from ponds in which Na2S0^ and MgSO^ are the main s a l t s have been shown to drink and assimilate the M g , SO" and presumably the Na i n such + +  water and to excrete excess M g  ++  +  via the urine (Kiceniuk and P h i l l i p s ,  1974; Maddrell and P h i l l i p s , 1975).  Larvae of the same species thrive  i n the strongly hyperosmotic waters i n which the dominant s a l t s are  either  NaHCO^ of NaC1 and produce urine with an osmotic concentration 2-4 times that of the hemolymph.  They can develop as well i n fresh water and  hyposmotic waters with ionic ratios s i m i l a r to those described above. ( P h i l l i p s and Meredith, 1969a, b; P h i l l i p s and Bradley, 1976). It  was not known whether A. campestris larvae which inhabit  environments with r a d i c a l l y different physiologically races.  i o n i c ratios represented d i s t i n c t  In the present chapter, I have examined this  question by determining whether A., campestris from one pond type could survive in other types of saline water.  The r e c t a l secretions from l a r -  vae i n each of these media were analyzed to determine to what extent rectal secretion can be adjusted by the animal to match the s p e c i f i c i o n i c c h a r a c t e r i s t i c s of each external medium.  MATERIALS AND METHODS Larvae of the mosquito Aedes campestris were collected on A p r i l 19 and May 14,1975 from Ctenocladus pond, a saline pond located near Kamloops, B.C. ( B l i n n , 1969).  Specimens were approximately evenly d i s -  tributed between the four instars at the time of the f i r s t c o l l e c t i o n but t h i r d and fourth instars predominated during the second.  Osmolalit-  40  ies of 277 mOsm and 414 mOsm, respectively were measured on water sampl c o l l e c t e d with the larvae on the above two dates.  The depression im-  mediately east of Ctenocladus pond was also flooded on A p r i l 19 and £ . campestris larvae were c o l l e c t e d from i t as w e l l .  The concentration  of this pool was 75 mOsm. Larvae were maintained in the pond water i n which they were c o l l e c t e d , kept at 10°C and fed dried Brewer's yeast.  Three experiment  a l media were prepared which varied in i o n i c composition but which a l l had an osmolality of 700 mOsm. (Table 3).  Ctenocladus pond water was  f i l t e r e d using Beckman #1 f i l t e r paper, concentrated by evaporation at room temperature  to 1000 mOsm and then diluted back to 700 mOsm with  d i s t i l l e d water, hereafter  referred to as (Na and Mg)S0^ medium.  Sea  water c o l l e c t e d l o c a l l y at Vancouver was f i l t e r e d as above and diluted to 700 mOsm with d i s t i l l e d water (NaC1 medium).  The NaHCO  medium was  prepared as follows and then diluted to 700 mOsm with d i s t i l l e d water: NaHC0 , 4 6 . 2 g / l j KC1, 0 . 7 5 g / l j CaC1 , 0 . 6 7 g / l ; 3  2  MgC1 , 3 . 9 7 g / l . 2  Larvae  were acclimated for at least four days i n these media at 10°C with Brewer's yeast supplied as food, before the r e c t a l f l u i d was sampled. During experiments, larvae were maintained at room temperature  (=22°C).  The Aedes taeniorhynchus larvae used f o r some of the experiments descri bed in this Chapter were reared and experiments were carried out as described previously (Bradley and P h i l l i p s 1975J and Chapter  II).  Samples of rectal secretion and hemolymph were obtained and the sodium, potassium, magnesium, chloride and osmotic concentrations of these samples were determined as previously described (Bradley and  41  Table 3  Ionic concentrations (mM) of external media a l l of which had a t o t a l osmolality of 700 mOsm.  Na* K  +  Ng  ++  CI" SO/ 4 HCO ~  Sea water medium  NaHC0 medium  (Na+Mg)S0 medium  347  438  640  8  12  2  39  8  126  368  26  18  21  0  377  2.1  3  529  4  20.5  42  P h i l l i p s , 1975; and C h a p t e r l l ) . 35 using  Sulphate concentrations were estimated  = SO^ (New England Nuclear) counted with a Nuclear Chicago  "Isocap 300" l i q u i d s c i n t i l l a t i o n system using the channels ratio method of quench c o r r e c t i o n .  S p e c i f i c a c t i v i t y was determined by measur-  ing the r a d i o a c t i v i t y of the external s o l u t i o n , the sulphate concentration of which was determined using the barium p r e c i p i t a t i o n method (Maddrell and P h i l l i p s , 1975).  Larvae were exposed to the labelled ex-  ternal medium f o r at least three days, a time period s u f f i c i e n t to 35 permit  = S0^ s p e c i f i c a c t i v i t y i n the body f l u i d s to reach equilibrium  with that of the external medium.  (Maddrell and P h i l l i p s , 1975).  Samples of rectal f l u i d from whole larvae were obtained by micropuncture 1 h after the anus was blocked with tissue adhesive (Eastman 910; Eastman Kodak).  Isolated recta were prepared by l i g a t i n g larvae  with fine s i l k thread around the anal segment of the seventh abdominal segment, to eliminate f l u i d entry from the Malphigian tubules and to  pre-  vent excretion via the anus (Bradley and P h i l l i p s , 1975; and Chapter  II).  The portion of the larvae anterior to the forward ligature was cut away. The external c u t i c l e was torn between the ligatures and this preparation was suspended by the respiratory siphon i n approximately 0.1  ml of  arti-  f i c i a l hemolymph placed in small depressions which had been cut into paraffin wax on the bottom of 15 x 60 mm petri dishes.  The l i d of the  p e t r i dish was placed over the bottom half during the duration of the experiment.  The concentration of the a r t i f i c i a l hemolymph was found to  change less than 10 mOsm during the course of experiments (2 Two a r t i f i c i a l hemolymphs were used (Table 4).  h).  Based on a  43  Table 4  The i o n i c (mM) and osmotic concentrations (mOsm) of the  artificial  hemolymphs.  Normal Hemolymph Na K  +  +  <  +  Cl"  mOsm  *  Low Chloride  149  150  14  14  5  5  102  20  27  47  346  337  For other constituents common to both these a r t i f i c i a l hemolymphs see Table 7 (Chapter  IV).  44  complex saline described previously (Bradley and P h i l l i p s , 1975; and Chapter Ii)  they differed only in the s a l t s which were used to adjust  the f i n a l sodium and chloride concentrations.  In the high-chloride hemo-  lymph, chloride concentration was adjusted using NaC1 and sodium levels were then adjusted further with Na^SO^.  In the low chloride hemolymph  only Na^SO^ was used to adjust sodium concentration l e v e l s resulting i n a higher (S0~)/(C1~) r a t i o .  A f u l l description of the procedure f o r the  production of these a r t i f i c i a l  hemolymphs i s available i n Appendix IV.  RESULTS Survival of A,, campestris i n various media. Marked differences were found in the a b i l i t y  of larvae from  Ctenocladus pond (277 mOsm) to withstand increases i n external concentrat i o n , as compared to those larvae c o l l e c t e d only a few hundred feet away i n a less s a l i n e pond (75 mOsm).  Although the t o t a l osmotic concentration of  the ponds differed considerably, the r e l a t i v e concentrations of the i n d i vidual ions were s i m i l a r (Table 3).  When larvae from Ctenocladus pond  were taken from their pond water and placed immediately i n 700 Mosm sea water medium, 4$ of the larvae died (n=25) within 4 days.  When acclimated  gradually over four days by replacing the pond water with sea water medium, no larvae died.  By contrast, larvae from the less s a l i n e pond showed a  mo r t a l i t y of 33$ (n=48) over 4 days when transferred immediately and 11.1% (n=27) when acclimated.  Clearly and not s u r p r i s i n g l y , larvae can better  withstand transfer to a strongly hyperosmotic medium i f the solution from which they come i s already f a i r l y concentrated, i n this case about isosmotic to the hemolymph, and i f t r a n s f e r r a l  i s gradual.  No difference i n s u r v i v a l  45  mas detectable i n the two groups of larvae c o l l e c t e d from Ctenocladus pond at different  times, although i t was observed that t h i r d and fourth  i n s t a r larvae can withstand changes of medium better than f i r s t and second i n s t a r s , perhaps due to the difference in the surface area to volume r a t i o .  In a l l subsequent experiments only t h i r d and fourth i n -  s t a r larvae c o l l e c t e d from Ctenocladus pond were used. Table 5 shows the percent mortality  rate of larvae  follow-  ing transfer d i r e c t l y from pond water to the three experimental media of Table 1.  It  can be seen that mortality was highest i n larvae  transferred to 700 mOsm (Na + Mg)S0^ medium as opposed to the 700 mOsm +  NaHCO^ and NaCl media. S0~ and low i n K  ++  The former s o l u t i o n , high i n Na , f?lg  and  and C l " , was c l e a r l y the most toxic of the three media  +  even though the larvae come from natural waters of i d e n t i c a l i o n i c composition but with a t o t a l osmolality approximately isosmotic to the hemolymph (340 mOsm). The results suggest that the larvae found in Ctenocladus pond do not represent a physiological race unable to survive in waters high in NaCl or NaHCO  but rather that the water from which larvae were  o  c o l l e c t e d i s less conducive to their s u r v i v a l than that of other habitats of high s a l i n i t y and d i f f e r i n g i o n i c composition.  natural  Mortality  must be high in dry years when rapid evaporation leads to increasing ion concentrations.  This conclusion i s born out by observations in the  f i e l d (Kiceniuk and P h i l l i p s , 1975).  46  Table 5  Total mortality  observed four days a f t e r A_. campestris larvae  were transferred to three experimental media (700m0sm) of  differ-  ent i o n i c composition.  (Na+IY!g)S0-  4  NaHC0  3  NaC1  medium  medium  medium  149  123  25  original # of larvae % that died  49.8  2.4  4  47  Survival of A,, taeniorhynchus in Various Media The larvae of A. taeniorhynchus are most commonly found in saline-water swamps i n close proximity to the sea (Provost, 1969). They never occur naturally i n fresh water and only occasionally i n inland saline ponds.  It  was of interest therefore,  to test whether  A., taeniorhynchus could survive i n the same wide range of saline water as can A., campestris. A., taeniorhynchus larvae can develop i n any concentration of sea water from d i s t i l l e d water to 300% seawater (Nayar and Sauerman, 1974).  We found that they also can survive and develop in NaHCO^ med-  ium from 0 to 700m0sm but were unable to develop in either Ctenocladus medium diluted to 140m0sm or in a mixture of 80$ sea water and 20$ 700m0sm (Na+Mg)S0^ medium.  This indicates that some f a c t o r or factors  i n Ctencladus medium prevents these larvae from both hypo- and hyperosmoregulating. To determine what the toxic elements i n Ctenocladus pond water might be,  3 groups of 200 larvae each were raised to t h i r d i n s t a r i n  f u l l strength sea water.  At that time one group received an additional  200mM/1 NaC1 the second 100mM/1 MgC1 and the t h i r d 100mm/1 N a S 0 2  2  4<  After 24 hours, no larvae had died i n the seawater + NaC1, 16.5$ died in seawater + MgCI^ and a l l the larvae died in the seawater Na2S0^.  The  control pan containing sea water + NaC1 excludes the p o s s i b i l i t y that the increased mortality in either of the two solutions could be due either to increased osmotic, chloride or sodium concentrations. Mg  ++  The  concentration in* the sea water + MgC1 solution was higher than ?  48  that in (Na+Mg)SO^ medium, suggesting that the t o x i c i t y of the l a t t e r medium to A,, taeniorhynchus larvae i s not due to the high Mg  ++  tration.  concen-  Only the sea water + Na^SO^ solution showed the same high  l e v e l of t o x i c i t y to A,, taeniorhynchus larvae as did (Na + Mg)S0^ medium, suggesting that the toxic factor in Ctenocladus medium i s the high s u l phate concentration.  Of i n t e r e s t i s the observation that the sulphate  proves toxic even when chloride ions are the major anion present.  Hemolymph Anion Concentrations in A_. campestris When A_. campestris larvae are transferred to waters of  differ-  ent s a l i n i t i e s , new steady-state levels of ions in the hemolymph are e s tablished within two days ( P h i l l i p s and Meredith, 1969aj Kiceniuk and P h i l l i p s , 1974? Claddrell and P h i l l i p s , 1975).  In the present study,  concentrations of sulphate and chloride ions i n the hemolymph of A,. campestris were determined after acclimation to the three media f o r four days.  experimental  While chloride levels i n these media varied from  18 to 368mM (Table 3),  those i n the hemolymph were regulated within a  narrow range from 49 - 6 i n NaHC0 to 53 * 5mffl i n (Na + Mg) SO^ and 3  78 - 2mM (n=6)  i n sea water medium; (n=6)  f o r a l l media.  Not s u r p r i s -  ingly only traces of sulphate ions ( 0.1 mM, n = 5) were detected i n the hemolymph of larvae acclimated to the experimental medlumtNaHCO^) lacking this anion.  In sea water medium (20mM S0~) and (Na + MgJSO^  medium (377mM S0~), concentrations of sulphate ions i n the hemolymph were maintained at hypotonic levels of 6.9^0.9 and 135 - 15mM respectively (n=5).  These results agree with those of Maddrell  49  and P h i l l i p s (1975) who found that when i n t e r n a l levels of sulphate ions were varied between 2.5 and 73 mM, the hemolymph concentrations were maintained between 1.5 and 6.6 mM.  However, hemolymph levels  increased sharply and tended to p a r a l l e l external levels when external sulphate exceeded 100 mM, probably due to saturation of the s u l phate transport processes which they demonstrated in the Malphigian tubules.  Composition of Rectal Secretion in Different Media The recta of larvae reared in a l l three experimental media exhibited marked swelling within 1.5 h of l i g a t i o n and analysis of f l u i d c o l l e c t e d from the lumen at that time indicated that the contents were i n a l l cases considerably hyperosmotic (844-968 mOsm; Table 6) the a r t i f i c i a l  hemolymph (346 mOsm; Table 4).  to  Clearly hyperosmotic  urine i s formed in A,, campestris by secretion of a concentrated f l u i d into the r e c t a l lumen, as previously demonstrated f o r larvae of A.. taeniorhynchus l i v i n g in sea water.  The concentrations of each ion in  r e c t a l secretions from larvae adapted to different experimental media are compared in Table 6 and depicted in F i g . 9 to show the ionic composition of the rectal secretion from larvae adapted to a particular experimental medium.  Sodium In a l l three experimental media, the sodium concentration in the r e c t a l f l u i d from whole intact larvae equalled or exceeded that of  Table 6 .  A comparison of the external i o n i c and osmotic concen-  trations in the three acclimation media, with the same parameters i n r e c t a l secretion from two r e c t a l preparations, mean - S . E . , n = 6 except where otherwise indicated: 1)  ligated recta bathed in normal a r t i f i c i a l  2)  recta i n whole larvae.  chloride a r t i f i c i a l  hemolymph and  Ligated recta were bathed i n high-  hemolymph, except f o r some preparations, i n -  dicated by a s t e r i s k s , which were exposed to low-chloride hemolymph (Table  4).  artificial  51  Acclimation Media  Concentrations External Media  Rectal Secretion Ligated recta in a r t i f i c i a l hemolymph  Whole animal with anus blocked  a."~~~Sodium(mM) NaCl medium NaHCOg medium (Na + Mg)S0^ medium  b.  736  +  379  +  640  520 619  +  8  73  347 438  NaHCO  medium  (Na + Mg)S0  11.5  medium  4  1.5  40  NaHCOj medium (Na • Mg) SO^ medium  NaHCOg medium (Na • Mg)S0  4  medium  •  136  +  32  +  8  29 12  126  124  368  498  26 1  fl  225  48  34 71*  639  17  Not measured  12  130  13  + • •  3 1  49  27 +  29  Not measured 7  8  100  NaHCO^ medium (Na • Mg)S0^ medium  21 0  •  53  294  •  41  230 + 15 40  Sulphate (mM) NaCl medium  f.  37  +  + •  1 12  Chloride (mM) NaCl medium  e.  510  +  Magnesium (mM) NaCl medium  d.  39  46 (n=  524  Potassium (mM) NaCl medium  c.  59  6  377  5 6 8  700  1037  ± + •  6* 2 3  + 2 + 2*  55 70  8 0.7 132  • • •  • •  +  55 18 15  2 0.1 32  Osmolality (mOsm) NaCl medium NarlCO^ medium (Na • Mg)S0„  low chloride Ringer  700 700  938 1007 769  •  + •  75 49 51  + 46*  968 844 943  • • •  43 18 47  52  the external media by as much as 1.5 times (Table 6a).  Significant  differences in the sodium ion content of r e c t a l f l u i d from the three groups of whole larvae could not be demonstrated.  Sodium ion levels  in r e c t a l secretions collected from ligated larvae adapted to either NaHC0 or (Na + Ng)SC< media were s i g n i f i c a n t l y lower (P<0.01) as 3  4  compared to sea water adapted larvae.  Consequently the sodium l e v e l  was almost twice as high in f l u i d from l i g a t e d recta of sea water adapted larvae as in NaHCO^-adapted animals. This may indicate that the a r t i f i c i a l  hemolymph did not  contain the appropriate concentrations of sodium and bicarbonate ions for optimum rates of sodium ion secretion by recta of larvae adapted to the NaHCO^ medium.  However, the secretion of the l a t t e r cation was  i f anything, enhanced when the chloride l e v e l in the a r t i f i c i a l hemolymph bathing i s o l a t e d recta from (Na + MgJSO^ adapted larvae was r e duced from 102 to 20 mM. The concentration of sodium in the a r t i f i c i a l  hemolymph was  150 mM, which i s close to the normal hemolymph l e v e l of 135 mM observed i n NaHCOj-adapted larvae ( P h i l l i p s , unpublished observations)! therefore this cation i s secreted into the r e c t a l lumen against 2 to 5 - f o l d concentration differences under a l l of the experimental conditions of Table 6a. Since sodium concentrations in the three experimental media varied two-fold, these experiments do not demonstrate the large changes i n the r e c t a l secretion of this cation which have been observed under appropriate conditions (Chapter  IV).  53  F i g . 7 shows the values obtained when paired determinations were made of osmolality and Na* concentration of the same samples -of rectal secretion. tionality  It  i s clear from these data that no direct propor-  exists between rectal f l u i d Na* concentration and the t o t a l  osmolality of that f l u i d .  Sodium concentrations between various r e c -  t a l f l u i d samples can vary by a factor of 2.5, while osmotic concentration varies only by a factor of 0,5.  This suggests the osmotic  concentration of the rectal secretion may be more s t r i c t l y c o n t r o l l e d , or l i m i t e d , than the Na* concentration.  Potassium Bradley and P h i l l i p s (1975; Chapter II)  showed that the rec-  t a l secretion from A., taeniorhynchus larvae raised i n 100% sea water was 12 times higher in potassium content than was the a r t i f i c i a l lymph and natural hemolymph of whole larvae. campestris (Table 6b); i . e .  hemo-  The same i s true f o r A,.  the potassium concentration in the  rectal  secretion i s 2-10x higher than i n either the hemolymph (14 mM) or the external medium (2 - 12mM).  No s i g n i f i c a n t differences were found be-  tween r e c t a l secretions from ligated larvae versus whole larvae.  The  potassium concentration in the r e c t a l secretion increased proportiona l l y with that of the acclimation medium, and this relationship prevailed even for i s o l a t e d recta which were bathed in the same a r t i f i c i a l hemolymph.  Clearly the level of potassium in the rectal secre-  tion i s dependent on the external conditions to which larvae are adapted.  Figure 7.  Paired determinations of the osmotic concentration  (mOsm) and sodium concentration (mM) in the r e c t a l f l u i d from whole larvae.  Osmotic Concentration of Rectal Secretion(mOsm) 00 o o  — I o o o  1  1  1  o o  1  o o  1  O o  1  O O  4^ O O  0-i O O  00 o  o  o o  o  ©  I-  1  fO  o o  1  o o  — r  55  Magnesium As f o r potassium, the magnesium concentrations i n the  rectal  secretions were found to be proportional to those in the external media, even in recta placed in a r t i f i c i a l this cation (Table 6c).  hemolymph having a constant l e v e l of  Although there were s l i g h t differences in the  concentrations between f l u i d from ligated and intact  r e c t a , the values  correspond approximately to those in the external medium.  The values  f o r A,, campestris i n (Na + Mg)S0^ medium agree f a i r l y well with those of Kiceniuk and P h i l l i p s (197,4). range of external M g  ++  These authors found that over a  concentrations, the urine Mg  ++  concentrations  equalled those of the external medium. Since Mg  ++  levels in the a r t i f i c i a l  hemolymph (5mM) and  natural hemolymph of larvae i n Ctenocladus pond water ( 2 - 4 Kiceniuk and P h i l l i p s , 1974)  mM;  were very low, this cation was secreted  into the r e c t a l lumen against 2 to 25 f o l d concentration differences.  Chloride As might be expected, the highest chloride concentrations in r e c t a l s e c r e t i o n s , whether from preparations bathed in a r t i f i c i a l hemolymph or from whole larvae, were observed in larvae acclimated to sea water medium, which was the only external medium high in chloride (Table 3).  The chloride concentration in the a r t i f i c i a l  hemolymph (l02mM)  was higher than i n the l a r v a l hemolymph (49 - 78 mM, pg. 48) and accordingly, secretions from the recta bathed i n a r t i f i c i a l hemolymph also contained considerably more chloride than the corresponding f l u i d  56  from whole larvae.  This suggests that the chloride concentration  i n the r e c t a l f l u i d i s influenced by hemolymph chloride concentration, as subsequently shown (Chapter IV).  This conclusion i s  further supported by the results obtained when an a r t i f i c i a l lymph with low chloride levels was used.  A change in  hemo-  artificial  hemolymph concentration from 102 mM to 20 mM led to a decrease i n chloride content of r e c t a l secretion from 230 mM to 40 mM, even though a l l the other i o n i c concentrations i n the two hemolymphs, except SO", were i d e n t i c a l .  artificial  This difference in the  chloride concentration of secretions from recta bathed in  artificial  hemolymphs of high and low chloride concentration occurred in spite of the fact that rates of f l u i d secretion (Chapter IV) trations (Table 6a) were r e l a t i v e l y  unchanged.  and Na* concen-  C l e a r l y , the other  anions substitute f o r chloride in the r e c t a l secretions when levels of chloride i n the hemolymph, a r t i f i c i a l  or r e a l , are low.  Chloride  levels are so low i n some of the hypersomotic waters i n which iA. campestris i s found that a problem of chloride retention must occur. It  i s therefore perhaps surprising to find^that, under these condi-  t i o n s , the chloride concentrations in r e c t a l f l u i d from whole ( 5 5 - 7 0 mM; Table 6a)  larvae  remain s l i g h t l y higher, or at most, equal to  l e v e l s i n the hemolymph (49 - 56 mffl; Table 4).  In such waters,  rectal  secretion c l e a r l y results i n a net loss of c h l o r i d e , which must be compensated f o r at other regulatory s i t e s ( e . g . anal papillae)  because  hemolymph levels of this anion are maintained well above those p r e v a i l ing in NaHC0 or (Na + MgJSO^ external media (26 and 18 mM C l " respect3  i v e l y , Table  3).  57  Sulphate - A_. campestris As f o r other ions discussed above, levels of sulphate i n r e c t a l f l u i d from whole larvae (0.7  to 132 mM; Table 6e)  those in the external media (0 to 377 mM, Table 3).  parallel  However, s u l -  phate ions are the only ions we have studied to date which are not found to be more concentrated in the r e c t a l secretion of s a l i n e water mosquito larvae than in the hemolymph, under conditions where the rectum i s isolated from midgut input and where the hemolymph concentration can be s t r i c t l y c o n t r o l l e d .  This f i n d i n g , that the  rectum probably does not contribute substantially to regulation of hemolymph sulphate concentrations, came as a considerable surprise because of the a b i l i t y  of A,, campestris to l i v e in water in which  S0~ represents over 90% of t o t a l anions. The f a i l u r e of the rectum to concentrate SO^ i s most c l e a r l y demonstrated when one examines the rectal f l u i d concentration of s u l phate in larvae acclimated to (Na + MgJSO^ medium. tum bathed in a r t i f i c i a l  The isolated r e c -  hemolymph of high chloride content produced  a f l u i d with a sulphate concentration of 6 mM, yet r e c t a l f l u i d from whole larvae was found to contain 132 mM.  This difference might be  due to i o n i c differences between the a r t i f i c i a l  and real hemolymphs,  p a r t i c u l a r l y with regard to chloride concentrations since the r e c t a l epithilium might prefer C l " to SO^. of the a r t i f i c i a l  However when the chloride content  hemolymph was lowered from 102 to 20 mM, no s i g n i f i -  cant increase was observed in the concentration of sulphate in the rectal secretion (Table  6e).  58  On the other hand, sulphate levels in r e c t a l f l u i d removed from whole larvae exceeded those i n the hemolymph.  Paired samples  were taken from the hemolymph and rectal lumen of larvae and in  all  cases except one, which was i s o t o n i c , the rectal f l u i d was hypertonic to the hemolymph with regard to sulphate ( F i g . 8 ) . This suggests that, although the urine i s hypertonic to the hemolymph with regard to sulphate, this sulphate apparently does not enter with the  rectal  secretion.  Sulphate- A_. taeniorhynchus A_. campestris larvae are pnly available during the spring because of the d i f f i c u l t y species.  of establishing laboratory colonies of this  For this reason we used jA. taeniorhynchus larvae to answer  two questions concerning sulphate transport;  1) Could the apparently  low sulphate levels i n f l u i d from l i g a t e d recta (Table 6a) be due to 35 lower s p e c i f i c a c t i v i t y of  SO^ i n the lumen?  2)  If  the answer to  this question i s negative, i s the low sulphate concentration in f l u i d from ligated recta a consequence of separating the rectum from the rest of the animal, thereby i n t e r f e r i n g with normal neural or endocrine stimulation of sulphate secretion? One group of A., taeniorhynchus larvae was raised in unlabelled 100$ sea water and a second group i n 100$ sea water containing radioactive sulphate with a s p e c i f i c a c t i v i t y equal to that of the artificial  hemolymph.  In this group then, the s p e c i f i c a c t i v i t y of  sulphate i n the a r t i f i c i a l  hemolymph was i d e n t i c a l to that i n the  hemolymph and rectal t i s s u e ; and therefore i n the r e c t a l secretion as  Figure 8.  Paired determinations of the sulphate concentration  the hemolymph and r e c t a l f l u i d of whole larvae. represents the line of sulphate i s o t o n i c i t y .  The dashed l i n e  O •P ra U •P d)  •iH  300  0  C  O U +J  <o  •a r-l  VJ1  3  200  W  o  •H -U 0)  9  o w iH f0  100  o  2  0  50  100  150  Hemolymph Sulphate Concentration  200  (mjyi)  60  well.  The sulphate concentration in the r e c t a l secretion from the  f i r s t group was 8.7 - 2.6 mM(n=5), in the second i t was 16.3 mM(n=7).  The hemolymph contained 27 mM sulphate.  s i m i l a r to those for A,, campestris (Table 6e).  2.5  These results are  C l e a r l y , differences  35 in s p e c i f i c a c t i v i t y of  SO^ do not explain the low levels of s u l -  phate observed i n the f l u i d from i s o l a t e d r e c t a . To answer the second question, A,, taeniorhynchus were raised i n a r t i f i c i a l sea water (Prosser, 1973) with a sulphate concentration (89mM) which was elevated by the addition of Na^O^.  The Malpighian  tubules of these animals show enhanced S0~ transport as compared to those of animals reared i n a r t i f i c i a l sea water with normal sulphate levels (Maddrell, 1976a,b).  To obtain an estimate of the actual  sulphate concentration in the normal rectal secretion of whole larvae, samples of r e c t a l f l u i d were removed from larvae either or 1.5 hours after the anus was plugged.  immediately  The rectum was i n i t i a l l y  found to contain a small amount of f l u i d (maximum volumes removed 11-14 nl) which was yellow and thick with suspended s o l i d material. The f l u i d was presumably derived from the Malpighian tubules either d i r e c t l y , or perhaps after modification by resorption in the i n t e s t i n e or anterior rectum.  The average sulphate concentration was calculated  to be 66.3 - 5.7 mM/l (n=6).  After 1.5 hours the rectum was found  to be highly distended with a c l e a r f l u i d (maximum volumes removed 30-34 n l ) .  This f l u i d had a lower sulphate concentration  (36.3 - 5.5 mM/l, n=7)  and was thought to be a d i l u t i o n of the o r i g i n -  a l f l u i d resulting from secretion of r e c t a l f l u i d lower in sulphate  61  content.  Assuming that a l l the o r i g i n a l f l u i d remained i n the rectum  and allowing for a f l u i d secreted during the experimental period which was twice the o r i g i n a l volume, the expected sulphate concentration of the r e c t a l f l u i d was calculated to be 21 mM/l.  This value i s only  s l i g h t l y higher than that found f o r recta i n a r t i f i c i a l (16.3 - 2.5 mW/l, n=7).  It  would seem therefore,  hemolymph  that r e c t a l secre-  tion in whole larvae reared i n high external concentrations of s u l phate i s so low in sulphate concentration as to be unimportant the regulation of hemolymph sulphate l e v e l s .  in  Most of the sulphate i n  the urine undoubtedly derives from Malpighian tubule s e c r e t i o n .  Osmolality A l l three of the experimental external media had an osmotic concentration of 700 mOsm.  If  the osmotic concentration of the  rectal  f l u i d were controlled simply by the osmotic concentration of the externa l medium, one might expect the r e c t a l f l u i d from a l l groups of larvae to have i d e n t i c a l osmotic concentrations.  In f a c t , the osmotic concen-  trations of the r e c t a l f l u i d from larvae acclimated to sea water and (Na + Mg)S0^ medium were e s s e n t i a l l y i d e n t i c a l and about 40$ above external o s m o l a l i t i e s , but that from NaHCO^ larvae was about 10-15$ lower (Table 6 f ) .  It  would seem that the p a r t i c u l a r ions present  i n the external medium have a small effect on the t o t a l concentration of the r e c t a l f l u i d . (p>0.4) Chloride levels could be important i n this regard.  When  experiments with ligated recta of larvae adapted to (Na + Mg)S0  med-  62  ium were repeated using a r t i f i c i a l  hemolymph low i n c h l o r i d e , the  osmotic concentration of the r e c t a l f l u i d was markedly reduced (Table 6 f ) .  As previously shown, (page  £8) larvae acclimated to  different media have different hemolymph chloride concentrations. This v a r i a b i l i t y  may be of importance i n influencing the osmolality  of the r e c t a l s e c r e t i o n . ide in a r t i f i c i a l  Moreover, the much higher levels of c h l o r -  hemolymph as compared to the natural hemolymph of  c p e s t r i s might explain the higher osmolalities of r e c t a l a m  from ligated as opposed to whole  larvae.  fluid  63  DISCUSSION  The results for A,, campestris are in agreement with a previous study on A,, taeniorhynchus which demonstrated that s a l i n e water mosquito larvae produce concentrated urine by the secretion of hyperosmotic f l u i d into the rectal lumen.  The r e c t a l secretions  from these two species are s i m i l a r in their mode and location of formation.  However, secretion from A. campestris larvae acclimated  to 700 mOsm sea water ( t h i s study) i s higher in t o t a l osmotic concentration  than that from A_, taeniorhynchus larvae grown in 832 mOsm  sea water (Bradley and P h i l l i p s , 1975; and Chapter II) ratios are very s i m i l a r .  but the ion  In both s p e c i e s , r e c t a l secretion contains  a l l the major ions found in sea water in approximately  the same con-  centrations as sea water with the exception of potassium which i s some 10-18 times higher.  Such s e c r e t i o n , i f  produced i n s u f f i c i e n t volumes,  could account f o r the excretion of a l l the ions ingested by drinking but would create a potassium d e f i c i e n c y . The excellent s u r v i v a l of A_. campestris larvae from Ctenocladus pond in waters of various chemical types (700 mOsm NaCl, NaHCO^ and (Na & Mg)S0^; also in fresh water) indicates remarkable adaptabili t y of regulatory processes in this species and excludes the a l t e r n a t i v e suggestion that d i s t i n c t physiological races are r e s t r i c t e d to p a r t i c u l a r kinds of natural water.  This was confirmed by a comparison  of rectal secretions from larvae adapted to hyperosmotic waters of different  chemical composition. Figure 9 summarizes both the location and the r e l a t i v e con-  Figure 9.  A diagrammatic representation of the proposed locations  of ion transport processes in the Malpighian tubules, rectum and anal papillae of A., campestris larvae, in each of three media used in this study.  The thickness of the arrows represents the r e l a t i v e rate  of net transport as demonstrated by an increased concentration of an ion in the f l u i d secreted by the Malpighian tubules and rectum.  The  indicated rates and direction of ion transport in the anal papillae are based on observations of drinking and urine excretion rates (see text).  AP, anal p a p i l l a e ; AR, anterior rectum segment;  MT, Malpighian tubule; PR, posterior rectal segment.  MG, midgut;  Dashed arrows  represent postulated ion transport pathways which are suggested by observations to date, but which have not been d i r e c t l y demonstrated.  64b  NaCl MEDIUM  ^  —  C  ^  M  ^  /Ztt*^-MT^S  65  centrations of ions in rectal secretion from A,, campestris larvae in different  types of water.  The thickness of arrows represents the r e -  l a t i v e rate of net transport as indicated by an increased concentration of an ion in the r e c t a l s e c r e t i o n .  This secretion can show elevated  levels of sodium, potassium, magnesium and chloride when the larvae are acclimated to solutions high i n these i o n s . recta were bathed i n i d e n t i c a l a r t i f i c i a l  Even when ligated  hemolymphs, the i o n i c  corn-  composition of the secretions tended to r e f l e c t those of the water to which the larvae were adapted.  Clearly the transport rate f o r various  ions i s adjusted during adaption either by changes in the amount of protein c a r r i e r s present i n the epithelium (induction) on of c a r r i e r s ( e . g . decreasing  or by turning  Km) already present through hormonal  and neural control mechanisms. The experiments with chloride indicate that the sharp i n c r e a s es in chloride concentration of the r e c t a l secretion occur when hemolymph levels are raised substantially above normal.  This f i n d i n g probably re-  f l e c t s the fact that both Na and C l ~ transport processes, as observed +  in recta of A_. taeniorhynchus, (Chapter IV) of a l l o s t e r i c rather than c l a s s i c a l enzymes.  exhibit kinetics l i k e those That i s , a sharp increase  in ion secretion rate occurs when hemolymph levels of the ion are high. The s i t u a t i o n i s d i f f e r e n t ,  however, for sulphate ions which  are neither concentrated in the r e c t a l secretion of whole larvae, nor i n larvae acclimated to (Na + Mg)S0. medium (377 mffl SO") and placed in 4 4 artificial  high-S0~, low-Cl  hemolymph to stimulate sulphate transport.  In every preparation in which the sulphate concentration of the  rectal  66  secretion was measured, as d i s t i n c t from Malpighian tubule f l u i d , was found to tbe hypotonic to the hemolymph.  it  The r e c t a l wall i s thus  impermeable enough to sulphate to allow the sulphate concentration gradient between the Malpighian tubule f l u i d and the hemolymph to be maintained, but this gradient i s not increased by the r e c t a l s e c r e t i o n . I  therefore propose that most of the ingested sulphate i s actively  secreted by the Malpighian tubules as shown by Maddrell and P h i l l i p s (1975).  The f l u i d from the Malpighian tubules must be modified by the  i n t e s t i n e or more probably (on u l t r a s t r u c t u r a l  grounds)  i n the anter-  i o r rectum, as a consequence of ion and water resorption to achieve the concentrations found i n the urine of larvae i n (Na + Mg)SO^ medium. The following events are thought to lead to the formation of urine i n saline-water larvae adapted to hyperosmotic waters.  It  is  known that the Malpighian tubules, l i k e those of other insects (Maddrell, 1971)  produce isosmotic f l u i d by transporting potassium and chloride  into the tubule lumen, producing an osmotic gradient which drives the movement of water and hemolymph s o l u t e s . high levels of Mg  ++  If  the natural water contains  and S0~ ( e . g . sea water and (Na + Mg) SO^ medium),  active secretion of these ions accounts for a proportionally f r a c t i o n of the t o t a l solute ( P h i l l i p s and Maddrell, 1975; and P h i l l i p s , 1975).  The t o t a l transport capacity ( i . e .  greater Maddrell  amount of  c a r r i e r ) f o r sulphate and possibly magnesium i s increased over a period of a day i n response to prolonged exposure to high external levels of these ions (Maddrell, 1976a, b ) . The primary excretory f l u i d from the Malpighian tubules i s  67  modified in the intestine and/or anterior  rectum and most of the ions  and organic compounds useful to the larvae are returned to the hemolymph at these s i t e s .  Meredith and P h i l l i p s (1974a) present u l t r a -  s t r u c t u r a l observations that suggest that this i s the role of the anterior rectum, and further evidence w i l l be presented i n t h i s gard in Chapter V.  re-  I believe that this excretory cycle involving the  Malpighian tubules and anterior rectum occurs i n laryae i n a l l types off-,saline .media as well" as i n .f resh^-water. The secretion of a hyperosmotic f l u i d occurs in the posteri o r rectum.  The r e l a t i v e rates at which various ions ( N a , K , M g , +  +  + +  C l ~ and probably HCGj") are transported depends on t h e i r concentration both i n the hemolymph and in the external medium to which the have been adapted.  larvae  Only low levels of sulphate are present i n the  secretion and the movement of sulphate across the rectum i s presumed to be passive (see Chapter VI).  Saline-water mosquito larvae must be  able not only to remove ions which reach abnormally high concentrations i n the hemolymph, but also to conserve p h y s i o l o g i c a l l y required ions in waters low in these ions ( e . g . C a  + +  C l " i n NaHC0 and (Na + Mg)S0 media). 3  4  and Mg j,in NaHCO^ medium or ++  It  i s useful therefore to ex-  amine the a b i l i t y of the rectum to l i m i t the loss of such ions in the rectal secretion. The r e c t a l secretions of A., taeniorhynchus (Bradley and P h i l l i p s , 1975; Chapter II)  and A_. campestris (present study) are  characterized under a l l conditions by a potassium concentration higher than both the external and the hemolymph concentrations.  As an extreme  68  example, in A. campestris larvae acclimated to NaHCO^ medium, the concentration of potassium i n r e c t a l secretion from whole larvae i s 11.3 times higher than that i n the external medium. suggested by Bradley and P h i l l i p s (1975J? Chapter II)  Therefore, as f o r A,.  taeniorhynchus and supported by this study on A,, campestris, I propose that one of the functions of the anal papillae i n s a l i n e water mosquito larvae i s the uptake of potassium possibly i n exchange f o r hemolymph sodium, as i s thought to occur i n the g i l l s of marine teleosts (fflaetz, 1971). In media low in chloride ( e . g . NaHCO^ and (Na + Mg)S0  4  media)  the chloride concentration of the r e c t a l secretion i s very much reduced compared to that from larvae reared i n seawater.  Nevertheless chloride  concentrations are at best i s o t o n i c to the hemolymph and thus hypertoni c to the external medium with regard to c h l o r i d e .  The r e c t a l s e c r e -  t i o n , therefore represents a constant drain of chloride from the hemolymph in media where chloride concentrations are lower than the hemolymph.  It  i s proposed that i n media extremely low in c h l o r i d e , the  larvae may use the anal papillae for the uptake of c h l o r i d e , much as has been shown f o r the same species acclimated to dilute media ( P h i l l i p s and Meredith, 1969b). F i g . 9.  This role i s shown i n the appropriate media  The direction of this chloride transport may be reversed in  media high i n chloride ( P h i l l i p s and Meredith, 1969b) and this cont r i b u t i o n i s depicted as well i n F i g . 9 f o r larvae acclimated to s e a water medium. Sulphate ions are apparently neither needed by the  larvae  69  i n t h e i r inorganic form nor p a r t i c u l a r l y toxic i n high concentrations i n the hemolymph (Maddrell and P h i l l i p s , 1975).  They are  therefore  not conserved at low concentrations and probably regulated at high concentrations to l i m i t osmotic concentration. The model of i o n i c regulation i n saline-water mosquito larvae outlined above and depicted i n F i g . 9, explains the ences i n survival i n the various media.  differ-  Media high in N a , Mg**, +  C l " and/or HCO^ are not toxic i n osmotic concentrations at or above sea water because the larvae can excrete these ions i n the posterior rectum as part of a hyperosmotic s e c r e t i o n , containing the proper ratios of these i o n s .  In the case of media high i n sulphate,however,  the Malpighian tubules and anterior rectum combine to form a f l u i d isosmotic to the hemolymph and high i n sulphate. of sulphate which can be excreted i s therefore  The concentration  limited by the  toler-  ance of the larvae to elevations i n hemolymph osmotic pressure, the cations associated with sulphate in the Malpighian tubules, and the rate of Malpighian tubule s e c r e t i o n .  One or more of these parameters  may be the explanation f o r the lesser tolerance of A,* taeniorhynchus to media high i n sulphate compared to i t s close r e l a t i v e A. campestris.  Only the l a t t e r species can survive i n (Na + Mg)S0^ medium,  i n d i c a t i n g that high-sulphate inland waters can be much more toxic to mosquito larvae than sea water having a higher osmotic pressure.  CHAPTER IV THE EFFECT OF EXTERNAL SALINITIES ON DRINKING RATE AND RECTAL  SECRETION  IN THE LARVAE OF THE SALINE-WATER MOSQUITO AEDES TAENIORHYNCHUS  70  71  INTRODUCTION  Hyperosmotic urine i s formed in the l a r v a l rectum of the s a l i n e uiater mosquitoes A_. taeniorhynchus and A_. campestris by the secretion of a hyperosmotic f l u i d into the lumen.  This secretion has an i o n i c composition  closely resembling the hyperosmotic external medium, suggesting that the rectum i s the major osmoregulatory organ i n these larvae (Chapters II  and  III).  These observations were made on larvae adapted to 100$ sea water or i n the case of .A. campestris, other s a l i n e waters of s i m i l a r osmolality. I wished to determine how the composition of r e c t a l secretion was affected by changes i n environmental osmolality, and how such changes might relate to osmoregulation in whole larvae.  I therefore measured the drinking rates,  hemolymph ion concentrations, and concentrations of r e c t a l secretions in A_. taeniorhynchus larvae adapted to various concentrations of sea water. To discover whether i n t r i n s i c regulatory  responses might be inherent in the  ion transport process, as suggested by previous results f o r CI (page  55),  the k i n e t i c s of Na and CI +  secretion  secretion were studied.  MATERIALS AND METHODS  The larvae were raised according to the method of Nayar (1967) and starved 1 - 2  days before use.  Four rearing media were used;  Vancouver sea water (832" 8.8 mOsm; Mean * S . E . ) 10$ sea water, 50$ sea water and sea water concentrated to one-half o r i g i n a l volume by evaporation at room temperature (200$ sea water).  100$  72  Drinking rates were determined according to the procedure of Bradley and P h i l l i p s (1975? Chapter II).  In t h i s study, however,  larvae were removed and weighed i n d i v i d u a l l y a f t e r one hour i n the 14 C-carboxy i n u l i n s o l u t i o n .  A further difference was that special  care was taken not only to mince the larvae before KOH d i g e s t i o n , but to cut the midgut into several pieces to achieve better release of i n u l i n .  Drinking rates were determined i n the same concentrations  of sea water as those i n which the larvae were r a i s e d . The length and diameter of larvae, p a r t i a l l y  immobilized  on moist f i l t e r paper, were measured using an eyepiece micrometer. The length was measured from the mouthparts to posterior edge of the base of the siphon.  The diameter of the larvae was measured across  the f i r s t abdominal segment. from equation ( l )  The volume of larvae was calculated  and surface area from equation (2).  tions are based on the assumption that the  3hape  These estima-  of larvae approximates  that of a c y l i n d e r .  3 Volume  2  (mm ) = f r  1  Equation 1  2 Surface area (mm ) = 2flr (r+1)  Equation 2  The symbols r and 1 refer to radius and length r e s p e c t i v e l y , both i n mm.  The estimated volume and surface area of larvae of known weight  were used to generate equations r e l a t i n g these values. Hemolymph samples were obtained, osmolality was determined, and concentrations of Na , fig  , K  and CI  were measured as described  previously (Bradley and P h i l l i p s , 1975; Chapter II).  Calcium  concen-  73  trations were measured using a "Techtron AA 120" atomic absorption spectrophotometer i n the absorption mode. pooled 5 pi  Determinations were made on  samples of hemolymph diluted i n 1ml of 0.5$ LaCl^.  Sul-  phate concentrations were determined by measuring the r a d i o a c t i v i t y  of  hemolymph samples of known volume from larvae raised in media c o n t a i n 35 ing radioactive  = SO^.  S p e c i f i c a c t i v i t y was determined by measuring  the r a d i o a c t i v i t y of the external medium, the sulphate concentration of which was known (Prosser,  1973).  Rectal samples were taken either from whole larvae with the anus blocked or from ligated larvae bathed i n a r t i f i c i a l  hemolymph as  described previously (Bradley and P h i l l i p s , 1975; Chapter II). artificial  The  hemolymphs used were based on the o r i g i n a l formula from  Berridge (1966) with modifications as previously described (Bradley and P h i l l i p s , 1975).  The composition of these a r t i f i c i a l hemolymphs  i s shown i n Table 7. To examine the effect of hemolymph o s m o l a l i t y , one s e r i e s of a r t i f i c i a l  hemolymphs contained a l l the const!tutents of the nor-  mal hemolymph except sucrose, the concentration of which was varied to achieve various f i n a l o s m o l a l i t i e s . i n chloride concentrations. tuents except NaCl.  A second s e r i e s varied only  The stock solution contained a l l  consti-  Sodium was added as Na^SO^ and various levels of  chloride were obtained by replacing d i f f e r e n t amounts of sucrose with choline chloride so that osmotic concentrations did not vary. t h i r d series of a r t i f i c i a l  A  hemolymphs was devised to examine the  fect of varying sodium levelst  ef-  NaCl was omitted from the stock s o l -  74  Table 7 .  The i o n i c (mM) and osmotic (mOsm) concentrations in the  a r t i f i c i a l hemolymphs used to study the effects of varying hemolymph parameters.  The variable osmotic and chloride concentration series  had the same organic constitutents as normal hemolymph (Bradley 4 P h i l l i p s , 1975; Chapter II).  The variable sodium concentration  series had reduced levels of acids (mg/100 ml): malic a c i d , 50; c i t r i c a c i d , 25.  Na succinate and Na glutamate were omitted.  Variable Chloride Concentration Series Na K  +  +  Mg Cl"  mOsm  Variable Osmotic Concentration Series  Variable Sodium Concentration Series  150  150  variable  18  15  18  5  5  5  variable  100  100  variable  27  variable  350  variable  348  75  ution and chloride was added as the choline s a l t . varied by s u b s t i t u t i n g  Ha^SO^  Sodium levels were  f o r d i f f e r i n g amounts of sucrose in the  a r t i f i c i a l hemolymph.  RESULTS  Drinking. Rates The drinking rate per mg. wet weight of larvae did not vary s i g n i f i c a n t l y when the osmotic concentration of the sea water in which larvae were reared ranged between 10$ and 200$ ( F i g , 10). —1 ingestion rate was very high indeed (B.Apl • larvae  The average  —1 day  ) f o r larvae  of average weight (3.5 mg). This r e s u l t was a surprising one because salt-water mosquitoes l i v i n g in hyperosmotic waters must drink to replace water l o s t by osmos i s across the body wall and by excretion.  If  such losses were the  only f a c t o r c o n t r o l l i n g drinking rate, then drinking should increase as the s a l i n i t y i n external media i s r a i s e d .  Moreover drinking should  cease in hyposmotic media, as observed f o r freshwater mosquitoes (Wigglesworth, 1933), where the direction of osmotic water flow across the integument i s into the hemolymph.  In order to better understand  the factors c o n t r o l l i n g drinking, the drinking rate of i n d i v i d u a l l y weighed larvae was determined. Nicholson and Leader (1974) obtained two nearly i d e n t i c a l estimates f o r the surface area of the mosquito l a r v a e , Qpifex fuscus, f i r s t l y by measuring i t s diameter and length and assuming i t  to be a  c y l i n d e r , and secondly by measuring the t o t a l content of c u t i c u l a r  Figure 10.  The drinking rate of larvae in the media i n which  they were reared. squares method.  The regression line was f i t t e d by the  least  180  External Concentration (% seawater)  77  wax.  These values agreed to within 3%.  I therefore made s i m i l a r mea-  surements of the dimensions of Aedes taeniorhynchus larvae and c a l c u lated the body volume and surface area for larvae over a wide range of sizes. The relationship between the weight of the larvae (x) and the calculated volume of the same i n d i v i d u a l (y)  can be accurately  described by the equation (y = - 0.19 + 1.07 x) ( P i g . 11).  The y i n -  tercept i s very close to zero and the slope i s nearly one.  This r e -  s u l t i s very close to that expected i f  one assumes that a l l the larvae  have a s p e c i f i c gravity close to one and that the method used to c a l culate body volume gives an accurate estimate. The relationship between the calculated surface area (y) weight (x)  and  of the larvae i s adequately expressed by the equation  0.75 y = 0.13 + x ' ( F i g . 12).  The relationship between surface (y) of 0 67 any object r e l a t i v e to i t s weight (x) can be expressed by y = x if the object retains the same shape as i t increases in s i z e (Schmidt Nielsen, K.; 1975).  The relationship found f o r mosquito larvae  0.75 (yotx *  ) suggests that the larvae do not grow merely by increasing in  size while maintaining the same shape, but rather that they are i n c r e a s ing in length proportionally faster than in width. These relationships ( F i g s . 11 and 12) were used to calculate the volumes and surface areas of i n d i v i d u a l l y weighed larvae from 10%, 50$, 100$ and 200$ sea water whose drinking rates were subsequently determined.  The empirical relationship between the drinking rates  (y) of i n d i v i d u a l animals and their volume (x)  i s shown i n F i g . 13.  a  Figure 11.  The relationship between the weight and volume of A,.  taeniorhynchus larvae.  The regression l i n e (Y = -0.19 + 1.07X)  was f i t t e d by the least squares method (R * 0.98).  Figure 12.  The relationship between the weight and surface area of 0.75  A,, taeniorhynchus larvae.  The regression l i n e (Y = 0.13 • X *  was f i t t e d by the least squares method (R = 0.98).  )  Figure 13.  The relationship between the volume and drinking rate  of larvae i n four s a l i n i t i e s ; 0  100$ sea water, •  •  10$ sea water,  200$ sea water.  A  50$ sea water,  Drinking rates were measur-  ed in the media i n which the larvae were r a i s e d .  The regression  0.72 l i n e (Y = 0.01 (R = 0.80).  + X  ) was f i t t e d by the least squares method  Drinking R a t e ( n l - l a r v a ' - 1  Q.08  h" ) 1  81  The regression l i n e i s expressed by the equation y = 0.01  + x0 * 72  The same relationship exists between drinking rate and the weight of the larvae since weight and volume are l i n e a r l y related ( F i g .  11).  C l e a r l y , the drinking rate of the larvae i n a l l s a l i n i t i e s studied i s related to their weight or volume in some complex f a s h i o n , for the exponent of x i s 0.72, surface area.  close to the relationship between weight and  F i g , 14 shows the relationship between the drinking  rate and surface area of the larvae.  The equation f o r the regression 1.05  l i n e shown i s expressed by the equation y = 0.08 + x close to l i n e a r .  , i.e.  very  The correlation c o e f f i c i e n t i s high (r = 0.80,  P<0.01). In summary, the drinking rate of A,, taeniorhynchus larvae i s not s i g n i f i c a n t l y affected by the s a l i n i t y of the external tions over the range of the s a l i n i t i e s tested.  Instead,  of the larvae determines the rate at which they drink.  solu-  the s i z e The change  in this rate with increasing s i z e of larvae p a r a l l e l s the rate of change of their surface area.  Hemolymph Ion Levels Hemolymph ion levies were measured in larvae raised i n 10%, 100% and 200;^ sea water. strictly  Sodium, magnesium and potassium were  regulated in that no s t a t i s t i c a l l y s i g n i f i c a n t concentration  differences could be detected between larvae reared i n these three concentrations of sea water ( F i g . 15).  Hemolymph calcium levels i n -  crease s i g n i f i c a n t l y (P<0.001) however, with each increase i n exter-  Figure 14.  The r e l a t i o n s h i p between the surface area and drinking  rate of larvae in four s a l i n i t i e s ; 0  100$ sea water, •  •  200$ sea water.  10$ sea water, A  50$ sea water,  Orinking rates were measured  i n the s a l i n i t y in which the larvae were r a i s e d .  The regression l i n e  1 05 (Y = 0.08 + X ) was f i t t e d by the least squares method (R = 0.08).  Drinking Rate (nl- l a r v a " ! .h~l)  <128  Figure 15* magnesium  The mean concentrations of sodium ^ |  and calcium  A  , potassium O  i n the hemolymph of larvae  i n sea reared i n sea water of d i f f e r i n g s a l i n i t y .  )  reared  V e r t i c a l bars denote  S . E . of the means unless these are smaller than the symbol. i s an average f o r 10 (Na , K , fflg ) or 5(Ca  •  larvae.  Each point  8 3 b  84  nal s a l i n i t y .  The t o t a l concentrations of these cations at  the  three external concentrations were 180 mM (10$ sea water), 172 mM (100$ sea water) and 165 mM (200$ sea water), i n d i c a t i n g , i f thing a s l i g h t decrease with increasing external  any-  salinity.  Anion regulation was less precise than that of the c a t i o n s . Chloride concentrations and osmolalities showed p a r a l l e l trends ( F i g . 16).  Relatively  low hemolymph chloride and osmotic concentrations  were found in animals in 10$ sea water, while larvae in 100$ and 200$ sea water showed somewhat elevated levels ( l e s s than 25$ i n crease) which were not s t a t i s t i c a l l y each other.  s i g n i f i c a n t l y d i f f e r e n t from  Nayar and Sauerman (1974) have measured chloride and  osmotic pressure i n A,, taeniorhynchus larvae aa well and found the the same pattern of regulation.  Sulphate concentrations on the other  hand were low i n 10$ and 100$ sea water but increased 3 . 4 - f o l d in 200$ sea water.  This suggests that sulphate i s c l o s e l y regulated at  lower levels but less so at higher concentrations (discussed by Madd r e l l and P h i l l i p s , 1975).  The t o t a l concentrations of these 2 anions  were 80mM (10$ sea water), 101 mM (100$ see water) and 113 mM (200$ sea water.  Minimum estimates of unmeasured anions required to b a l -  ance measured cations are 100 mM (10$ sea water), 71 mM (100$ sea water) and 52 mM (200$ sea water). Total measured ion concentrations i n the hemolymph increase less than does the hemolymph osmolality when larvae l i v i n g i n 10$ and 200$ sea water are compared.  Unless i o n i c a c t i v i t i e s undergo consider-  able changes, which has been shown in some insects (Treherne, Buchan  Figure 16. concentration  The mean chloride 4)  * sulphate  O  a  n  d  osmotic  i n the hemolymph of larvae reared in three  media of d i f f e r i n g s a l i n i t y .  V e r t i c a l bars denote S . E . of the  means unless these are smaller than the symbol. average 10 ( C l ~ , mOsm) or 9 (S0~)  larvae.  Each point i s an  85b  !0%  100%  200%  External Concentration (% seawater)  66  and Bennett, 1975)  the contribution of inorganic ions to t o t a l osmol-  a l i t y of the hemolymph decreases in the more s a l i n e external media.  Ionic and Osmotic Concentrations of Rectal Secretion a)  In 10$ Sea water When l i g a t e d recta from larvae raised i n 10$ sea water were  placed i n a r t i f i c i a l  hemolymph, the recta did not swell with secreted  f l u i d as did those from larvae raised in hyperosmotic media.  Not only  do the recta from 10$ sea water larvae not s e c r e t e , but the small amount of f l u i d observed in the rectal lumen i n i t i a l l y during the 1.5 hour incubation period, that i t  i s so reduced  is difficult  to obtain  a f l u i d sample. When whole larvae from 10% sea water had their anuses blocked with tissue adhesive, f l u i d accumulated in the rectum, presumably due to f l u i d entry from the Malpighian tubules.  This f l u i d was so  d i l u t e and such small quantities were a v a i l a b l e ,  that only the osmotic  concentration of the f l u i d was measured ( f i g .  17).  The average  osmolality of this f l u i d was found to be 170± 11 mOsm (n = 8) or 50$ of hemolymph values.  This agrees f a i r l y well with the measurements  of Ramsay (1950) f o r the r a c t a l f l u i d osmotic concentration of A_. detritus  larvae (0.33$ NaCl, 105 mOsm).  The f a c t that the A., taenior-  hynchus larvae were acclimated to a more concentrated medium (10% sea water) than the A_. detritus ( d i s t i l l e d water) might account f o r the higher r e c t a l f l u i d concentration found in the former species, but n e i ther saline-water species achieves the low r e c t a l f l u i d concentrations  a  Figure 17.  The i o n i c and osmotic concentrations (mean - S.E., n = 6)  of r e c t a l f l u i d from whole larvae in 10$ sea water water  and 200$ sea water  100$ sea  The arrows indicate the  respective concentrations in the external medium,  1100  CO  a  1000  2000  900  1800  800  1600  700  1400  600  1200  o +>  (0 M •P 0) O O  u o  500 400  1  1000 800  300  600  200  400  100  200  •rl  o  H  0 Na+  Mg++  ci-  S0 " 4  mOsm  88  found in Aedes aeovoti acclimated to d i s t i l l e d water (0.10$ NaCl, 35 mOsm) (Ramsay 1950). These results support the hypothesis that the rectum i s the s i t e of s a l t resorption i n larvae acclimated to hyposmotic media (Ramsay, 1950).  b)  In 100$ sea water The ion concentrations in the r e c t a l f l u i d from whole  lar-  vae reared i n 100$ sea water were described i n d e t a i l by Bradley and P h i l l i p s (1975) (Chapter I i ) .  It  was suggested that the apparent  anion d e f i c i t observed i n the rectal f l u i d (68 mEq) might be due to sulphate i o n s .  For this reason, the sulphate concentration i n  f l u i d was measured and found to be 17 - 3 mM (n=10); that i s ,  rectal this  anion accounts f o r only one-half of the apparent anion d e f i c i t . However, I presented evidence in Chapter III  that most of the ingested  sulphate i s removed from the hemolymph by the Malpighian tubules r a ther than the rectum and t h i s might be the source of much of the s u l phate observed i n r e c t a l f l u i d from whole larvae.  Therefore, the  anion d e f i c i t which i s observed in the r e c t a l f l u i d from i s o l a t e d recta that contain l i t t l e of this anion must be due to some other unmeasured anionic component ( e . g . bicarbonate or negatively charged macromolecules.). The i o n i c and osmotic concentrations of r e c t a l f l u i d from larvae adapted to 100$ sea water are close to those in sea water ( F i g . 17), with the exception of potassium as previously reported (Bradley and P h i l l i p s , 1975; and Chapter II).  It  was of i n t e r e s t to determine  89  whether concentrations of r e c t a l secretion are adjusted to match those of more concentrated external media, just as ion ratios i n the secreted f l u i d are regulated to match those in various chemical types of hyperosmotic waters of equal osmotic concentration (Chapter  c)  III).  In 200$ sea water For those ions which are at much higher concentrations ex-  ternally  than in the hemolymph (Na , fflg , CI , S0~), and therefore  might pose a regulatory stress on the larvae, the l e v e l s i n the r e c t a l secretion were higher in whole larvae from 200$ sea water than i n those from 100$ sea water ( F i g . 17).  Sodium and chloride concentra-  tions in the r e c t a l secretion of larvae reared i n 200$ sea water were s l i g h t l y lower than those in the external medium, although the standard errors overlapped the l a t t e r values. ions they i n g e s t , i t the removal of Na  +  Since larvae must excrete  follows that either some other organ i s aiding in  and C l " from the hemolymph ( e . g . anal papillae)  or  the r e c t a l preparation used in this study does not accurately indicate the maximum concentrations developed i n undistributed larvae. The l e v e l s of magnesium and sulphate observed i n the secretion equalled those i n 200$ sea water. (1975) (Chapter II)  rectal  Bradley and P h i l l i p s  found that the potassium concentration was 16 times  higher in rectal secretion than in 100$ sea water.  In the present  study, the l e v e l of this cation in rectal secretion from larvae  living  in 200$ sea water was found to be lower than that i n larvae from 100$ sea water, unlike a l l other ions measured.  However, the r e c t a l sec-  90  retion level was s t i l l 3 . 6 - f o l d higher than the external concentration.  Factors Regulating Ionic and Osmotic Concentrations of Rectal Secretion Having established that ion concentrations of r e c t a l secretion are influenced by the concentration of the external medium ( t h i s study) and by the r e l a t i v e concentrations of ions in the medium (Chapter  III).  I wished to obtain more information concerning the regulation of the ionic and osmotic composition of r e c t a l s e c r e t i o n .  Since the composi-  tion of this f l u i d r e f l e c t s concentrations in the external medium, larvae obviously monitor changes i n external ion l e v e l s .  The rectum i s  bathed i n hemolymph and the most direct method of regulatory response would be a s e n s i t i v i t y of the r e c t a l secretion to changes i n hemolymph ion levels brought about by the ingestion of the external medium.  The  s i m i l a r i t y of hemolymph ion levels i n larvae adapted to 100$ and 200$ sea water ( F i g s . 15 and 16) suggest that there i s l i t t l e basis for such a proposal.  However, upon transfer of larva from one medium to another  substantial transient changes in hemolymph ion levels occur  initially  and concentrations only return to normal over a 12 to 24 hour period (Nayar and Sauerman, 1974; P h i l l i p s , unpublished observations).  Rapid  changes in the rate and concentration of r e c t a l secretion may be one form of short-term response to such abrupt external concentration changes.  Therefore, the effect on rectal secretion of varying only one  parameter of a r t i f i c i a l ted. water.  hemolymph composition at a time was i n v e s t i g a -  A l l larvae used in these experiments were raised in 100$ sea  91  Figure 18 shows the effect of varying hemolymph osmotic concentration through the addition of sucrose, on the osmotic concentration of r e c t a l f l u i d produced by ligated recta bathed in artificial  hemolymph.  In a r t i f i c i a l  hemolymph of low osmotic con-  centration (173 mOsm; Table 7) the rectum i s unable to produce a f l u i d as concentrated as that produced in more concentrated a r t i f i c i a l hemolymph ranging between 250 and 500 mOsm or in whole animals. This lower concentration of r e c t a l f l u i d may be due to tissue s w e l l ing in dilute media with a subsequent reduction i n  intracellular  osmotic and i o n i c concentrations, or increased osmotic permeability. In a l l the other a r t i f i c i a l  hemolymphs of higher osmolality, the  r e c t a l f l u i d had the same osmotic concentration as sea water (about 800 mOsm), which was s i m i l a r to that found in whole animals.  This  suggests that the osmotic concentration of the hemolymph as d i s t i n c t from the i o n i c concentrations, does not have a s i g n i f i c a n t effect on the osmotic concentration of rectal s e c r e t i o n .  The physiological  range of hemolymph osmotic concentrations observed by us i n A. taeniorhynchus (303 - 427 mOsm; F i g . 6) i s s u b s t a n t i a l l y than the experimental range of F i g . 18.  narrower  The rate of r e c t a l s e c r e t i o n ,  as judged by unquantified observations of rectal s w e l l i n g , did not appear to change substantially even in a r t i f i c i a l  hemolymph with an  osmotic concentration of 500 mOsm. Since the osmotic concentration of r e c t a l secretion proved to be i n s e n s i t i v e to that in the hemolymph over the normal physiol o g i c a l range, I considered the influence of changing hemolymph c h l o r ide levels when a l l other parameters were held constant, except for  F i g 18.  The effect of a r t i f i c i a l  hemolymphs d i f f e r i n g only i n  osmotic (sucrose) concentration on the osmotic concentration (mean - S . E . , n = 6) of r e c t a l s e c r e t i o n . eye.  The l i n e was drawn by  93  sulphate concentrations.  Changes in the hemolymph concentrations of  chloride seemed a l i k e l y basis for control of r e c t a l secretion because of the v a r i a b i l i t y  in hemolymph levels of t h i s ion ( F i g .  16).  The r e c t a l secretion was found to be hypertonic to hemolymph with regard to chloride at every hemolymph chloride concentration tested ( F i g . 19).  However, the chloride concentration i n  rectal  f l u i d increased more sharply at higher hemolymph chloride l e v e l s .  Ths  steep slope for the increase in this relationship when hemolymph concentration of chloride rises above 100 mM i s of p a r t i c u l a r since this i s the normal hemolymph l e v e l .  Thus in larvae  interest, introduced  to media higher in c h l o r i d e , the hemolymph chloride might  initially  r i s e above the average range observed f o r adapted animals.  This would  cause the chloride concentration of the r e c t a l secretion to increase substantially, hemolymph.  thereby reducing the chloride concentration in the  That i s , a degree of i n t r i n s i c regulation  incorporated  within the transport process i s possible. Figure 19 also shows that increasing hemolymph c h l o r i d e , i n the absence of osmotic concentration  changes leads to s l i g h t l y  evated osmolality of r e c t a l secretion.  However, this change i s not  s i g n i f i c a n t over the physiological range (50 - 100 mM).  el-  As with the  concentration of c h l o r i d e , the osmotic concentration of rectal  fluid  shows the steepest increase at hemolymph chloride levels above 100 mM. The rate of r e c t a l s e c r e t i o n , as estimated by the s i z e of the  largest  three samples out of 15, obtained by micro-puncture, shows a positive c o r r e l a t i o n to the hemolymph chloride concentration as well ( F i g .  19).  Figure 19.  The e f f e c t of varying chloride concentration in a r t i f i -  c i a l hemolymph on the volume of rectal secretion c o l l e c t e d after 1.5 ti  A  ( n = 3 ) , osmotic concentration  ide concentration means•  £  (n = 6).  O  (n = 6) and c h l o r -  V e r t i c a l bars denote S . E . of the  Maximum Volume of Rectal Secretion Removed (nl)  95  The product o f f l u i d s e c r e t i o n r a t e and c o n c e n t r a t i o n y i e l d s an e s t i m ate of c h l o r i d e s e c r e t i o n rate ( F i g . 21). The k i n e t i c s of the process a r e n o t o f t h e type.  Michaelis-Menten  The above f i n d i n g s i n d i c a t e t h a t the c h l o r i d e c o n c e n t r a t i o n o f  the r e c t a l s e c r e t i o n i n s a l i n e - w a t e r mosquitoes i s i n f l u e n c e d and p e r haps c o n t r o l l e d by changes i n hemolymph c h l o r i d e l e v e l s . both J\. t a e n i o r h y n c h u s  and A,, c a m p e s t r i s  However,  can s u r v i v e i n w a t e r s low i n  c h l o r i d e ( e . g . NaHCO^ medium, Chapter I I I ) and produce a r e c t a l  fluid  of a p p r o p r i a t e l y low c h l o r i d e c o n c e n t r a t i o n under these c o n d i t i o n s , even though hemolymph c o n c e n t r a t i o n s of c h l o r i d e a r e m a i n t a i n e d above 50 mM.  at or  C l e a r l y , some o t h e r parameters o t h e r than hemolymph  c h l o r i d e l e v e l s must r e g u l a t e r e c t a l f u n c t i o n as w e l l . F i g u r e 20 shows the r e s u l t s o f v a r y i n g hemolymph sodium l e v e l s on sodium and osmotic c o n c e n t r a t i o n s of r e c t a l s e c r e t i o n .  No  s i g n i f i c a n t d i f f e r e n c e ( P ^ O . 0 2 ) was found between the sodium concent r a t i o n i n the s e c r e t i o n produced by r e c t a bathed i n normal hemolymph h a v i n g a sodium c o n c e n t r a t i o n of 150 mM (285 - 29 mM, n * 11) and t h a t from r e c t a i n hemolymph w i t h the same sodium l e v e l i n t h i s s e r i e s (325 - 23 mM, n = 7) i n which the l e v e l s of some o r g a n i c a c i d s were r e duced ( T a b l e 7 ) . As w i t h c h l o r i d e , the c o n c e n t r a t i o n o f sodium i n r e c t a l f l u i d i n c r e a s e d as hemolymph l e v e l s o f sodium were r a i s e d . sodium 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 (128 mM) remained n e a r l y a t hemolymph sodium c o n c e n t r a t i o n s between 5 mM and 50 mM,  The constant  Although  the sodium c o n c e n t r a t i o n of hemolymph was s u b s t a n t i a l l y reduced t o 5 mM i n some e x p e r i m e n t s ,  a hyper o s m o t i c r e c t a l s e c r e t i o n was  still  Figure 20.  The effect of varying sodium concentration i n a r t i -  f i c i a l hemolymph on the volume of r e c t a l secretion c o l l e c t e d a f t e r 1.5 h A  (n = 6 ) , osmotic concentration Q  concentration means.  ^  (n = 6).  (n = 6) and sodium  V e r t i c a l bars denote S . E . of the  Rectal Secretion Sodium Concentration  (mM)  97  formed under these conditions (634 - 31 mOsm, n = 7 ) .  The potassium  ion concentration i n this f l u i d was very high (280 - 70 mOsm, n = 7) suggesting that potassium ions can p a r t i a l l y substitute f o r sodium ions in the secreted f l u i d when levels of the l a t t e r ion are low. The net rate of ion secretion can be calculated by m u l t i plying the volume of f l u i d secreted times the concentration of the ion i n the r e c t a l secretion ( F i g . 21).  Since the volume secreted has been  assumed to be equal to the volume removed from the r e c t a , and may therefore be subject to s l i g h t underestimation, the curves i n F i g . 21 are meant to show the r e l a t i v e rather than absolute transport  rates.  These curves indicate that the response of sodium and chloride transport rate to increasing levels of these ions i n the hemolymph does not follow Michaelis-Menten k i n e t i c s .  Instead, over the range of  hemolymph ion levels observed in, v i v o , the curves show increasing conc e n t r a t i o n , suggesting kinetics l i k e those of a l l o s t e r i c enzymes.  Figure 21. chloride  The relationship between the concentration of O  or sodium 0  in a r t i f i c i a l hemolymph  bathing the rectum and the rate of transport of that i o n .  <i86  99  DISCUSSION  Beadle (1939) was the f i r s t to propose that larvae of s a l i n e water mosquitoes drink the external medium as a means of replacing water l o s t by osmosis across the c u t i c l e and by excretory processes. The present study confirms this f i n d i n g , but in examining the  relation-  ship between drinking rate and external concentration some unexpected results were observed.  I have shown the drinking rate of A. taenior-  hynchus to be related to the surface area of the larvae ( F i g . 14) and to be unaffected by external s a l i n i t y ( F i g . 10).  Decreased osmotic  permeability of the body wall and changing concentration of the excreta might possibly compensate f o r a s i m i l a r drinking rate by larvae i n the two hyperosmotic media.  However, no change i n body wall perm-  e a b i l i t y was observed by P h i l l i p s i n A., campestris ( P h i l l i p s and Bradl e y , in press).  In 10% sea water, moreover, water moves into  both by osmosis across the integument and by d r i n k i n g .  larvae  Under these  conditions high rates of ingestion are c l e a r l y detrimental  to osmotic  homeostasis and must serve some other f u n c t i o n , such as nutrient energy) procurement.  (i.e.  I suggest that the drinking rate of s a l i n e -  water mosquito larvae i s proportional to the surface area of the larvae because of the commonly observed link between surface area and the metabolic rate of animals (Hemmingsen, 1960). The larvae used i n this study were starved f o r 24 hours p r i o r to the determination of drinking rate, and under these conditions show many signs of hunger ( e . g . constantly moving mouthparts  100  and foraging along s u r f a c e s ) .  Aedes taeniorhynchus larvae are able  to take up dissolved nutrients by drinking (Nayar, 1966).  In the  present study, drinking rate experiments were conducted with waters which were f i l t e r e d prior to use and the i n u l i n was f u l l y dissolved by heating to 90  o  C.  Therefore, any nutrients and  up had to be i n dissolved form.  14 C - i n u l i n taken  We suggest that these larvae drink  the external medium even when particulate food i s not available in order to gain nutrients which might be dissolved i n the medium.  external  The rate of this a c t i v i t y i s proportional to the metabolic  rate of the larva and hence proportional to surface area, and i s unaffected by the s a l i n i t y of the external medium.  Such a c t i v i t y may  be stimulated by certain organic solutes in natural waters, and variations i n concentration of the l a t t e r might explain  different  estimates of ingestion at the same s a l i n i t y . The estimate f o r the drinking rate f o r larvae reared in —1 100$ sea water in this study (100 n l . mg  —1 . hr  .)  i s three times  higher than that found in a previous study (Bradley and P h i l l i p s , 1975; Chapter II).  Larvae were handled i d e n t i c a l l y in the two  studies with the exception that in the present procedure the  larvae  were weighed i n d i v i d u a l l y and the midgut was cut into 3-4 pieces i n addition to mincing the whole larva before K0H d i g e s t i o n .  Whether  this aided the release of i n u l i n from the gut contents and accounted for the whole three-fold difference It  i s not known.  may be calculated from values observed in this study,  that larvae drink an amount of f l u i d equivalent to t h e i r  total  101  weight every 10 hours.  The turnover times for t o t a l body contents i n  100% and 200% sea water are 3.B and 1.9 hours respectively f o r sodium; and 4.B and 2.4 hours respectively f o r c h l o r i d e .  These animals c l e a r l y  face a considerable osmoregulatory load due to the rapid ingestion of the external medium.  High rates of f l u i d ingestion could serve a  useful function not only in nutrient uptake but also i n reducing the r e c t a l f l u i d concentration necessary to achieve homeostasis i n hyperosmotic media ( P h i l l i p s and Bradley, i n p r e s s ) .  This i s because the  greater the r a t i o of the f l u i d ingested to water l o s t across the i n tegument via osmosis, the c l o s e r the r e c t a l f l u i d concentration r e q u i r ed to achieve osmotic balance approaches that of the external medium. If  the permeability of the external c u t i c l e of A,, taeniorhynchus i s  s i m i l a r to that of Opifex fuscus (Nicholson and Leader, 1974)  then the  calculated osmotic water loss f o r a 3 mg larva i s 38 n l . h  i n 100%  sea water (Bradley and P h i l l i p s , 1975).  In this study, the drinking  rate of such larvae was shown to be approximately 300 n l . h  .  This  necessitates a r e c t a l secretion only 14% more concentrated than the external medium f o r osmotic balance, whereas the average value observed f o r whole larvae (Bradley and P h i l l i p s , 1975)  was 11% higher.  observed osmotic concentration of the r e c t a l secretion i s  The  therefore  very close to the predicted value necessary f o r osmotic homeostasis. As discussed below, some ions cannot be regulated by means of the r e c t a l secretion and other regulatory s i t e s must play a v i t a l role ( e . g . Malpighian tubules, anal p a p i l l a e ) . Aedes taeniorhynchus larvae show impressive a b i l i t i e s  to  102  r e g u l a t e hemolymph i o n c o n c e n t r a t i o n s i n e x t e r n a l media v a r y i n g from 10$ t o 200$ sea w a t e r . Mg  I n p a r t i c u l a r , the l e v e l s o f N a , K +  and  +  i n the hemolymph show no s i g n i f i c a n t v a r i a t i o n s o v e r t h i s wide  range of e x t e r n a l c o n c e n t r a t i o n s .  Only c a l c i u m i n c r e a s e s  cantly with increasing external concentration. hemolymph c a l c i u m l e v e l may be n e c s s s a r y hyperosmotic waters.  signifi-  This increase i n  f o r osmoregulation  i n more  F o r example i n the f r e s h w a t e r mosquito l a r v a ,  Culex p i p i e n s , i n c r e a s e d e x t e r n a l c a l c i u m promotes b e t t e r s u r v i v a l of l a r v a e p l a c e d i n hyperosmotic media h i g h i n sodium ( B r a d l e y and P a r k i n s , 1975).  Hemolymph a n i o n l e v e l s and o s m o l a l i t y show much more  change than do those of c a t i o n s , but even these i n c r e a s e by l e s s than 25$.  Nayar and Sauerman (1974) showed t h a t t h e c h l o r i d e c o n c e n t r a -  t i o n i n the hemolymph of A,, t a e n i o r h y n c h u s  i s not l i n e a r l y r e l a t e d to  the e x t e r n a l c o n c e n t r a t i o n but i s s t r i c t l y  r e g u l a t e d a t one l e v e l i n  a l l hyposmotic d i l u t i o n s of s e a water and e q u a l l y s t r i c t l y a t a s l i g h t l y h i g h e r l e v e l i n a l l hyperosmotic ones, i . e . an a b r u p t change i n hemolymph c o n c e n t r a t i o n o c c u r s over a narrow range of e x t e r n a l concentration. Sulphate  i o n s show a d i f f e r e n t p a t t e r n o f r e g u l a t i o n .  hemolymph l e v e l s a r e found i n animals  r e a r e d i n 10$ and 100$ s e a w a t e r ,  w h i l e h i g h e r l e v e l s a r e found i n l a r v a e from 200$ s e a w a t e r . i s excreted  l a r g e l y by the M a l p i g h i a n  tubules (Chapter  This i o n  I I I ) and t h i s  p a t t e r n of r e g u l a t i o n r e f l e c t s the f a c t t h a t t h e M a l p i g h i a n can o n l y produce a s e c r e t i o n i s o s m o t i c t o the hemolymph. p a t t e r n of s u l p h a t e  Low  tubules  A similar  r e g u l a t i o n was observed i n £ . c a m p e s t r i s  by  103  Waddrell and P h i l l i p s (1975). The results c i t e d in this chapter shpui that the concentrations of a l l but one ion were higher in the r e c t a l f l u i d from larvae  reared  i n 200% sea water than in that from animals l i v i n g i n 100% sea water. If  larvae are raised i n a medium with increased l e v e l of either N a ,  +  +  —  K ,C1  ++  or Mg  , then the r e c t a l secretion contains elevated levels of  the ion in question (Chapter III).  It  i s therefore c l e a r that the  secretion of r e c t a l f l u i d i s a major mechanism i n the osmoregulation of saline-water mosquito larvae.  The only ion whose secretion i s not  greater in larvae from 200% sea water than in those from 100% sea water i s potassium.  In both media, the potassium concentration of the  rectal secretion i s several times higher than that i n the medium.  Bradley and P h i l l i p s (1975); also Chapter II  external  suggest that t h i s  potassium loss from the hemolymph through secretion may be replaced by potassium uptake from the medium by the anal p a p i l l a e . It  i s i n t e r e s t i n g to speculate why the potassium concentra-  tion of the r e c t a l secretion i s lower i n 200% than in 100% sea water. In a recent paper, Bodil Schmidt-Nielsen (1975) proposed that c e l l shrinkage in hyperosmotic media i s accompanied by the excretion of e x t r a c e l l u l a r potassium.  This mechanism might be important  in some short-term experiments involving rapid changes i n hemolymph concentrations.  artificial  However the composition of normal rectal  f l u i d from larvae adapted to 100% and 200% sea water involved animals which had been reared in these solutions since hatching and thus v o l ume adjustments should not have changed during experiments.  A more  104  +  l i k e l y explanation i s that Na mechanism in the rectum.  +  and K  compete f o r the same transport  This explanation gains some support from  the observation that rectal secretion i s very high in potassium (280*. 70 mM, n = 5), when recta are incubated in a r t i f i c i a l which has a low Na  +  hemolymph  concentration.  In 200$ sea water, magnesium levels i n the r e c t a l secretion were higher than i n the external medium but sodium chloride and osmotic concentrations were on the average s l i g h t l y lower. i b l e explanations f o r this phenomenon come to mind.  Two poss-  A considerable  amount of evidence has been c i t e d in previous papers ( P h i l l i p s and Meredith, 1969; Bradley and P h i l l i p s , 1975; P h i l l i p s and Bradley, in press)  suggesting that the anal papillae are a s i t e of chloride and  possibly sodium excretion i n water high i n these i o n s .  The low con-  centrations of these ions in rectal secretion from larvae l i v i n g i n 200$ sea water are consistent with such a conclusion.  Alternatively,  blockage of the anus i n whole larvae, as discussed previously by Bradley and P h i l l i p s (1975) and Chapter II  could lead to a backflux  of ions from the r e c t a l lumen to the hemolymph and increased osmotic flow of water in the reverse direction due to excessive swelling of the rectum under these experimental c o n d i t i o n s . Some of the factors influencing mechanisms c o n t r o l l i n g the i o n i c and osmotic concentrations of the r e c t a l secretion have been c l a r i f i e d during the course of this study.  A,, taeniorhynchus larvae  can survive rapid transfer from 10$ to 200$ sea water and vice versa at any time during their development.  In 10$ sea water the rectum  105  does not secrete but rather seems to remove f l u i d from the lumen. In hyperosmotic media, r e c t a l secretion i s a major means of ion excretion.  Obviously, some neural or hormonal control exists which  dictates whether the rectum w i l l function as a net resorptive or secretory s i t e .  Long-term changes in r e c t a l function may be i n i t i a t e d  by external parameters ( e . g . via external osmoreceptors or s a l t recept o r s ) , or control mechanisms which respond to hemolymph ion levels over a time course longer than our experimental period (1.5 - 2 h r s ) . While the r e l a t i v e l y constant levels of the ions observed in hemolymph of adapted larvae ( F i g s . 15 and 16) might seem inconsistent with internal receptors, i t  i s reasonable to assume that abrupt changes  in external s a l i n i t y do in fact result in s i z a b l e transient changes i n hemolymph ion l e v e l s .  Variations in t o t a l hemolymph osmotic con-  c e n t r a t i o n , within physiological l e v e l s , have no e f f e c t on the osmotic concentration of the r e c t a l s e c r e t i o n .  The hemolymph levels of at  least two ions sodium and c h l o r i d e , have a profound influence not only on the secretion concentrations of these two ions but also on the osmotic concentration of the r e c t a l f l u i d and i t s rate of s e c r e t i o n . Increases i n sodium or chloride concentrations In the hemolymph lead to a higher concentration of this ion i n the r e c t a l s e c r e tion ( F i g s . 19 and 20).  This in turn leads to an increase in the  osmotic concentration of the secretion since sodium and chloride are major contributors to the osmotic concentration.  Simultaneously, the  volume of f l u i d secretion increases at higher hemolymph concentrations of these i o n s .  106  The  s l o p e s of the c u r v e s i n f i g u r e 21 i n d i c a t e t h a t  r e l a t i o n s h i p between i o n c o n c e n t r a t i o n s  the  of sodium and c h l o r i d e i n the  hemolymph and i o n t r a n s p o r t r a t e do not f o l l o w M i c h a e l i s - M e n t e n k i n e t i c s but demonstrate an a l l o s t e r i c type k i n e t i c c u r v e . property  Whether t h i s i s a  of the t r a n s p o r t mechanisms themselves o r i s a f u n c t i o n of  changing r a t e s of a c c e s s of the i o n s to these mechanisms ( e . g . t o v a r i a b l e membrane p e r m e a b i l i t i e s ) i s not p r e s e n t l y known.  due Regard-  l e s s of the a c t u a l mechanism i n v o l v e d , the normal l e v e l s of sodium and c h l o r i d e i n the hemolymph c o r r e s p o n d to the s t e e p l y r i s i n g p a r t of the c u r v e f o r the t r a n s p o r t k i n e t i c s ( F i g . 2 1 ) .  C l e a r l y , any  r i s e i n hemolymph sodium o r c h l o r i d e l e v e l s w i l l a u t o m a t i c a l l y i n c r e a s e d s e c r e t i o n of these i o n s by the  rectum.  transient result in  CHAPTER V  THE USE OF AN IN VITRO RECTAL PREPARATION TO DIFFERENTIATE THE FUNCTIONS OF THE ANTERIOR AND POSTERIOR RECTAL SEGMENTS IN AEDES TAENIORHYNCHUS  107  108  INTRODUCTION  The  r e c t a of a l l s a l i n e - w a t e r mosquito l a r v a e examined to  date have two m o r p h o l o g i c a l l y d i s t i n c t segments of the rectum (Aedes d e t r i t u s , Ramsay, 1950; fl . c a m p e s t r i s . M e r e d i t h and L  A,, t a e n i o r h y n c h u s ,  B r a d l e y and P h i l l i p s ,  1975).  Phillips,  1973;  Ultrastructural  e v i d e n c e s u g g e s t s t h a t the p o s t e r i o r r e c t a l p o r t i o n i s the s i t e of h y p e r o s m o t i c f l u i d s e c r e t i o n ( M e r e d i t h and P h i l l i p s ,  1973a).  I there-  f o r e m o n i t o r e d changes i n f l u i d volume and o s m o t i c c o n c e n t r a t i o n i n i s o l a t e d a n t e r i o r and p o s t e r i o r r e c t a l segments i n an a t t e m p t to d i s t i n g u i s h the p h y s i o l o g i c a l process  o c c u r i n g i n the two p a r t s of  the  rectum, p a r t i c u l a r l y w i t h r e g a r d to the l o c a t i o n of the s i t e of h y p e r osmotic f l u i d s e c r e t i o n .  MATERIALS AND Aedes t a e n i o r h y n c h u s p r e v i o u s l y d e s c r i b e d (Chapter use.  METHODS  l a r v a e were r a i s e d i n 100$ I I ) and s t a r v e d f o r 1 - 2  An in v i t r o p r e p a r a t i o n of the rectum was  l a r v a e i n normal hemolymph ( C h a p t e r  I I ) . The  l a r v a e and l i g a t e d w i t h f i n e s i l k t h r e a d a t any t h r e e p o i n t s as a p p r o p r i a t e :  days p r i o r to  prepared  gut was  sea water as  by d i s s e c t i n g  removed from the  two of the f o l l o w i n g  the p o s t e r i o r p a r t of the i l e u m ,  the  j u n c t i o n a l r e g i o n between the a n t e r i o r and p o s t e r i o r r e c t a l segmants, o r the a n a l c a n a l near the anus.  These l i g a t u r e s thus i s o l a t e d e i t h e r  the e n t i r e rectum, o r the p o s t e r i o r o r a n t e r i o r segments i n d i v i d u a l l y . The  r e g i o n s of the gut not i s o l a t e d between the l i g a t u r e s were removed.  These l i g a t e d r e c t a were p l a c e d i n a hanging drop ( c a . 10 u l ) of normal  109  a r t i f i c i a l hemolymph suspended from the c o v e r of a 60 p e t r i d i s h (Marks and Holman, 1974). was  The  x 15 mm  bottom of the p e t r i  f i l l e d w i t h d i s t i l l e d water to r e t a r d e v a p o r a t i o n .  The  glass dish  osmotic  c o n c e n t r a t i o n of the hanging drop i n c r e a s e d by l e s s than 15 mOsm d u r i n g the two hour e x p e r i m e n t a l  period.  A f t e r two h o u r s , r e c t a  were removed from such drops w i t h an eyedropper and p l a c e d on a scope s l i d e , where the a d h e r i n g f l u i d was The  rectum was  micro-  b l o t t e d up w i t h f i l t e r  paper.  p u n c t u r e d u s i n g a g l a s s m i c r o p i p e t t e and a sample of  r e c t a l f l u i d was  removed.  measured i m m e d i a t e l y Physics, Ltd.).  The  osmotic c o n c e n t r a t i o n of the sample  u s i n g a n a n o l i t e r osmometer ( C l i f t o n  F o r a more d e t a i l e d d e s c r i p t i o n of t h i s  was  Technical procedure  see Appendix I I .  RESULTS  S e c r e t i o n by r e c t a l segments The differentiate of the rectum.  i n , v i t r o p r e p a r a t i o n of the l a r v a l rectum was the f u n c t i o n s of the a n t e r i o r and T h i s was  most e a s i l y  used to  p o s t e r i o r segments  done by p l a c i n g a l i g a t u r e  tween the two segments and m o n i t o r i n g f l u i d c o n c e n t r a t i o n s and umes i n the two p o r t i o n s w i t h time.  The  bevol-  r e s u l t s of such an e x p e r i -  ment c o u l d not be compared to p r e v i o u s o b s e r v a t i o n s , i n v i v o , because of i n t e r f e r e n c e w i t h n e u r a l and  tracheal connections  to the rectum.  t h i s reason an i n v i t r o p r e p a r a t i o n of the whole rectum s e r v e d a c o n t r o l f o r comparison w i t h i s o l a t e d  a n t e r i o r and p o s t e r i o r  as  For  110  rectal segments.  In a l l three preparations, neural connections had  been broken, and oxygen entered the tissue from the surrounding f l u i d , rather than by the tracheae. Photographs 1 and 2 of Plate 2 compare a single jLn v i t r o preparation of a whole i s o l a t e d rectum 5 minutes and 2 hours a f t e r d i s s e c t i o n , respectively.  The rectum swells with f l u i d during the  2 hour experimental period as previously observed for in, vivo preparations of the rectum (Bradley and P h i l l i p s , 1975 and Chapter Similar results were observed for 23 preparations.  II).  Photographs 3 and  4 of Plate 2 show the posterior segment of a single rectum at 5 minutes and 2 hours a f t e r d i s s e c t i o n , respectively.  When ligatured such  that only the posterior rectum f i l l s with f l u i d , the preparation i s not capable of greatly increasing in volume due to a greater tension on the rectal w a l l s . obvious, i f  Nevertheless  some swelling of this segment i s  less marked than for the whole rectum (11  observations).  No change in volume could be discerned i n any of the 13 preparations of the anterior r e c t a l segment observed.  Because of the  small volume of the anterior rectum, i t i s impossible to conclude absolutely from photographic data alone that the anterior r e c t a l either secretes or resorbs any f l u i d i n v i t r o .  portion  However since both  the whole rectum and posterior rectal portion do f i l l with f l u i d when i s o l a t e d , we were interested in determining whether the secretion in v i t r o was hyperosmotic to the hemolymph as previously demonstrated i n vivo  (Bradley and P h i l l i p s , 1975 and Chapter  II).  Lla  Plate 2.  Photographs 1 and 2 show a s i n g l e , whole in, v i t r o  rectum 5 min and 2 h after l i g a t i o n , respectively.  In photo-  graph 2 the rectum can be seen to be f i l l e d with secreted fluid.  Photographs 3 and 4 show a s i n g l e , i s o l a t e d posterior  r e c t a l preparation 5 min and 2 h after l i g a t i o n ,  respectively.  In photograph 4 of the posterior segment can be seen to be swollen with secreted f l u i d . outlined i n white.  The i n i t i a l shape of the segment  Similar photographs f o r anterior  segments showed no change in volume.  rectal  XUb  112  Osmotic Concentrations of r e c t a l secretion i n v i t r o The same procedures as were used to obtain photographic e v i dence of r e c t a l secretion were followed to obtain f l u i d from the lumina of whole recta and i s o l a t e d posterior and anterior r e c t a l segments (Table 8).  When whole recta were dissected from l a r v a e , l i g a t e d , and  immediately sampled by micropuncture, the r e c t a l f l u i d was found to be hyperosmotic to the a r t i f i c i a l hemolymph by an average value of 127 mOsm. This i n i t i a l r e c t a l f l u i d concentration was high compared to that previously observed with i s o l a t e d in, vivo preparations possibly because of the following difference i n procedure.  Larvae used for i n v i t r o ex-  periments were k i l l e d rapidly by grasping the thorax with forceps which may not have allowed time for complete defecation of r e c t a l contents.  During preparation of ligated in, vivo preparations, f o r which  initial  r e c t a l concentration values are available (Bradley and P h i l l i p s .  1975; and Chapter II),  the rectum completely empties and p a r t i a l l y  re-  f i l l s with midgut f l u i d while the larva i s being blotted dry on f i l t e r paper, resulting in an i n i t i a l  rectal  f l u i d concentration which i s  isosmotic to the hemolymph. While the volume of f l u i d i n whole irj. v i t r o recta increased over 2 h (Photographs 1 and 2 of Plate 2 ) ,  the f i n a l osmotic concentra-  tion of the secreted f l u i d was not s i g n i f i c a n t l y d i f f e r e n t from that initially  present (Table 8).  The i s o l a t e d posterior segment of the  rectum f i l l s with a f l u i d which was s i g n i f i c a n t l y hyperosmotic to the initial  rectal f l u i d , the a r t i f i c i a l  hemolymph, and also secretion  from i n v i t r o whole recta a f t e r 2 hours ( P < 0 . 0 2 ) .  In the i s o l a t e d  113  Table 8.  The osmotic concentrations of r e c t a l f l u i d removed  from lumina of ^n v i t r o preparations of the rectum.  Concentra-  tions are given i n the two l e f t hand columns f o r samples from whole recta taken i n i t i a l l y  or 2 h after l i g a t i o n .  The two  right hand columns show the osmotic concentration of f l u i d removed 2 h after l i g a t i o n to i s o l a t e the posterior from the anterior  rectal segment.  Osmotic concentration of the  artificial  hemolymph used i n a l l experiments was 355 mOsm.  Whole Rectum (mOsm) Serial  0 hr  Rectal Segments (mOsm)  2 hr  Anterior  403  457  446  753  2  409  522  452  640  3  551  503  473  1183  4  511  441  425  742  500  462  419  672  495  468  484  424  505  444  711  6 7  Mean S  (2  1  5  1  Posterior  (2 hr)  ' ' E  475 1  2 9  480 1  13  !  8  t 88  hr)  114  anterior r e c t a l portion where no volume change was discernable, a s i g n i f i c a n t reduction (P<0.05) in osmotic concentration occurred during the two hour experimental time period.  P o s s i b l y , the  failure  of the whole rectum ^n v i t r o to secrete f l u i d as concentrated as the posterior rectum alone i s due to solute reabsorption i n the ant e r i o r rectum.  This i s consistent with the decline i n f l u i d concen-  tration in i s o l a t e d anterior r e c t a l segments. The r e l a t i v e l y  low concentration of the r e c t a l secretion i n  v i t r o could r e f l e c t lack of a natural neural or hormonal stimulus. Compounds known to stimulate secretion i n some other insect tissues were therefore added to the a r t i f i c i a l rectal preparations.  hemolymph bathing in, v i t r o  No s i g n i f i c a n t increase i n r e c t a l f l u i d concen-  tration was found upon adding c y c l i c 3* - 5' adenosine monophosphate (10  —2 —2 M) with or without theophylline (10** W), or 5 hydroxytryptamine  (10" M); (Table 9 ) . 4  The results of these experiments using in, v i t r o r e c t a l preparations indicate that the posterior rectal segment i s a s i t e , i f the only one, of hyperosmotic f l u i d s e c r e t i o n .  The anterior  not  rectal  segment seems to reduce the concentration of the r e c t a l f l u i d i n v i t r o , which i s consistent with the suggestion that this part of the rectum i s involved i n solute reabsorption (Meredith and P h i l l i p s , 1974).  115  Table 9.  The osmotic concentration (mOsm) of r e c t a l secretion  produced by whole, in vitro recta in a r t i f i c i a l hemolymph with the addition of potential stimulatory agents.  Serial  Cyclic  Cyclic AMP(lO~ in)  5-Hydroxy-  + Theophylline (l0" m)  Tryptamine  2  AiriP(lO- m) 2  2  1  466  478  2  462  349  3  898  392  559  4  769  505  602  5  430  6  495  Wean  587  1  S.E.  1 80  392 626  454 435  431  1 36  5 1 1  +40  116  DISCUSSION  In v i t r o preparations of the l a r v a l rectum used in this study secreted a much less concentrated f l u i d than do the in, vivo preparations of Bradley and P h i l l i p s (1975) and Chapter II. the same a r t i f i c i a l  Since  hemolymph bathed both preparations the poorer  performance of in, v i t r o recta i s probably due to disruption of tracheal connections and possibly the absence of neural and hormona l stimulation.  However, s u f f i c i e n t transport a c t i v i t y  remains in  v i t r o i n the posterior rectum to produce a secretion 366 mOsm more concentrated than the hemolymph (Table 8).  This portion of the  rectum i s therefore c l e a r l y one, i f not the only, area where hyperosmotic secretion occurs. No firm conclusions can be drawn from the present data alone concerning the function of the anterior rectum.  I_n v i t r o ,  this r e c t a l segment appears to reduce the concentration of the f l u i d in the lumen of both whole recta and i s o l a t e d anterior This could occur by means of three separate mechanisms.  recta.  The anter-  i o r rectum in, vitro may transport s a l t s out of the r e c t a l lumen at a rate equal to the rate of secretion by the posterior rectum, or might allow s a l t to leak back by simple d i f f u s i o n in, v i t r o .  Alter-  n a t i v e l y , the anterior r e c t a l portion may be more permeable to water in, v i t r o than in, v i v o . The u l t r a s t r u c t u r a l observations of Meredith and P h i l l i p s (1973a) indicated that the anterior rectum of saline-water  larvae  117  resembles the rectum of freshwater mosquito larvae where s a l t resorption results in the formation of hyposmotic excreta. rectum possesses a c e l l u l a r structure r e s t r i c t e d to  The posterior saline-water  larvae and therefore i s implicated in the formation of hyperosmotic fluid.  The evidence to date suggests that the two r e c t a l portions  serve separate functions.  They show morphological  differentiation,  e l e c t r i c a l potentials d i f f e r i n g both in magnitude and p o l a r i t y , and opposite effects of rectal f l u i d concentrations jjn v i t r o .  This sup-  ports the hypothesis of P h i l l i p s and Meredith (1969a) and Meredith and P h i l l i p s (1973a) that the anterior rectum i s the s i t e of s a l t and nutrient resorption in both fresh and saline-water and that the posterior rectum i s the location of the hyperosmotic secretion of s a l t s for osmoregulation in saline waters.  Another saline-water  i n s e c t , Ephydrella possesses two c e l l types in the hind-gut and Wright, 1974).  (Marshall  The two c e l l types, rather than existing in sep-  arate regions as in the saline-water mosquito rectum, are i n t e r s p e r s ed throughout the hind-gut.  The rectum of Ephydrella i s seemingly  i n a c t i v e , but the hindgut contains hyperosmotic f l u i d .  Marshall and  Wright propose that the two c e l l types perform separate functions, either s a l t resorption or s a l t secretion by the small c e l l s , and water resorption by the large c e l l s . Based on our present understanding of r e c t a l function in two species of saline-water mosquito larvae (Bradley and P h i l l i p s , 1975; Chapters II,  III  (Prusch, 1974),  and IV) and the blowfly larva Sarcophaoa bullata I suggest that most aquatic larvae capable of hyposmot-  118  i c regulation produce a concentrated urine by the secretion of a hyperosmotic f l u i d in some part of the hindgut.  In accordance with  t h i s function the portion of the gut in which f i n a l urine modification occurs w i l l show two c e l l types, one engaged i n resorption of essential metabolites and some ions and the other in secretion of hyperosmotic f l u i d when the animal i s in a hyperosmotic medium.  CHAPTER VI  THE MECHANISM OF HYPEROSMOTIC FLUID SECRETION IN THE RECTUM OF LARVAE OF THE SALINE-WATER MOSQUITO, AEDES TAENIORHYNCHUS  119  120  INTRODUCTION  The production of a concentrated urine i n saline-water mosquito larvae i s achieved by the secretion of a hyperosmotic f l u i d into the rectal lumen (Bradley and P h i l l i p s , 1975; and Chapter II). f l u i d contains N a , M g , CI +  ++  This  and probably HC0~ in ratios and concen-  trations resembling the external medium to which the larvae i s acclimated (Chapters III  and IV).  However, the r e c t a l f l u i d i s hypertonic to the  hemolymph and external medium with regard to potassium under every experimental condition which has been studied.  I wished to determine  which of these ions are actively transported during the process of r e c tal secretion.  Aedes taeniorhynchus larvae raised i n 100% sea water  were chosen for this study because the i o n i c concentration differences across the rectal epithelium are known (Bradley and P h i l l i p s 1975; Chapter II).  In the present study, e l e c t r i c a l potential differences across  the rectal epithelium were determined under the same experimental cond i t i o n s in order to determine whether the d i s t r i b u t i o n of any of the ions could be explained by passive forces alone ( i . e . potential d i f f e r e n c e ) .  A model for t r a n s - e p i t h e l i a l  by electrochemical transport of ions i s  prooosed, based on these observations and those from Chapters II,  III,  IV.  MATERIALS AND METHODS  The e l e c t r i c a l potential difference across the r e c t a l epithe-  and  121  Hum was measured using two separate methods.  Tn one case, (hereafter  referred to as e l e c t r i c a l oreoaration #1) the larvae were ligated between the sixth and seventh abdominal segments and the portion of the larvae anterior to the ligature was removed.  The larva was placed on a p e t r i  d i s h , the bottom of which was lined with paraffin wax into which a hole 2mm x 5mm and 3mm deep had been made, such that the siphon extended into t h i s hole.  A glass microscope s l i d e coverslip was placed over the hole,  allowing a space f o r the siphon and the edge was sealed with melted paraffin wax.  The siphon was thereby i s o l a t e d and exposed to the pocket  of a i r under the c o v e r s l i p .  The area immediately adjacent to the larva  was sealed by melting the wax with a hot probe so that only a very small amount of melted wax touched the larva to hold i t  in place.  The p e t r i  dish was then f i l l e d with paraffin o i l which was prevented from entering the siphon by the sealed c o v e r s l i p .  The exposed c u t i c l e of the larvae  was torn and a drop of a r t i f i c i a l hemolymph was placed on i t .  Under these  circumstances the rectum was bathed in a solution of known composition while supplied with oxygen via the tracheae.  E l e c t r i c a l potentials were  measured by introducing the recording electrode into the lumen of the rectum through the anus. E l e c t r i c a l preparation #2  was used to measure both i n t r a -  c e l l u l a r and t r a n s - e p i t h e l i a l e l e c t r i c a l potential d i f f e r e n c e s , by impaling r e c t a l c e l l s from the hemolymph side with glass microelectrodes. To this end, larvae were placed on moist f i l t e r paper and ligated between the s i x t h and seventh abdominal segments and at the anal segment so as to i s o l a t e the rectum.  The portion of the larva anterior to the  first  122  ligature to  was r e m o v e d .  paraffin  the  The r e m a i n i n g l a r v a l p o r t i o n  wax l i n i n g  ligatures  into  the  bottom of  the wax.  a petri  the a i r o r  the s u r f a c e of  was b a t h e d i n supply v i a the few minutes only f o r  tracheae.  Since  artificial  Electrical  S y s t e m (Ul-P I n s t r u m e n t s , voltmeter  trode  was c o n n e c t e d t o  of  t h e s i p h o n was o p e n  to  hemolymph, the  rectum oxygen  of  Inc.)  with potentials  the a r t i f i c i a l by means o f  r e c o r d e d on an e x p a n d e d  The i n d i f f e r e n t  hemolymph b a t h i n g  a salt  calomel  the  recta in  bridge c o n s i s t i n g of  filled  elec-  w i t h 3M KC1 i n  c a s e the  tip  (electrical  through  r e s i s t a n c e was l o w ( 1 - 2 p r e p a r a t i o n #2)  The a r t i f i c i a l to  the anus ( e l e c t r i c a l  3% A g a r .  choline,  in  hemolymphs u s e d i n  the s u b s t i t u t i o n  through  using high resistance t i p s  those d e s c r i b e d i n Chapter IV.  ed o n l y  p r e p a r a t i o n #1)  megohms) o r  of  this  the  s t u d y were  in  into  which  rectal  (10-17  both  polyethy-  The r e c o r d i n g g l a s s e l e c t r o d e s w h i c h c o n t a i n e d 3M KC1 w e r e i n s e r t e d rectum e i t h e r  this  w e r e m e a s u r e d u s i n g a M701 M i c r o p r o b e  l e n e t u b i n g ( P . E . 5 0 , C l a y Adams, I n c . )  the  every  T h i s p r e p a r a t i o n was u s e d  F o r a more d e t a i l e d d e s c r i p t i o n  (Radiometer, Copenhagen).  preparations  was t o r n a n d a d r o p  of  II.  potentials  scale  ends  The hemolymph d r o p was r e m o v e d a n d r e p l a c e d  measurements.  procedure see Appendix  the  known c o m p o s i t i o n a n d h a d a n o r m a l  t o a v o i d c h a n g e s due t o e v a p o r a t i o n .  short-term  electrical  the drop of  a s o l u t i o n of  d i s h by m e l t i n g  The l a r v a l c u t i c l e  hemolymph was p l a c e d on t h e p r e p a r a t i o n .  was a n c h o r e d s e c u r e l y  wall  megohms).  identical  T h e s e v a r i o u s hemolymph s o l u t i o n s  a single ion.  differ-  T h u s s o d i u m was r e p l a c e d w i t h  p o t a s s i u m was a d d e d a s ^ S O ^ t o n o r m a l h e m o l y m p h , o r c h l o r i d e was  replaced with sulphate.  F o r a d e t a i l e d d e s c r i p t i o n of  the p r e p a r a t i o n  of  123  t h e s e hemolymphs s e e A p p e n d i x  IV.  RESULTS Trans-rectal Electro-potential Recta of  s a l i n e - w a t e r mosquito  containing concentrations hypertonic  to  Differences  of  Na , K , Mg  t h e hemolymph by 2-18  Chapters II,  III  and I V ) ,  transported,  a study  was u n d e r t a k e n o f  the  l a r v a e a d a p t e d t o 100$  m i c r o e l e c t r o d e was a d v a n c e d i n t o different  successively  and c o n s i s t e n t l y  only d i f f e r e n t relative cisely, P.D.  P.D. values for  to  potentials  actively  potential  differ-  water.  rectal  T h e s e two  in  to  measuring  the anus  two  t h e hemolymph w e r e  r e g i o n s showed not  cases,  locate  was u s e d ,  i n which the  lumen t h r o u g h  lumen r e l a t i v e  In o r d e r t o  1975;  opposite  polarity  t h e s e r e g i o n s more which  the  pre-  trans-epithelial  was m e a s u r e d by p a s s i n g g l a s s m i c r o e l e c t r o d e s f r o m t h e hemolymph s i d e rectal wall into  r e c t a l segment s u c h t h a t was k n o w n , ( T a b l e wa y s p o s i t i v e -12  mV.  mV.  10).  relative  the  the  lumen o f  either  the p r e c i s e l o c a t i o n The lumen o f  to  The l u m e n o f  w i t h r e s p e c t to  20.9  electrical  i n a m a j o r i t y of  e l e c t r i c a l p r e p a r a t i o n #2  through the  of  these ions are  was u s e d ,  observed.  but,  the hemolymph.  the  and P h i l l i p s ;  d u r i n g s e c r e t i o n by A_.  saa  the  fluid  a n d p r o b a b l y HCO^  To d e t e r m i n e w h i c h o f  When e l e c t r i c a l p r e p a r a t i o n #1  quite  , CI  times ( B r a d l e y  e n c e (P.D.) a c r o s s t h e w h o l e r e c t a l w a l l taeniorhynchus  larvae can s e c r e t e  of  the a n t e r i o r  the a n t e r i o r the  lumen o f  rectal  rectal  tip  s e g m e n t was a l one  r e c t a l s e g m e n t was a l w a y s  the a n t e r i o r  posterior  microelectrode  t h e hemolymph w i t h t h e e x c e p t i o n o f the p o s t e r i o r  or  value positive  s e g m e n t by an a v e r a g e  of  124  Table 10. potential  P a i r e d m e a s u r e m e n t s of difference  in  the a n t e r i o r  of  r e c t a bathed i n  of  dissection (electrical  shows the  the  trans-rectal  and p o s t e r i o r  the normal a r t i f i c i a l  electrical  difference  rectal  hemolymph w i t h i n  preparation #2).  potential  electrical  The r i g h t  segments ten  hand column  between the  two  rectal  segments.  Serial  Anterior Rectal Potential (lumen s i d e )  Posterior Rectal Potential (lumen s i d e )  Posterior Potential R e l a t i v e to Anterior  -22  +6  + 28  -11  +9  + 20  -30  -12  +18  0  +40  + 40  -7  +15  + 22  6  -2  +4  +6  7  -6  +14  +20  8  -6  +14  • 20  9  -10  +5  + 15  Wean  -10.4  +10.5  t S.E.  -  *  3.0  4.6  minutes  20.9  i  3.0  125  To a s c e r t a i n w h i c h i o n s were a c t i v e l y ion concentration  ratios  and e l e c t r i c a l p o t e n t i a l  m e a s u r e d u n d e r t h e same c o n d i t i o n s . t o measure the  Therefore  electrical potential  h o u r s , so t h a t c o n d i t i o n s were i d e n t i c a l ionic  c o m p o s i t i o n of  1 9 7 5 ; and C h a p t e r I I ) .  ior  rectal  portion  to  between the  d u c e d (mean o f  8.2mV).  region (Meredith resistance  continuity  the  ligated  anterior  1973)  between the l u m i n a of  the  used  two to  Phillips, swol-  and the  to  the  recta f i l l e d  the f a c t rectal  that  at  hemolymph with  the  portions the  s e c r e t i o n leads to  sec-  potential was  re-  junctional  high  electrical while  increased  and the p o s t e r i o r  the o p p o s i n g p o t e n t i a l  and  poster-  the u n s w o l l e n c o n d i t i o n ,  the a n t e r i o r of  was u s e d  r e c t u m was  provides a r e l a t i v e l y  2 hours of  be  showed b o t h n e g a t i v e  constriction  r e c t a l segments i n  rectum a f t e r  for  and p o s t e r i o r  Perhaps a s l i g h t  and hence to a s h o r t - c i r c u i t i n g the  when t h e  relative  reflect  must  hemolymph f o r  those p r e v i o u s l y  portion  The l o w e r p o t e n t i a l s  and P h i l l i p s ,  between the  s w e l l i n g of  rectal  rectum,  the w a l l of  t h e hemolymph (mean = 1.8mV)  versus those r e c e n t l y  difference  to  lumen was a l w a y s p o s i t i v e  by +6.4mV ( T a b l e 1 1 ) . retion  artificial  Under these c o n d i t i o n s  values r e l a t i v e  across  r e c t a l s e c r e t i o n ( B r a d l e y and  l e n w i t h s e c r e t i o n , the a n t e r i o r positive  differences  p r e p a r a t i o n #2  differences  r e c t a w h i c h had been l i g a t e d and p l a c e d i n  determine  s e c r e t e d by t h e  rectum  differences  in  two s e g m e n t s .  Influence  of I  across  the  Hemolymph I o n C o n c e n t r a t i o n s £ n t h e was i n t e r e s t e d  rectal wall  in  determining  and w h e t h e r  they  Trans-epithelial P.P.  how p o t e n t i a l  differences  are a s s o c i a t e d with a c t i v e  arise trans-  126  Table 11.  Paired measurements of the t r a n s - r e c t a l e l e c t r i c a l  potential difference i n the anterior and posterior r e c t a l segments of recta bathed in normal hemolymph two hours a f t e r dissection ( e l e c t r i c a l preparation #2).  The right hand column shows the e l e c t r i c a l  potential difference between the two r e c t a l segments.  Serial  Anterior Rectal Potential (lumen side)  Posterior Rectal Potential (lumen side)  Posterior Potential Relative to Anterior  1  +8  +8  2  -9  +7  + 16  3  -3  +16  + 19  4  -17  +4  +21  0  5  +5  +4  -1  6  -5  +6  + 11  7  -2  +6  +8  8  +4  +4  0  9  +3  +3  0  Wean  -1.8  +6.4  +8.2  - S.E.  -2.6  ±1.0  ±3.0  127  p o r t of  ions responsible for  retion.  Electrical potential  terior  profiles  stantially cellular  in  although  the a n t e r i o r  potentials  p l a s m a membrane)  through  and p o s t e r i o r  relative  to  hemocoel ( i . e .  was n o t s i g n i f i c a n t l y  in  different  in  the  of  of  the c e l l d u r i n g  p o s s i b l e to measure the e f f e c t  of  b a s a l p l a s m a membrane o f  the  posterior  w h i c h was a l w a y s n e g a t i v e  c o u l d be made l e s s n e g a t i v e  rectal  (cell  intrabasal  importance  reached  procedure.  varying ion  difference  segment.  across  This  relative  to  hemolymph)  by i n c r e a s i n g h e m o l y m p h p o t a s s i u m l e v e l s ;  63mM K  D e c r e a s i n g the c h l o r i d e c o n c e n t r a t i o n  observation).  t h e hemolymph a l s o r e d u c e d t h e  potential  membrane, b e i n g r e d u c e d by 7i0mV i n 1 . 3 of  difference.  in  39mMK a n d 13mV  difference  +  across the  mM C l " ( n = 3 ) .  t h e hemolymph f r o m 200mM t o  s t i t u t i n g c h o l i n e d i d not  the  potential  B 1 0.7mV (n=4)  sodium c o n c e n t r a t i o n  It  concentra-  b e i n g r e d u c e d by an a v e r a g e o f (single  is  hemolymph s u c h  entire  interior  sub-  s p e c i f i c ions  potentials  the  12).  two s e g m e n t s  critical  transport  When i n t r a c e l l u l a r  remained i n  the  P.O. across the  t h e hemolymph on t h e e l e c t r i c a l p o t e n t i a l  difference  P.O. d i f f e r s  was p o s s i b l e t o c h a n g e t h e a r t i f i c i a l  electrode  was t h e r e f o r e  sec-  and p o s -  and s i g n  portions,  These v a l u e s are o f  (see D i s c u s s i o n ) .  steady v a l u e s , i t the  the s i z e  rectal  when d e t e r m i n i n g a t w h i c h membrane a c t i v e occurring  rectal  the a n t e r i o r  the t r a n s - e p i t h e l i a l  a n d -49mV r e s p e c t i v e l y ) .  tions  hyperosmotic  s t e p s a c r o s s a p i c a l and b a s a l p l a s m a membranes ( T a b l e  T h e s e d a t a show t h a t  that  of  r e c t a l c e l l s were m e a s u r e d t o d e t e r m i n e  the p o t e n t i a l  (-39  the formation  have a s i g n i f i c a n t  effect  Varying  in  basal the  5mM Na by s u b on t h i s  potential  of  128  T a b l e 12.  P a i r e d measurements of  the  r e c t a l c e l l s and the  the  anterior  rectal  and p o s t e r i o r  on r e c t a b a t h e d i n  the  electrical potentials  lumen ( r e l a t i v e  rectal portions.  normal a r t i f i c i a l  to  within  t h e hemolymph)  M e a s u r e m e n t s w e r e made  hemolymph w i t h i n 10 m i n u t e s  dissection.  Serial  Anterior  Rectum  Cell Interior  Lumen  Posterior Cell Interior  Rectum Lumen  1  -34  -16  -37  +14  2  -50  -16  -50  +17  3  -40  -12  -41  +6  4  -60  -12  -27  +2  5  -61  -7  -41  +15  Mean  -49  -13  -39  +11  4 S.E.  i  12  ± 4  5  in  1  3  of  129  Since  earlier  e x p e r i m e n t s i n C h a p t e r V w i t h i_n v i t r o  parations  of  the  is  of  hyperosmotic f l u i d  a site  effect  of  secretion, I  generated a c r o s s the  potentials  in  the  two p o r t i o n s  of  rectum are o f t e n  and tend to c n a c e l each o t h e r o u t ,  originating  observed)  in  the a n t e r i o r  rectal  d i d not  p o t a s s i u m o r s o d i u m ( 2 0 0 mm,  Na )  T h i s change i n  a s s o c i a t e d w i t h the  low i n c h l o r i d e  posterior  that  not a f f e c t  the a n t e r i o r  cal short-circuit  in  ( F i g . 22).  the  of  the  Of  h a d any lumen.  therefore  The o b s e r v a t i o n  levels in  between the  r e c t a l segments i n on t h e  as be that  t h e hemolymph do  r e c t a l P.D. argue a g a i n s t a s u b s t a n t i a l  s i n c e t h e s e p a r a m e t e r s have marked e f f e c t s rectal  the  t h e same d i r e c t i o n  region.  h i g h p o t a s s i u m and s o d i u m , and low c h l o r i d e  in  not  hemolymph h i g h  r e c t a l segment and might  P . D . change i n  were  preparations  +  was i n  in  important  portion  t h a t l o w i n s o d i u m (5mM N a )  difference  The  was  The p o t e n t i a l  c a u s i n g an i n c r e a s e d n e g a t i v i t y  the p o t e n t i a l  that observed i n the  or  +  v a r i o u s hemolymphs u s e d , o n l y  rectal  r e c t u m was b a t h e d i n  the  opposite it  t o t h e hemolymph ( e i g h t  v a r y when t h e  appreciable effect,  the p o s t e r i o r  detail  segment.  therefore  rectal portion.  lumen r e l a t i v e  this  portion  electrical  c e l l of  the  rectal  examined i n  entire  t o show t h a t c h a n g e s o b s e r v e d i n  anterior  the p o s t e r i o r  v a r y i n g hemolymph i o n c o n c e n t r a t i o n s on t h e  potential  polarity  rectum demonstrated t h a t  pre-  the unswollen P.D. i n  the  electrirectum,  posterior  segment. Electrical  placed through  p r e p a r a t i o n #1 was u s e d w i t h t h e  the anus i n t o  F i g u r e 2 3 shows t h e e f f e c t  t h e most p o s i t i v e  on t h e  part  of  electrical potential  micro-electrode the  rectum.  difference  of  the  L30a  F i g u r e 22.  The t r a n s - r B c t a l e l e c t r i c a l p o t e n t i a l  (lumen r e l a t i v e  t o hemolymph)  observed i n a n t e r i o r  ments b a t h e d i n n o r m a l a r t i f i c i a l fering  rectal  seg-  hemolymph o r h e m o l y m p h s  dif-  i n the c o n c n e t r a t i o n of only  preparation  #1).  difference  the i o n i n d i c a t e d  (electrical  130b  La  Figure 23.  The t r a n s - r e c t a l  (lumen r e l a t i v e  electrical potential  t o hemolymph) o b s e r v e d i n  segment b a t h e d i n  normal a r t i f i c i a l  hemolymphs d i f f e r i n g i n  difference  the p o s t e r i o r  hemolymph o r  the c o n c e n t r a t i o n of  rectal  artificial  the i o n  indicated. -3  CN  i n d i c a t e s normal a r t i f i c i a l  bean a d d e d .  hemolymph t o w h i c h 10  W KCN h a s  10  20  30  J_ _50  40 Time  (min)  60  70  80  90  100  132  posterior rectum of a variety of different a r t i f i c i a l hemolymphs in which the concentration of only an i n d i v i d u a l ion and i t s replacement are varied.  This procedure was repeated with twenty different  preparations.  rectal  The posterior r e c t a l segments showed variable differences  in t r a n s - r e c t a l e l e c t r i c a l potential after dissection in normal hemolymph (Table 10).  artificial  Therefore, the effects of a r t i f i c i a l hemolymph  changes are expressed as mean changes in e l e c t r i c a l potential differences - S . E . relative  to the o r i g i n a l potential difference observed i n recta  bathed i n normal hemolymph ( F i g s . 24, 25, and 26), The t r a n s - r e c t a l e l e c t r i c a l potential difference in the post e r i o r r e c t a l segment became more positive (lumen r e l a t i v e to hemolymph) with increasing potassium concentration ( F i g s . 23 and 24).  The poten-  t i a l increase was porportional to the potassium concentration increase and was completely reversible upon return to normal hemolymph. The effects of varying a r t i f i c i a l hemolymph concentrations of sodium on a t r a n s - r e c t a l potential are shown in F i g . 25.  This potential  difference increases (lumen more positive) with increasing levels of sodium in the hemolymph.  The potential differences observed in 5mW Na  +  and 200mM Na' are s i g n i f i c a n t l y different (P<0.01), with the other sodium concentrations observed producing intermediate differences.  The effects of a r t i f i c i a l  trans-rectal  potential  hemolymph containing 5mM Na  lowering the e l e c t r i c a l potential were completely and immediately  +  re-  versible upon return to normal hemolymph. In a r t i f i c i a l hemolymphs containing variable amounts of c h l o r i d e , the t r a n s - r e c t a l potential difference decreased (lumen less  in  5a  F i g u r e 24. of  The e f f e c t  the a r t i f i c i a l  difference  of  the p o s t e r i o r  recta bathed i n  point.  i n c r e a s i n g the potassium  hemolymph on t h e t r a n s - r e c t a l  a s t h e mean c h a n g e i n  n=10).  of  r e c t a l segment.  potential  electrical  to  hemolymph ("^T)  The n u m b e r s i n b r a c k e t s i n d i c a t e  potential  A l l values are expressed  - S.E. relative  normal a r t i f i c i a l  concentration  that observed (+  10.5 -  the sample s i z e f o r  in  4 . 6 mV, each  133 c  LEAF 134 OMITTED IN PAGE NUMBERING.  Figure 25.  The e f f e c t  artificial  hemolymph on t h e  trans-rectal  difference  of  r e c t a l segment.  the  of  posterior  v a r y i n g the sodium c o n c e n t r a t i o n  e x p r e s s e d a s t h e mean c h a n g e i n observed i n (+  r e c t a bathed i n  1 0 . 5 - 4 . 6 mV, n = 1 0 ) .  sample s i z e  for  each  potential  electrical All  values  are  hemolymph (  The n u m b e r s i n b r a c k e t s i n d i c a t e  point.  th  potential  ± S.E. relative  normal a r t i f i c i a l  of  to  that )  the  Sodium Concentration of A r t i f i c i a l Hemolymph  (mM)  Figure 26,  The e f f e c t of varying the chloride concentration of  a r t i f i c i a l hemolymph on the t r a n s - r e c t a l e l e c t r i c a l difference of the posterior r e c t a l segment.  potential  A l l values are ex-  pressed as the mean change in potential - S . E . r e l a t i v e to that observed i n recta bathed in normal a r t i f i c i a l (+ 10.5 - 4.6mV, n=10).  hemolymph (  The numbers in brackets indicate the  sample s i z e for each point.  )  137  positive) s i g n i f i c a n t l y with increasing hemolymph chloride concentration (P<0.01).  Nearly a two-fold increase  i n the mean of the trans-  r e c t a l potential was observed i n the posterior segments of recta transferred from normal hemolymph to a r t i f i c i a l hemolymph ing 1.3 mM CI  ( F i g . 26). This potential increase  contain-  was maintained  f o r 20 to 30 minutes, after which time a p a r t i a l or even t o t a l reduction i n potential occurred ( F i g . 27). Upon return to normal hemolymph the trans-rectal potential was more negative by 3 to 4 mV than previously observed i n normal hemolymph. In Chapter IV, I presented evidence that high levels of either sodium (200 mM, Na ) or chloride (150 mM, Cl~) i n a r t i f i c i a l +  hemolymph stimulated  the rate of secretion of these ions.  The rate  of f l u i d secretion and i n the case of sodium, the osmotic concentration of the r e c t a l f l u i d also increased normal a r t i f i c i a l  hemolymph.  as compared to values with  Therefore, i t i s of p a r t i c u l a r i n t e r e s t  to observe the e f f e c t of these a r t i f i c i a l hemolymphs on the transrectal p o t e n t i a l .  High levels of sodium (200 mM) i n the a r t i f i c i a l  hemolymph were found to increase  the trans-rectal potential i n a manner  which was not reversible within a one hour period ( F i g . 28). High levels of chloride (150 mM) i n the a r t i f i c i a l hemolymph, on the other hand did not change the trans-rectal potential (Fig.  appreciably  26.). Although, the effect of a r t i f i c i a l hemolymph high i n  chloride shown i n F i g . 28 i s not completely t y p i c a l i n that a slow increase  i n potential can be observed a f t e r the i n i t i a l dip, the  increase  i n potential upon return to normal hemolymph as compared  Figure 27.  The t r a n s - r e c t a l  (lumen r e l a t i v e  t o hemolymph)  segments bathed i n  electrical potential observed i n  normal a r t i f i c i a l  hemolymphs d i f f e r i n g i n  difference  two p o s t e r i o r  hemolymph o r  the c o n c e n t r a t i o n of  rectal  artificial  the i o n  indicated.  138b  Time (min)  >9a  F i g u r e 28.  The t r a n s - r e c t a l e l e c t r i c a l p o t e n t i a l  (lumen r e l a t i v e  to  hemolymph)  observed i n  segment b a t h e d i n n o r m a l a r t i f i c i a l hemolymphs d i f f e r i n g i n  difference  the p o s t e r i o r  hemolymph o r  the c o n c e n t r a t i o n of  rectal  artificial  the i o n  indicated.  E l e c t r i c a l Potential (mV)  q6£t  140  to  previous  levels in  the a p p l i c a t i o n slight  decline in  upon r e t u r n t o higher  of  than  v a l u e of  n o r m a l hemolymph i s  artificial trans-rectal  that previously  3 . 0 ± 1.0  when c o m p a r e d t o 4  AMP ( 1 0  2  of  rectal  an e f f e c t l a c k of  of  (-2.0 i  o u a b a i n (10  15 minutes  0^,  (+ 0 . 8  M) t o  the in, v i t r o this  0.6  the  - 0 . 5 mV, n = 4 ) . 7 M) t o  (10  electrical  potentials  hemolymph mV,  - 0.6  on t h e  condition  n=3)  Serotonin  trans-rectal  P.O.  The a d d i t i o n  normal  of  hemolymph  mV, n = 3 ) . rectal osmotic or  These  preparation concentration 5-HT.  Lack  c o u l d p o s s i b l y be a t t r i b u t e d t o the p r e s e n t e x p e r i m e n t s .  the s i p h o n and the  were u n a f f e c t e d  of a  In  t r a c h e a a were i n t a c t ,  yet  by t h e s e p h a r m a c o l o g i c a l a g e n t s .  f r o m t h e s e two s e p a r a t e e x p e r i m e n t s  suggest that  l a r v a e c a n n o t be s t i m u l a t e d  b a s i s by t h e s e hormone a n a l o g s a p p l i e d on t h e  the  but  leval  *• 0 . 6  c y c l i c AMP + t h e o p h y l l i n e  s e c r e t i o n i n s a l i n e - w a t e r mosquito short-term  effect  c a n be e x c l u d e d i n  p r e p a r a t i o n #1  The r e s u l t s  a  the normal  affect  change i n P . D . ( 1 . 0  t o show a s t i m u l a t o r y  but  a  mV, n = 4 ) ,  rose to  c h a n g e i n P . D . (+  M) a n d t h e o p h y l l i n e  s e c r e t i o n by 3 - 5 * in  1,6  led to  t h a t o b s e r v e d i n n o r m a l hemolymph a l o n e .  electrical  s i d e of  chloride  c o n f i r m o b s e r v a t i o n s u s i n g t h e _in v i t r o  which f a i l e d  Generally,  mV ( n = 4 ) .  i n d u c e d no s i g n i f i c a n t findings  shown.  n o r m a l hemolymph by a mean  a l s o d i d not s i g n i f i c a n t l y  o v e r a p e r i o d of cyclic  potential  observed i n  produce a s i g n i f i c a n t  ( 1 0 ~ M 5-HT)  hemolymph h i g h i n  n o r m a l hemolymph, the p o t e n t i a l  The a d d i t i o n d i d not  clearly  rectal on a  hemocoel  rectum.  The p o t e n t i a l  difference  observed a c r o s s the p o s t e r i o r  rectal  141  segment was substantially and i r r e v e r s i b l y reduced upon addition of —3 KCN (10" W).  This rapid decline in potential indicates the depend-  ence of the potential producing mechanisms on energy supplied by the c e l l s . DISCUSSION  The e l e c t r i c a l potentials measured in this study can be compared to the i o n i c gradients previously measured under i d e n t i c a l conditions (Bradley and P h i l l i p s , 1975; Chapterll)  to determine  which ions are a c t i v e l y transported across the r e c t a l wall of /U taeniorhynchus l i v i n g in 100% sea water.  The e l e c t r i c a l  potential  required to support an ionic concentration difference across a diffusion b a r r i e r , in this case the r e c t a l c e l l , i s described by the Nernst equation!  E* ~  1n (c^A^)  where E equals the e l e c t r i c a l potential difference observed, R the gas constant, T the temperature in K e l v i n , E the charge of the i o n , F the Faraday constant and c^A^  the concentration ratio which can be  maintained by the e l e c t r i c a l p o t e n t i a l .  At the experimental  temperature  used ( 2 5 ° C ) , and f o r monovalent i o n s , the equation can be s i m p l i f i e d !  E= 59 log  (c^/c ) 2  The ratio of the r e c t a l f l u i d to hemolymph concentration observed for each ion as well as the potential to maintain that concentration ratio (lumen relative  to hemolymph) difference required i n the  ligated recta i n vivo i s as follows! Na 1.9 (-16mV), K +  +  9.9 (-57.7mV),  142  Mg  5.0 (-58mV), CI 4.3 (+37mV).  The concentration r a t i o s represent  average values 2 h after l i g a t i o n and must therefore  be compared to  average values f o r p o t e n t i a l differences observed across the r e c t a l w a l l at the same time.  At the s t a r t of the experimental period when l i t t l e  f l u i d has yet accumulated, the e l e c t r i c a l p o t e n t i a l difference i s -10.4 and +10.5 mV i n the a n t e r i o r and p o s t e r i o r r e c t a l segments r e s p e c t i v e l y . After 2h, the p o t e n t i a l differences for these two segments of recta had f a l l e n to -1.8 and +6.4 mV, r e s p e c t i v e l y .  Therefore, under these e x p e r i +  +  ++  mental conditions i t i s necessary to postulate that Na , K , Mg CI  and  are a l l a c t i v e l y transported across the r e c t a l w a l l from the hemolymph  to the r e c t a l lumen.  In p a r t i c u l a r , i f one considers the evidence that  hyperosmotic s e c r e t i o n occurs i n the p o s t e r i o r r e c t a l segment where the lumen i s p o s i t i v e , then c l e a r l y a l l three cations are transported only against concentration but also e l e c t r o p o t o n t i a l  not  differences.  I examined the p o t e n t i a l across the p o s t e r i o r r e c t a l w a l l i n more d e t a i l because the morphological and p h y s i o l o g i c a l evidence i n d i c a t e s that hyperosmotic s e c r e t i o n occurs i n t h i s region.  I have made three  assumptions i n constructing a model for ion transport across p o s t e r i o r r e c t a l epithelium ( F i g . 29)i  the  1) As i n d i c a t e d by e l e c t r o n  micrographs, only two important b a r r i e r s to ion d i f f u s i o n occur i n the p o s t e r i o r r e c t a l w a l l , namely the basal (hemolymph side) and a p i c a l (lumen side) plasma membranes.  2) The f l u i d secreted across the p o s t e r i o r  r e c t a l w a l l i s s i m i l a r to or more concentrated than that c o l l e c t e d from the lumen of the whole, l i g a t e d rectum.  3) The i n t r a c e l l u l a r concentra-  tions of ions are t y p i c a l of those i n most other c e l l s i . e . high K , +  t  Figure 29.  A model of  the  rectal  posterior  fluid.  segment d u r i n g  The u p p e r r e g i o n i s  ultrastructure sites  the processes o c c u r i n g i n  of  ion  of  the c e l l .  transport  present active  the s e c r e t i o n of  a diagrammatic The c e n t r a l  while  block d e p i c t s the  The b o t t o m d i a g r a m s h o w s t h e a v e r a g e e l e c t r i c a l m e a s u r e d a c r o s s e a c h membrane a n d a c r o s s t h e epithelium.  of  the  proposed  S o l i d arrows  dashed ones d e p i c t  of  hyperosmotic  representation  during f l u i d secretion.  transport  the c e l l s  re-  p a s s i v e movements.  potential  entire  differences  posterior  rectal  143b  HEMOLYMPH  LUMEN  ^•K , +  Na +  •  4-  144  lout Na  and CI .  Like most c e l l s , those of the posterior r e c t a l segment  have an i n t e r i o r which i s negative to the hemolymph.  This i s  largely  attributed to a potassium d i f f u s i o n p o t e n t i a l , with the potassium concentration gradient maintained by an active Na -K (reviewed by Schwarz, Lindenmayer and A l l e n , 1972). when an a r t i f i c i a l  exchange mechanism In support of  this,  hemolymph abnormally high i n potassium was placed  on the posterior rectum, the i n t e r i o r of the c e l l became less negative. A reduction of the potential across the basal membrane should increase the t r a n s - r e c t a l p o t e n t i a l , since thB e l e c t r i c a l potentials across the basal and a p i c a l membranes oppose each other. observed when a r t i f i c i a l  This was experimentally  hemolymphs high i n potassium were placed on  the posterior rectum ( F i g s . 23 and 24).  On the basis of these observa-  tions an outward d i f f u s i o n of potassium to the hemocoel which i s opposed by active transport involving a t y p i c a l N a - K +  +  exchange pump i s depicted  i n F i g . 29. The potential difference across the basal membrane was found to decrease upon reduction of the chloride concentration of the artificial  hemolymph.  A decrease i n the rate of chloride d i f f u s i o n into  the c e l l , which might be expected on reducing hemolymph C l " l e v e l s , should have the opposite effect on the potential  to that observed.  We therefore  suggest that an electrogenic chloride "pump" e x i s t s on the basal membrane which transports chloride into the c e l l during hyperosmotic f l u i d port.  trans-  Active transport of chloride i s necessary i n this location not  only to explain the offset of low chloride hemolymph on the  electrical  potential difference across the basal membrane, but also to ensure the entry of chloride into the c e l l against the steep e l e c t r i c a l  potential  145  gradient across the basal membrane (Table 12). +  potential would f a c i l i t a t e the movement of Na  This same e l e c t r i c a l ++  and Mg  into the c e l l .  These ions are generally present i n c e l l s at lower a c t i v i t i e s than i n the blood (Palaty and Friedman). 1974).  These cations would therefore  enter the c e l l by passing down both an e l e c t r i c a l and chemical gradient. Schmidt-Nielsen, B. (1975) has reviewed evidence that i n some e p i t h e l i a secreting hyperosmotic f l u i d , the barrier to the d i f f u s i o n of water i s apparently at the basal membrane, since the i n t r a c e l l u l a r osmotic concentrations are very high (1000-2000 mOsm). the c e l l s are isosmotic to the blood*  In other such e p i t h e l i a ,  We have no such estimates f o r  the l a r v a l r e c t a l t i s s u e , and knowledge of the location of the b a r r i e r to water d i f f u s i o n must await further  experimentation.  The e l e c t r i c a l potential difference across the a p i c a l membrane of the posterior r e c t a l c e l l s (34-61 mV) i s approximately equal to that across the basal (37-50 mV), the c e l l being negative to i t s exterior each membrane.  It  at  i s at the a p i c a l membrane that the passive transport of  the cations i s opposed.  It  i s therefore necessary to propose one or more  cation pumps at this location to account for the ion concentrations observed in the hyperosmotic s e c r e t i o n .  This membrane also has the great-  est surface area i n the c e l l and i s associated with most of the mitochondria.  Fig 29 shows separate cation pumps f o r the monovalent and  divalent cations.  In some vertebrate tissues magnesium i s thought to be  transported by a separate mechanism from that f o r other c a t i o n s , with this  146  transport being dependent on a sodium gradient d r i v i n g N a - M g +  (Baker and Crawford, 1973; Palaty, 1974).  ++  exchange  Whether this applies to the  l a r v a l rectum as well i s presently unknown. When the sodium concentration of the a r t i f i c i a l  hemolymph bath-  ing the rectum i s lowered from 150 mM to 5 mM, the t r a n s - r e c t a l i s reduced s l i g h t l y but s i g n i f i c a n t l y . (5 mM Na ) +  potential  In the hemolymph containing  the r e c t a l secretion was found to have an osmotic concentration  of 634 * 31 mOsm (n=7), a sodium concentration of 128 * 25 mM (n=7) a potassium concentration of 280 ± 70 mM (n=5)  and  i n d i c a t i n g that potassium  can substitute f o r sodium in the formation of a hyperosmotic r e c t a l secretion (Chapter IV).  It  i s therefore possible that these two ions  compete f o r the same transport mechanism i n the rectum, as has been postulated f o r the Malpighian tubules of Rhodnius (Maddrell, 1971).  For  the sake of s i m p l i c i t y , potassium, and sodium are shown sharing the same monovalent transport mechanism ( F i g . 28),  Further studies may indicate  that these ions actually use separate transport mechanisms. The e l e c t r i c a l potential across the apical membrane i s s u f f i c i e n t to support a 10-fold gradient f o r chloride ions.  The normal  i n t r a c e l l u l a r chloride concentration f o r e p i t h e l i a secreting hyperosmotic f l u i d i s 80-120 mM (Schmidt-Nielsen, B . ; 1975), a concentration only half this value would be s u f f i c i e n t i n the r e c t a l c e l l to allow chloride to pass across the apical membrane and accumulate in the lumen by passive means.  The i n t r a c e l l u l a r chloride concentration could be  maintained at this l e v e l by the active chloride transport proposed across the basal membrane i n F i g . 29.  It  i s of interest that the  147  rectal secretion has always been found to be hypertonic to the hemolymph with regard to chloride (Chapters II,  III  and Iv)  demonstrating that the  movement of chloride from the hemolymph to the r e c t a l lumen i s incapable of being completely eliminated, even when CI environment at very low l e v e l s .  i s present in the  external  I have followed the convention of not  proposing an active transport mechanism where no thermodynamic  requirement  e x i s t s , and therefore propose that chloride and other anions cross the apical membrane by passive means ( F i g .  29).  The transfer of other anions by the posterior rectal segment must be considered for larvae l i v i n g in several natural waters. l i k e l i h o o d , bicarbonate largely substitutes for chloride i n the  In  all  rectal  secretion of animals reared in hyperosmotic media low in chloride (Chapter III)  resulting in hyperosmotic secretion with chloride concen-  trations as low as 40mm.  In waters high in NaHCO^, bicarbonate ions  apparently accompany the active transport of cations at the a p i c a l membrane when c e l l u l a r chloride concentrations are depressed.  Bicarbonate  may be transported by the chloride pump postulated for the a p i c a l membrane since both must enter the c e l l against an e l e c t r i c a l  gradient.  In (Na + Mg)S0^ ponds the concentration of chloride i s also low and the hemolymph chloride concentration i s correspondingly reduced (Chapter III).  This situation i s s i m i l a r to that in NaHCO^ waters with  the exception that bicarbonate ions may not enter the hemolymph in large quantities from the external medium unless taken up by the anal p a p i l l a e . The r e c t a l c e l l presumably responds in the same manner however, with bicarbonate accompanying the cations transported.  Bicarbonate ions in  148  the absence of uptake via anal papillae are presumed to arise in the c e l l through the action of carbonic anhydrase. rectal secretion in quantities  Sulphate ions occur in the  i s o - or hypo- tonic to the hemolymph.  This agrees with the model which shows no active secretion of sulphate, although some sulphate might be swept along by the movement of water. The model of transport processes in c e l l s of the posterior r e c t a l segment i s consistent with several previous observations regarding the stimulation of r e c t a l secretion. that while an a r t i f i c i a l  In Chapter IV I showed  hemolymph with normal ionic concentrations but  increased osmotic (sucrose) concentration does not increase the osmotic concentration of r e c t a l s e c r e t i o n , increased l e v e l s of sodium or chloride in the hemolymph do. t r a n s - r e c t a l potential  Raising hemolymph levels of sodium increases the ( F i g . 25).  This i s predicted by the model in  which increased sodium transport hyperpolarizes the apical membrane and thereby increases the t r a n s - r e c t a l p o t e n t i a l .  Raising hemolymph levels  of chloride does not appreciably change the t r a n s - r e c t a l  potential,  generally causing a s l i g h t reduction i n potential presumably due to hyperpolarization of the basal membrane by increased chloride  transport.  Perhaps the i n i t i a l depolarization brought about by increased chloride i s countered by the stimulatory  effect that high hemolymph levels of chloride  have on hyperosmotic f l u i d secretion ( F i g . 19).  Thus, the  trans-rectal  potential i s only s l i g h t l y affected in hemolymph high in chloride because cation transport i s stimulated ( F i g . 29).  This interpretation  i s sup-  ported by the observation that, upon return to normal hemolymph from hemolymph high in c h l o r i d e , the t r a n s - r e c t a l potential difference  in-  149  creases over that observed before in normal hemolymph, suggesting that cation transport has been increased. The t r a n s - e p i t h e l i a l  potential differences across the posterior  r e c t a l segment were unaffected by ouabain.  This i s not unusual f o r i n -  sect e p i t h e l i a and i s variously interpreted as i n d i c a t i n g either  the  lack of Na -K ATPase in the electrogenic process or a membrane impermeable +  +  to ouabain (reviewed by, Maddrell, 1971).  Owing to the apparent presence  of a K* d i f f u s i o n potential on the hemocoel side of the rectal t i s s u e , I prefer the l a t t e r explanation. The potential i n the posterior rectal portion i s not by the short-term application (20 min.) of 5-HT(10" M). 4  I hypothesize that the long-term a c t i v i t y  affected  In Chapter  of the rectum, i . e .  IV,  the f r e s h -  water versus saline-water mode of function, i s controlled hormonally or neurally, while the rate and osmotic concentration of the rectal secretion in the short-term i s modified by hemolymph ion concentrations ( i . e . t r i n s i c regulation; P h i l l i p s and Bradley, 1976).  in-  The e l e c t r i c a l and i n  v i t r o secretion experiments in this study support that hypothesis.  CHAPTER  VII  GENERAL SUMMARY  150  151  SUMMARY  This concluding chapter w i l l summarize the o v e r a l l process of osmoregulation i n saline-water mosquito larvae as now envisaged from previously published work and the results of this t h e s i s .  Saline-  water mosquito larvae drink the external medium at a rapid rate -1 (lOOnl'hr  —1 *mg  ) regardless of the s a l i n i t y of the external medium.  This rate of drinking i s probably proportional to the metabolic rate of the animal and seems to serve to supply the larva with both necessary water and dissolved organic nutrients.  Because the f l u i d intake by  drinking greatly exceeds the volume of water l o s t by osmosis across the body w a l l , i t determines the osmoregulatory load imposed on the animals and dictates that the composition of the urine must c l o s e l y resemble that of the external medium. Osmoregulation i n saline-water mosquito larvae adapted to hyposmotic waters i s very s i m i l a r to that previously described in s t r i c t l y fresh-water species of mosquito larvae with the exception that a much higher drinking rate i s observed.  As reviewed by P h i l l i p s and  Bradley (in p r e s s ) , the Malpighian tubules transport potassium and chloride into the tubule lumen and the concentration of these ions creates the osmotic gradient for the movement of water and subsequently other hemolymph constituents.  The resulting isosmotic secretion passes  from the Malpighian tubules down the ileum to the rectum.  In the anterior  rectum, ions and nutrients essential to the larva are resorbed from the luminal f l u i d to produce a hyposmotic f l u i d containing largely nitrogenous  152  and other waste products.  This process i s e s s e n t i a l l y i d e n t i c a l to that  found in other freshwater i n s e c t s .  The posterior rectum i s apparently  inactive in fresh water and the f l u i d which has been modified in the anterior rectum i s excreted via the anus.  The anal papillae of s a l i n e -  water mosquito larvae are active in fresh water and take up ions from the external medium ( P h i l l i p s and Meredith, 1969). In hyperosmotic media, additional transport of ions contributing to ion excretion i s i n i t i a t e d at several s i t e s i n the larvae.  In  the Malpighian tubules, the normal transport of potassium and chloride continues to be the mechanism of f l u i d production. high levels of M g  ++  In media containing  and SO", these two ions are a c t i v e l y secreted into  the tubule lumen, such that magnesium and sulphate account for a proportionally greater f r a c t i o n of the t o t a l solute (Maddrell and P h i l l i p s , 1975; P h i l l i p s and Maddrell, 1975).  Sulphate i o n s , in f a c t , do not  appear to be a c t i v e l y transported at any other excretory s i t e s .  It  this r e s t r i c t i o n which seems to l i m i t the a b i l i t y of saline-water  is larvae  to survive in waters rich i n sulphate, such as Ctenocladus pond in which Ae"des campestris larvae naturally occur.  Even f o r larvae reared in these  waters, hyperosmotic concentrations of sea water and NaHCO^-rich waters are less toxic than their own pond water of equal osmolality. In hyperosmotic media the anterior rectal segment continues to resorb KC1, nutrients and water, resulting in a  isosmotic f l u i d contain-  ing elevated levels of waste products and unresorbed i o n s , e . g . Mg SO".  It  and  i s at this point that sulphate ions secreted in the Malpighian  tubules are concentrated to levels above those in the external medium.  153  In hyperosmotic media, the posterior rectum i s a major s i t e , i f  not  the only one, of urine concentration by means of the secretion of a hyperosmotic f l u i d into the lumen.  The r e l a t i v e concentration of the  various ions transported ( N a , K , M g , Cl'+HCOj) +  external medium.  +  + +  resemble those i n the  In concentrated media, the transport of ions and the  volume of secretion both increase but not i n s t r i c t proportion, so that the osmotic concentration of the r e c t a l secretion rises as w e l l . Sodium and magnesium occur in the r e c t a l secretion in concentrations s i m i l a r to external l e v e l s .  Potassium concentration in the  rectal  secretion are always hypertonic to the medium and hemolymph.  In media  high in c h l o r i d e , this anion i s the major one secreted in the posterior r e c t a l segment.  In media low i n chloride ( e . g . HCO"  or SO" =-rich medi  chloride levels i n the secretion are depressed while cation concentrations are unchanged.  8icarbonate ions presumably accompany the cations  in this media into the r e c t a l lumen.  Larvae are capable of producing  a hyperosmotic secretion in these unbalanced media as can l a r v a l bathed in a r t i f i c i a l Mg  hemolymphs with very low levels of either  recta  Na , +  or C l " .  ++  The levels of potassium ( i n a l l media) or chloride ( i n  low-  chloride media) may be lower in the external medium than i n the  rectal  s e c r e t i o n , resulting i n a depletion of hemolymph levels of these ions through the action of the rectum. of K  +  and C l  This loss may be balanced by uptake  from the external medium through the anal p a p i l l a e .  In  sea water, where sodium and chloride levels are both high, the uptake of chloride i s turned off but potassium uptake in the papillae may  154  continue, being perhaps coupled to sodium extrusion as occurs i n the g i l l s of marine t e l e o s t s . Measurements of the e l e c t r i c a l potential differences across each of the plasma membranes in the c e l l s of the posterior r e c t a l segment, as well as across the entire epithelium, have demonstrated that +  +  ++  Ma , K , Mg lumen.  and CI  are a l l actively transported from hemolymph to  Chloride and potassium are probably a c t i v e l y transported across  the basal membrane into the c e l l .  Sodium and magnesium are presumed to  enter the c e l l p a s s i v e l y , moving down both t h e i r chemical and e l e c t r i c a l gradients.  The e l e c t r i c a l potential difference at the basal membrane  i s possibly due to both a potassium d i f f u s i o n potential and an e l e c t r o genic chloride pump. + K  It  i s proposed that at the a p i c a l membrane, Na ,  ++ and Mg  are electrogenically transported into the lumen against  their electrochemical gradients, while chloride ions follow passively across this membrane.  In media, low i n c h l o r i d e , the anion crossing  this membrane appears to be largely HCO^ although C l ~ always appears in the r e c t a l secretion i n concentrations i s o t o n i c to the hemolymph or higher. The bicarbonate ions accompanying the cations across the apical membrane may derive from the hemolymph or within the c e l l through the action of carbonic anhydrase. Several levels of control seem to e x i s t f o r these transport processes*  1) Larvae in hyposmotic media do not form urine by secretion  in the rectum, while those in hyperosmotic media do, therefore  external  conditions must d i r e c t l y or i n d i r e c t l y affect a hormonal or neural control of rectal function which i n i t i a t e s or halts r e c t a l s e c r e t i o n .  2) Larvae  adapted over four days to d i f f e r e n t media show d i f f e r e n t relative ion  155  concentrations i n the r e c t a l secretion even when bathed in the same a r t i f i c i a l hemolymph, i n d i c a t i n g that the transport mechanisms have either changed i n capacity or a f f i n i t y during the adaption process. 3)  The ion pumps in the Malpighian tubules and rectum show immediate  increased transport rates ( i n t r i n s i c control) with increasing hemolymph ion l e v e l s , the Malpighian tubules showing Michaelis-Menten type responses and the rectum a l l o s t e r i c - t y p e k i n e t i c s . Separate control mechanisms therefore porbably exist to i n i t i a t e and coordinate hypo-vvsrsus hyper-regulation and to modify the t o t a l capacity of the transport mechanisms f o r various ions.  These  adaptations are separate from the immediate responses of the transport mechanisms to changing hemolymph ion l e v e l s .  This thesis has examined  the mechanisms of rectal secretion and described the importance of this process in the osmoregulation of saline-water mosquito larvae.  A very  i n t e r e s t i n g f i e l d of enquiry remains that of determining how these processes are controlled to allow the s u r v i v a l of the larvae i n the extremely variable environments which they i n h a b i t .  156  REFERENCES  Baker, P.F. & Crawford, A . C . (1973) Sodium-dependent transport of magnesium ions in giant axons of Lolioo f o r b e s i . J_. Physiol. Lond. 21>6: 33P-39P. Beadle, L . C . (1939) Regulation of the hemolymph i n the s a l i n e water mosquito larva Aedes detritus Edw. J.. exp. B i o l . 16: 346-362. Beadle, L . C . (1943) Osmotic regulation and the fauna of inland waters. B i o l . Rev. 18: 172-183. Berridge, M.J. (1966) Metabolic pathways of i s o l a t e d Malpighian tubules of the blowfly functioning i n an a r t i f i c i a l medium. J . Insect P h y s i o l . 12* 1523-1538. Berridge, M.J. & Oschman, J . L . (1972) Transport E p j t h e l i a Academic Press N.Y. and Lond. B l i n n , D.W. (1969) Autoecology of Ctenocladus (Chlorophyceae) i n saline environments. Ph.D. t h e s i s , University of B r i t i s h Columbia. Bradley, T . J . & Perkins, O . L . (1975) The role of calcium i n the osmoregulation of mosquito larvae, Culex pipiens. Comp. Biochem. P h y s i o l . 52A* 403-407. Bradley, T . J . & P h i l l i p s , J . E . (1975) The secretion of hyperosmotic f l u i d by the rectum of a saline-water mosquito l a r v a , Aedes taeniorhynchus. J . exp. B i o l . 63* 331-342. Cameron, J . (1971) Rapid method f o r determination of t o t a l carbon dioxide i n small blood samples. J_. applied P h v s i p l . 31(4)* 632-634. Copeland, E . (1964) A mitochondrial pump i n the c e l l s of the anal papillae of mosquito larvaa. J_. C e l l . B i o l . 23* 253-263. Harvey, W.R. & Zerahn, K. (1972) Active transport of potassium and other a l k a l i metals by the i s o l a t e d midgut of the s i l k worm, i n Current Topics i n Membranes and Transpprt. 3* 368-410. Hemmingsen, A.M. (1960) Metabolism i n r e l a t i o n to body s i z e . Rep. Steno. Mem. Hpsp. Nordisk Insulin Lab. 9* 1-110. I r v i n e , H.B. (1969) Sodium and potassium secretion by i s o l a t e d Malpighian tubules. Am. J . P h y s i o l . 217: 1520-1527. Kaufman, W.R. & P h i l l i p s , J . E . (1973) Ion and water balance i n the Ixodid t i c k , Dermacentor andersoni. J . exp. B i o l . 58: 549-564. Kiceniuk, J.W. & P h i l l i p s , J . E . (1974 Magnesium regulation i n mosquito larvae, Aedes campestris. l i v i n g i n waters of high MgS0 content. J . exp. B i o l . 61: 749-760. Leader, J . P . (1972) Osmoregulation i n the larva of the marine caddis f l y , Philanensis plebius (Walk) ( T r i c h o p t e r s ) . J . exp. B i o l . 57: 821-838. Maddrell, S.H.P. (1971) The mechanisms pf insect excretcry systems. Adv. Insect P h v s i e l . 8: 199-331. 4  157  Maddrell, S.H.P. (1976a) Transport across insect e p i t h e l i a . In Transport across B i o l o g i c a l Membranes (eds. Ussing, H . ; Tosteson, D.C. & Giebisch, G.) Springer Verlag 0in p r e s s ) . Maddrell, S.H.P. (1976b) ^Malpighian tubules. In Transpprt of Ions and Water in Animal Tissues. Gupta, B . L . ; Moreton, R . B . ; •schman, J . L . & Wall, B . J . (eds.) Academic press ( i n press). Maddrell, S . H . P . ; Gardiner, B . O . C . ; P i l c h e r , D.E.M. & Reynolds, S . E . (1974) Active transport by insect Malpighian tubules of acidic dyes and of acylamides. J . exo. B i o l . 61: 357-377. Maddrell, S . H . P . & P h i l l i p s , J . E . (1975) Active transport of sulphate ions by the Malpighian tubules of the larvae of the mosquito Aedes campestris. J_. exp. B i o l . 62: 367-378. Maddrell, S.H.P. & P h i l l i p s , J . E . (1975b) Secretion of hyposmotic f l u i d by the lower Malpighian tubules of Rhodnius p r o l i x u s . ( H e t . , Reduviidae). J,. exp. B i o l . 62(3): 671-683. Maetz, J . (1971) Fish g i l l s : mechanisms of s a l t transfer i n fresh water and sea water. P h i l . Trans. Roy. Soc. Lond. B. 262: 209-249. Marks, E . P . & Holman, G.M. (1974) Release from brain and acquisition by corpus cardiacum of a neurohormone, in, v i t r o . J_. Insect P h y s i o l . 20 (10): 2087. Marshall, A . T . & Wright, A. (1974) U l t r a s t r u c t u r a l changes associated with osmoregulation in the hindgut c e l l s of a saltwater i n s e c t , Ephydrella s p . (Ephydridae: Diptera). Tissue and C e l l 6(2): 301-318. ^ Meredith, J . & P h i l l i p s , J . E . (1973a) Rectal ultrastrueture i n s a l t and freshwater mosquito larvae in relation to physiological s t a t e . L» Z e l l f o r s c h . 138: 1-22. Meredith, J . & P h i l l i p s , J . E . (1973b) Ultrastructure of the anal papillae of salt-water mosquito l a r v a , Aedes campestris. J_. Insect P h y s i o l . 19: 1157-1172. Nayar, J . K . (1966) A Method of rearing salt-marsh mosquito larvae i n a defined s t e r i l e medium. Ann. Ent. Soc. Am. 59(6): 1283-1285. Nayar, J . K . (1967) The pupation rhythum i n Aedes taeniorhynchus (JDiptera, C u l i c i d a e ) . II Ann. Ent. Soc. Amer. 60: 946-971. Nayar, J . K . & Sauerman, D.M. (1975) Osmoregulation i n larvae of the salt-marsh mosquito Aedes taeniorhynchus Wiedemann, ( i n p r e s s ) . Nicholson, S.W. & Leader, J . P . ( 1 9 7 4 ) T h e permeability to water of the c u t i c l e of the larva of Opifex fucus (Hutton) (Diptera, C u l i c i d a e ) . J . exo. B i o l . 60: 593-604. Noble-Nesbitt, J . (1973) Rectal uptake of water i n i n s e c t s . In Comparative Physiology B o l i s , Schmidt-Nielsen and Maddrell (eds.) North-Holland Pub. Co. Noirot, C.H. & Noirot-Thimotee, C. (1967) L'epithelium absorbant de la pause d'un termite superieur. Ultrastructures et rapport avec la symbiose bacterienne. Ann. Soc. ent. F r . 3: 577-592. Palaty, V. (1974) regulation of the c e l l magnesium i n vascular smooth muscle. J_. P h y s i o l . 242: 555-569. Palaty, V. & Friedman, S.M. (1975) Estimation of the state of ions i n smooth muscle; i n Methods in Pharmacoloov, V o l . 3. Eds. D a n i e l s , E . E . & Paton, D.M. Plenum Pub. C o . ; N.Y.  158  P h i l l i p s . J . E . (1964a) Rectal absorption i n the desert locust Schistocerca oreqaria F o r s k a l . I. Water. J . exp. B i o l . 41: 15-38. P h i l l i p s , J . E . (1964b) Rectal absorption i n the desert locust Schistocerca areoaria F o r s k a l . II Sodium, potassium and c h l o r i d e . J . exp. B i o l . 41: 39-67. P h i l l i p s , J . E . (1964c) Rectal absorption i n the desert locust Schistocerca qreqaria. F o r s k a l . III. The nature of the excretory process. J» exp. B i o l . 41: 67-80. P h i l l i p s , J . E . (1970) Apparent transport of water by insect excretory systems. Am. Z o o l . 10: 413-436. P h i l l i p s , J . E . & Bradley T . J . ( i n press) Osmotic and i o n i c regulation i n saline-water mosquito larvae. In Transpprt of Ions and Water in Animal Tissues. Gupta, B . L . ; Moreton R . B . : Oschman, J . L . & Wall, B . J . (eds.) Academic Press. P h i l l i p s , J . E . & D o c k r i l l , A.A. (1968) Molecular seiving of hydrophilic molecules by the r e c t a l intima of the desert locust (Schistocerca oreqaria) J . exp. B i o l . 48: 521-532. P h i l l i p s , J . E . & Maddrell, S.H.P. (1975) Active transport of magnesium by the Malpighian tubules of the larvae of the mosquito Aedes campestris. J_. exp. B i o l . 61: 761-771. P h i l l i p s , J . E . & Meredith, J . (1969a) Osmotic and i o n i c regulation i n a salt-water mosquito larva Aedes campestris. Amer. Zool. 9: 588. P h i l l i p s , J . E . & Meredith J . (1969b) Active sodium and chloride transport by anal papillae of a salt-water mosquito larva (Aedes campestris). Nature. 222: 168-169. Prpsser, C L . (1973) Comparative Animal Physiology. W.B. Saunders Co. P h i l a . , London & Toronto. Provost, M.W. (1967) Managing impounded s a l t marsh f o r mosquito control and estuarine resource conservation. LSU Marsh & Estuary Symposium pp 163-171. Prusch, R.D. (1971) The s i t e of ammonia excretion in the blowfly l a r v a , Sarcophaoa b u l l a t a . Comp. Biochem. P h y s i o l . 39A: 761-767. Prusch, R.D. (1972) Secretion of NH C l by the hindgut of Sarcophaoa b u l l a t a . Cpmp. Biochem. P h y s i o l . 41 A: 215-223. Prusch, R.D. (1973) Secretion of hyperosmotic excreta by the blowfly l a r v a , Sarcophaoa b u l l a t a . Comp. Biochem. P h y s i o l . 46A: 691-698. Prusch, R.D. (1974) Active ion transport i n the l a r v a l hindgut of Sarcophaoa bullata (Diptera: Sarcophagidae). J_. exp. B i o l . 61: 95-109. Ramsay, J . A . (1949) A new method of freezing point determination f e r small q u a n t i t i e s . J_. exp. B i o l . 26: 57-64. Ramsay, J . A . (1950) Osmotic regulation in mosquito larvae. J . exp. B i o l . 27: 145-157.  159  Ramsay, J . A . (1951) Osmotic regulation i n mosquito larvae; the role of the Malpighian tubules. J_. exp. B i o l . 28s 62-73. Ramsay, J . A . (1955a) The excretpry system pf the s t i c k insect* Dixippus morosus (Orthpptera, Phasmidae). J . exp. B i o l . 32: 183-199. Ramsay, J . A . (1955b) The excretion of sodium, potassium and water by the Malpighian tubules of the s t i c k i n s e c t , Dixippus morosus (Orthoptera, Phasmidae). J . exp. B i o l . 32: 200-216. Ramsay, J . A . ( I 9 6 l j Excretion of i n u l i n by Malpighian tubules. Nature 191: 1115. Ramsay, J.A.jji- Brown, R . H . J . & Croghan, P.C. (1955) Electrometric t i t r a tion of chloride in small volumes. J_. exp. B i o l . 32: 822-829. Schmidt-Nielsen, B. (1975) Comparative physiology of c e l l u l a r ion and volume regulation. J_. exp. Z o o l . 194: 207-220. Schmidt-Nielsen, K. (1975) Scaling in biology. The consequences of s i z e . J . exp. Z o o l . 194: 287-307. Schwartz, A . ; Lindenmayer, G.E. & A l l e n , J . C . (1972) The Na*k ATPase membrane transport system: Importance i n c e l l u l a r function. In Current Topics i n Membranes and Transport! Bronner and K l e i n z e l l e r (eds.) Scudder, G.G.E. (1969b) The tauna of saline lakes on the Fraser Plateau i n B r i t i s h Columbia. Verh. Internat. Verein. Limnol. 17: 430-439. Scudder, G.G.E. (1969a) The d i s t r i b u t i o n of two species of Cenocorixa i n inland saline lakes of B r i t i s h Columbia. J,. ent. Soc. B. C,. 66: 32-41. Scudder, G . G . E . ; J a r i a l , M.S. and Choy, S.K. (1972) Osmotic and i o n i c balance in two species of Cenocorixa (Hemiptera). J . Insect P h y s i o l . 18: 883-895. ~* Shaw, J . & Stobbart, R.H. (1963))Osmotic and i o n i c regulation in i n s e c t s . In Advances i n Insect Physiology. Beament, Treherne and Wigglesworth (eds.) Academic Press, N.Y. and Lond. Sohal, R.S. & Copeland, E. (1966) U l t r a s t r u c t u r a l variations i n anal papillae of Aedes aeovoti (L.) at d i f f e r e n t environmental s a l i n i t i e s . J . Insect P h y s i o l . 12: 429-439. Stobbart, R.H. (1965) The effect of some anions and cations upon the fluxes and net uptake of sodium in the larvae of Aedes aeovptl (L.). J . exp. B i o l . 42: 29-43. Stpbbart, R.H. & Shaw, J . (1964) Salt and water balance: Excretion. In They Physiology of Insecta. 3s 190-258. M. Rochstein (ed.) Academic Press, N.Y. S u t c l i f f e , D.W. (i960) Osmotic regulation i n the larvae of some euryhaline Diptera. Nature 187: 331-332. S u t c l i f f e , D.W. (1961a) Studies on s a l * ; and water balance i n caddis larvae (Trichoptera): I. Osmotic and i o n i c regulation of body f l u i d s i n Limnephilus a f f i n i s C u r t i s . J.. exp. B i o l . 38 : 501-519. S u t c l i f f e , D.W. (1961b) Studies on s a l t and water balance in caddis larvae (Trichoptera). II. Osmotic and i o n i c regulation of body f l u i d s i n Limnephilus stioma Curtis and Anabolis nervosa Leach. ! • sxp» B i o l . 38: 521-530.  160  Treherne, J . E . J Buchan, P.B. & Bennett, R.R. (1975) Sodium a c t i v i t y of insect blood: Physiological significance and relevance to the design of physiological s a l i n e . J,. exp. B i o l . 62: 721-732. Wall, B . J . & Oschman, J . L . (1975) Structure and function of the rectum in i n s e c t s . In Forthschritte i n Zooloaie. Band 23, Heft 2 / 3 / Custav Fischer Verlag. Stuttgart. Wessing, A. (1967) Funktionsmorphologie von exkretionsorganen bei insekten. Verh. Dtsh. Z o o l . Ges. Heidelberg, 633-681. Wigglesworth, V . B . (1933) The function of the anal g i l l s of the mosquito larva.  J_. exp. B i o l . 10: 16-26.  161  APPENDIX I  )\  A quantity of Aedes taeniorhynchus eggs were obtained from Dr. J . K . Nayar at the Entomological Research Center i n Vero Beach, F l o r i d a and used to e s t a b l i s h a colony. While the basic rearing techniques used followed Nayar (196?) some major revisions were made. The i n structions below describe the rearing procedures used f o r a l l A,, 'm taeniorhynchus. 1.  Remove eggs from storage (10°C) one to two days before hatching and place at room temperature.  2.  On day of hatching wash eggs from f i l t e r paper into a crucible with d i s t i l l e d water. Concentrate eggs i n center of bowl by s w i r l i n g . Sprinkle Fleischmann's Active Dry Yeast on surface. Leave 29-30 minutes.  3.  Remove larvae with eyedropper and place in s e i v e . Wash with d i s t i l l e d water to remove yeast. Rinse larvae into crucible with d i s t i l l e d water.  4.  Using eyedropper, place 200 larvae i n p l a s t i c pan containing 500 mis of growing medium. Add 320 mg dried brewers yeast. Cover pans loosely.  5.  Feed 49 mg dried l i v e r on second day. Feed 160 mg dried brewers yeast on successive days. T i l t pans repeatedly after feeding to disperse fjood.  6.  If used i n experiments larvae were poured into seive and rinsed with d i s t i l l e d water on f i f t h day, and placed i n clean medium. Fourth i n s t a r larvae were used f o r experiments on the s i x t h and seventh days.  7.  If adults are required, pupae are removed and placed in bowls lined with moist paper towels. No standing water must be present. Place bowls i n cages 1 f t by 6 in by 6 i n with screen top. Allow 500 adults per cage.  8.  As adults emerge, offer them 10$ sucrose i n d i s t i l l e d water on f i l t e r paper tents immersed i n p e t r i dishes. Change sugar water and f i l t e r paper three times a week.  9.  Larvae and adults are kept at 27°C with 12 hours f u l l l i g h t , 1 hr subdued l i g h t , 10.5 hrs. darkness and 0.5 hrs subdued l i g h t .  10.  Feed adults with mouse f i v e days a f t e r emergence by restraining mouse on screen top of cage. Feed f o r one hour i n t e r v a l three times a week.  162  11.  C o l l e c t eggs four days a f t e r blood meal on f i l t e r paper tent in p e t r i dish f i l l e d with 25% sea water.  12.  Wash eggs from f i l t e r paper into fine s i e v e . Rinse well with d i s t i l l e d water. Wash into bowl with d i s t i l l e d water. C o l l e c t eggs with eyedropper.  13.  Place eggs i n water drops onto f i l t e r paper l y i n g on the bottom of p e t r i dishes. Place eggs i n scattered pattern to avoid hatching at warm temperatures due to self-induced reduction i n oxygen tension. Keep f i l t e r paper moist during storage.  14.  Cover eggs containers and incubate 5 days in adult or l a r v a l environmental chamber.  15.  After incubation period, store egg containers at 10°C u n t i l needed.  163  APPENDIX  II  Whole larvae with blocked anuses Larvae were removed from the rearing pan with an eyedropper and placed on a dry, 7 cm diameter piece of f i l t e r paper (Whatman No* 1). The abdomen of the larvae was held l i g h t l y between needle-tipped f o r ceps to keep the larvae stationary and a small drop of Eastman 910 tissue adhesive (Eastman Kodak) was placed on the anus and anal papillae with a minuten p i n . The excess adhesive was allowed to flow off the anus into the f i l t e r paper, but care was taken not to l e t the glue contact either the siphon or head. If this occured, care had to be taken to separate these parts subsequently in the water, but this procedure often led to irreparable damage to the larvae. To avoid d i f f i c u l t i e s , therefore, the larvae were quickly transferred using a l i g h t grip on the abdomen„with the forceps, to a s t a i n l e s s s t e e l planchei (Nuclear Chicago) f i l l e d about one-half f u l l with one ml. of the rearing pan medium. This contact with water caused the adhesive to s e t . If contact between the anUs and the siphon or head had been avoided, the larvae were then able to swim f r e e l y and drink and r e spire normally. The planchets were placed on a metal d i s c , the i n t e r i o r of which was bathed by water from a water bath maintained at 27°C. The p l a n chets were covered with microscope s l i d e covers with a small aperture left for ventilation. The whole disc was placed i n a covered p l a s t i c container, the bottom of which was f i l l e d with water to increase humidity. Under these circumstances sea water changed concentration by a minimum of 30 mOsm during a two hour period. An experimental period of two hours was used i n i t i a l l y (Bradley & P h i l l i p s , 1975) but this was reduced i n l a t e r experiments to 1.5 hrs to reduce the amount of swelling the rectum was forced to undergo. After this time the larvae were removed from the planchet with an eyedropper and placed again on f i l t e r paper. Using fine s i l k thread, a ligature was placed around the larva between the s i x t h and seventh abdominal segments. This assured that f l u i d then i n the rectum remained there and was available f o r sampling after the following manipulations. The c u t i c l e of the larva posterior to the ligature was ripped using needle-tipped forceps, with care being taken not to pinch too deeply and r i p the swollen rectum. Under t h i s c u t i c l e the rectum was c l e a r l y v i s i b l e and could be punctured with a micropipette f o r f l u i d sampling. Glass micropipettes were made by p u l l i n g microelectrodes on a v e r t i c a l electrode p u l l e r (David Kopf Instruments) and breaking off the t i p to y i e l d a sharp yet small-bored t i p . This pipette was s i l i c o n treated inside and out by being dipped to a depth of 2 cm in Repelcote  1614  s i l i c o n coating l i q u i d and sucking the l i q u i d into the bore and blowing i t out again. The Repelcote was allowed to a i r dry inside and out, whereupon the pipette was f i l l e d with paraffin o i l by sucking. The o i l f i l l e d pipette was then used to puncture the swollen rectum and extract a f l u i d sample. The best results were obtained when the larvae were held by the anal segment and the pipette was forced i n through the posterior end of the posterior rectum. The anterior r e c t a l c e l l s are more e a s i l y torn than the posterior and do not seal around the pipette as well allowing more f l u i d to escape unsampled. The sample of r e c t a l f l u i d was quickly transferred i n the pipette and blown out into a p e t r i dish l i n e d with paraffin wax and f i l l e d with paraffin o i l . The volume of this drop could be measured using an eyepiece micrometer (See Appendix III). The drops could be coalesced or s p l i t under o i l using a very fine glass probe. When the drops were ready f o r analysis they were picked up with a pipette s i m i l a r to the one used in puncturing the rectum (for osmotic cone analysis) or with a drawn out Pasteur pipette (for ion cone a n a l y s i s ) . Between uses the p e t r i dishes l i n e d with paraff i n wax were emptied of paraffin o i l , rinsed with water and stored to dry on a s l a n t . Liaatuyed larvae In order to investigate the action of the rectum when separated from Malpighian tubule f l u i d input and i n a variety of a r t i f i c i a l hemolymphs, a preparation was designed which involved the l i g a t i o n s , both posteriorly and a n t e r i o r l y , of the rectum. Larvae were removed from the rearing pan with an eyedropper and placed on f i l t e r paper as described above. One ligature was placed between the s i x t h and seventh abdominal segment, i s o l a t i n g the rectum from the midgut. The second ligature was placed around the anal segment, preventing the rectum from emptying out the anus. The portion of the larva anterior to the f i r s t ligature was cut away. The c u t i c l e between the ligatures was t o r n , being c a r e f u l not to pinch or tear either the rectum or the trachea i n the process. The whole preparation was then l i f t e d , using the ends of the thread used i n l i g a t u r i n g and placed i n a r t i f i c i a l hemolymph. Small p e t r i dishes f i l l e d 1 cm deep with paraffin wax into which small wells had been dug were placed on the temperature controlled disc described above. A r t i f i c i a l hemolymph was placed in these w e l l s , the preparations were placed therein such that the t i p of the siphon made contact with the air-water interface and the petri dish l i d was replaced. The disc with petri dishes was placed i n a covered p l a s t i c container the bottom of which was f i l l e d with water to increase the humidity. After 1.5-2 hours the r e c t a l preparations were removed with forceps using the end of the thread ligatures and placed on dry f i l t e r paper. The c u t i c l e was torn back to reveal the swollen rectum and samples were taken as described above.  165  In Vitro Rectal  Preparation  Larvae were grabbed by the anterior half of the body using needletipped forceps and transferred to a r t i f i c i a l hemolymph in a shallow dish on a microscope stage. The anal segment was cut off using microscissors (John Weiss & Son, L t d . ) . Two cuts into the c u t i c l e were made on opposite sides of the body at the location of the posterior end of the midgut. While one pair of forceps retained a hold on the anterior end of the animal a second pair was used to p u l l the posterior half gently using the siphon as a handle. In three cases out of four the c u t i c l e would break where the two i n c i s i o n s had been made and the entire gut could be pulled from the posterior half of the c u t i c l e , as an arm i s pulled from a sleeve. The rectum could then be ligatured as required by the experiment i n progress, usually at any two of the following three l o c a t i o n s ; the i n t e s t i n e , the anal canal or between the anterior and posterior r e c t a l portions. Small p e t r i dishes were washed thoroughly i n detergent, rinsed s i x times in tap water, s i x times in d i s t i l l e d water and a i r d r i e d . On the inside of the p e t r i dish top a c i r c l e of s i l i c o n grease was smeared such that a c i r c u l a r spot of ungreased glass approximately 5 mm in diameter was l e f t in the center of the glass top. From this central spot a small drop of hemolymph was hung using the "hanging drop method" (Marks, 1975) The ligated rectum described above was picked up by the thread ends of the ligature and placed in the hanging drop. The p e t r i dish top was inverted so as to be right side up and placed over the bottom of a petri dish which contained d i s t i l l e d water to maintain a high humidity. Under these conditions the small hanging drop was found to change osmotic concentration by 10 mOsm during the two hour experimental period. After two hours i n the hanging drop the rectum was removed. If swollen with f l u i d , the rectum can be very delicate and easily b u r 3 t , therefore, the rectum was taken up i n a Pasteur pipette with a portion of the f l u i d in the hanging drop. A drop of f l u i d containing the rectum was placed on a glass microscope s l i d e on the stage of a microscope. The f l u i d was then gently blotted away using the edge of a piece of f i l i t e r paper. While holding the rectum with forceps by means of the end of the thread l i g a t u r e , the rectum could be punctured with a micropipette as described for whole larvae, and the sample handled as with other preparations. Rectal Preparation for E l e c t r i c a l Studies Larvae were treated as in the ligatured preparation up to the point where the f i r s t ligature had been applied and the anterior half of the animal cut away. Half of a glass p e t r i dish was f i l l e d with paraffin wax to a depth of 1 cm. A depression was made i n the wax 2 mm wide, 5 mm long and 3 mm deep. The siphon of the ligated posterior portion of  166  the larvae was placed i n the depression such that the larvae l a / dorsal side down. A glass microscope s l i d e c o v e r s l i p was s l i d into place covering most of the depression but allowing the siphon to remain i n place. The edge of the c o v e r s l i p was sealed with hot paraffin max and the wax around the larva was made into a seal by heating with a hot probe. The larva i t s e l f was thereby sealed into place but care was taken not to overheat the t i s s u e . The entire petri dish was then flooded with paraffin o i l . The larva was therefore immersed in o i l but the siphon was safely sealed into the socket of a i r under the c o v e r s l i p . The c u t i c l e of the ventral side of the larva was torn and a drop of hemolymph was placed over the l a r v a , thereby bathing the rectum i n a solution of choice. The e l e c t r i c a l potentials generated across the rectum were measured using a Radiometer voltmeter and M 701 Amplifier (W-P Instruments, Hamden, Conn.). The i n d i f f e r e n t electrode was connected via a KC1 agar bridge in PE 50 tubing (intramedic) to the drop of hemolymph around the rectum. The measuring electrode was a glass electrode f i l l e d with 3MKC1 attached to the probe on the M 701 a m p l i f i e r . The electrodes were pulled on a v e r t i c a l glass electrode p u l l e r and f i l l e d by b o i l i n g i n 37 MKC1 under vacuum. Unless i n t r a c e l l u l a r potentials were required, the t i p was broken off to reduce t i p p o t e n t i a l s . The e l e c t r i c a l potential difference between the hemolymph and the rectal lumen was determined in two ways. 1) The measuring electrode was s l i d through the anus, into the rectum and the potentials were r e corded. Using this preparation the hemolymph could be changed to follow i t s e f f e c t on the e l e c t r i c a l potential across the rectum. 2) The e l e c trode was used to pierce the rectum and measure the potential i n the anterior or posterior portion of the rectum. I n t r a c e l l u l a r potentials were obtained by using a f i n e - t i p p e d microelectrode (resistance 10 Meg Ohms) and slowly approaching the rectum u n t i l an abrupt change of potential occurred. This potential was recorded, the electrode was advanced u n t i l a potential was observed i n d i c a t i n g that the rectal lumen had been reached. The electrode was then backed off again so that the t i p was i n the c e l l again, and t h i s potential was recorded as w e l l . Hemolymph Samples Larvae were removed from the rearing pans in an eyedropper and placed on a 7 cm diameter of piece of Whatman No. 1 f i l t e r paper. After 5?10 seconds the larvae had blotted r e l a t i v e l y dry and were transferred with needle-tipped forceps to a small piece of "Parafilm" on the stage of a microscope. The c u t i c l e of the larvae was torn with two pairs of needle-tipped forceps with care being taken not to breakthe gut. The hemolymph was immediately taken up i n 1 u l Drummond pipettes  167  In most cases, one m i c r o l i t e r of hemolymph could be i s o l a t e d * When a lesser volume was taken up, the length of the f l u i d column i n the micropipettes was measured and the ratio of that length to the t o t a l length of the pipette was considered to be the r a t i o of the volume of the sample relative to one m i c r o l i t e r * For osmotic concentration readings, the hemolymph on the *Parafilm* was taken up d i r e c t l y into the pipette used f o r loading the osmometer. For ion concentration measurements, the Drummond microcap pipettes were emptied into the proper solutions f o r analysis on an atomic absorption spectrophotometer.  16®  APPENDIX  III  Volume Measurements The volumes of small drops on the order of 1-200 pi mere estimated by measuring their diameters under paraffin o i l ( F i s h e r ) . The drops were placed in half a p e t r i dish which had been l i n e d with paraffin wax and f i l l e d with paraffin o i l . When placed under the surface of this o i l , the drops would sink slowly to the bottom and would appear as round spheres with very d i s t i n c t edges i f illuminated from below. The diameters of the drops were measured at 50 times magnification using an eyepiece micrometer i n a Wild stereo binocular microscope. This method was calibrated with a solution of radioactive i n u l i n C-carboxy dissolved in 10$ sea water. Drops were placed i n o i l and their diameters were measured. These drops were then taken up i n drawn-out Pateur pipettes and placed i n 10 ml of KentEluor s c i n t i l l a t i o n f l u i d (Kent Laboratories, Ltd.) and counted on an Isocap/300 l i q u i d s c i n t i l l a t i o n counting system (Nuclear Chicago). Several samples of the i d e n t i c a l f l u i d were taken up i n 1 m i c r o l i t e r Drummond s e l f - f i l l i n g micropipettes and these samples were used to calculate the r a d i o a c t i v i t y of a known volume. Figure 1 shows the results of this c a l i b r a t i o n . A l l volumes measured for experimental purposes were smaller than 100 u l .  163  APPENDIX IV The Production of A r t i f i c i a l Hemolymph A r t i f i c i a l hemolymphs were produced to enable the components of r e c t a l secretion produced from a hemolymph solution of known i o n i c and osmotic c h a r a c t e r i s t i c s . In a d d i t i o n , i t was of interest to vary the i o n i c parameters and monitor the effect of these changes on r e c t a l secretion and components and t r a n s - r e c t a l e l e c t r i c a l p o t e n t i a l s . A l l of the a r t i f i c i a l hemolymphs used were based on Berridge's insecit* Ringer (Berridge, 1966). The compounds used were l i s t e d in Chapter 1. The purpose of this appendix i s the c l a r i f i c a t i o n of the s p e c i f i c order in which the hemolymph components wexe added so as to optimize the match with the measured parameters of real mosquito larva hemolymph. In a d d i t i o n , these procedures are c r i t i c a l i f the a r t i f i c i a l hemolymphs d e f i c i e n t i n one ion are to resemble the complete one in every i o n i c concentration save two, the missing ion and i t s replacement. -  The production of the complete a r t i f i c i a l hemolymph was begun by adding a l l the organic ingredients and Ca C l to the proper volume of d i s t i l l e d water. T'he pH was then adjusted to 6.8 with s o l i d NaOH p e l l e t s , dipped into the solution and removed as a means of gradual t i t r a t i o n . The magnesium and potassium concentrations were measured and adjusted to the proper l e v e l with MgCl2 ' KC1, r e s p e c t i v e l y . Chloride concentration was then measured and adjusted with NaCl. Next, the sodium concentration was' determined and adjusted with Na2Su"4. F i n a l l y , the osmotic concentration was measured and adjusted with sucrose. a n c  Low magnesium a r t i f i c i a l hemolymph was achieved by simply omitting the addition of MgC^. Hemolymphs with variable potassium concentrations were produced by adding more KC1 in the proper place in the sequence so that less NaCl but more Na2S04 was required. The a r t i f i c i a l hemolymphs with variable osmotic concentrations were produced l i k e the normal a r t i f i c i a l hemolymphs. When no sucrose was added, the lowest osmotic concentration i n the series was produced. Other concentrations were produced by the addition of sucrose. The a r t i f i c i a l hemolymph with variable chloride concentrations used i n Chapters 2 and 3 contained the normal organic components, CaCl2, MgC^ and NaOH to pH 6.8. The sodium levels were adjusted with Na2S0 . This gave a more elevated sulphate concentration than the regular ringer and a chloride concentration of 20 mCl. Lower chloride concentrations were required for the e l e c t r i c a l studies in Chapter 4, therefore CaSO^ and MgSO^ were substituted f o r the chloride s a l t s of these metals. 4  17.0  A r t i f i c i a l hemolymphs with variable sodium concentrations were produced using a more r e s t r i c t e d set of organic compounds (See Chapter A) namely those which were not sodium s a l t s . The pH was adjusted with KOH and fortunately, exactly the proper amount of potassium was added i n this way, largely because the amount of c i t r i c and malic acid were restricted. I suggest that the reader, i f he wishes to produce t h i s hemolymph, omit the malic acid when the organics are added, adjust potassium with KOH, and then t i t r a t e down to pH 6.8 with malic a c i d . This gives an uncertain malic acid concentration but a l l the inorganic ions w i l l be at t h e i r proper concentrations and the use of NaOH can be avoided. In my preparation procedure, as stated above, the pH and potassium concentrations were adjusted simultaneously. Chloride concentration l e v e l s were brought to the proper l e v e l with choline c h l o r i d e , y i e l d i n g a hemolymph with a low sodium concentration (5mW), and with unadjusted osmotic concentration. Half of this solution was r e moved and adjusted to a high sodium concentration (200mM) with Na S04. Both solutions were then adjusted to their proper osmotic concentration with sucrose. Intermediate sodium concentrations were achieved by mixing appropriate volumes of the two stock s o l u t i o n s . 2  PUBLICATIONS B r a d l e y , T.J. & P e r k i n s , D.L. (1975) I o n i c Antagonism i n Mosquito L a r v a e , Culex p i p l e n s . Comp. Biochem. P h y s i o l . 52A: 403-407. B r a d l e y , T.J. (1975) R e c t a l S e c r e t i o n i n S a l i n e - w a t e r Mosquito L a r v a e . Am. Z o o l . 15 ( 3 ) : 794. B r a d l e y , T.J. & P h i l l i p s , J.E. (1975) The S e c r e t i o n o f Hyperosmotic F l u i d by the Rectum o f a S a l i n e - w a t e r Mosquito L a r v a , Aedes t a e n i o r h y n c h u s . J_. exp. B i o l . .63: 331-342. P h i l l i p s , J.E. & B r a d l e y , T.J. (1976) Osmotic, and I o n i c R e g u l a t i o n i n S a l i n e - w a t e r Mosquito L a r v a e . In Transport o f Ions and Water i n Animal T i s s u e s . (Eds. Gupta, Moreton, Oschman and W a l l ) Academic P r e s s ( i n p r e s s . )  

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