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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 University, 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 p r e s e n t i n g t h i s t h e s i s in p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an advanced degree at the U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by the Head o f my Department o r by h i s r e p r e s e n t a t i v e s . It i s u n d e r s t o o d tha t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t ten pe rm i ss ion . Department o f The U n i v e r s i t y o f B r i t i s h Co lumbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 i ABSTRACT The osmoregulatory function of the larval recta of two sa l ine-water mosquitoes, Aedes campestris and A,, taeniorhynchus, was examined. In hyperosmotic waters the rectum was shown to be the s i te of formation of a concentrated urine by secretion of a hyperosmotic f lu id into the rectal lumen. In similar concentrations of sea water, both species produced a rectal secretion having an osmotic concentration and ionic composition similar to that of sea water, with the exception that potassium levels are elevated 18- to 21-fold in the secretion. Osmoregulatory strategies in both species involve the rapid ingestion of the external medium. In taeniorhynchus this drinking —1 —1 rate, (100 nl mg h ) did not vary s igni f icant ly in sa l in i t i es between 10$ and 200$ sea water. It i s suggested that two purposes are served by a rapid rate of drinking; 1) dissolved nutrients can be taken up when particulate food is l imited and 2) when the uptake of external medium is large relative 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 is close to that of the external medium. Rectal function is A., campestris was examined in three media, a l l with an osmotic concentration of 700 mOsm but varying in their ionic rat ios. These media contained the ionic ratios found in 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 i i to those ionic conditions. I demonstrated that changes in the relative transport rates of ions in the rectum are very s igni f icant in the acclimation process. The rectum was found to be the major si te of Na , K , Mg , CI 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 ra t ios , exclusive of K + found in the rectal secretion matched those in the external medium, with the ex-ception that an unidentified anion (probably HC0~) substituted for SO" in (l\)a + Wg)S0~ medium. A model i s proposed showing the si tes 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 1 0 $ , 100$ and 200$ sea water. In • hyposmotic• media the rectum does not secrete a f lu id and i t i s postulated that sal t and water resorp-tion occur in the rectum under these conditions as in s t r i c t l y f resh-+ + water species. In hyperosmotic media the concentrations of Na , K , Mg + + and E l 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 . Due to the high rate of K + secretion by the rectum, potassium uptake by the anal papillae i s postulated. Sodium and chloride may be excreted at this s i te as wel 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 loster ic relationship rather than the Michaelis-Mentin kinetics observed with most transport processes (e .g . the Mal-pighian tubules). i i i An in v i t ro preparation (lacking tracheal and neural con* nections) of the larval rectum of A,, taeniorhynchus was used to examine the function of the anterior and posterior recta l segments. These two regions, which d i f f e r morphologically, were shown to have separate functions in. v i t ro , the posterior segment secreting a hyperosmotic f l u i d while the f l u i d in the lumen of the anterior segment decreased in osmotic concentration and showed no change in volume. E l e c t r i c a l potential differences were measured across the basal and apical membranes as well as across the entire rectal wall in vivo. Based on these observations in a r t i f i c i a l hemolymphs of various ionic compositions, a model i s presented of ion transport processes occuring in the posterior rectal segments during the secre-tion of a hyperosmotic f l u i d . The model accounts for the ion concen-trations and ionic ratios observed in rectal secretion from larvae reared in dif ferent media. iv TABLE OF CONTENTS Page Chapter I GENERAL INTRODUCTION The Formation of Urine in Insects 1 Rectal Function in Insects 2 Chapter II THE SECRETION OF HYPEROSMOTIC FLUID BY THE 8 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 luid 22 Changes in volume of the Rectal Contents 25 Composition of Rectal Secretion 29 Discussion 33 Chapter III REGULATION OF RECTAL SECRETION IN SALINE- 37 WATER MOSQUITO LARVAE IN ENVIRONMENTS OF VARYING IONIC COMPOSITION Introduction 38 Materials and Methods 39 Results 44 Survival of A. campestris in various 44 media Survival of A,, taeniorhynchus in various 47 media V Hemolymph Anion Concentrations in 48 A., campestris Composition of Rectal Secretion in Different Media 49 Sodium 49 Potassium 53 Magnesium 55 Chloride 55 Sulphate - A. campestris 57 Sulphate - A. taeniorhynchus 58 Osmolality 61 Discussion 63 Chapter IV THE EFFECT OF EXTERNAL SALINITIES ON DRINK- 70 ING RATE AND RECTAL SECRETION IN THE LARVAE OF THE SALINE-WATER MOSQUITO AEDES  TAENIORHYNCHUS Introduction 71 Materials and Methods 71 Results 75 Drinking Rates 75 Hemolymph Ion Levels 81 Ionic and Osmotic Concentrations of 86 Rectal Secretion In 10$ Sea water 86 In 100$ Sea water 88 In 200$ Sea water 89 Factors Regulating Ionic and Osmotic 90 Concentrations of Rectal Secretion Discussion 99 vi Chapter V THE USE OF AN IN VITRO RECTAL PREPARATION 107 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 112 Secretion in, vi tro Discussion 116 Chapter VI THE MECHANISM OF HYPEROSMOTIC FLUID SECRETION 119 IN THE RECTUM OF LARVAE OF THE SALINE-WATER MOSQUITO AEDES TAENIORHYNCHUS Introduction 120 Materials and Methods 120 Results 123 Trans-rectal E lec t r i ca l Potential 123 Difference Influence of Hemolymph Ion Concentrations 124 on the Trans-rectal P.D. Discussion 141 Chapter VII GENERAL SUMMARY 150 REFERENCES 156 APPENDICES 161 v i i LIST OF TABLES Table Page 1 Ionic concentration (mlfl) and osmolality 17 (mOsm) of a r t i f i c i a l and natural hemolymph. 2 The determination of increase in volume 27 of rectal contents following l igat ion o§ larvae using ^ C - i n u l i n a s a volume marker. 3 Ionic concentrations of external media 41 (NaCl, NaHC03 and (Na + Mg)S04) a l l of which had a total osmolality of 700 mOsm. 4 The ionic (mfll) and osmotic concentrations 43 (mOsm) of normal and low chloride hemolymph. 5 Total mortality observed four days after A,. 46 campestris larvae were transferred to three experimental media (700 mOsm) of different ionic composition. 6 A comparison of the external ionic and osmotic 50 concentrations in the three acclimation media with the same parameters in rectal secretion from two rectal preparations. 7 The ionic (mfll) and osmotic (mOsm) concentre- 74 tions in the a r t i f i c i a l hemolymphs used to study the effects of varying hemolymph parameters. 8 The osmotic concentrations of rectal f l u i d 113 removed from lumina of in, vi tro preparations of whole recta and isolated anterior and posterior rectal segments. 9 The osmotic concentration (mOsm) of rectal 115 secretion produced by whole, iti v i tro recta in a r t i f i c i a l hemolymph with the addition of potential stimulatory agents. v i i i 10 Paired measurements of the trans-rectal 124 e lec t r ica l potential difference in the an-ter ior and posterior rectal segments of recta bathed in normal a r t i f i c i a l hemolymph uiithin ten minutes of dissect ion. 11 Paired measurements of the trans-rectal 126 e lec t r ica l potential difference in the anterior and posterior rectal segments of recta bathed in normal hemolymph, two hours after dissect ion. 12 Paired measurements of the trans-rectal 128 potential difference within the rectal ce l ls and the rectal lumen in the anterior and posterior rectal portions. ix LIST OF FIGURES Figure Page Measurements demonstrating the l inear 14 relationship between the radioactivity of drops of I^C-inulin solution and the calculated volume of those drops based on their measured diameter. A schematic diagram showing the three most 16 posterior segments of the mosquito larva with the position of ligatures used to isolate the rectum. The amount of external medium consumed 21 with time as estimated from the increase in whole body radioactivity with time. The change in osmotic concentration of 23 rectal f l u i d and hemolymph with time after the rectum was l igated. The increase in osmotic concentration of 24 rectal f l u i d with time in preparations bathed in a r t i f i c i a l hemolymph. A comparison of the ionic and osmotic 31 concentrations of rectal f l u i d with hemolymph and sea water. Paired determinations of the osmotic con- 54 centration (mOsm) and sodium concentration (mm) in the rectal f l u i d from whole larvae. Paired determinations of the sulphate con- 59 centration in the hemolymph and rectal f l u i d of whole larvae. A diagrammatic representation of the pro- 64 posed locations of ion transport processes in the Malpighian tubule, rectum and anal papillae of A_. campestris larvae, in each of three media used in this study. X 10 The drinking rate of larvae in the media in 76 which they were reared 11 The relationship between the weight and v o l - 78 ume of A,, taeniorhynchus larvae* 12 The relationship between the weight and 79 surface area of A,, taeniorhynchus larvae* 13 The relationship between the volume and 80 drinking rate of larvae in four s a l i n i t i e s , 10$, 50$ 100$ and 200$ sea water. 14 The relationship between the surface area and 82 drinking rate of larvae in four s a l i n i t i e s ; 10$, 50$ 100$ and 200$ sea water. 15 The mean concentrations of sodium, potassium 83 magnesium and calcium in the hemolymph of larvae reared in sea water of d i f fer ing s a l i n i t y . 16 The mean chloride, sulphate and osmotic 85 concentrations in the hemolymph of larvae reared in three media of d i f fer ing s a l i n i t y . 17 The ionic and osmotic concentrations of 87 rectal f l u i d from whole larvae in 10$j 100$, and 200$ sea water. 18 The effect of a r t i f i c i a l hemolymph di f fer ing 92 only in osmotic (sucrose) concentration on the osmotic concnetration of rectal secretion. 19 The effect of varying chloride concentration 94 in a r t i f i c i a l hemolymph on the volume, chloride concentration and osmotic concentration of rectal secretion collected after 1.5 h. 20 The effect of varying sodium concentration in 96 a r t i f i c i a l hemolymph on the volume, sodium concentration and osmotic concentration of rectal secretion collected after 1.5 h. 21 The relationship between the concentration of 98 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 ion. 22 The trans-rectal e lec t r ica l potential d i f - 130 ference observed in anterior rectal seg-ments bathed in normal a r t i f i c i a l hemolymphs or a r t i f i c i a l hemolymphs di f fer ing in the concentrations of the ion indicated. 23 The trans-rectal e lec t r ica l potential d i f - 131 ference observed in the posterior rectal segment 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 di f fer ing in the concentrations of the ion indicated. 24 The effect of increasing the potassium con- 133 centration of the a r t i f i c i a l hemolymph on the trans-rectal e lec t r ica l potential difference of the posterior rectal segment. 25 The effects of varying the sodium concentra- 135 tion of the a r t i f i c i a l hemolymph on the trans-rectal e lec t r ica l potential difference of the posterior rectal segment. 26 The effect of varying the chloride concentra- 136 tion of the a r t i f i c i a l hemolymph on the trans-rectal e lec t r ica l potential difference of the posterior rectal segment. 27 The trans-rectal e lec t r ica l potential d i f - 138 ference observed in two posterior rectal seg-ments 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". 28 The trans-rectal e lec t r ica l potential d i f - 139 ference observed in the posterior rectal segments bathed in normal a r t i f i c i a l hemo-lymph or a r t i f i c i a l hemolymphs di f fer ing in the concentration i f the ion indicated. 29 A model of the process occurring in the ce l l s 143 of the posterior rectal segment during the secretion of hyperosmotic f l u i d . x i i LIST OF PLATES Plates Page Photographs of in, vivo rectal preparations 5 min. and 2 h after l igation 26 Photographs of in, vitro rectal preparations 10 min. and 2 h after dissection and l igat ion 111 x i i i ACKNOWLEDGEMENTS I would l ike to thank Dr. John Phi l l ips for his assistance and guidance, his fa i th in 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 Gosline, Dave Randall and G.G.E. Scudder for their useful comments regarding the form of this thesis. I wish to thank M.S. Haswell for technical assistance in measuring ionic concentrations, Dr. J .K . Nayar for sharing his knowledge of culturing techniques and Dr. E.P. Marks for information regarding the in vi tro preparation. I am grateful to Joan Martin and Doug Williams for interesting discussions, pertinent and otherwise. I wish to thank my wife, L isa , for 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 in most of those insects which have been stud-ied , i s a two-step process, involving non-selective secretion of a f l u i d followed by selective resorption of solutes and/or water in appropriate amounts prior to excretion. The formation of the primary excretory f lu id occurs in the Malpighian tubules (Ramsay, 1950-1961) by a process of K + secret ion, which forms an osmotic gradient leading to diffusion of water into the lumen of the tubule. Ramsay suggested that other solutes enter the lumen following their respective concentration gradients, re-sult ing in 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 secretion, 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 acidic dyes (Maddrell et a l , 1974), Mg + + (Ph i l l ips and Maddrell, 1975) 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 tub-ules 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; Irvine, 1969; Maddrell and P h i l l i p s , 1975b). Upon reaching the alimentary t ract , the excretory f lu id i s directed posteriorly into the rectum via the intest ine. Maddrell (1971) reviewed the evidence point-ing to resorption of ions and water in the intestine of several insects. 2 He suggests a signif icant role for this region of the gut, part icularly in insect species such as the locust where molecular sieving by the rectal cut ic le may reduce the rate at which large organic molecules are absorbed in the rectum. (Phi l l ips and D o c k r i l l , 1968). With the exception of those cases already c i ted , 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 insects, "primary urine" from Malpighian tubules i s modified in the hindgut or rectum to maintain osmotic homeostasis (Madd-r e l l , 1971). The urine i s concentrated in the rectum in ter rest r ia l and saline-water insects and made more dilute in freshwater insects. Hyper-osmotic excreta are formed in terrestr ia l insects,by the resorption of water in 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 local osmotic gradients formed by ions that are actively resorbed from the rectal lumen, and concentrated within the lateral intercel lu lar 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 rectal ce l ls occurs. This results in a di lut ion of the f l u i d moving through the rectal epithelium, such that in dehydrated insects, the resorbate which f ina l l y enters the hemo-3 lymph at the mouth of the lateral channels i s not only hyposmotic to the rectal f l u i d but may be more dilute than the hemolymph as well . In conditions of excess ingestion of water, this mechanism would obviously be inappropriate for the maintenance of osmotic ho-meostasis. In these circumstances, fewer ions are presumably resorbed as the f l u i d passes along the intercel lu lar channels, resulting in a hyperosmotic resorbate, and thus a hyposmotic excreta. The desert locust Schistocerca greaaria forms hyperosmotic excreta under a l l conditions, but other insects, 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 di lut ion of the urine in these insects i s believed to occur in the rectum by means of selective ion resorption from the primary excretory f l u i d . The- term fresh-water insect when applied to some species refers more to an inab i l i ty 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 ight ly hyposmotic to the hemolymph. This separation of niches may occur at the level of genera, Limneohilus s p . , Anabolia sp. (Sutcl i f fe 1961a» b)j species, Cenocorixa b i f ida . C. exoleta (Scudder, 1969a, b; Scudder ejb a l , 1972); or that of physiological races of the same spe-c i e s , Cricotoous vitr ioennis (Sutcl i f fe 1960). Yet a l l of these insects, 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 se . 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 lu ids in the saline-water larvae of Aedes detr i tus. raised in sea water. The f lu id from the Malpighian tubules was very s l ight ly hyperosmotic to the hemo-lymph and the rectal f l u id was markedly hyperosmotic, with a mean concen-tration near that of sea water. Nayar (1975) has shown that Aedes taeniorhynchus is capable of maintaining hemolymph levels of sodium(173-218mM) and chloride(48-78mM) within narrow l imits in the face of a wide range of external concentra-tions(0-300$ sea water). The fflalpighian tubules of this species produce a f l u i d which i s very s l ight ly 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. detr i tus, whereas in A,. campestris the Malpighian tubule f lu id is isosmotic to the hemolymph (Phi l l ips and Waddrell, 1975). Therefore, the extreme concentration of the urine to a level 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 ter rest r ia l insects, i . e . resorption of water without proportional amounts of solute. Meredith and Ph i l l ips (1973a) investigated the rect-a l ultrastructure of the saline-water larva A. campestris in both hypo-5 and hyperosmotic environments. They found the ultrastructure of the anterior rectal segment of A. campestris which contains only one c e l l type to be very similar to that of the rectum of A,, aegypti and suggest-ed that this portion resorbs ions from the primary excretory f l u i d when the larva i s in hyposmotic environments. This would mean that this act-i v i ty i s associated with straight lateral membranes, moderately in fo ld -ed apical ones, extensively infolded basal membranes and mitochondria distributed evenly throughout the c e l l . Based on the ultrastructural dissimi lar i ty to the recta of ter rest r ia l insects, Meredith and Ph i l l ips also suggested that the posterior rectum forms a concentrated excreta by the secretion of a hyperosmotic f l u i d into the lumen. Whereas the rect-um of te r rest r ia l insects i s characterized by relat ively straight basal membranes', moderately infolded apical ones, and highly infolded latera l membranes in close association with most of the c e l l ' s mitachondria, the posterior rectum of A_. campestris showed straight lateral membranes, moderately infolded basal ones, and a highly infolded apical membrane associated with most of the mitochondria. The only terrestr ia l insect having a rectum ultrastructural ly similar to this i s Thermobia domestica. This surely represents a superf ic ia l resemblance, since the latter rectum has been implicated in the uptake of water from subsaturated a i r (Noble-Nesbitt, 1973). A similar 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 bacterial symbionts. (Noirot +Noirot-Thimotee, 1967). In summary, for the three regulatory epithel ia just discussed, 6 sal t resorption to form a hyposmotic urine i s associated with wel l -developed apical membranes and evenly distributed mitochondria. Water resorption to form a hyperosmotic urine i s found in recta having high-ly folded lateral membranes associated with many mitochondria. Hyper-osmotic secretion i s hypothesized for tissues showing extensive apical infolding associated with many mitochondria (reviewed by Meredith and P h i l l i p s , 1973a; Wall and Oschman, 1975). The research described in Chapter II was undertaken to obtain direct experimental evidence for or against the hypothesis of Ph i l l ips and Meredith (1969a) and Meredith and Phi l l ips (1973a) that the rectal epithelium of saline-water mosquito larvae produces a hyperosmotic ex-creta by sa l t secretion into the rectal lumen, rather than by water re-sorption, as occurs in ter rest r ia l insects. Once i t had been establ ish-ed that hyperosmotic urine i s formed by sa l t secretion the ion concentra-tions of the rectal secretion was measured to demonstrate that the f l u i d produced was contributing to osmoregulation. These measurements were made for larvae reared in sea water of varying concentration and also (Na+fflg)S0^ and NaHCO^ waters to investigate the ab i l i ty of the larvae to adjust secretion to varying ionic environments in which they are naturally found (Chapters III + IV). Meredith and Phi l l ips (1973a) also suggest that the anterior rectum is the s i te of sal t resorption from primary excretory f l u i d in fresh and saline water and that the posterior rectum is the s i te of sal t secretion in saline water. This hypothesis was tested using an iri v i tro rectal preparation (Chapter V). In addition} e lec t r ica l potential mea-7 surements were carried out in order to ascertain which ions are act ive-ly transported in the larval rectum and in what region secretion may occur. A study of the kinetics of the transport mechanisms for sodium and chlor-ide in the rectum was carried out. F i n a l l y , I propose a model for the organization of transport processes in epi the l ia l ce l ls of the posterior rectum, which i s consistent with experimental observations presented in this thesis (Chapter VI). A word of explanation concerning the organization of this thesis i s in order. Chapter II i s presented as published (Bradley and P h i l l i p s , 1975). Chapters III-VI are s l ight ly modified from the form in 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 in separate publications. 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 te of selective reabsorption from the primary excretory f l u i d produced by the Malpighian tubules. This act iv i ty has been shown in ter rest r ia l (Ph i l -l i p s , 1964a-cj 1970) and freshwater insects (reviewed by Stobbart and Shaw, 1964) and i s thought to be ultimately responsible for osmotic regu-lation in most insects. A similar mechanism has been postulated for saline-water i n -sects. 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 concentra-tion similar to sea water and up to three times the osmotic concentration of the hemolymph. It has since been suggested that the f lu id became con-centrated in the rectum in the same manner as in te r rest r ia l insects (Stobbart & Shaw, 1964), that is by selective reabsorption of water with-out proportional amounts of solute (reviewed by P h i l l i p s , 1970; Maddrell, 1971). Ramsay associated the ab i l i ty of this saline-water larva to pro-duce hyperosmotic excreta with an additional rectal segment absent in freshwater species of mosquito larvae. However, Ph i l l ips and Meredith (1969a), and Meredith and P h i l -l ips (1973a) found that the ultrastructural features associated with the production of hyperosmotic urine in the rectum of most terrestr ia l i n -10 sects, namely elaborate development of the lateral membranes which are associated with most of the mitochondria of the c e l l s , were absent in the rectal epithelium of another saline-water mosquito larva, Aedes  campestris. Instead, the apical membrane facing the lumen was most highly developed. 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 id 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 apical 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 Sarco-phaoa 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 isolated adaptation to an environment r ich in this ion , 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 direct physiological evidence for or against the suggestion of Meredith and Phi l l ips (1973a), that mosquito larvae l iv ing in saline water pro-duce hyperosmotic urine by secretion of ions into the rectum. Aedes  taeniorhynchus» a mosquito native to coastal sea water swamps in 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 ca -pable of maintaining hemolymph levels of sodium (l73-218mffl) and chlor-11 ide (48-78mM) within narrow l imits in the face of a wide range of ex-ternal concentrations (0-300$ sea water). 12 MATERIALS AND METHODS A culture of Aedes taeniorhynchus was obtained from the Ento-mological Research Center, Vero Beach, Florida and maintained according to the method of Nayar (1967) in sea water obtained in the Vancouver area (832 - 8.8 mOsm). Fourth instar larvae were starved for 1-2 days before being used in 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 l inear increase in whole-body act iv i ty following transfer of larvae to sea water 14 / \ containing C- inul in (.New England Nuclear Corp. ) . Larvae were removed in groups of 5 after various time intervals , weighed, placed in 1 ml of 10$ KOH, macerated, incubated for one hour at 90°C and allowed to cool . The solution was then neutralized with 1 ml of appropriate strength H^SO^ and 1 ml aliquots were added to 10 ml of 'Scintiverse* (Fisher Sc ient i f ic Co.) and counted in a Nuclear Chicago *Isocap 300' l iqu id sc in t i l l a t ion system, Hemolymph was obtained from larvae blotted dry on f i l t e r pap-er and torn open so as to allow the hemolymph to flow onto a sheet of •Paraf i lm' . No difference in estimates of hemolymph composition was observed between larvae removed directly from sea water and those f i r s t rinsed in d i s t i l l e d water. The hemolymph was immediately taken up in 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 rectal f l u id 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 iquid 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 petri dish coated with paraffin wax. The volume of the drop was estimated by measuring i t s diameter. This volumetric technique was 14 calibrated by estimating radioactivity in drops of a standard C-inul in solution (F ig . 1). Ionic concentrations of hemolymph and rectal f lu ids were measured by transferring the samples to vials 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 in the trans-mission (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 (Clifton Technical Physics Ltd.) or the cry-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, approx-imately between the sixth and seventh abdominal segments, thus el imin-ating delivery of f lu id from the midgut and Malpighian tubules. A second ligature around the anal segment prevented any f lu id from enter-ing or leaving the rectum other than by passing through the wall of the rectum and a short portion of the intest ine. The larva was severed in front of the anterior ligature to produce the experimental preparation Figure 1. Measurements demonstrating the l inear relationship between 14 the radioactivity of drops of C- inul in solution and the calculated volume of those drops based on their measured diameter. The radio-act iv i ty of drops of 1u1 size measured by this method and by 1JJ1 "Microcap" pipettes were also in 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. Phi l l ips (unpublished observation) found that chloride was actively ab-sorbed by the anal papillae of Aedes campestris adapted to fresh water at comparable rates in intact larvae and in posterior segments prepared in the manner outlined above. The above preparation was suspended in sea water, and hemolymph and rectal samples were taken after varying periods of time. In i t i a l studies indicated that the concentration of the l im i t -ed volume of hemolymph in the above preparation increased drast ical ly with time. To eliminate this problem in later experiments, the same preparation was floated in an a r t i f i c i a l hemolymph solution and the i n -tegument was torn open. Under these circumstances the rectum was bathed in a relat ively large volume of a r t i f i c i a l medium but was s t i l l connected to i t s normal tracheal supply. 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 in mg/l00ml of water: NaC1 175.3, Na ci trate 69.7, KC1 44.8, CaC1 2.2H 20 29.0, ClgC12. 6H20 264, T.C. Yeastolate 400, Pen ic i l l i n "G" 10, Strepto-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 rectal contents in two ways. Photographs were taken of the recta of larvae immediately af ter , or two hours af ter , l iga t ion . In Figure 2. A schematic diagram shouting the three most posterior segments of the mosquito larva with the position of ligatures used to isolate the rectum. Abbreviations: A anus, AC anal canal, AP anal papi l lae, AR anterior rectum, H hemolymph, I intest ine, M Malpighian tubules, PR posterior rectum, S siphon, T tracheae, X anterior l igature, Y posterior l igature. 16b 17 Table 1. Ionic concentrations (mfll) and osmolality (mOsm) of a r t i f i c i a l and natural hemolymph (mean - S . E . ) . Hemolymph A r t i f i c i a l hemolymph Na+ 1 4 9 - 3 148 K + 1 6 - 1 17 |Ylg++ 1 0 - 1 24 C l " 9 8 - 5 97 Osmolality 348 - 17 315 18 the second method, after the anterior l igature mas in place but before 14 m the posterior one had been applied, a known volume of C- inul in in 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 radioactivity of the resul t -:ing drop was measured for comparison with the known values for the f l u i d injected. Assuming uniform distr ibution of i n u l i n , the percent-age of the injected radioactivity recovered from the rectum corresponds to the percentage of the rectal f l u id 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" isolate the rectum, the exterior cut ic le torn, and the preparation floated in a r t i f i c i a l hemolymph for 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 ina l estimates of volume gave a minimum estimate of the i n -crease in the volume of the rectal contents, i . e . total secretion. The composition of the rectal f l u i d secreted by ligated pos-ter ior segments was compared with that from intact animals. Determining the composition of the rectal contents from normal, unligated larvae pre-sented some d i f f i c u l t i e s . Larvae empty the rectum in response to almost any disturbance, making i t impossible to introduce a pipette into the rectum via the anus before the rectal contents have been voided. For this reason the anus must be closed to enable the rectal f lu id to accumu-late and remain in the rectum unt 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 for one hour, at which time the rectal contents were removed as described above. 20 RESULTS Estimation of Osmotic Load The saline-water mosquito larva, Aedes campestris, ingests large quantities of external medium (Phi l l ips & 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 sa l t entry into this species (Phi l l ips + Bradley, in press) and hence largely establishes the rate of ion secretion required to achieve ionic balance in this animal. The drinking rate of Aedes taeniorhynchus in sea water was therefore measured to provide a minimum estimate of ion and water turn-over. 14 The i n i t i a l accumulation of C- inul in by whole larvae in sea water (F ig . 3) indicated that they ingested 33.5 n l mg body weight -1 + -1 _1 + h , or 100 - 2 n l h larva mean - S .E . for larvae having a mean weight of 3.0 - 0.5 mg. Thus, £ . taeniorhynchus larvae drink their own body weight every 29.6 h. This i s comparable to the rates for A.. campestris in saline media reported by Kiceniuk & Ph i l l ips (1974). Since the sodium concentration of sea water i s nearly 3 times that of larval hemolymph, the hemolymph content of this ion must be turned over in approx-imately 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 ionic 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 radioactivity with time after placing mosquito larvae in sea water containing 14 C- inu l in . Vert ical bars denote S .E . of the means, N=6 for each point. 22 Osmolality of Rectal Fluid 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 prior to l igat ion 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 surpr is ingly, therefore, samples of rectal f l u id collected within a few minutes of l igat ion were isosmotic with the hemolymph (F ig . 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 after one and two hours showed a continual increase. Hemolymph osmotic concentration did not change s i g -ni f icant ly over the f i r s t hour, but increased s igni f icant ly during the second. Indeed, over the second hour, the osmotic concentration d i f fe r -ence across the rectal wall did not change appreciably. This increase in hemolymph concentration was attributed to passive net exchange of water and ions across the body wall of the larva. I wished to separate the increase in rectal f l u i d concentration from the influence of the increasing hemolymph concentration. To this end, the same type of preparation was placed in a large volume of a r t i f i c i a l hemolymph (Table 1) and the body wall was torn open to expose the rect-um to this external medium. Under these conditions the osmotic pressure of the rectal 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 igni f icant ly over the next 1.5 h (F ig . 5). The osmotic gradients produced by the two types of preparation Figure 4. The change in osmotic concentration of rectal f l u id and hemolymph 0 with time after the rectum was ligated as shown in F ig . 1 . The preparation was placed in sea water. Vert ical bars denote S .E . of the means. (n=10) Time (h) Figure 5. The increase in osmotic concentration of rectal f l u i d uiith time. The rectum was ligated as shown in F i g . 1, the i n -tegument was torn, and the preparation was placed in a r t i f i c i a l hemolymph. Vert ical bars denote S .E . of the means. 1000 0 25 did not d i f fer s igni f icant ly at 1 h (P<0.1), indicating 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 selective water resorption from the small volume of isosmotic f l u i d i n -i t i a l l y present in the lumen, or by secretion of a hyperosmotic f l u i d into the rectum. These poss ib i l i t i es were differentiated by measuring the change in volume of the rectal contents under the experimental con-ditions described above. Changes in Volume of the Rectal Contents Increases in rectal volume during the production of hyperos-motic urine by ligated recta were apparent from the swelling of the rectum and the larger volume of f lu id which could be recovered, using micropipettes, at the end of a l l experiments. This considerable i n -crease in rectal volume was documented by comparing photographs of recta taken within 5 min or at 2 h after isolat ion of the rectum by l igation (Plate 1). The rectum, which i s nearly empty after being l igated, swells considerably with f l u i d which can only have been produced by way of rect-a l secretion. In addit ion, d ist inct posterior and anterior segments of the rectum are clearly 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 ) . Assuming the inul in to be evenly distributed in the rectum, the i n i t i a l volume of rectal Explanation of Plate 1 Photographs of recta 5 minutes ( left ) and 2 h (right) after l iga t ion . 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 different recta are shown in each photograph at the same magnification. Abbreviations! AR anterior rectum, M Malpighien tubules, PR posterior rectum. Table 2. The determination of increase in volume of rectal contents following l igat ion of larvae, as shown in F i g . 1. Recta were injected 14 with a known amount of C- inul in by means of a micropipette inserted through the anus before the posterior ligature was applied. The same pipette was used to retrieve as much f l u i d from the rectum as possible .af ter 20 seconds had elapsed ( In i t i a l ) . The percent of the i n i t i a l rad-ioact iv i ty recovered was proportional to the percent of rectal contents removed, allowing an estimate of volume of remaining rectal f l u i d to be made. The anal segment was then ligated and after 2 hours the rectal contents were measured for volume and osmotic concentration (F ina 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. In i t ia l Final (2 h) Series 1 2 3. 4 5 Volume recovered from rectum (nl) 46 17 113 17 36 % Radio act iv i ty recovered 103 92 103 73 91 Calculated volume remaining in rectum (nl) 0 1.5 0 6.3 3.6 Volume of rectal f l u id removed (nl) 65 34 45 24 23 Minimum volume Osmolality secreted by of rectal rectum (n1) 65 32.5 45 17.7 '19.4 f l u i d (mOsm) 834 651 1039 794 1106 Mean S .E . 45.8 17.7 92.4 5.5 2.3 1.2 38.2 7.8 35.9 8.8 884 186 29 f lu id could be estimated from the percent recovery of injected C-inu l in , and was found to be very small (2.3 - 1.2 n l ) . Within 2 h of l igat ion , the volume of rectal f l u i d had increased to a mean value of 38.2 - 7.8 n l . The largest volume increase was 65 nl after 2 h and a l l recta showed an increase. In every case the volume increase was asso-ciated with a large increase in osmotic concentration of the rectal f l u i d which did not d i f fer s igni f icant ly (P>0.2) from previous estim-ates (F ig . 5). Clear ly , 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 solute. Composition of Rectal Secretion If the rectum is solely responsible for ionic regulation and i f a l l ingested ions are absorbed in 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 relative ionic composition of the rectal secretion should ref lect that of sea water and the total concentration of the former f lu id should be greater to compen-sate for osmosis across the body wall . The small volume of f lu id secret-ed by individual recta made i t necessary to pool the f l u i d from 7-10 pre-parations for a single chemical determination. The average ion concen-trations in rectal f l u i d 2 h after l igation of recta, which were bathed in a r t i f i c i a l hemolymph, were! 285 mW-Na+ (n=1l), 158 mM-K+ (n = 5), 25 mM-Mg++ (n=5), 425 mW-C1- (n=5), 818 mOsm (n=18). Standard errors are recorded in F i g . 6 , which compares the mean values for rectal secretion to those of sea water. Rectal secretion would seem inadequate to explain completely 30 the maintenance of ionic 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 can be accounted for by drinking. The larvae were starved prior 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 in the food. It was f e l t that the unexpected Na:K ratio may have been due to decreased act iv i ty of the preparation, or lack of hormonal or neural stimulation of secretion. With these poss ib i l i t i es in mind, rectal f l u id was collected from intact , whole larvae reared in sea water, 1 h after the anus was blocked with tissue adhesive to allow the accumulation of suf f ic ient rectal f l u i d for analysis. The mean concentrations of a l l ions in this rectal f l u i d were s l ight ly higher (Fig 6), but not s igni f icant ly so (P>0.05 for a l l ions measured), and the ion concentration ratios remained unchanged: 435 mM-Na+ (n=5), 192 mm-K+ (n=5), 36 mm-Mg++ (n=5), 468 mlV)-C1= (n=6), 920 mOsm (n=6). Occasionally during the sampling of the rectal contents, the rectum was punctured in 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 pipette. 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 intact innervations, and receive f lu id from the midgut. The f lu id derived under these conditions i s very similar to that secreted by the isolated, l igated rectal prepar-at ion. Both preparations demonstrate that the larval rectum of Aedes  taeniorhynchus in sea water secretes a f lu id which, though 16 to 19 Figure 6. A comparison of rectal f l u i d with hemolymph and sea water. The ionic and osmotic concentrations of natural hemolymph j secretion from ligated rectal preparations (F ig . 1) with a torn cu t ic le , in a r t i f i c i a l hemolymph ; rectal f l u i d from intact larvae with the anus plugged and placed in sea water and Vancouver sea water Vertical bars denote the S . E . of the means. Osmolality (mOsm) 32 times higher in potassium than sea water, i s otherwise nearly ident ical to sea water in i t s ionic and osmotic characterist ics (F ig . 6 ) . 33 DISCUSSION The results clearly confirm the hypothesis (Phi l l ips & Meredith, 1969a) that saline-water mosquito larvae produce hyper-osmotic urine by ion secretion into the lumen rather than by water reabsorption, as occurs in ter rest r ia 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 similar act iv i ty in the hind-gut of another dipteran larva, Sarcophaga bul lata. Since the anterior and posterior rectum were not isolated from one another in the present experiments i t is not yet possible to assign the secretory act iv i ty to a speci f ic segment of the rectum, although Meredith and Phi l l ips (1973a) have presented ultrastructural evidence which implicates the posterior rectum in this ac t iv i ty . They have suggested that the anterior rectum is involved in selective reabsorp-tion when larvae are in hyposmotic environments. The observed concentrations of ions in the secretion from ligated recta were not s igni f icant ly different from those of sea water, except that potassium concentration was much higher in the secretion. Using cryoscopic coeff icients for pure manovalent sa l t solut ions, the mean levels of K + , Na* and C l~ w i l l account for 96% of the observed osmotic pressure of the rectal f l u i d (818 - 37 mOsm, n=18). However, when the magnesium concentration is included, there i s an excess of cations over anions tota l l ing 68 mEquiv. 1 \ suggesting that other anions prevalent in sea water (e .g . S0A) may be present (suggested by 34 Maddrell & P h i l l i p s , 1975). If, to simplify calculat ions, one assumes that the entire anion def ic i t i s sulphate ions associated with magnes-ium and sodium ions, the calculated osmotic concentration of the rectal f l u id using cryoscopic coeff icients i s 856 mOsm.1 which is within the standard error of the observed osmotic pressure. When the same ca lcula-tions are made using the ionic concentrations in the rectal f l u id from intact larvae with blocked anuses, the anion de f ic i t i s 231 mEquiv. 1 and the total calculated osmotic pressure i s 1144 mOsm 1 , which ex-ceeds the standard error of the observed osmotic concentration ( i . e . equal to 137$ sea water). It is therefore necessary to postulate some ion binding to polyvalent anions, e .g. macromolecules of fecal material, as reported for magnesium in Ades campestris (Kiceniuk & P h i l l i p s , 1974). In summary, the major components of the rectal secretion have been ident i f ied with the exception of a small anion d e f i c i t . The sec-retion of a l l of these ions (Na , K , fflg , C1 ) occurs against large concentration differences of 2 to 10-fold. E l e c t r i c a l potentials across the rectal wall must be measured to determine which of these transport processes are act ive. Aedes taeniorhynchus larvae, in 100$ sea water, drink and probably assimilate 100 nl of external f l u id per larva per hour. Some of this water i s presumably lost by osmosis across the body wall . Nicholson and Leader (1974) have estimated this loss for another sa l ine-water mosquito (Opifex fuscus) of similar s i z e , l igatured at the neck and -2 -1 anus, at 0.0026 jul mm h . Assuming a similar 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 nl h for water loss through the excretory system. The largest _1 volume of rectal secretion observed was 96 nl h but the mean was _1 19 nl h . While secretion of Malpighian tubule f l u i d might account for 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 (Phi l l ips + Maddrell, 1975; Maddrell +, P h i l l i p s , 1975). The same is true for any f l u i d which might pass down the intestine from the midgut (Ramsay, 1950; Kiceniuk + P h i l l i p s , 1974). It would be premature to conclude that rectal secretion i s inadequate to account for osmotic regulation, since the calculations described above for larvae with the anus blocked suggest that the effective concentration of the secretion can reach 137$ sea water. In order to col lect enough f l u i d for analysis, the anus was blocked in a l l experiments. The accumulation of f lu id in the rectum might lead to under-estimation of both ion concentrations and volume of the rectal secretion for various reasonst 1) Stretching of the rectal wall might lead either d i rect ly , or indirect ly through stretch receptors, to i n -creased passive permeability of the rectal wal l . 2) The buildup of hydrostatic pressure in the rectum might oppose further f l u i d secretion. 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 ight ly hyposmotic to the sea water medium. A. 36 taeniorhynchus can survive in 300$ sea water. If the rectal f l u i d is isosmotic to the external environment in this s i tuat ion, then the rectum is clearly capable of creating larger gradients than have been observed. If Ramsay's observations with intact larvae and the present values for larvae with the anus ligated indicate the true upper l imit for hyperosmosity of rectal secretion then other s i tes of sal t secre-t ion, such as the anal papi l lae, must be invoked. Phi l l ips & Meredith (1969b) have presented preliminary evidence that the anal papillae of A,, campestris larvae l iv ing in hyperosmotic media might actively secrete chloride to the external medium. If sodium is also secreted by anal papillae in exchange for an inward movement of potassium, (e .g . as in the g i l l s of saltwater teleosts; Maetz, 1971), this might balance the loss of K + ions by rectal secretion (F ig . 6). Leadem (unpublished observation) in our laboratory has obtained some preliminary evidence for such a mechanism. A,, campestris larvae which l ive in waters of high NaHCO^ or high MgSO^ content are also known to produce hyperosmotic urine although f lu id leaving their Malpighian tubules is isosmotic to the hemolymph (Phi l l ips & Meredith, 1969aj Kiceniuk & P h i l l i p s , 1974; Ph i l l ips & Maddrell, 1975; Maddrell & P h i l l i p s , 1975). Presumably the dominant ions in the rectal secretion of this species, which can also l ive in sea water, can be varied as dictated by the environment in which the larvae develop. Otherwise, i t i s necessary to postulate d ist inct physiological races of this species in saline waters having different dominant ions. •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 mosquito larvae were f i r s t investigated by Beadle (1939). 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 lu id from various body compartments in detritus larvae adapted to sea water and showed that hyperosmotic urine was formed largely during pas-sage through the rectum. In a previous paper (Bradley and P h i l l i p s , 1975; and Chapter Ii) we presented evidence that the urine was concen-trated in another saline-water mosquito larva, Aedes taeniorhynchus. by secretion of a hyperosmotic f l u i d into the rectal lumen. It was of i n -terest to extend these observations to another euryhaline species of mosquito larva, Aedes campestris, which l ives in a lka l i salt- lakes having a wide range of ionic compositions. I wished to determine wheth-er the ionic composition of rectal secretion ref lects that of the natural water to which larvae are adapted; i . e . can the ion ratios of the secretion be radically altered from those observed in A. taenio-rhynchus. Such changes would suggest elaborate control of ion transport mechanisms and possibly even induction of new protein carr iers , a pro-cess rare amongst metazoans. Scudder (1969a) examined the distr ibution of several insect species, including A., campestris, found in saline-water ponds in Br i t -ish Columbia. 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 di f fer ing ionic composition as well . Larvae from ponds in which Na2S0^ and MgSO^ are the main sal ts have been shown to drink and assimilate the M g + + , SO" and presumably the Na+ in such water and to excrete excess Mg + + 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 in the strongly hyperosmotic waters in which the dominant sal ts are either NaHCO^ of NaC1 and produce urine with an osmotic concentration 2-4 times that of the hemolymph. They can develop as well in fresh water and hyposmotic waters with ionic ratios similar to those described above. (Phi l l ips and Meredith, 1969a, b; Phi l l ips and Bradley, 1976). It was not known whether A. campestris larvae which inhabit environments with radical ly different ionic ratios represented dist inct physiologically races. 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 rectal secretions from la r -vae in each of these media were analyzed to determine to what extent rectal secretion can be adjusted by the animal to match the speci f ic ionic characterist ics of each external medium. MATERIALS AND METHODS Larvae of the mosquito Aedes campestris were collected on Apri l 19 and May 14,1975 from Ctenocladus pond, a saline pond located near Kamloops, B.C. (Bl inn, 1969). Specimens were approximately evenly d i s -tributed between the four instars at the time of the f i r s t col lect ion but third and fourth instars predominated during the second. Osmolalit-40 ies of 277 mOsm and 414 mOsm, respectively were measured on water sampl collected with the larvae on the above two dates. The depression im-mediately east of Ctenocladus pond was also flooded on Apri l 19 and £ . campestris larvae were collected from i t as wel l . The concentration of this pool was 75 mOsm. Larvae were maintained in the pond water in which they were col lected, kept at 10°C and fed dried Brewer's yeast. Three experiment a l media were prepared which varied in ionic composition but which a l l had an osmolality of 700 mOsm. (Table 3). Ctenocladus pond water was f i l t e red 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 collected local ly at Vancouver was f i l t e red 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: NaHC03, 46.2g/lj KC1, 0.75g/lj CaC1 2 , 0 .67g/ l ; MgC12, 3.97g/l . Larvae were acclimated for at least four days in these media at 10°C with Brewer's yeast supplied as food, before the rectal f l u i d was sampled. During experiments, larvae were maintained at room temperature (=22°C) . The Aedes taeniorhynchus larvae used for some of the experiments descri bed in this Chapter were reared and experiments were carried out as described previously (Bradley and Phi l l ips 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 total osmolality of 700 mOsm. Sea water NaHC03 (Na+Mg)S04 medium medium medium Na* 347 438 640 K + 8 12 2 Ng + + 39 8 126 CI" 368 26 18 S O / 21 0 377 4 HCO ~ 2.1 529 20.5 42 Ph i l l i ps , 1975; and Chapterl l ) . Sulphate concentrations were estimated 35 = using SO^ (New England Nuclear) counted with a Nuclear Chicago "Isocap 300" l iquid sc in t i l l a t ion system using the channels ratio me-thod of quench correction. Specif ic act iv i ty was determined by measur-ing the radioactivity of the external solut ion, the sulphate concentra-tion of which was determined using the barium precipitation method (Maddrell and P h i l l i p s , 1975). Larvae were exposed to the labelled ex-ternal medium for at least three days, a time period suff ic ient to 35 = permit S0^ speci f ic act iv i ty in the body f lu ids 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 (East-man 910; Eastman Kodak). Isolated recta were prepared by l igat ing larvae with fine s i l k thread around the anal segment of the seventh abdominal segment, to eliminate f lu id 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 cut ic le was torn between the ligatures and this preparation was suspended by the respiratory siphon in approximately 0.1 ml of a r t i -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 petri 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 h). Two a r t i f i c i a l hemolymphs were used (Table 4). Based on a 43 Table 4 The ionic (mM) and osmotic concentrations (mOsm) of the a r t i f i c i a l hemolymphs. Normal Low Hemolymph Chloride Na+ 149 150 K+ 14 14 <+ 5 5 C l " 102 20 27 47 mOsm 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 salts which were used to adjust the f ina 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 levels resulting in a higher (S0~)/(C1~) rat io . A f u l l description of the procedure for the production of these a r t i f i c i a l hemolymphs is available in Appendix IV. RESULTS Survival of A,, campestris in various media. Marked differences were found in the abi l i ty of larvae from Ctenocladus pond (277 mOsm) to withstand increases in external concentra-t ion , as compared to those larvae collected only a few hundred feet away in a less saline pond (75 mOsm). Although the total osmotic concentration of the ponds differed considerably, the relative concentrations of the i n d i -vidual ions were similar (Table 3). When larvae from Ctenocladus pond were taken from their pond water and placed immediately in 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 saline pond showed a mo r ta l i ty of 33$ (n=48) over 4 days when transferred immediately and 11.1% (n=27) when acclimated. Clearly and not surpr is ingly, larvae can better withstand transfer to a strongly hyperosmotic medium i f the solution from which they come i s already fa i r l y concentrated, in this case about isosmotic to the hemolymph, and i f transferral i s gradual. No difference in survival 45 mas detectable in the two groups of larvae collected from Ctenocladus pond at different times, although i t was observed that third and fourth instar larvae can withstand changes of medium better than f i r s t and second instars , perhaps due to the difference in the surface area to volume rat io . In a l l subsequent experiments only third and fourth i n -star larvae collected from Ctenocladus pond were used. Table 5 shows the percent mortality rate of larvae follow-ing transfer directly from pond water to the three experimental media of Table 1. It can be seen that mortality was highest in larvae transferred to 700 mOsm (Na + Mg)S0^ medium as opposed to the 700 mOsm + ++ NaHCO^ and NaCl media. The former solut ion, high in Na , f?lg and S0~ and low in K + and C l " , was clear ly the most toxic of the three media even though the larvae come from natural waters of ident ical ionic com-position but with a total 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 collected i s less conducive to their survival than that of other natural habitats of high sa l in i ty and di f fer ing ionic composition. 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 after A_. campestris larvae were transferred to three experimental media (700m0sm) of d i f fe r -ent ionic composition. (Na+IY!g)S0-4 NaHC03 NaC1 medium medium medium original # of larvae 149 123 25 % 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 in close proximity to the sea (Provost, 1969). They never occur naturally in fresh water and only occasionally in inland saline ponds. It was of interest therefore, to test whether A., taeniorhynchus could survive in the same wide range of saline water as can A., campestris. A., taeniorhynchus larvae can develop in 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 factor or factors in Ctencladus medium prevents these larvae from both hypo- and hyper-osmoregulating. To determine what the toxic elements in Ctenocladus pond water might be, 3 groups of 200 larvae each were raised to third instar in f u l l strength sea water. At that time one group received an additional 200mM/1 NaC1 the second 100mM/1 MgC12 and the third 100mm/1 Na 2 S0 4 < After 24 hours, no larvae had died in 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 possib i l i ty that the increased mortality in either of the two solutions could be due either to increased osmotic, chloride or sodium concentrations. The Mg + + concentration in* the sea water + MgC1? solution was higher than 48 that in (Na+Mg)SO^ medium, suggesting that the toxicity of the lat ter medium to A,, taeniorhynchus larvae i s not due to the high Mg+ + concen-trat ion. Only the sea water + Na^SO^ solution showed the same high level of toxici ty 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 interest 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 d i f f e r -ent s a l i n i t i e s , new steady-state levels of ions in the hemolymph are es-tablished within two days (Ph i l l ips 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 in the hemolymph of A,. campestris were determined after acclimation to the three experimental media for four days. While chloride levels in these media varied from 18 to 368mM (Table 3), those in the hemolymph were regulated within a narrow range from 49 - 6 in NaHC03 to 53 * 5mffl in (Na + Mg) SO^ and 78 - 2mM (n=6) in sea water medium; (n=6) for a l l media. Not surpr is-ingly only traces of sulphate ions ( 0.1 mM, n = 5) were detected in 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 in 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 Phi l l ips (1975) who found that when internal 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 paral le l external levels when exter-nal 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 igation and analysis of f l u i d collected from the lumen at that time indicated that the contents were in a l l cases considerably hyperosmotic (844-968 mOsm; Table 6) to the a r t i f i c i a l hemolymph (346 mOsm; Table 4). Clearly hyperosmotic urine is formed in A,, campestris by secretion of a concentrated f lu id into the rectal lumen, as previously demonstrated for larvae of A.. taeniorhynchus l iv ing in sea water. The concentrations of each ion in rectal secretions from larvae adapted to different experimental media are compared in Table 6 and depicted in F i g . 9 to show the ionic com-position 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 rectal f lu id from whole intact larvae equalled or exceeded that of Table 6 . A comparison of the external ionic and osmotic concen-trations in the three acclimation media, with the same parameters in rectal secretion from two rectal preparations, mean - S . E . , n = 6 except where otherwise indicated: 1) l igated recta bathed in normal a r t i f i c i a l hemolymph and 2) recta in whole larvae. Ligated recta were bathed in high-chloride a r t i f i c i a l hemolymph, except for some preparations, i n -dicated by asterisks, which were exposed to low-chloride a r t i f i c i a l hemolymph (Table 4). 51 Acclimation Media a."~~~Sodium(mM) NaCl medium NaHCOg medium (Na + Mg)S0^ medium b. Potassium (mM) NaCl medium NaHCO medium (Na + Mg)S04 medium c . Magnesium (mM) NaCl medium NaHCOj medium (Na • Mg) SO^ medium d. Chloride (mM) NaCl medium NaHCOg medium (Na • Mg)S04 medium e. Sulphate (mM) NaCl medium NaHCO^ medium (Na • Mg)S0^ medium f . Osmolality (mOsm) NaCl medium NarlCO^ medium (Na • Mg)S0„ Concentrations External Rectal Secretion Media Ligated recta Whole animal in a r t i f i c i a l with anus hemolymph blocked 347 736 + 59 524 46 (n= 438 379 + 39 510 + 37 640 520 + 34 639 + 48 619 71* 8 73 • 17 Not measured 11.5 136 + 12 130 27 1.5 32 + 13 49 + 29 40 29 + 3 Not measured 8 12 • 1 7 + 1 126 124 • 8 100 • 12 368 498 • 53 294 • 55 26 225 • 41 55 • 18 1 f l 230 + 15 70 • 15 40 ± 6 * 21 6 + 2 8 • 2 0 5 • 3 0.7 • 0.1 377 6 8 + + 2 2* 132 + 32 700 1037 • 75 968 • 43 700 938 + 49 844 • 18 700 1007 • 51 943 • 47 769 + 46* low chloride Ringer 52 the external media by as much as 1.5 times (Table 6a). Signif icant differences in the sodium ion content of rectal f l u i d from the three groups of whole larvae could not be demonstrated. Sodium ion levels in rectal secretions collected from ligated larvae adapted to either NaHC03 or (Na + Ng)SC<4 media were s igni f icant ly lower (P<0.01) as compared to sea water adapted larvae. Consequently the sodium level was almost twice as high in f lu id from ligated 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 latter cation was i f anything, enhanced when the chloride level in the a r t i f i c i a l hemo-lymph bathing isolated recta from (Na + MgJSO^ adapted larvae was re-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 level of 135 mM observ-ed in NaHCOj-adapted larvae ( P h i l l i p s , unpublished observations)! therefore this cation i s secreted into the rectal lumen against 2 to 5-fold concentration differences under a l l of the experimental con-ditions of Table 6a. Since sodium concentrations in the three experimental media varied two-fold, these experiments do not demonstrate the large changes in the rectal 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. It is clear from these data that no direct propor-t ional i ty exists between rectal f l u id Na* concentration and the total osmolality of that f l u i d . Sodium concentrations between various rec-tal f lu id samples can vary by a factor of 2.5, while osmotic concen-tration 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 control led, or l imited, than the Na* concentration. Potassium Bradley and Phi l l ips (1975; Chapter II) showed that the rec-tal secretion from A., taeniorhynchus larvae raised in 100% sea water was 12 times higher in potassium content than was the a r t i f i c i a l hemo-lymph and natural hemolymph of whole larvae. The same i s true for A,. campestris (Table 6b); i . e . the potassium concentration in the rectal secretion i s 2-10x higher than in either the hemolymph (14 mM) or the external medium (2 - 12mM). No signif icant differences were found be-tween rectal secretions from ligated larvae versus whole larvae. The potassium concentration in the rectal secretion increased proportion-al ly with that of the acclimation medium, and this relationship pre-vailed even for isolated recta which were bathed in the same a r t i f i -c ia 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 rectal f l u i d from whole larvae. Osmotic Concentration of Rectal Secretion(mOsm) I—1 I-1 00 o fO o o O o o o o o o o o o o o 1 1 1 1 1 1 — r O O 4^ O O 0 - i O O 00 o o o o o © 55 Magnesium As for potassium, the magnesium concentrations in 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 hemolymph having a constant level of this cation (Table 6c). Although there were s l ight differences in the concentrations between f lu id from ligated and intact recta, the values correspond approximately to those in the external medium. The values for A,, campestris in (Na + Mg)S0^ medium agree f a i r l y well with those of Kiceniuk and Phi l l ips (197,4). These authors found that over a range of external Mg + + 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 in Ctenocladus pond water ( 2 - 4 mM; Kiceniuk and P h i l l i p s , 1974) were very low, this cation was secreted into the rectal lumen against 2 to 25 fold concentration differences. Chloride As might be expected, the highest chloride concentrations in rectal secretions, whether from preparations bathed in a r t i f i c i a l hemo-lymph or from whole larvae, were observed in larvae acclimated to sea water medium, which was the only external medium high in chloride (Tab-le 3). The chloride concentration in the a r t i f i c i a l hemolymph (l02mM) was higher than in the larval hemolymph (49 - 78 mM, pg. 48) and accordingly, secretions from the recta bathed in 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 in the rectal f l u i d is influenced by hemolymph chloride con-centration, as subsequently shown (Chapter IV). This conclusion is further supported by the results obtained when an a r t i f i c i a l hemo-lymph with low chloride levels was used. A change in a r t i f i c i a l hemolymph concentration from 102 mM to 20 mM led to a decrease in chloride content of rectal secretion from 230 mM to 40 mM, even though a l l the other ionic concentrations in the two a r t i f i c i a l hemolymphs, except SO", were ident ica l . This difference in the chloride concentration of secretions from recta bathed in a r t i f i c i a l hemolymphs of high and low chloride concentration occurred in spite of the fact that rates of f lu id secretion (Chapter IV) and Na* concen-trations (Table 6a) were relat ively unchanged. Clear ly , the other anions substitute for chloride in the rectal secretions when levels of chloride in the hemolymph, a r t i f i c i a l or rea l , are low. Chloride levels are so low in some of the hypersomotic waters in which iA. campestris is found that a problem of chloride retention must occur. It i s therefore perhaps surprising to find^that, under these condi-t ions, the chloride concentrations in rectal f l u i d from whole larvae ( 5 5 - 7 0 mM; Table 6a) remain s l ight ly higher, or at most, equal to levels in the hemolymph (49 - 56 mffl; Table 4). In such waters, rectal secretion clearly results in a net loss of chlor ide, which must be compensated for at other regulatory s i tes (e .g . anal papillae) because hemolymph levels of this anion are maintained well above those prevai l -ing in NaHC03 or (Na + MgJSO^ external media (26 and 18 mM C l " respect-ive ly , Table 3). 57 Sulphate - A_. campestris As for other ions discussed above, levels of sulphate in rectal f l u id from whole larvae (0.7 to 132 mM; Table 6e) paral le l those in the external media (0 to 377 mM, Table 3). 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 rectal secretion of sa l ine-water mosquito larvae than in the hemolymph, under conditions where the rectum is isolated from midgut input and where the hemolymph concentration can be s t r i c t l y control led. This f inding, that the rectum probably does not contribute substantially to regulation of hemolymph sulphate concentrations, came as a considerable surprise because of the ab i l i ty of A,, campestris to l ive in water in which S0~ represents over 90% of total anions. The fai lure of the rectum to concentrate SO^ is most clearly demonstrated when one examines the rectal f l u id concentration of s u l -phate in larvae acclimated to (Na + MgJSO^ medium. The isolated rec-tum bathed in a r t i f i c i a l hemolymph of high chloride content produced a f l u i d with a sulphate concentration of 6 mM, yet rectal f l u id from whole larvae was found to contain 132 mM. This difference might be due to ionic differences between the a r t i f i c i a l and real hemolymphs, part icularly with regard to chloride concentrations since the rectal epithilium might prefer C l " to SO^. However when the chloride content of the a r t i f i c i a l 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 rectal f l u i d removed from whole larvae exceeded those in the hemolymph. Paired samples were taken from the hemolymph and rectal lumen of larvae and in a l l cases except one, which was isotonic , the rectal f lu id was hypertonic to the hemolymph with regard to sulphate (F ig . 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 cu l ty of establishing laboratory colonies of this species. For this reason we used jA. taeniorhynchus larvae to answer two questions concerning sulphate transport; 1) Could the apparently low sulphate levels in f lu id from ligated recta (Table 6a) be due to 35 lower speci f ic act iv i ty of SO^ in the lumen? 2) If the answer to this question is negative, i s the low sulphate concentration in f lu id from ligated recta a consequence of separating the rectum from the rest of the animal, thereby interfering with normal neural or endo-crine stimulation of sulphate secretion? One group of A., taeniorhynchus larvae was raised in unlabel-led 100$ sea water and a second group in 100$ sea water containing radioactive sulphate with a speci f ic act iv i ty equal to that of the a r t i f i c i a l hemolymph. In this group then, the speci f ic act iv i ty of sulphate in the a r t i f i c i a l hemolymph was ident ical to that in the hemolymph and rectal t issue; and therefore in the rectal secretion as Figure 8. Paired determinations of the sulphate concentration the hemolymph and rectal f l u id of whole larvae. The dashed l ine represents the line of sulphate isotonic i ty . O •iH •P ra U •P d) 0 C O U 300 +J <o •a r - l W o •H -U 0) o w iH f0 o 2 200 V J 1 3 9 100 0 50 100 150 200 Hemolymph Sulphate Concentration (mjyi) 60 wel l . The sulphate concentration in the rectal 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 - 2.5 mM(n=7). The hemolymph contained 27 mM sulphate. These results are similar to those for A,, campestris (Table 6e). Clear ly , differences 35 in speci f ic act iv i ty of SO^ do not explain the low levels of s u l -phate observed in the f lu id from isolated recta. To answer the second question, A,, taeniorhynchus were raised in 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 in 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 rectal f l u id were removed from larvae either immediately or 1.5 hours after the anus was plugged. The rectum was i n i t i a l l y found to contain a small amount of f l u id (maximum volumes removed 11-14 nl) which was yellow and thick with suspended so l id material. The f lu id was presumably derived from the Malpighian tubules either d i rect ly , or perhaps after modification by resorption in the intestine 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 clear f lu id (maximum volumes removed 30-34 n l ) . This f lu id had a lower sulphate concentration (36.3 - 5.5 mM/l, n=7) and was thought to be a di lut ion of the o r ig in -al f l u i d resulting from secretion of rectal f l u id lower in sulphate 61 content. Assuming that a l l the or iginal f l u id remained in the rectum and allowing for a f l u i d secreted during the experimental period which was twice the or iginal volume, the expected sulphate concentration of the rectal f l u i d was calculated to be 21 mM/l. This value i s only s l ight ly higher than that found for recta in a r t i f i c i a l hemolymph (16.3 - 2.5 mW/l, n=7). It would seem therefore, that rectal secre-tion in whole larvae reared in high external concentrations of s u l -phate i s so low in sulphate concentration as to be unimportant in the regulation of hemolymph sulphate levels . Most of the sulphate in the urine undoubtedly derives from Malpighian tubule secret ion. 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 extern-a l medium, one might expect the rectal f l u i d from a l l groups of larvae to have ident ical osmotic concentrations. In f ac t , the osmotic concen-trations of the rectal f l u i d from larvae acclimated to sea water and (Na + Mg)S0^ medium were essential ly ident ical and about 40$ above external osmolal i t ies, but that from NaHCO^ larvae was about 10-15$ lower (Table 6f ) . It would seem that the particular ions present in the external medium have a small effect on the total concentration of the rectal f l u i d . (p>0.4) Chloride levels could be important in 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 in chlor ide, the osmotic concentration of the rectal f l u i d was markedly reduced (Table 6f ) . As previously shown, (page £8) larvae acclimated to different media have different hemolymph chloride concentrations. This var iabi l i ty may be of importance in influencing the osmolality of the rectal secretion. Moreover, the much higher levels of chlor-ide in a r t i f i c i a l hemolymph as compared to the natural hemolymph of c a m p e s t r i s might explain the higher osmolalities of rectal f l u i d from ligated as opposed to whole larvae. 63 DISCUSSION The results for A,, campestris are in agreement with a pre-vious study on A,, taeniorhynchus which demonstrated that sa l ine-water mosquito larvae produce concentrated urine by the secretion of hyperosmotic f l u i d into the rectal lumen. The rectal secretions from these two species are similar in their mode and location of formation. However, secretion from A. campestris larvae acclimated to 700 mOsm sea water (this study) is higher in total osmotic concen-tration 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) but the ion ratios are very s imi lar . In both species, rectal 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 secretion, i f produced in suf f ic ient volumes, could account for the excretion of a l l the ions ingested by drinking but would create a potassium deficiency. The excellent survival of A_. campestris larvae from Cteno-cladus pond in waters of various chemical types (700 mOsm NaCl, NaHCO^ and (Na & Mg)S0^; also in fresh water) indicates remarkable adaptabil-i ty of regulatory processes in this species and excludes the alterna-t ive suggestion that d ist inct physiological races are restricted to part icular 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 relative 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 relative rate of net transport as demonstrated by an increased concentration of an ion in the f lu id 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 papi l lae; AR, anterior rectum segment; MG, midgut; MT, Malpighian tubule; PR, posterior rectal segment. Dashed arrows represent postulated ion transport pathways which are suggested by observations to date, but which have not been direct ly demonstrated. 64b NaCl ^ — C ^ M ^ MEDIUM /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 re-lat ive rate of net transport as indicated by an increased concentration of an ion in the rectal secretion. This secretion can show elevated levels of sodium, potassium, magnesium and chloride when the larvae are acclimated to solutions high in these ions. Even when ligated recta were bathed in identical a r t i f i c i a l hemolymphs, the ionic corn-composition of the secretions tended to ref lect those of the water to which the larvae were adapted. Clearly the transport rate for various ions i s adjusted during adaption either by changes in the amount of protein carriers present in the epithelium (induction) or by turning on of carriers (e .g . decreasing Km) already present through hormonal and neural control mechanisms. The experiments with chloride indicate that the sharp increas-es in chloride concentration of the rectal secretion occur when hemolymph levels are raised substantially above normal. This f inding probably re-f lec ts the fact that both Na+ and Cl~ transport processes, as observed in recta of A_. taeniorhynchus, (Chapter IV) exhibit kinetics l ike those of a l loster ic rather than c lass ica l enzymes. That i s , a sharp increase in ion secretion rate occurs when hemolymph levels of the ion are high. The situation i s di f ferent , however, for sulphate ions which are neither concentrated in the rectal secretion of whole larvae, nor in larvae acclimated to (Na + Mg)S0. medium (377 mffl SO") and placed in 4 4 a r t i f i c i a l 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 dist inct from Malpighian tubule f l u i d , i t was found to tbe hypotonic to the hemolymph. The rectal 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 is not increased by the rectal secretion. I therefore propose that most of the ingested sulphate i s actively secreted by the Malpighian tubules as shown by Maddrell and Ph i l l ips (1975). The f l u i d from the Malpighian tubules must be modified by the intestine or more probably (on ultrastructural grounds) in the anter-ior rectum, as a consequence of ion and water resorption to achieve the concentrations found in the urine of larvae in (Na + Mg)SO^ medium. The following events are thought to lead to the formation of urine in saline-water larvae adapted to hyperosmotic waters. It i s known that the Malpighian tubules, l ike 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 solutes. If the natural water contains high levels of Mg + + and S0~ (e .g . sea water and (Na + Mg) SO^ medium), active secretion of these ions accounts for a proportionally greater fraction of the total solute (Phi l l ips and Maddrell, 1975; Maddrell and P h i l l i p s , 1975). The total transport capacity ( i . e . amount of carrier) for sulphate and possibly magnesium is increased over a per-iod of a day in 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 hemo-lymph at these s i t e s . Meredith and Ph i l l ips (1974a) present u l t ra -structural observations that suggest that this i s the role of the anterior rectum, and further evidence w i l l be presented in this re-gard in Chapter V. I believe that this excretory cycle involving the Malpighian tubules and anterior rectum occurs in laryae in a l l types off-,saline .media as well" as in .f resh^-water. The secretion of a hyperosmotic f l u i d occurs in the poster-ior rectum. The relative rates at which various ions (Na + , K + , M g + + , C l~ and probably HCGj") are transported depends on their concentration both in the hemolymph and in the external medium to which the larvae have been adapted. Only low levels of sulphate are present in the secretion and the movement of sulphate across the rectum is presumed to be passive (see Chapter VI). Saline-water mosquito larvae must be able not only to remove ions which reach abnormally high concentrations in the hemolymph, but also to conserve physiologically required ions in waters low in these ions (e .g . C a + + and Mg + +j,in NaHCO^ medium or C l " in NaHC03 and (Na + Mg)S04 media). It i s useful therefore to ex-amine the ab i l i ty of the rectum to l imit the loss of such ions in the rectal secretion. The rectal 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 in rectal secretion from whole larvae i s 11.3 times higher than that in the external medium. Therefore, as suggested by Bradley and Phi l l ips (1975J? Chapter II) for A,. taeniorhynchus and supported by this study on A,, campestris, I propose that one of the functions of the anal papillae in saline water mosquito larvae i s the uptake of potassium possibly in exchange for hemolymph sodium, as i s thought to occur in the g i l l s of marine teleosts (fflaetz, 1971). In media low in chloride (e .g . NaHCO^ and (Na + Mg)S04 media) the chloride concentration of the rectal secretion i s very much reduced compared to that from larvae reared in seawater. Nevertheless chloride concentrations are at best isotonic to the hemolymph and thus hyperton-i c to the external medium with regard to chlor ide. The rectal secre-t ion , therefore represents a constant drain of chloride from the hemo-lymph in media where chloride concentrations are lower than the hemo-lymph. It is proposed that in media extremely low in chlor ide, the larvae may use the anal papillae for the uptake of chlor ide, much as has been shown for the same species acclimated to dilute media (Ph i l l ips and Meredith, 1969b). This role i s shown in the appropriate media F i g . 9. The direction of this chloride transport may be reversed in media high in chloride (Phi l l ips and Meredith, 1969b) and this con-tribution i s depicted as well in F i g . 9 for larvae acclimated to sea-water medium. Sulphate ions are apparently neither needed by the larvae 69 in their inorganic form nor part icularly toxic in high concentrations in 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 imit osmotic concentration. The model of ionic regulation in saline-water mosquito larvae outlined above and depicted in F i g . 9, explains the d i f fe r -ences in survival in the various media. Media high in Na + , Mg**, C l " and/or HCO^ are not toxic in osmotic concentrations at or above sea water because the larvae can excrete these ions in the posterior rectum as part of a hyperosmotic secretion, containing the proper ratios of these ions. In the case of media high in sulphate,however, the Malpighian tubules and anterior rectum combine to form a f l u i d isosmotic to the hemolymph and high in sulphate. The concentration of sulphate which can be excreted i s therefore limited by the to ler -ance of the larvae to elevations in hemolymph osmotic pressure, the cations associated with sulphate in the Malpighian tubules, and the rate of Malpighian tubule secretion. One or more of these parameters may be the explanation for the lesser tolerance of A,* taeniorhynchus to media high in sulphate compared to i t s close relat ive A. campes-t r i s . Only the lat ter species can survive in (Na + Mg)S0^ medium, indicating 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 7 0 71 INTRODUCTION Hyperosmotic urine i s formed in the larval rectum of the sa l ine-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 ionic composition closely resembling the hyperosmotic external medium, suggesting that the rectum i s the major osmoregulatory organ in these larvae (Chapters II and III). These observations were made on larvae adapted to 100$ sea water or in the case of .A. campestris, other sal ine waters of similar osmolality. I wished to determine how the composition of rectal secretion was affected by changes in 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 rectal secretions in A_. taeniorhynchus larvae adapted to various concentrations of sea water. To discover whether in t r ins ic regulatory responses might be inherent in the ion transport process, as suggested by previous results for CI secretion (page 55), the kinetics of Na+ and CI 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; 100$ Vancouver sea water (832" 8.8 mOsm; Mean * S.E.) 10$ sea water, 50$ sea water and sea water concentrated to one-half or ig inal volume by evaporation at room temperature (200$ sea water). 72 Drinking rates were determined according to the procedure of Bradley and Ph i l l ips (1975? Chapter II). In this study, however, larvae were removed and weighed individually after one hour in the 14 C-carboxy inul in solut ion. A further difference was that special care was taken not only to mince the larvae before KOH digestion, but to cut the midgut into several pieces to achieve better release of inu l in . Drinking rates were determined in the same concentrations of sea water as those in which the larvae were raised. The length and diameter of larvae, par t ia l ly 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. The volume of larvae was calculated from equation ( l ) and surface area from equation (2). These estima-tions are based on the assumption that the 3 h a p e of larvae approximates that of a cyl inder. 3 2 Volume (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 respectively, both in mm. The estimated volume and surface area of larvae of known weight were used to generate equations relating 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 in the absorption mode. Determinations were made on pooled 5 pi samples of hemolymph diluted in 1ml of 0.5$ LaCl^. Su l -phate concentrations were determined by measuring the radioactivity of hemolymph samples of known volume from larvae raised in media contain-35 = ing radioactive SO^. Specif ic act iv i ty was determined by measuring the radioactivity 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 in 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). The a r t i f i c i a l hemolymphs used were based on the or iginal 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 in Table 7. To examine the effect of hemolymph osmolality, one series 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 osmolal i t ies. A second series varied only in chloride concentrations. The stock solution contained a l l const i -tuents except NaCl. Sodium was added as Na^SO^ and various levels of chloride were obtained by replacing different amounts of sucrose with choline chloride so that osmotic concentrations did not vary. A third series of a r t i f i c i a l hemolymphs was devised to examine the ef -fect of varying sodium levelst NaCl was omitted from the stock s o l -74 Table 7 . The ionic (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 ac id , 50; c i t r i c ac id , 25. Na succinate and Na glutamate were omitted. Variable Chloride Concentration Series Variable Osmotic Concentration Series Variable Sodium Concentration Series Na+ 150 150 variable K + Mg 18 5 15 5 18 5 C l " variable 100 100 variable 27 variable mOsm 350 variable 348 75 ution and chloride was added as the choline s a l t . Sodium levels were varied by substituting Ha^SO^ for di f fer ing 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 igni f icant ly when the osmotic concentration of the sea water in which larvae were reared ranged between 10$ and 200$ (F ig , 10). The average —1 —1 ingestion rate was very high indeed (B.Apl • larvae day ) for larvae of average weight (3.5 mg). This result was a surprising one because salt-water mosquitoes l iv ing in hyperosmotic waters must drink to replace water lost by osmo-s is across the body wall and by excretion. If such losses were the only factor control l ing drinking rate, then drinking should increase as the sa l in i ty in external media i s raised. Moreover drinking should cease in hyposmotic media, as observed for 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 control l ing drinking, the drinking rate of individual ly weighed larvae was determined. Nicholson and Leader (1974) obtained two nearly identical estimates for the surface area of the mosquito larvae, Qpifex fuscus, f i r s t l y by measuring i t s diameter and length and assuming i t to be a cyl inder, and secondly by measuring the total content of cuticular Figure 10. The drinking rate of larvae in the media in which they were reared. The regression line was f i t t ed by the least squares method. 180 External Concentration (% seawater) 77 wax. These values agreed to within 3%. I therefore made similar mea-surements of the dimensions of Aedes taeniorhynchus larvae and calcu-lated the body volume and surface area for larvae over a wide range of s izes . The relationship between the weight of the larvae (x) and the calculated volume of the same individual (y) can be accurately described by the equation (y = - 0.19 + 1.07 x) (P ig . 11). The y i n -tercept i s very close to zero and the slope i s nearly one. This re-sult i s very close to that expected i f one assumes that a l l the larvae have a speci f ic 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) and weight (x) of the larvae i s adequately expressed by the equation 0.75 y = 0.13 + x ' (F ig . 12). The relationship between surface (y) of 0 67 any object relative to i t s weight (x) can be expressed by y = x i f the object retains the same shape as i t increases in size (Schmidt -Nielsen, K.; 1975). The relationship found for 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 increas-ing in length proportionally faster than in width. These relationships (Figs. 11 and 12) were used to calculate the volumes and surface areas of individually weighed larvae from 10%, 50$, 100$ and 200$ sea water whose drinking rates were subsequent-ly determined. The empirical relationship between the drinking rates (y) of individual animals and their volume (x) is shown in F i g . 13. a Figure 11. The relationship between the weight and volume of A,. taeniorhynchus larvae. The regression l ine (Y = -0.19 + 1.07X) was f i t ted 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 ine (Y = 0.13 • X * ) was f i t ted by the least squares method (R = 0.98). Figure 13. The relationship between the volume and drinking rate of larvae in four s a l i n i t i e s ; • 10$ sea water, A 50$ sea water, 0 100$ sea water, • 200$ sea water. Drinking rates were measur-ed in the media in which the larvae were raised. The regression 0.72 l ine (Y = 0.01 + X ) was f i t ted by the least squares method (R = 0.80). Drinking Rate(nl- l a r v a - 1 ' h" 1) Q.08 81 0 72 The regression l ine i s expressed by the equation y = 0.01 + x * The same relationship exists between drinking rate and the weight of the larvae since weight and volume are l inearly related (F ig . 11). Clear ly , the drinking rate of the larvae in 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 fashion, for the exponent of x is 0.72, close to the relationship between weight and surface area. F i g , 14 shows the relationship between the drinking rate and surface area of the larvae. The equation for the regression 1.05 l ine shown is expressed by the equation y = 0.08 + x , i . e . very close to l inear . The correlation coeff icient i s high (r = 0.80, P<0.01). In summary, the drinking rate of A,, taeniorhynchus larvae is not s igni f icant ly affected by the sa l in i ty of the external so lu -tions over the range of the sa l in i t i es tested. Instead, the size of the larvae determines the rate at which they drink. The change in this rate with increasing size of larvae paral le ls the rate of change of their surface area. Hemolymph Ion Levels Hemolymph ion levies were measured in larvae raised in 10%, 100% and 200;^  sea water. Sodium, magnesium and potassium were s t r i c t l y regulated in that no s t a t i s t i c a l l y s igni f icant concentration differences could be detected between larvae reared in these three concentrations of sea water (F ig . 15). Hemolymph calcium levels i n -crease s igni f icant ly (P<0.001) however, with each increase in exter-Figure 14. The relationship between the surface area and drinking rate of larvae in four s a l i n i t i e s ; • 10$ sea water, A 50$ sea water, 0 100$ sea water, • 200$ sea water. Orinking rates were measured in the sa l in i ty in which the larvae were raised. The regression l ine 1 05 (Y = 0.08 + X ) was f i t t ed by the least squares method (R = 0.08). Drinking Rate (nl- larva"! .h~l) <128 Figure 15* The mean concentrations of sodium ^ , potassium O • magnesium | and calcium A in the hemolymph of larvae reared in sea reared in sea water of di f fer ing s a l i n i t y . Ver t ica l bars denote S . E . of the means unless these are smaller than the symbol. Each point is an average for 10 (Na , K , fflg ) or 5(Ca ) larvae. 83b 84 nal sa l in i t y . The total 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), indicat ing, i f any-thing a s l ight decrease with increasing external s a l i n i t y . Anion regulation was less precise than that of the cations. Chloride concentrations and osmolalities showed para l le l trends (F ig . 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 ( less than 25$ i n -crease) which were not s ta t i s t i ca l l y s igni f icant ly different from each other. Nayar and Sauerman (1974) have measured chloride and osmotic pressure in A,, taeniorhynchus larvae aa well and found the the same pattern of regulation. Sulphate concentrations on the other hand were low in 10$ and 100$ sea water but increased 3.4-fold in 200$ sea water. This suggests that sulphate i s closely regulated at lower levels but less so at higher concentrations (discussed by Mad-dre l l and P h i l l i p s , 1975). The total 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 ba 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 in the hemolymph increase less than does the hemolymph osmolality when larvae l iv ing in 10$ and 200$ sea water are compared. Unless ionic ac t iv i t i es undergo consider-able changes, which has been shown in some insects (Treherne, Buchan Figure 16. The mean chloride 4) * sulphate O a n d osmotic concentration in the hemolymph of larvae reared in three media of d i f fer ing s a l i n i t y . Vertical bars denote S . E . of the means unless these are smaller than the symbol. Each point i s an average 10 (Cl~ , mOsm) or 9 (S0~) larvae. 85b ! 0 % 100% 2 0 0 % External Concentration (% seawater) 66 and Bennett, 1975) the contribution of inorganic ions to total osmol-a l i ty of the hemolymph decreases in the more saline external media. Ionic and Osmotic Concentrations of Rectal Secretion a) In 10$ Sea water When l igated recta from larvae raised in 10$ sea water were placed in 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 secrete, but the small am-ount of f l u id observed in the rectal lumen i n i t i a l l y is so reduced during the 1.5 hour incubation period, that i t i s d i f f i c u l t to obtain a f lu id sample. When whole larvae from 10% sea water had their anuses block-ed with tissue adhesive, f l u id accumulated in the rectum, presumably due to f lu id entry from the Malpighian tubules. This f l u i d was so dilute and such small quantities were avai lable, that only the osmotic concentration of the f lu id was measured ( f i g . 17). The average osmolality of this f lu id was found to be 170± 11 mOsm (n = 8) or 50$ of hemolymph values. This agrees fa i r l y well with the measurements of Ramsay (1950) for the ractal f lu id osmotic concentration of A_. detritus larvae (0.33$ NaCl, 105 mOsm). The fact that the A., taenior-hynchus larvae were acclimated to a more concentrated medium (10% sea water) than the A_. detritus (d is t i l l ed water) might account for the higher rectal f l u id concentration found in the former species, but ne i -ther saline-water species achieves the low rectal f l u i d concentrations a Figure 17. The ionic and osmotic concentrations (mean - S.E., n = 6) of rectal f l u id from whole larvae in 10$ sea water 100$ sea water and 200$ sea water The arrows indicate the respective concentrations in the external medium, CO a o +> (0 M •P 0) O O u o • r l o H 1100 1000 900 800 700 600 500 400 300 200 100 0 1 Na+ 2000 1800 1600 1400 1200 1000 800 600 400 200 Mg++ c i - S 0 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 is the s i te of sa l t resorption in larvae acclimated to hyposmotic media (Ramsay, 1950). b) In 100$ sea water The ion concentrations in the rectal f l u i d from whole l a r -vae reared in 100$ sea water were described in deta i l by Bradley and P h i l l i p s (1975) (Chapter I i ) . It was suggested that the apparent anion def ic i t observed in the rectal f l u i d (68 mEq) might be due to sulphate ions. For this reason, the sulphate concentration in rectal f l u i d was measured and found to be 17 - 3 mM (n=10); that i s , this anion accounts for 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 ra-ther than the rectum and this might be the source of much of the s u l -phate observed in rectal f l u i d from whole larvae. Therefore, the anion de f ic i t which i s observed in the rectal f l u i d from isolated recta that contain l i t t l e of this anion must be due to some other un-measured anionic component (e .g . bicarbonate or negatively charged macromolecules.). The ionic and osmotic concentrations of rectal f l u i d from larvae adapted to 100$ sea water are close to those in sea water (F ig . 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 interest to determine 89 whether concentrations of rectal secretion are adjusted to match those of more concentrated external media, just as ion ratios in the sec-reted f l u i d are regulated to match those in various chemical types of hyperosmotic waters of equal osmotic concentration (Chapter III). c) 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 levels in the rectal secretion were higher in whole larvae from 200$ sea water than in those from 100$ sea water (F ig . 17). Sodium and chloride concentra-tions in the rectal secretion of larvae reared in 200$ sea water were s l ight ly lower than those in the external medium, although the stand-ard errors overlapped the latter values. Since larvae must excrete ions they ingest, i t follows that either some other organ i s aiding in the removal of Na+ and C l " from the hemolymph (e .g . anal papillae) or the rectal preparation used in this study does not accurately indicate the maximum concentrations developed in undistributed larvae. The levels of magnesium and sulphate observed in the rectal secretion equalled those in 200$ sea water. Bradley and Ph i l l ips (1975) (Chapter II) found that the potassium concentration was 16 times higher in rectal secretion than in 100$ sea water. In the present study, the level of this cation in rectal secretion from larvae l iv ing in 200$ sea water was found to be lower than that in larvae from 100$ sea water, unlike a l l other ions measured. However, the rectal sec-90 retion level was s t i l l 3.6-fold higher than the external concentration. Factors Regulating Ionic and Osmotic Concentrations of Rectal Secretion Having established that ion concentrations of rectal secretion are influenced by the concentration of the external medium (this study) and by the relat ive concentrations of ions in the medium (Chapter III). I wished to obtain more information concerning the regulation of the ionic and osmotic composition of rectal secretion. Since the composi-tion of this f l u i d ref lects concentrations in the external medium, larvae obviously monitor changes in external ion leve ls . The rectum is bathed in hemolymph and the most direct method of regulatory response would be a sensi t iv i ty of the rectal secretion to changes in hemolymph ion levels brought about by the ingestion of the external medium. The simi lar i ty of hemolymph ion levels in larvae adapted to 100$ and 200$ sea water (Figs. 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 i n i t i a l l y 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 rectal secretion may be one form of short-term response to such abrupt external concentration chan-ges. Therefore, the effect on rectal secretion of varying only one parameter of a r t i f i c i a l hemolymph composition at a time was investiga-ted. A l l larvae used in these experiments were raised in 100$ sea water. 91 Figure 18 shows the effect of varying hemolymph osmotic concentration through the addition of sucrose, on the osmotic con-centration of rectal f lu id produced by ligated recta bathed in a r t i f i c i a l hemolymph. In a r t i f i c i a l hemolymph of low osmotic con-centration (173 mOsm; Table 7) the rectum is 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 rectal f l u id may be due to tissue swel l -ing in dilute media with a subsequent reduction in in t race l lu lar osmotic and ionic 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 rectal f l u id had the same osmotic concentration as sea water (about 800 mOsm), which was similar to that found in whole animals. This suggests that the osmotic concentration of the hemolymph as dist inct from the ionic concentrations, does not have a s igni f icant effect on the osmotic concentration of rectal secretion. The physiological range of hemolymph osmotic concentrations observed by us in A. taeniorhynchus (303 - 427 mOsm; F ig . 6) is substantially narrower than the experimental range of F i g . 18. The rate of rectal secret ion, as judged by unquantified observations of rectal swell ing, 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 rectal secretion proved to be insensit ive to that in the hemolymph over the normal physio-logical range, I considered the influence of changing hemolymph chlor-ide levels when a l l other parameters were held constant, except for Fig 18. The effect of a r t i f i c i a l hemolymphs di f fer ing only in osmotic (sucrose) concentration on the osmotic concentration (mean - S . E . , n = 6) of rectal secretion. The l ine was drawn by eye. 93 sulphate concentrations. Changes in the hemolymph concentrations of chloride seemed a l ike ly basis for control of rectal secretion be-cause of the var iab i l i ty in hemolymph levels of this ion (F ig . 16). The rectal secretion was found to be hypertonic to hemo-lymph with regard to chloride at every hemolymph chloride concentra-tion tested (F ig . 19). However, the chloride concentration in rectal f l u i d increased more sharply at higher hemolymph chloride levels . Ths steep slope for the increase in this relationship when hemolymph con-centration of chloride rises above 100 mM i s of part icular interest , since this is the normal hemolymph leve l . Thus in larvae introduced to media higher in chloride, the hemolymph chloride might i n i t i a l l y r ise above the average range observed for adapted animals. This would cause the chloride concentration of the rectal secretion to increase substant ial ly , thereby reducing the chloride concentration in the hemolymph. That i s , a degree of in t r ins ic regulation incorporated within the transport process is possible. Figure 19 also shows that increasing hemolymph chloride, in the absence of osmotic concentration changes leads to s l ight ly e l -evated osmolality of rectal secretion. However, this change i s not s igni f icant over the physiological range (50 - 100 mM). As with the concentration of chloride, the osmotic concentration of rectal f l u i d shows the steepest increase at hemolymph chloride levels above 100 mM. The rate of rectal secretion, as estimated by the size of the largest three samples out of 15, obtained by micro-puncture, shows a positive correlation to the hemolymph chloride concentration as well (F ig . 19). Figure 19. The effect of varying chloride concentration in a r t i f i -c i a l hemolymph on the volume of rectal secretion collected after 1.5 ti A ( n = 3 ) , osmotic concentration O (n = 6) and chlor -ide concentration £ (n = 6). Vert ical bars denote S .E . of the means• Maximum Volume of Rectal Secretion Removed (nl) 95 The product of f l u i d s e c r e t i o n rate and c o n c e n t r a t i o n y i e l d s an es 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 are not of the Michaelis-Menten type. The above f i n d i n g s i n d i c a t e that the c h l o r i d e c o n c e n t r a t i o n of 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 per-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 . However, both J\. taeniorhynchus and A,, campestris can s u r v i v e i n waters 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 f l u i d of a p p r o p r i a t e l y low c h l o r i d e concentration under these c o n d i t i o n s , even though hemolymph concentrations of c h l o r i d e are maintained at or above 50 mM. C l e a r l y , some other parameters other than hemolymph c h l o r i d e l e v e l s must regulate r e c t a l f u n c t i o n as w e l l . Figure 20 shows the r e s u l t s of va r y i n g hemolymph sodium l e v e l s on sodium and osmotic concentrations 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.02) was found between the sodium concen-t 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 having a sodium con c e n t r a t i o n of 150 mM (285 - 29 mM, n * 11) and that from r e c t a i n hemolymph with 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 organic a c i d s were r e -duced (Table 7 ) . As with c h l o r i d e , the c o n c e n t r a t i o n of sodium i n rec-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 of sodium were r a i s e d . The sodium c o n c e n t r a t i o n of r e c t a l f l u i d (128 mM) remained n e a r l y constant at hemolymph sodium concentrations between 5 mM and 50 mM, Although the sodium concentration of hemolymph was s u b s t a n t i a l l y reduced to 5 mM i n some experiments, a hyper osmotic r e c t a l s e c r e t i o n was s t i l l Figure 20. The effect of varying sodium concentration in a r t i -f i c i a l hemolymph on the volume of rectal secretion collected after 1.5 h A (n = 6) , osmotic concentration Q (n = 6) and sodium concentration ^ (n = 6). Vert ical bars denote S .E . of the means. Rectal Secretion Sodium Concentration (mM) 97 formed under these conditions (634 - 31 mOsm, n = 7). The potassium ion concentration in this f lu id was very high (280 - 70 mOsm, n = 7) suggesting that potassium ions can part ia l ly substitute for sodium ions in the secreted f lu id when levels of the lat ter ion are low. The net rate of ion secretion can be calculated by multi-plying the volume of f l u i d secreted times the concentration of the ion in the rectal secretion (F ig . 21). Since the volume secreted has been assumed to be equal to the volume removed from the recta, and may therefore be subject to s l ight underestimation, the curves in F i g . 21 are meant to show the relative rather than absolute transport rates. These curves indicate that the response of sodium and chloride trans-port rate to increasing levels of these ions in the hemolymph does not follow Michaelis-Menten k inet ics. Instead, over the range of hemolymph ion levels observed in, vivo, the curves show increasing con-centration, suggesting kinetics l ike those of a l los te r ic enzymes. Figure 21. The relationship between the concentration of chloride 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 ion . <i86 99 DISCUSSION Beadle (1939) was the f i r s t to propose that larvae of sal ine-water mosquitoes drink the external medium as a means of replacing water lost by osmosis across the cut ic le and by excretory processes. The present study confirms this f inding, 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 ig . 14) and to be unaffected by external sa l in i ty (F ig . 10). Decreased osmotic permeability of the body wall and changing concentration of the ex-creta might possibly compensate for a simi lar drinking rate by larvae in the two hyperosmotic media. However, no change in body wall perm-eabi l i ty was observed by Phi l l ips in A., campestris (Ph i l l ips and Brad-ley , in press). In 10% sea water, moreover, water moves into larvae both by osmosis across the integument and by drinking. Under these conditions high rates of ingestion are clearly detrimental to osmotic homeostasis and must serve some other function, such as nutrient ( i . e . energy) procurement. I suggest that the drinking rate of sa l ine-water mosquito larvae is 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 in this study were starved for 24 hours prior to the determination of drinking rate, and under these condi-tions show many signs of hunger (e .g. constantly moving mouthparts 100 and foraging along surfaces). 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 red prior to use and the inu l in was fu l l y dissolved o 14 by heating to 90 C. Therefore, any nutrients and C- inul in taken up had to be in dissolved form. 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 in the external medium. The rate of this act iv i ty i s proportional to the metabolic rate of the larva and hence proportional to surface area, and i s un-affected by the sa l in i ty of the external medium. Such act iv i ty may be stimulated by certain organic solutes in natural waters, and variations in concentration of the latter might explain different estimates of ingestion at the same s a l i n i t y . The estimate for the drinking rate for larvae reared in —1 —1 100$ sea water in this study (100 n l . mg . 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 ident ical ly in the two studies with the exception that in the present procedure the larvae were weighed individually and the midgut was cut into 3-4 pieces in addition to mincing the whole larva before K0H digestion. Whether this aided the release of inul in from the gut contents and account-ed for the whole three-fold difference i s not known. It may be calculated from values observed in this study, that larvae drink an amount of f l u id equivalent to their total 101 weight every 10 hours. The turnover times for total body contents in 100% and 200% sea water are 3.B and 1.9 hours respectively for sodium; and 4.B and 2.4 hours respectively for chlor ide. These animals clear ly 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 in reducing the rectal f l u i d concentration necessary to achieve homeostasis in hyper-osmotic media (Ph i l l ips and Bradley, in press). This i s because the greater the ratio of the f l u i d ingested to water lost across the i n -tegument via osmosis, the closer the rectal f l u i d concentration requir-ed to achieve osmotic balance approaches that of the external medium. If the permeability of the external cut ic le of A,, taeniorhynchus i s similar to that of Opifex fuscus (Nicholson and Leader, 1974) then the calculated osmotic water loss for a 3 mg larva i s 38 nl . h in 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 rectal secretion only 14% more concentrated than the external medium for osmotic balance, whereas the average value observ-ed for whole larvae (Bradley and P h i l l i p s , 1975) was 11% higher. The observed osmotic concentration of the rectal secretion i s therefore very close to the predicted value necessary for osmotic homeostasis. As discussed below, some ions cannot be regulated by means of the rectal secretion and other regulatory s i tes must play a v i t a l role (e .g . Malpighian tubules, anal papi l lae) . Aedes taeniorhynchus larvae show impressive a b i l i t i e s to 102 regulate hemolymph i o n concentrations i n e x t e r n a l media varying from 10$ to 200$ sea water. In p a r t i c u l a r , the l e v e l s of Na +, K + and Mg 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 over 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 calcium i n c r e a s e s s i g n i f i -c a n t l y with i n c r e a s i n g e x t e r n a l c o n c e n t r a t i o n . This i n c r e a s e i n hemolymph calcium l e v e l may be necsssary f o r osmoregulation i n more hyperosmotic waters. For example i n the f r e s h water mosquito l a r v a , Culex p i p i e n s , increased e x t e r n a l calcium promotes b e t t e r s u r v i v a l of larv a e placed i n hyperosmotic media high i n sodium (Bradley and P a r k i n s , 1975). Hemolymph anion 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$. Nayar and Sauerman (1974) showed th a t the c h l o r i d e concentra-t i o n i n the hemolymph of A,, taeniorhynchus i s not l i n e a r l y r e l a t e d to the e x t e r n a l c o n c e n t r a t i o n but i s s t r i c t l y r e g u l a t e d a t one l e v e l i n a l l hyposmotic d i l u t i o n s of sea water and e q u a l l y s t r i c t l y at a s l i g h t -l y higher l e v e l i n a l l hyperosmotic ones, i . e . an abrupt change i n hemolymph co n c e n t r a t i o n occurs over a narrow range of e x t e r n a l concen-t r a t i o n . Sulphate ions show a d i f f e r e n t p a t t e r n of r e g u l a t i o n . Low hemolymph l e v e l s are found i n animals reared i n 10$ and 100$ sea water, while higher l e v e l s are found i n larvae from 200$ sea water. This i o n i s excreted l a r g e l y by the Malpighian tubules (Chapter I I I ) and t h i s pattern of r e g u l a t i o n r e f l e c t s the f a c t that the Malpighian tubules can only produce a s e c r e t i o n isosmotic to the hemolymph. A s i m i l a r p a t t e r n of sulphate r e g u l a t i o n was observed i n £. campestris by 103 Waddrell and Ph i l l ips (1975). The results cited in this chapter shpui that the concentrations of a l l but one ion were higher in the rectal f l u i d from larvae reared in 200% sea water than in that from animals l iv ing in 100% sea water. If larvae are raised in a medium with increased level of either Na + , + — ++ K ,C1 or Mg , then the rectal secretion contains elevated levels of the ion in question (Chapter III). It i s therefore clear that the secretion of rectal f l u id is a major mechanism in the osmoregulation of saline-water mosquito larvae. The only ion whose secretion is 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 in the external medium. Bradley and Ph i l l ips (1975); also Chapter II suggest that this potassium loss from the hemolymph through secretion may be replaced by potassium uptake from the medium by the anal papi l lae . It is interesting to speculate why the potassium concentra-tion of the rectal secretion i s lower in 200% than in 100% sea water. In a recent paper, Bodil Schmidt-Nielsen (1975) proposed that c e l l shrinkage in hyperosmotic media is accompanied by the excre-tion of extracellular potassium. This mechanism might be important in some short-term experiments involving rapid changes in a r t i f i c i a l hemolymph concentrations. However the composition of normal rectal f l u id from larvae adapted to 100% and 200% sea water involved animals which had been reared in these solutions since hatching and thus vo l -ume adjustments should not have changed during experiments. A more 104 + + l ike ly explanation i s that Na and K compete for the same transport mechanism in the rectum. This explanation gains some support from the observation that rectal secretion is very high in potassium (280*. 70 mM, n = 5), when recta are incubated in a r t i f i c i a l hemolymph which has a low Na+ concentration. In 200$ sea water, magnesium levels in the rectal secretion were higher than in the external medium but sodium chloride and osmotic concentrations were on the average s l ight ly lower. Two poss-ib le explanations for this phenomenon come to mind. A considerable amount of evidence has been cited in previous papers (Phi l l ips and Meredith, 1969; Bradley and P h i l l i p s , 1975; Ph i l l ips and Bradley, in press) suggesting that the anal papillae are a s i te of chloride and possibly sodium excretion in water high in these ions. The low con-centrations of these ions in rectal secretion from larvae l iv ing in 200$ sea water are consistent with such a conclusion. Alternatively, blockage of the anus in whole larvae, as discussed previously by Bradley and Phi l l ips (1975) and Chapter II could lead to a backflux of ions from the rectal lumen to the hemolymph and increased osmotic flow of water in the reverse direction due to excessive swelling of the rectum under these experimental conditions. Some of the factors influencing mechanisms control l ing the ionic and osmotic concentrations of the rectal 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, rectal secretion i s a major means of ion ex-cret ion. Obviously, some neural or hormonal control exists which dictates whether the rectum wi l l function as a net resorptive or secretory s i t e . Long-term changes in rectal function may be in i t ia ted by external parameters (e .g . via external osmoreceptors or sa l t recep-tors) , or control mechanisms which respond to hemolymph ion levels over a time course longer than our experimental period (1.5 - 2 hrs) . While the relat ively constant levels of the ions observed in hemo-lymph of adapted larvae (Figs. 15 and 16) might seem inconsistent with internal receptors, i t is reasonable to assume that abrupt changes in external sa l in i ty do in fact result in sizable transient changes in hemolymph ion levels . Variations in total hemolymph osmotic con-centration, within physiological levels , have no effect on the osmotic concentration of the rectal secretion. The hemolymph levels of at least two ions sodium and chloride, have a profound influence not only on the secretion concentrations of these two ions but also on the osmotic concentration of the rectal f l u i d and i t s rate of secretion. Increases in sodium or chloride concentrations In the hemo-lymph lead to a higher concentration of this ion in the rectal secre-tion (Figs. 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 ions. 106 The slopes of the curves i n f i g u r e 21 i n d i c a t e t h a t the r e l a t i o n s h i p between i o n concentrations 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 rate do not f o l l o w Michaelis-Menten 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 curve. Whether t h i s i s a property of the t r a n s p o r t mechanisms themselves or i s a f u n c t i o n of changing r a t e s of access of the ions to these mechanisms (e.g. due to 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. Regard-le 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 correspond to the s t e e p l y r i s i n g part of the curve f o r the t r a n s p o r t k i n e t i c s ( F i g . 21). C l e a r l y , any t r a n s i e n t r i s e i n hemolymph sodium or 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 r e s u l t i n increased s e c r e t i o n of these ions by the rectum. 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 morphologically d i s t i n c t segments of the rectum (Aedes  d e t r i t u s , Ramsay, 1950; flL. campestris. Meredith and P h i l l i p s , 1973; A,, taeniorhynchus, Bradley and P h i l l i p s , 1975). U l t r a s t r u c t u r a l evidence suggests that 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 hyperosmotic f l u i d s e c r e t i o n (Meredith and P h i l l i p s , 1973a). I there-f o r e monitored changes i n f l u i d volume and osmotic 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 attempt 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 occuring i n the two p a r t s of the rectum, p a r t i c u l a r l y with regard to the l o c a t i o n of the s i t e of hyper-osmotic f l u i d s e c r e t i o n . MATERIALS AND METHODS Aedes taeniorhynchus larvae were r a i s e d i n 100$ sea water as p r e v i o u s l y described (Chapter I I ) and s t a r v e d f o r 1 - 2 days p r i o r to use. An in v i t r o p r e p a r a t i o n of the rectum was prepared by d i s s e c t i n g larvae i n normal hemolymph (Chapter I I ) . The gut was removed from the larvae and l i g a t e d w i t h f i n e s i l k thread at any two of the f o l l o w i n g three p o i n t s as a p p r o p r i a t e : the p o s t e r i o r part of the i l e u m , the j u n c t i o n a l region 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, or the anal canal 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, or the p o s t e r i o r or a n t e r i o r segments i n d i v i d u a l l y . The regions 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 placed 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 cover of a 60 x 15 mm glass p e t r i d i s h (Marks and Holman, 1974). The bottom of the p e t r i d i s h was f i l l e d with d i s t i l l e d water to r e t a r d evaporation. The osmotic concentration of the hanging drop increased by l e s s than 15 mOsm during the two hour experimental p e r i o d . A f t e r two hours, r e c t a were removed from such drops with an eyedropper and placed on a micro-scope s l i d e , where the adhering f l u i d was b l o t t e d up w i t h f i l t e r paper. The rectum was punctured using a glass 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. The osmotic c o n c e n t r a t i o n of the sample was measured immediately using a n a n o l i t e r osmometer ( C l i f t o n T e c h n i c a l P h y s i c s , L t d . ) . For 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 procedure see Appendix I I . RESULTS Se c r e t i o n by r e c t a l segments The in, v i t r o preparation of the l a r v a l rectum was used to d i f f e r e n t i a t e the f u n c t i o n s of the a n t e r i o r and p o s t e r i o r segments of the rectum. This was most e a s i l y done by p l a c i n g a l i g a t u r e be-tween the two segments and monitoring f l u i d c o n c e n t r a t i o n s and v o l -umes i n the two p o r t i o n s with time. The r e s u l t s of such an e x p e r i -ment could not be compared to previous 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 neural and t r a c h e a l connections to the rectum. For t h i s reason an i n v i t r o preparation of the whole rectum served as a c o n t r o l f o r comparison with 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 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 vitro preparation of a whole isolated rectum 5 minutes and 2 hours after dissection, respectively. The rectum swells with f l u i d during the 2 hour experimental period as previously observed for in, vivo pre-parations of the rectum (Bradley and P h i l l i p s , 1975 and Chapter II). Similar results were observed for 23 preparations. Photographs 3 and 4 of Plate 2 show the posterior segment of a single rectum at 5 min-utes and 2 hours after dissection, 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 walls. Nevertheless some swelling of this segment i s obvious, i f less marked than for the whole rectum (11 observations). No change in volume could be discerned in any of the 13 pre-parations of the anterior rectal segment observed. Because of the small volume of the anterior rectum, i t i s impossible to conclude ab-solutely from photographic data alone that the anterior rectal portion either secretes or resorbs any f l u i d in v i t ro . However since both the whole rectum and posterior rectal portion do f i l l with f lu id when isolated, we were interested in determining whether the secretion in vitro was hyperosmotic to the hemolymph as previously demonstrated in 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 ingle , whole in, vitro rectum 5 min and 2 h after l igat ion, respectively. In photo-graph 2 the rectum can be seen to be f i l l e d with secreted f l u i d . Photographs 3 and 4 show a s ingle , isolated posterior rectal preparation 5 min and 2 h after l iga t ion , respectively. In photograph 4 of the posterior segment can be seen to be swollen with secreted f l u i d . The i n i t i a l shape of the segment outlined in white. Similar photographs for anterior rectal segments showed no change in volume. XUb 112 Osmotic Concentrations of rectal secretion in v i t ro The same procedures as were used to obtain photographic e v i -dence of rectal secretion were followed to obtain f l u i d from the lumina of whole recta and isolated posterior and anterior rectal segments (Table 8). When whole recta were dissected from larvae, l igated, and immediately sampled by micropuncture, the rectal 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 rectal f l u i d concentration was high compared to that pre-viously observed with isolated in, vivo preparations possibly because of the following difference in procedure. Larvae used for in vitro 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 rectal con-tents. During preparation of ligated in, vivo preparations, for which i n i t i a l rectal concentration values are available (Bradley and P h i l l i p s . 1975; and Chapter II), the rectum completely empties and part ia l ly 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 lu id in whole irj. v i tro recta increased over 2 h (Photographs 1 and 2 of Plate 2), the f ina l osmotic concentra-tion of the secreted f l u i d was not s igni f icant ly dif ferent from that i n i t i a l l y present (Table 8). The isolated posterior segment of the rectum f i l l s with a f lu id which was signi f icant ly hyperosmotic to the i n i t i a l rectal f l u i d , the a r t i f i c i a l hemolymph, and also secretion from in vi tro whole recta after 2 hours (P<0.02). In the isolated 113 Table 8. The osmotic concentrations of rectal f l u i d removed from lumina of ^n vi tro preparations of the rectum. Concentra-tions are given in the two le f t hand columns for samples from whole recta taken i n i t i a l l y or 2 h after l igat ion. The two right hand columns show the osmotic concentration of f l u i d re-moved 2 h after l igation to isolate the posterior from the an-terior rectal segment. Osmotic concentration of the a r t i f i c i a l hemolymph used in a l l experiments was 355 mOsm. Whole Rectum Rectal Segments (mOsm) (mOsm) Serial 0 hr 2 hr Anterior Posterior (2 hr) (2 hr) 1 403 457 446 753 2 409 522 452 640 3 551 503 511 441 462 419 495 468 4 5 500 6 7 Mean 475 480 473 1183 425 742 672 484 424 505 444 711 1 S ' E ' 1 2 9 1 13 ! 8 t 88 114 anterior rectal portion where no volume change was discernable, a s igni f icant reduction (P<0.05) in osmotic concentration occurred during the two hour experimental time period. Possibly, the fai lure of the whole rectum ^n vitro to secrete f l u i d as concentrated as the posterior rectum alone i s due to solute reabsorption in the an-ter ior rectum. This i s consistent with the decline in f l u i d concen-tration in isolated anterior rectal segments. The relat ively low concentration of the rectal secretion in  vi tro could ref lect lack of a natural neural or hormonal stimulus. Compounds known to stimulate secretion in some other insect tissues were therefore added to the a r t i f i c i a l hemolymph bathing in, v i t ro rectal preparations. No signi f icant increase in rectal f l u i d concen-tration was found upon adding cyc l ic 3* - 5' adenosine monophosphate —2 —2 (10 M) with or without theophylline (10** W), or 5 hydroxytryptamine (10"4M); (Table 9 ) . The results of these experiments using in, v i t ro rectal pre-parations indicate that the posterior rectal segment i s a s i t e , i f not the only one, of hyperosmotic f lu id secretion. The anterior rectal segment seems to reduce the concentration of the rectal f l u i d in  v i t ro , which is consistent with the suggestion that this part of the rectum is involved in solute reabsorption (Meredith and P h i l l i p s , 1974). 115 Table 9. The osmotic concentration (mOsm) of rectal 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. Ser ia l Cycl ic Cyclic AMP(lO~2in) AiriP(lO-2m) + Theophylline (l0"2m) Tryptamine 1 466 478 2 462 349 3 898 392 4 769 505 5 430 6 495 Wean 587 5-Hydroxy-392 626 559 602 454 435 431 5 1 1 1 S .E . 1 80 1 36 +40 116 DISCUSSION In vi tro preparations of the larval rectum used in this study secreted a much less concentrated f l u i d than do the in, vivo preparations of Bradley and Phi l l ips (1975) and Chapter II. Since the same a r t i f i c i a l hemolymph bathed both preparations the poorer performance of in, vi tro recta i s probably due to disruption of tracheal connections and possibly the absence of neural and hormon-al stimulation. However, suff ic ient transport act iv i ty remains in  vitro in the posterior rectum to produce a secretion 366 mOsm more concentrated than the hemolymph (Table 8). This portion of the rectum is therefore clearly one, i f not the only, area where hyper-osmotic 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 ro , this rectal segment appears to reduce the concentration of the f l u i d in the lumen of both whole recta and isolated anterior recta. This could occur by means of three separate mechanisms. The anter-ior rectum in, vitro may transport salts out of the rectal lumen at a rate equal to the rate of secretion by the posterior rectum, or might allow sal t to leak back by simple diffusion in, v i t ro . Al ter-natively, the anterior rectal portion may be more permeable to water in, v i t ro than in, vivo. The ultrastructural observations of Meredith and Ph i l l ips (1973a) indicated that the anterior rectum of saline-water larvae 117 resembles the rectum of freshwater mosquito larvae where sal t resorp-tion results in the formation of hyposmotic excreta. The posterior rectum possesses a ce l lu lar structure restr icted to saline-water larvae and therefore is implicated in the formation of hyperosmotic f l u i d . The evidence to date suggests that the two rectal portions serve separate functions. They show morphological d i f ferent iat ion, e lect r ica l potentials dif fer ing both in magnitude and polar i ty , and opposite effects of rectal f lu id concentrations jjn v i t ro . This sup-ports the hypothesis of Phi l l ips and Meredith (1969a) and Meredith and Phi l l ips (1973a) that the anterior rectum is the s i te of sa l t and nutrient resorption in both fresh and saline-water and that the posterior rectum is the location of the hyperosmotic secretion of salts for osmoregulation in saline waters. Another saline-water insect , Ephydrella possesses two ce l l types in the hind-gut (Marshall and Wright, 1974). The two c e l l types, rather than existing in sep-arate regions as in the saline-water mosquito rectum, are interspers-ed throughout the hind-gut. The rectum of Ephydrella is seemingly inactive, 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 sa l t resorption or sal 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 rectal function in two species of saline-water mosquito larvae (Bradley and P h i l l i p s , 1975; Chapters II, III and IV) and the blowfly larva Sarcophaoa bullata (Prusch, 1974), I suggest that most aquatic larvae capable of hyposmot-118 i c regulation produce a concentrated urine by the secretion of a hyperosmotic f lu id in some part of the hindgut. In accordance with this function the portion of the gut in which f ina l urine modifica-tion occurs w i l l show two c e l l types, one engaged in resorption of essential metabolites and some ions and the other in secretion of hyperosmotic f l u i d when the animal is 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 in saline-water mos-quito larvae is achieved by the secretion of a hyperosmotic f lu id into the rectal lumen (Bradley and P h i l l i p s , 1975; and Chapter II). This f l u i d contains Na + , Mg + + , CI 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 rectal f l u i d i s hypertonic to the hemolymph and external medium with regard to potassium under every ex-perimental condition which has been studied. I wished to determine which of these ions are actively transported during the process of rec-ta l secretion. Aedes taeniorhynchus larvae raised in 100% sea water were chosen for this study because the ionic concentration differences across the rectal epithelium are known (Bradley and Phi l l ips 1975; Chap-ter II). In the present study, e lect r ica l potential differences across the rectal epithelium were determined under the same experimental con-dit ions in order to determine whether the distr ibut ion of any of the ions could be explained by passive forces alone ( i . e . by electrochemical potential difference). A model for trans-epithel ial transport of ions is prooosed, based on these observations and those from Chapters II, III, and IV. MATERIALS AND METHODS The e lec t r ica l potential difference across the rectal epithe-121 Hum was measured using two separate methods. Tn one case, (hereafter referred to as e lect r ica 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 petri d ish, 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 this hole. A glass microscope sl ide coverslip was placed over the hole, allowing a space for the siphon and the edge was sealed with melted paraffin wax. The siphon was thereby isolated and exposed to the pocket of a i r under the coversl ip. 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 petr i dish was then f i l l e d with paraffin o i l which was prevented from enter-ing the siphon by the sealed coversl ip. The exposed cut ic le 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 in t ra -ce l lu lar and trans-epithel ia l e lect r ica l potential differences, by im-paling rectal ce l ls 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 sixth and seventh abdominal segments and at the anal segment so as to isolate the rectum. The portion of the larva anterior to the f i r s t 122 l i g a t u r e was r e m o v e d . The r e m a i n i n g l a r v a l p o r t i o n was a n c h o r e d s e c u r e l y t o p a r a f f i n wax l i n i n g the bo t tom o f a p e t r i d i s h by m e l t i n g t he ends o f t h e l i g a t u r e s i n t o t h e wax . The l a r v a l c u t i c l e was t o r n and a d rop o f hemolymph was p l a c e d on t h e p r e p a r a t i o n . S i n c e t h e s i p h o n was open t o t h e a i r o r t he s u r f a c e o f t he d rop o f a r t i f i c i a l hemolymph, t he r e c t u m was b a t h e d i n a s o l u t i o n o f known c o m p o s i t i o n and had a n o r m a l oxygen s u p p l y v i a t h e t r a c h e a e . The hemolymph d r o p was removed and r e p l a c e d e v e r y few m i n u t e s to a v o i d changes due to e v a p o r a t i o n . T h i s p r e p a r a t i o n was used o n l y f o r s h o r t - t e r m m e a s u r e m e n t s . F o r a more d e t a i l e d d e s c r i p t i o n o f t h i s p r o c e d u r e s e e A p p e n d i x I I . E l e c t r i c a l p o t e n t i a l s were measured u s i n g a M701 M i c r o p r o b e S y s t e m (Ul-P I n s t r u m e n t s , I n c . ) w i t h p o t e n t i a l s r e c o r d e d on an expanded s c a l e v o l t m e t e r ( R a d i o m e t e r , C o p e n h a g e n ) . The i n d i f f e r e n t c a l o m e l e l e c -t r o d e was c o n n e c t e d t o the a r t i f i c i a l hemolymph b a t h i n g t h e r e c t a i n b o t h e l e c t r i c a l p r e p a r a t i o n s by means o f a s a l t b r i d g e c o n s i s t i n g o f p o l y e t h y -l e n e t u b i n g ( P . E . 5 0 , C l a y Adams, I n c . ) f i l l e d w i t h 3M KC1 i n 3% A g a r . 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 were i n s e r t e d i n t o t h e r e c t u m e i t h e r t h r o u g h the anus ( e l e c t r i c a l p r e p a r a t i o n #1) i n w h i c h c a s e the t i p r e s i s t a n c e was low ( 1 - 2 megohms) o r t h r o u g h t he r e c t a l w a l l ( e l e c t r i c a l p r e p a r a t i o n #2) u s i n g h i g h r e s i s t a n c e t i p s ( 1 0 - 1 7 megohms) . The a r t i f i c i a l hemolymphs u s e d i n t h i s s t u d y were i d e n t i c a l to t h o s e d e s c r i b e d i n C h a p t e r I V . These v a r i o u s hemolymph s o l u t i o n s d i f f e r -ed o n l y i n t h e s u b s t i t u t i o n o f a s i n g l e i o n . Thus s o d i u m was r e p l a c e d w i t h c h o l i n e , p o t a s s i u m was added as ^ S O ^ to n o r m a l hemolymph, o r c h l o r i d e was r e p l a c e d w i t h s u l p h a t e . F o r a d e t a i l e d d e s c r i p t i o n o f t he p r e p a r a t i o n o f 123 t h e s e hemolymphs s e e A p p e n d i x I V . RESULTS T r a n s - r e c t a l E l e c t r o - p o t e n t i a l D i f f e r e n c e s R e c t a o f s a l i n e - w a t e r m o s q u i t o l a r v a e c a n s e c r e t e f l u i d c o n t a i n i n g c o n c e n t r a t i o n s o f Na , K , Mg , CI and p r o b a b l y HCO^ h y p e r t o n i c t o t he hemolymph by 2-18 t i m e s ( B r a d l e y and P h i l l i p s ; 1975; C h a p t e r s I I , I I I and I V ) , To d e t e r m i n e w h i c h o f t h e s e i o n s a r e a c t i v e l y t r a n s p o r t e d , a s t u d y was u n d e r t a k e n o f the e l e c t r i c a l p o t e n t i a l d i f f e r -e n c e (P.D.) a c r o s s the who le r e c t a l w a l l d u r i n g s e c r e t i o n by A_. t a e n i o r h y n c h u s l a r v a e a d a p t e d t o 100$ saa w a t e r . When e l e c t r i c a l p r e p a r a t i o n #1 was u s e d , i n w h i c h t he m e a s u r i n g 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 the r e c t a l lumen t h r o u g h t he anus two q u i t e d i f f e r e n t P . D . v a l u e s f o r t he lumen r e l a t i v e to t h e hemolymph were s u c c e s s i v e l y and c o n s i s t e n t l y o b s e r v e d . These two r e g i o n s showed no t o n l y d i f f e r e n t p o t e n t i a l s b u t , i n a m a j o r i t y o f c a s e s , o p p o s i t e p o l a r i t y r e l a t i v e to t he hemolymph. In o r d e r t o l o c a t e t h e s e r e g i o n s more p r e -c i s e l y , e l e c t r i c a l p r e p a r a t i o n #2 was u s e d , i n w h i c h the t r a n s - e p i t h e l i a l P.D. was measured 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 t h r o u g h t h e r e c t a l w a l l i n t o t h e lumen o f e i t h e r t h e a n t e r i o r o r p o s t e r i o r r e c t a l segment s u c h t h a t t he p r e c i s e l o c a t i o n o f the m i c r o e l e c t r o d e t i p was known, ( T a b l e 10). The lumen of the a n t e r i o r r e c t a l segment was a l -ays p o s i t i v e r e l a t i v e t o t he hemolymph w i t h t h e e x c e p t i o n o f one v a l u e o f -12 mV. The lumen o f t h e p o s t e r i o r r e c t a l segment was a l w a y s p o s i t i v e w i t h r e s p e c t to t h e lumen o f the a n t e r i o r r e c t a l segment by an a v e r a g e o f 20.9 mV. w 124 T a b l e 1 0 . P a i r e d measurements of 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 d i f f e r e n c e i n t he 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 o f r e c t a b a t h e d i n the n o r m a l a r t i f i c i a l hemolymph w i t h i n t e n m i n u t e s o f d i s s e c t i o n ( e l e c t r i c a l p r e p a r a t i o n # 2 ) . The r i g h t hand co lumn shows t he e l e c t r i c a l p o t e n t i a l d i f f e r e n c e between t h e two r e c t a l s e g m e n t s . S e r i a l 6 7 8 9 A n t e r i o r R e c t a l P o t e n t i a l ( l u m e n s i d e ) - 22 -11 - 3 0 0 - 7 - 2 -6 - 6 - 1 0 P o s t e r i o r R e c t a l P o t e n t i a l ( l umen s i d e ) +6 +9 -12 +40 +15 + 4 +14 +14 + 5 P o s t e r i o r P o t e n t i a l R e l a t i v e t o A n t e r i o r + 28 + 20 +18 + 40 + 22 +6 +20 • 20 + 15 Wean t S . E . - 1 0 . 4 - 3 . 0 +10.5 * 4 . 6 2 0 . 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 s e c r e t e d by t h e r e c t u m , i o n c o n c e n t r a t i o n r a t i o s and e l e c t r i c a l p o t e n t i a l d i f f e r e n c e s must be measured u n d e r t he same c o n d i t i o n s . T h e r e f o r e p r e p a r a t i o n #2 was u s e d t o measure the e l e c t r i c a l p o t e n t i a l d i f f e r e n c e s a c r o s s t h e w a l l o f the 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 a r t i f i c i a l hemolymph f o r two 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 t o t h o s e p r e v i o u s l y u s e d t o d e t e r m i n e i o n i c c o m p o s i t i o n o f r e c t a l s e c r e t i o n ( B r a d l e y and P h i l l i p s , 1 9 7 5 ; and C h a p t e r I I ) . Unde r t h e s e c o n d i t i o n s when t he r e c t u m was s w o l -l e n w i t h s e c r e t i o n , t he a n t e r i o r r e c t a l p o r t i o n showed b o t h n e g a t i v e and p o s i t i v e v a l u e s r e l a t i v e t o the hemolymph (mean = 1.8mV) and t he p o s t e r -i o r r e c t a l p o r t i o n lumen was a l w a y s p o s i t i v e r e l a t i v e t o t h e hemolymph by +6.4mV ( T a b l e 1 1 ) . The l o w e r p o t e n t i a l s f o r r e c t a f i l l e d w i t h s e c -r e t i o n v e r s u s t h o s e r e c e n t l y l i g a t e d r e f l e c t t h e f a c t t h a t t he p o t e n t i a l d i f f e r e n c e 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 p o r t i o n s was r e -duced (mean o f 8 . 2 m V ) . P e r h a p s a s l i g h t c o n s t r i c t i o n a t t h e j u n c t i o n a l r e g i o n ( M e r e d i t h and P h i l l i p s , 1973) p r o v i d e s a r e l a t i v e l y h i g h e l e c t r i c a l r e s i s t a n c e between t h e r e c t a l segments i n the u n s w o l l e n c o n d i t i o n , w h i l e s w e l l i n g o f t he r e c t u m a f t e r 2 hou rs o f s e c r e t i o n l e a d s t o i n c r e a s e d c o n t i n u i t y be tween t h e l u m i n a o f the a n t e r i o r and t he p o s t e r i o r r e c t u m and hence t o a s h o r t - c i r c u i t i n g o f the o p p o s i n g p o t e n t i a l d i f f e r e n c e s i n the two s e g m e n t s . I n f l u e n c e o f Hemolymph Ion C o n c e n t r a t i o n s £ n t he T r a n s - e p i t h e l i a l P . P . I was i n t e r e s t e d i n d e t e r m i n i n g how p o t e n t i a l d i f f e r e n c e s a r i s e a c r o s s t he r e c t a l w a l l and w h e t h e r they a r e a s s o c i a t e d w i t h a c t i v e t r a n s -126 Table 11. Paired measurements of the trans-rectal e lec t r ica l potential difference in the anterior and posterior rectal segments of recta bathed in normal hemolymph two hours after dissection ( e l e c t r i -ca l preparation #2). The right hand column shows the e lec t r ica l potential difference between the two rectal segments. Ser ia l Anterior Rectal Potential (lumen side) Posterior Rectal Potential (lumen side) Posterior Potential Relative to Anterior 1 2 3 4 5 6 7 8 9 Wean - S .E . +8 -9 -3 -17 + 5 -5 -2 +4 +3 -1.8 -2.6 + 8 +7 +16 +4 +4 +6 +6 +4 + 3 +6.4 ±1.0 0 + 16 + 19 +21 -1 + 11 + 8 0 0 +8.2 ±3.0 127 p o r t o f i o n s r e s p o n s i b l e f o r t h e f o r m a t i o n o f h y p e r o s m o t i c r e c t a l s e c -r e t i o n . E l e c t r i c a l p o t e n t i a l p r o f i l e s t h r o u g h t he a n t e r i o r and p o s -t e r i o r r e c t a l c e l l s were measured t o d e t e r m i n e t he s i z e and s i g n o f t h e p o t e n t i a l 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 1 2 ) . T h e s e d a t a show t h a t a l t h o u g h t he t r a n s - e p i t h e l i a l P . O . d i f f e r s s u b -s t a n t i a l l y i n t he a n t e r i o r and p o s t e r i o r r e c t a l p o r t i o n s , the i n t r a -c e l l u l a r p o t e n t i a l s r e l a t i v e t o hemocoe l ( i . e . P . O . a c r o s s t h e b a s a l p l a s m a membrane) was no t s i g n i f i c a n t l y d i f f e r e n t i n t he two segments ( - 3 9 and -49mV r e s p e c t i v e l y ) . These v a l u e s a r e o f c r i t i c a l i m p o r t a n c e 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 t r a n s p o r t o f s p e c i f i c i o n s i s o c c u r r i n g ( s e e D i s c u s s i o n ) . When i n t r a c e l l u l a r p o t e n t i a l s r e a c h e d s t e a d y v a l u e s , i t was p o s s i b l e t o change t h e a r t i f i c i a l hemolymph s u c h t h a t the e l e c t r o d e r e m a i n e d i n t h e c e l l d u r i n g t h e e n t i r e p r o c e d u r e . I t was t h e r e f o r e p o s s i b l e t o measure t he e f f e c t o f v a r y i n g i o n c o n c e n t r a -t i o n s i n 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 d i f f e r e n c e a c r o s s t h e b a s a l p l a s m a membrane o f the p o s t e r i o r r e c t a l s e g m e n t . T h i s p o t e n t i a l d i f f e r e n c e w h i c h was a l w a y s n e g a t i v e ( c e l l i n t e r i o r r e l a t i v e t o hemolymph) c o u l d be made l e s s n e g a t i v e by i n c r e a s i n g hemolymph p o t a s s i u m l e v e l s ; b e i n g r e d u c e d by an a v e r a g e o f B 1 0.7mV (n=4) i n 39mMK + and 13mV i n 63mM K ( s i n g l e o b s e r v a t i o n ) . 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 o f the hemolymph a l s o r e d u c e d the p o t e n t i a l d i f f e r e n c e a c r o s s t h e b a s a l membrane, b e i n g r e d u c e d by 7i0mV i n 1 . 3 mM C l " ( n = 3 ) . V a r y i n g the s o d i u m c o n c e n t r a t i o n o f t h e hemolymph f r o m 200mM to 5mM Na by s u b -s t i t u t i n g c h o l i n e d i d no t have a s i g n i f i c a n t e f f e c t on t h i s p o t e n t i a l d i f f e r e n c e . 128 T a b l e 12. P a i r e d measurements o f t h e e l e c t r i c a l p o t e n t i a l s w i t h i n t h e r e c t a l c e l l s and t he r e c t a l lumen ( r e l a t i v e t o t h e hemolymph) i n t h e a n t e r i o r and p o s t e r i o r r e c t a l p o r t i o n s . Measu remen ts were made on r e c t a b a t h e d i n n o r m a l a r t i f i c i a l hemolymph w i t h i n 10 m i n u t e s o f d i s s e c t i o n . S e r i a l 1 2 3 4 5 A n t e r i o r Rectum Lumen C e l l I n t e r i o r - 3 4 - 5 0 - 4 0 -60 -61 -16 - 1 6 - 12 - 1 2 - 7 P o s t e r i o r Rectum Lumen C e l l I n t e r i o r - 3 7 - 5 0 -41 - 2 7 -41 +14 +17 +6 + 2 +15 Mean 4 S . E . - 49 - 1 3 i 5 1 2 - 3 9 +11 ± 4 1 3 129 S i n c e e a r l i e r 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 p r e -p a r a t i o n s o f the r e c t u m d e m o n s t r a t e d t h a t t he p o s t e r i o r r e c t a l p o r t i o n i s a s i t e o f h y p e r o s m o t i c f l u i d s e c r e t i o n , I e x a m i n e d i n d e t a i l t h e e f f e c t o f 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 the e l e c t r i c a l p o t e n t i a l g e n e r a t e d a c r o s s t he e n t i r e c e l l o f t h i s s e g m e n t . The p o t e n t i a l s i n the two p o r t i o n s o f the r e c t u m a r e o f t e n o p p o s i t e i n p o l a r i t y and t e n d t o c n a c e l e a c h o t h e r o u t , t h e r e f o r e i t was i m p o r t a n t t o show t h a t c h a n g e s o b s e r v e d i n the p o s t e r i o r r e c t a l p o r t i o n were no t o r i g i n a t i n g i n t he a n t e r i o r r e c t a l p o r t i o n . The p o t e n t i a l i n t h e a n t e r i o r r e c t a l lumen r e l a t i v e to the hemolymph ( e i g h t p r e p a r a t i o n s o b s e r v e d ) d i d n o t v a r y when t h e r e c t u m was b a t h e d i n hemolymph h i g h i n p o t a s s i u m o r s o d i u m (200 mm, N a + ) o r low i n c h l o r i d e ( F i g . 2 2 ) . Of t h e v a r i o u s hemolymphs u s e d , o n l y t h a t low i n s o d i u m (5mM N a + ) had any a p p r e c i a b l e e f f e c t , 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 o f t h e l u m e n . T h i s change i n t h e p o t e n t i a l d i f f e r e n c e was i n t h e same d i r e c t i o n a s t h a t o b s e r v e d i n t h e p o s t e r i o r r e c t a l segment and m i g h t t h e r e f o r e be a s s o c i a t e d w i t h the P . D . change i n t h a t r e g i o n . The o b s e r v a t i o n t h a t h i g h p o t a s s i u m and s o d i u m , and l ow c h l o r i d e l e v e l s i n t h e hemolymph do n o t a f f e c t t h e a n t e r i o r r e c t a l P . D . a r g u e a g a i n s t a s u b s t a n t i a l e l e c t r i -c a l s h o r t - c i r c u i t be tween t he r e c t a l segmen ts i n t h e u n s w o l l e n r e c t u m , 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 on t h e P . D . i n t he p o s t e r i o r r e c t a l s e g m e n t . E l e c t r i c a l p r e p a r a t i o n #1 was u s e d w i t h t h e m i c r o - e l e c t r o d e p l a c e d t h r o u g h the anus i n t o t he most p o s i t i v e p a r t o f the r e c t u m . F i g u r e 23 shows t he e f f e c t on t he e l e c t r i c a l p o t e n t i a l d i f f e r e n c e o f t h e 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 d i f f e r e n c e ( l umen r e l a t i v e t o hemolymph) o b s e r v e d i n a n t e r i o r r e c t a l s e g -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 hemolymph o r hemolymphs d i f -f e r i n g i n t h e c o n c n e t r a t i o n o f o n l y t h e i o n i n d i c a t e d ( e l e c t r i c a l p r e p a r a t i o n #1). 130b La F i g u r e 2 3 . 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 d i f f e r e n c e ( l umen r e l a t i v e t o hemolymph) o b s e r v e d i n the p o s t e r i o r r e c t a l 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 hemolymph o r a r t i f i c i a l hemolymphs d i f f e r i n g i n the c o n c e n t r a t i o n o f t he i o n i n d i c a t e d . - 3 CN i n d i c a t e s n o r m a l a r t i f i c i a l hemolymph t o w h i c h 10 W KCN has bean a d d e d . J_ 10 20 30 40 _50 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 individual ion and i ts replacement are varied. This procedure was repeated with twenty different rectal preparations. The posterior rectal segments showed variable differences in trans-rectal e lect r ica l potential after dissection in normal a r t i f i c i a l hemolymph (Table 10). Therefore, the effects of a r t i f i c i a l hemolymph changes are expressed as mean changes in e lec t r ica l potential differences - S .E . relative to the or iginal potential difference observed in recta bathed in normal hemolymph (Figs. 24, 25, and 26), The trans-rectal e lec t r ica l potential difference in the pos-terior rectal segment became more positive (lumen relative to hemolymph) with increasing potassium concentration (Figs. 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 trans-rectal 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 signif icant ly different (P<0.01), with the other sodium concentrations observed producing intermediate trans-rectal potential differences. The effects of a r t i f i c i a l hemolymph containing 5mM Na+ in lowering the e lect r ica 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 chloride, the trans-rectal potential difference decreased (lumen less 5a F i g u r e 2 4 . The e f f e c t o f i n c r e a s i n g t h e p o t a s s i u m c o n c e n t r a t i o n o f t h e a r t i f i c i a l hemolymph on t h e 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 d i f f e r e n c e o f t h e p o s t e r i o r r e c t a l s e g m e n t . A l l v a l u e s a r e e x p r e s s e d a s the mean change i n p o t e n t i a l - S . E . r e l a t i v e t o t h a t o b s e r v e d i n r e c t a b a t h e d i n n o r m a l a r t i f i c i a l hemolymph ("^T) (+ 1 0 . 5 - 4 . 6 mV, n = 1 0 ) . The numbers i n b r a c k e t s i n d i c a t e t h e s a m p l e s i z e f o r e a c h p o i n t . 133 c LEAF 134 OMITTED IN PAGE NUMBERING. F i g u r e 2 5 . The e f f e c t o f v a r y i n g t he s o d i u m c o n c e n t r a t i o n o f t h 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 p o t e n t i a l d i f f e r e n c e o f t h e p o s t e r i o r r e c t a l s e g m e n t . A l l v a l u e s a r e e x p r e s s e d as t h e mean change i n p o t e n t i a l ± S . E . r e l a t i v e to t h a t o b s e r v e d i n r e c t a b a t h e d i n n o r m a l a r t i f i c i a l hemolymph ( ) (+ 1 0 . 5 - 4 . 6 mV, n = 1 0 ) . The numbers i n b r a c k e t s i n d i c a t e t h e samp le s i z e f o r e a c h p o i n t . Sodium Concentration of A r t i f i c i a l Hemolymph (mM) Figure 26, The effect of varying the chloride concentration of a r t i f i c i a l hemolymph on the trans-rectal e lec t r ica l potential difference of the posterior rectal segment. A l l values are ex-pressed as the mean change in potential - S . E . relat ive to that observed in recta bathed in normal a r t i f i c i a l hemolymph ( ) (+ 10.5 - 4.6mV, n=10). The numbers in brackets indicate the sample size for each point. 137 positive) significantly with increasing hemolymph chloride concentra-tion (P<0.01). Nearly a two-fold increase in the mean of the trans-rectal potential was observed in the posterior segments of recta transferred from normal hemolymph to a r t i f i c i a l hemolymph contain-ing 1.3 mM CI (Fig. 26). This potential increase was maintained for 20 to 30 minutes, after which time a partial or even total reduc-tion in potential occurred (Fig. 27). Upon return to normal hemolymph the trans-rectal potential was more negative by 3 to 4 mV than pre-viously observed in normal hemolymph. In Chapter IV, I presented evidence that high levels of either sodium (200 mM, Na+) or chloride (150 mM, Cl~) in 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 in the case of sodium, the osmotic concentra-tion of the rectal f l u i d also increased as compared to values with normal a r t i f i c i a l hemolymph. Therefore, i t is of particular interest to observe the effect of these a r t i f i c i a l hemolymphs on the trans-rectal potential. High levels of sodium (200 mM) in the a r t i f i c i a l hemolymph were found to increase the trans-rectal potential in a manner which was not reversible within a one hour period (Fig. 28). High levels of chloride (150 mM) in the a r t i f i c i a l hemolymph, on the other hand did not change the trans-rectal potential appreciably (Fig. 26.). Although, the effect of a r t i f i c i a l hemolymph high in chloride shown in Fig. 28 i s not completely typical in that a slow increase in potential can be observed after the i n i t i a l dip, the increase in potential upon return to normal hemolymph as compared F i g u r e 2 7 . 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 d i f f e r e n c e ( l umen r e l a t i v e t o hemolymph) o b s e r v e d i n two p o s t e r i o r r e c t a l segments b a t h e d i n n o r m a l a r t i f i c i a l hemolymph o r a r t i f i c i a l hemolymphs d i f f e r i n g i n the c o n c e n t r a t i o n o f t he i o n i n d i c a t e d . 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 d i f f e r e n c e ( l umen r e l a t i v e t o hemolymph) o b s e r v e d i n the p o s t e r i o r r e c t a l 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 hemolymph o r a r t i f i c i a l hemolymphs d i f f e r i n g i n t he c o n c e n t r a t i o n o f t he i o n i n d i c a t e d . Electrical Potential (mV) q6£t 140 t o p r e v i o u s l e v e l s i n no rma l hemolymph i s c l e a r l y s h o w n . G e n e r a l l y , t h e a p p l i c a t i o n o f a r t i f i c i a l hemolymph h i g h i n c h l o r i d e l e d t o a s l i g h t d e c l i n e i n t r a n s - r e c t a l p o t e n t i a l (-2 .0 i 1 ,6 mV, n = 4 ) , bu t upon r e t u r n t o n o r m a l hemolymph, t he p o t e n t i a l r o s e t o a l e v a l h i g h e r than t h a t p r e v i o u s l y o b s e r v e d i n n o r m a l hemolymph by a mean v a l u e o f 3 . 0 ± 1.0 mV ( n = 4 ) . The a d d i t i o n of o u a b a i n (10 M) t o t h e n o r m a l hemolymph d i d not p r o d u c e a s i g n i f i c a n t change i n P . D . (+ 0 . 6 *• 0 . 6 mV, n=3) when compared t o t h a t o b s e r v e d i n no rma l hemolymph a l o n e . S e r o t o n i n ( 1 0 ~ 4 M 5-HT) a l s o d i d n o t s i g n i f i c a n t l y a f f e c t t h e t r a n s - r e c t a l P . O . o v e r a p e r i o d o f 15 m i n u t e s (+ 0 . 8 - 0 . 5 mV, n=4 ) . The a d d i t i o n o f 2 7 c y c l i c AMP (10 M) and t h e o p h y l l i n e (10 M) t o n o r m a l hemolymph i n d u c e d no s i g n i f i c a n t change i n P . D . ( 1 . 0 - 0 . 6 mV, n = 3 ) . These f i n d i n g s 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 he _in v i t r o r e c t a l p r e p a r a t i o n w h i c h f a i l e d t o show a s t i m u l a t o r y e f f e c t on t h e o s m o t i c c o n c e n t r a t i o n of r e c t a l s e c r e t i o n by 3 - 5 * c y c l i c AMP + t h e o p h y l l i n e o r 5 -HT . Lack o f an e f f e c t i n t he i n , v i t r o c o n d i t i o n 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 a l a c k o f 0 ^ , bu t t h i s can be e x c l u d e d i n t he p r e s e n t e x p e r i m e n t s . I n e l e c t r i c a l p r e p a r a t i o n #1 t he s i p h o n and the t r a c h e a a were i n t a c t , y e t e l e c t r i c a l p o t e n t i a l s were u n a f f e c t e d 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 . The r e s u l t s f rom 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 s u g g e s t t h a t r e c t a l s e c r e t i o n i n s a l i n e - w a t e r m o s q u i t o l a r v a e c a n n o t be s t i m u l a t e d on a s h o r t - t e r m 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 the hemocoe l s i d e o f the r e c t u m . The p o t e n t i a l d i f f e r e n c e o b s e r v e d a c r o s s t he p o s t e r i o r r e c t a l 141 segment was substantially and irreversibly 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 lec t r ica l potentials measured in this study can be compared to the ionic gradients previously measured under ident ical conditions (Bradley and P h i l l i p s , 1975; Chapterll) to determine which ions are actively transported across the rectal wall of /U taeniorhynchus l i v ing in 100% sea water. The e lec t r i ca l potential required to support an ionic concentration difference across a diffusion barr ier , in this case the rectal c e l l , i s described by the Nernst equation! E* ~ 1n (c^A^) where E equals the e lect r ica l potential difference observed, R the gas constant, T the temperature in Kelvin, E the charge of the ion , F the Faraday constant and c^A^ the concentration ratio which can be maintained by the e lec t r ica l potential . At the experimental temperature used (25°C) , and for monovalent ions, the equation can be s impl i f ied! E= 59 log (c^/c2) The ratio of the rectal f l u id to hemolymph concentration observed for each ion as well as the potential to maintain that concen-tration ratio (lumen relative to hemolymph) difference required in the ligated recta in vivo is as follows! Na+ 1.9 (-16mV), K + 9.9 (-57.7mV), 142 Mg 5.0 (-58mV), CI 4.3 (+37mV). The concentration ra t ios represent average values 2 h after l i g a t i o n and must therefore be compared to average values for potent ia l differences observed across the r ec t a l wal 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 potent ia l difference i s -10.4 and +10.5 mV i n the anterior and posterior r e c t a l segments respec t ive ly . After 2h, the potent ia l differences for these two segments of recta had fa l l en to -1.8 and +6.4 mV, respect ively . Therefore, under these exper i -+ + ++ mental conditions i t i s necessary to postulate that Na , K , Mg and CI are a l l ac t ive ly transported across the r ec t a l wal l from the hemolymph to the rec ta l lumen. In pa r t i cu l a r , i f one considers the evidence that hyperosmotic secret ion occurs i n the posterior r ec t a l segment where the lumen i s p o s i t i v e , then c l ea r ly a l l three cations are transported not only against concentration but also e lec t ropotont ia l differences. I examined the potent ia l across the poster ior rec ta l wal l i n more de t a i l because the morphological and phys io log ica l evidence indicates that hyperosmotic secret ion occurs i n th is region. I have made three assumptions i n constructing a model for ion transport across the posterior r ec t a l epithelium ( F i g . 29)i 1) As indicated by electron micrographs, only two important barr iers to ion d i f fus ion occur i n the poster ior r ec t a l w a l l , namely the basal (hemolymph side) and ap ica l (lumen side) plasma membranes. 2) The f l u i d secreted across the posterior recta l wal l i s s i m i l a r to or more concentrated than that co l lec ted from the lumen of the whole, l iga ted 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 in most other c e l l s i . e . high K + , t F i g u r e 2 9 . A model o f t h e p r o c e s s e s o c c u r i n g i n t h e c e l l s o f t h e p o s t e r i o r r e c t a l segment d u r i n g the s e c r e t i o n o f h y p e r o s m o t i c f l u i d . The uppe r r e g i o n i s a d i a g r a m m a t i c r e p r e s e n t a t i o n o f the u l t r a s t r u c t u r e o f the c e l l . The c e n t r a l b l o c k d e p i c t s t h e p r o p o s e d s i t e s o f i o n t r a n s p o r t d u r i n g f l u i d s e c r e t i o n . S o l i d a r r o w s r e -p r e s e n t a c t i v e t r a n s p o r t w h i l e dashed ones d e p i c t p a s s i v e movements . The b o t t o m d i a g r a m shows t he a v e r a g e e l e c t r i c a l p o t e n t i a l d i f f e r e n c e s measured a c r o s s e a c h membrane and a c r o s s t h e e n t i r e p o s t e r i o r r e c t a l e p i t h e l i u m . 143b H E M O L Y M P H L U M E N ^•K+, Na + • 4-144 lout Na and CI . Like most c e l l s , those of the posterior rectal segment have an inter ior which i s negative to the hemolymph. This i s largely attributed to a potassium diffusion potent ial , with the potassium con-centration gradient maintained by an active Na -K exchange mechanism (reviewed by Schwarz, Lindenmayer and A l len , 1972). In support of th is , when an a r t i f i c i a l hemolymph abnormally high in potassium was placed on the posterior rectum, the inter ior of the c e l l became less negative. A reduction of the potential across the basal membrane should increase the trans-rectal potential , since thB e lec t r ica l potentials across the basal and apical membranes oppose each other. This was experimentally observed when a r t i f i c i a l hemolymphs high in potassium were placed on the posterior rectum (Figs. 23 and 24). On the basis of these observa-tions an outward diffusion of potassium to the hemocoel which i s opposed by active transport involving a typical Na + -K + exchange pump i s depicted in F i g . 29. The potential difference across the basal membrane was found to decrease upon reduction of the chloride concentration of the a r t i f i c i a l hemolymph. A decrease in the rate of chloride diffusion into the c e l l , which might be expected on reducing hemolymph C l " leve ls , should have the opposite effect on the potential to that observed. We therefore suggest that an electrogenic chloride "pump" exists on the basal membrane which transports chloride into the c e l l during hyperosmotic f l u i d trans-port. Active transport of chloride i s necessary in this location not only to explain the offset of low chloride hemolymph on the e lec t r ica l potential difference across the basal membrane, but also to ensure the entry of chloride into the c e l l against the steep e lec t r ica l potential 145 gradient across the basal membrane (Table 12). This same e lec t r ica l + ++ potential would fac i l i t a te the movement of Na and Mg into the c e l l . These ions are generally present in ce l ls at lower ac t iv i t ies than in the blood (Palaty and Friedman). 1974). These cations would therefore enter the c e l l by passing down both an e lec t r ica l and chemical gradient. Schmidt-Nielsen, B. (1975) has reviewed evidence that in some epithel ia secreting hyperosmotic f l u i d , the barrier to the diffusion of water i s apparently at the basal membrane, since the int racel lu lar osmotic concentrations are very high (1000-2000 mOsm). In other such ep i the l ia , the ce l l s are isosmotic to the blood* We have no such estimates for the larval rectal t issue, and knowledge of the location of the barrier to water diffusion must await further experimentation. The e lec t r ica l potential difference across the apical membrane of the posterior rectal ce l ls (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 at each membrane. It i s at the apical 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 ob-served in the hyperosmotic secretion. This membrane also has the great-est surface area in the c e l l and is associated with most of the mito-chondria. Fig 29 shows separate cation pumps for the monovalent and divalent cations. In some vertebrate tissues magnesium is thought to be transported by a separate mechanism from that for other cations, with this 146 transport being dependent on a sodium gradient driving Na + -Mg + + exchange (Baker and Crawford, 1973; Palaty, 1974). Whether this applies to the larval 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 is lowered from 150 mM to 5 mM, the trans-rectal potential i s reduced s l ight ly but s ign i f icant ly . In the hemolymph containing (5 mM Na+) the rectal secretion was found to have an osmotic concentration of 634 * 31 mOsm (n=7), a sodium concentration of 128 * 25 mM (n=7) and a potassium concentration of 280 ± 70 mM (n=5) indicating that potassium can substitute for sodium in the formation of a hyperosmotic rectal secretion (Chapter IV). It is therefore possible that these two ions compete for the same transport mechanism in the rectum, as has been pos-tulated for the Malpighian tubules of Rhodnius (Maddrell, 1971). For the sake of s impl ic i ty , potassium, and sodium are shown sharing the same monovalent transport mechanism (F ig . 28), Further studies may indicate that these ions actually use separate transport mechanisms. The e lect r ica l potential across the apical membrane is suf-f ic ient to support a 10-fold gradient for chloride ions. The normal int racel lu lar chloride concentration for epithel ia secreting hyperos-motic f l u i d i s 80-120 mM (Schmidt-Nielsen, B.; 1975), a concentration only half this value would be suf f ic ient in the rectal c e l l to allow chloride to pass across the apical membrane and accumulate in the lumen by passive means. The int racel lu lar chloride concentration could be maintained at this level by the active chloride transport proposed across the basal membrane in F i g . 29. It is 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 rectal lumen is incapable of being completely eliminated, even when CI is present in the external environment at very low levels . I have followed the convention of not proposing an active transport mechanism where no thermodynamic requirement ex ists , and therefore propose that chloride and other anions cross the apical membrane by passive means (F ig . 29). The transfer of other anions by the posterior rectal segment must be considered for larvae l iv ing in several natural waters. In a l l l ike l ihood, bicarbonate largely substitutes for chloride in the 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 apical mem-brane when ce l lu lar chloride concentrations are depressed. Bicarbonate may be transported by the chloride pump postulated for the apical membrane since both must enter the c e l l against an e lec t r ica l gradient. In (Na + Mg)S0^ ponds the concentration of chloride is also low and the hemolymph chloride concentration i s correspondingly reduced (Chapter III). This situation is similar 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 papi l lae. The rectal 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. Sulphate ions occur in the rectal secretion in quantities 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 ce l ls of the posterior rectal segment i s consistent with several previous observations re-garding the stimulation of rectal secretion. In Chapter IV I showed that while an a r t i f i c i a l hemolymph with normal ionic concentrations but increased osmotic (sucrose) concentration does not increase the osmotic concentration of rectal secretion, increased levels of sodium or chloride in the hemolymph do. Raising hemolymph levels of sodium increases the trans-rectal potential (F ig. 25). This i s predicted by the model in which increased sodium transport hyperpolarizes the apical membrane and thereby increases the trans-rectal potential . Raising hemolymph levels of chloride does not appreciably change the trans-rectal potential , generally causing a s l ight reduction in 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 is countered by the stimulatory effect that high hemolymph levels of chloride have on hyperosmotic f lu id secretion (F ig . 19). Thus, the trans-rectal potential i s only s l ight ly affected in hemolymph high in chloride because cation transport is stimulated (F ig . 29). This interpretation i s sup-ported by the observation that, upon return to normal hemolymph from hemolymph high in chloride, the trans-rectal potential difference i n -149 creases over that observed before in normal hemolymph, suggesting that cation transport has been increased. The trans-epithel ial potential differences across the posterior rectal segment were unaffected by ouabain. This is not unusual for i n -sect epithel ia and is variously interpreted as indicating 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* diffusion potential on the hemocoel side of the rectal t issue, I prefer the lat ter explanation. The potential in the posterior rectal portion is not affected by the short-term application (20 min.) of 5-HT(10"4M). In Chapter IV, I hypothesize that the long-term act ivi ty of the rectum, i . e . the fresh-water versus saline-water mode of function, is controlled hormonally or neurally, while the rate and osmotic concentration of the rectal secretion in the short-term is modified by hemolymph ion concentrations ( i . e . i n -t r ins ic regulation; Phi l l ips and Bradley, 1976). The e lect r ica l and in  vitro secretion experiments in this study support that hypothesis. CHAPTER VII GENERAL SUMMARY 150 151 SUMMARY This concluding chapter w i l l summarize the overall process of osmoregulation in saline-water mosquito larvae as now envisaged from previously published work and the results of this thesis. Sal ine-water mosquito larvae drink the external medium at a rapid rate -1 —1 (lOOnl'hr *mg ) regardless of the sa l in i ty of the external medium. This rate of drinking is 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 lu id intake by drinking greatly exceeds the volume of water lost by osmosis across the body wal l , i t determines the osmoregulatory load imposed on the animals and dictates that the composition of the urine must closely resemble that of the external medium. Osmoregulation in saline-water mosquito larvae adapted to hyposmotic waters is very similar 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 is observed. As reviewed by Phi l l ips and Bradley (in press), 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 lu id to produce a hyposmotic f lu id containing largely nitrogenous 152 and other waste products. This process is essentially ident ical to that found in other freshwater insects. The posterior rectum is apparently inactive in fresh water and the f lu id which has been modified in the anterior rectum is excreted via the anus. The anal papillae of sa l ine-water mosquito larvae are active in fresh water and take up ions from the external medium (Phi l l ips and Meredith, 1969). In hyperosmotic media, additional transport of ions contribut-ing to ion excretion i s in i t ia ted at several s i tes in 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. In media containing high levels of Mg + + and SO", these two ions are actively secreted into the tubule lumen, such that magnesium and sulphate account for a pro-portionally greater fraction of the total solute (Maddrell and P h i l l i p s , 1975; Ph i l l ips and Maddrell, 1975). Sulphate ions, in fac t , do not appear to be actively transported at any other excretory s i t e s . It is this restr ict ion which seems to l imit the ab i l i ty of saline-water larvae to survive in waters rich in sulphate, such as Ctenocladus pond in which Ae"des campestris larvae naturally occur. Even for 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 ions, e .g . Mg and SO". It is 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 is 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 relative concentration of the various ions transported (Na + , K + , M g + + , Cl'+HCOj) resemble those in the external medium. In concentrated media, the transport of ions and the volume of secretion both increase but not in s t r i c t proportion, so that the osmotic concentration of the rectal secretion rises as wel l . Sodium and magnesium occur in the rectal secretion in concentrations similar to external levels . Potassium concentration in the rectal secretion are always hypertonic to the medium and hemolymph. In media high in chloride, this anion is the major one secreted in the posterior rectal segment. In media low in chloride (e .g . HCO" or SO" =-rich medi chloride levels in the secretion are depressed while cation concentra-tions are unchanged. 8icarbonate ions presumably accompany the cations in this media into the rectal lumen. Larvae are capable of producing a hyperosmotic secretion in these unbalanced media as can larval recta bathed in a r t i f i c i a l hemolymphs with very low levels of either Na + , Mg + + or C l " . The levels of potassium ( in a l l media) or chloride ( in low-chloride media) may be lower in the external medium than in the rectal secretion, resulting in a depletion of hemolymph levels of these ions through the action of the rectum. This loss may be balanced by uptake of K + and Cl from the external medium through the anal papi l lae. In sea water, where sodium and chloride levels are both high, the uptake of chloride is turned off but potassium uptake in the papillae may 154 continue, being perhaps coupled to sodium extrusion as occurs in the g i l l s of marine teleosts. Measurements of the e lec t r ica l potential differences across each of the plasma membranes in the ce l ls of the posterior rectal seg-ment, as well as across the entire epithelium, have demonstrated that + + ++ Ma , K , Mg and CI are a l l actively transported from hemolymph to lumen. Chloride and potassium are probably actively transported across the basal membrane into the c e l l . Sodium and magnesium are presumed to enter the c e l l passively, moving down both their chemical and e lec t r ica l gradients. The e lect r ica l potential difference at the basal membrane is possibly due to both a potassium diffusion potential and an e lectro-genic chloride pump. It i s proposed that at the apical membrane, Na , + ++ K and Mg are electrogenically transported into the lumen against their electrochemical gradients, while chloride ions follow passively across this membrane. In media, low in chlor ide, the anion crossing this membrane appears to be largely HCO^ although C l~ always appears in the rectal secretion in concentrations isotonic 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 exist for 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 direct ly or indirect ly affect a hormonal or neural control of rectal function which in i t ia tes or halts rectal secretion. 2) Larvae adapted over four days to different media show different relative ion 155 concentrations in the rectal secretion even when bathed in the same a r t i f i c i a l hemolymph, indicating that the transport mechanisms have either changed in capacity or a f f in i ty during the adaption process. 3) The ion pumps in the Malpighian tubules and rectum show immediate increased transport rates ( in t r ins ic control) with increasing hemolymph ion levels , the Malpighian tubules showing Michaelis-Menten type respon-ses and the rectum al loster ic- type k inet ics . Separate control mechanisms therefore porbably exist to i n i -t iate and coordinate hypo-vvsrsus hyper-regulation and to modify the total capacity of the transport mechanisms for various ions. These adaptations are separate from the immediate responses of the transport mechanisms to changing hemolymph ion levels . 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 interesting f i e l d of enquiry remains that of determining how these processes are controlled to allow the survival of the larvae in the extremely variable environments which they inhabit . 156 REFERENCES Baker, P.F. & Crawford, A.C. (1973) Sodium-dependent transport of magnesium ions in giant axons of Lolioo forbesi . J_. Physiol. Lond. 21>6: 33P-39P. Beadle, L.C. (1939) Regulation of the hemolymph in the sal ine-water mosquito larva Aedes detritus Edw. J.. exp. B i o l . 16: 346-362. Beadle, L.C. 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(1973) Comparative Animal Physiology. W.B. Saunders Co. P h i l a . , London & Toronto. Provost, M.W. (1967) Managing impounded sal t marsh for mosquito control and estuarine resource conservation. LSU Marsh & Estuary Symposium pp 163-171. Prusch, R.D. (1971) The s i te of ammonia excretion in the blowfly larva, Sarcophaoa bul lata. Comp. Biochem. Physiol . 39A: 761-767. Prusch, R.D. (1972) Secretion of NH Cl by the hindgut of Sarcophaoa bul lata. Cpmp. Biochem. Physiol. 41 A: 215-223. Prusch, R.D. (1973) Secretion of hyperosmotic excreta by the blowfly larva, Sarcophaoa bul lata. Comp. Biochem. Physiol . 46A: 691-698. Prusch, R.D. (1974) Active ion transport in the larval 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 fer small quantit ies. 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 in 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 st ick 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 st ick insect , Dixippus morosus (Orthoptera, Phasmidae). J . exp. B i o l . 32: 200-216. Ramsay, J .A . ( I96l j Excretion of inul in 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 ce l lu lar ion and volume regulation. J_. exp. Zool. 194: 207-220. Schmidt-Nielsen, K. (1975) Scaling in biology. The consequences of s i ze . J . exp. Zool. 194: 287-307. Schwartz, A . ; Lindenmayer, G.E. & A l len , J . C . (1972) The Na*k ATPase membrane transport system: Importance in ce l lu la r function. In Current Topics in Membranes and Transport! Bronner and Kle inzel ler (eds.) Scudder, G.G.E. (1969b) The tauna of saline lakes on the Fraser Plateau in Br i t ish Columbia. Verh. Internat. Verein. Limnol. 17: 430-439. Scudder, G.G.E. (1969a) The distr ibution of two species of Cenocorixa in inland saline lakes of Br i t ish 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 ionic balance in two species of Cenocorixa (Hemiptera). J . Insect Physiol . 18: 883-895. ~* Shaw, J . & Stobbart, R.H. (1963))Osmotic and ionic regulation in insects. In Advances in Insect Physiology. Beament, Treherne and Wiggles-worth (eds.) Academic Press, N.Y. and Lond. Sohal, R.S. & Copeland, E. (1966) Ultrastructural variations in anal papillae of Aedes aeovoti (L.) at different environmental s a l i n i t i e s . J . Insect Physiol . 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. Su tc l i f f e , D.W. (i960) Osmotic regulation in the larvae of some euryhaline Diptera. Nature 187: 331-332. Sutc l i f f e , D.W. (1961a) Studies on sal* ; and water balance in caddis larvae (Trichoptera): I. Osmotic and ionic regulation of body f lu ids in Limnephilus a f f in is Curt is . J.. exp. B i o l . 38 : 501-519. Sutc l i f f e , D.W. (1961b) Studies on sa l t and water balance in caddis larvae (Trichoptera). II. Osmotic and ionic regulation of body f lu ids in 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 act iv i ty of insect blood: Physiological significance and relevance to the design of physiological sa l ine . J,. exp. B i o l . 62: 721-732. Wall, B . J . & Oschman, J . L . (1975) Structure and function of the rectum in insects. In Forthschritte in Zooloaie. Band 23, Heft 2 /3 / Custav Fischer Verlag. Stuttgart. Wessing, A. (1967) Funktionsmorphologie von exkretionsorganen bei insekten. Verh. Dtsh. Zool. 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 in Vero Beach, Florida and used to establish 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 for 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 in center of bowl by swir l ing. Sprinkle Fleischmann's Active Dry Yeast on surface. Leave 29-30 minutes. 3. Remove larvae with eyedropper and place in seive. 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 in plast ic pan containing 500 mis of growing medium. Add 320 mg dried brewers yeast. Cover pans loosely. 5. Feed 49 mg dried l iver 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 in 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 in clean medium. Fourth instar larvae were used for experiments on the sixth and seventh days. 7. If adults are required, pupae are removed and placed in bowls l ined with moist paper towels. No standing water must be present. Place bowls in cages 1 f t by 6 in by 6 in with screen top. Allow 500 adults per cage. 8. As adults emerge, offer them 10$ sucrose in d i s t i l l e d water on f i l t e r paper tents immersed in petri 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 gh t , 1 hr subdued l igh t , 10.5 hrs. darkness and 0.5 hrs subdued l igh t . 10. Feed adults with mouse f ive days after emergence by restraining mouse on screen top of cage. Feed for one hour interval three times a week. 162 11. Collect eggs four days after blood meal on f i l t e r paper tent in petri dish f i l l e d with 25% sea water. 12. Wash eggs from f i l t e r paper into fine sieve. 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. Col lect eggs with eyedropper. 13. Place eggs in water drops onto f i l t e r paper lying on the bottom of petri dishes. Place eggs in scattered pattern to avoid hatch-ing at warm temperatures due to self-induced reduction in oxygen tension. Keep f i l t e r paper moist during storage. 14. Cover eggs containers and incubate 5 days in adult or larval en-vironmental chamber. 15. After incubation period, store egg containers at 10°C unt 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 ight ly between needle-tipped for -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 pin. 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 let 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 ight grip on the abdomen„with the forceps, to a stainless steel 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 set . If contact between the anUs and the siphon or head had been avoided, the larvae were then able to swim freely and drink and re-spire normally. The planchets were placed on a metal d isc , the inter ior of which was bathed by water from a water bath maintained at 27°C. The plan-chets were covered with microscope sl ide covers with a small aperture le f t for venti lat ion. The whole disc was placed in a covered plast ic container, the bottom of which was f i l l e d with water to increase humid-i t y . 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 in later 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 eye-dropper 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 sixth and seventh ab-dominal segments. This assured that f l u i d then in the rectum remained there and was available for sampling after the following manipulations. The cut ic le of the larva posterior to the ligature was ripped using needle-tipped forceps, with care being taken not to pinch too deeply and r ip the swollen rectum. Under this cut ic le the rectum was clear ly v is ible and could be punctured with a micropipette for f l u i d sampling. Glass micropipettes were made by pull ing microelectrodes on a ver t ica l electrode puller (David Kopf Instruments) and breaking off the t ip to yield a sharp yet small-bored t ip . 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 con coating l iquid and sucking the l iquid into the bore and blow-ing 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 in through the posterior end of the posterior rectum. The anterior rectal ce l ls are more easily 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 rectal f l u i d was quickly transferred in the pipette and blown out into a petri dish l ined 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 for analysis they were picked up with a pipette similar to the one used in puncturing the rectum (for osmotic cone analysis) or with a drawn out Pasteur pipette (for ion cone analysis) . Between uses the petri dishes l ined with paraf-f i n wax were emptied of paraffin o i l , rinsed with water and stored to dry on a s lant . Liaatuyed larvae In order to investigate the action of the rectum when separated from Malpighian tubule f l u i d input and in a variety of a r t i f i c i a l hemolymphs, a preparation was designed which involved the l igat ions, both posteriorly and anter ior ly, 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 sixth and seventh abdominal segment, isolat ing 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 l igature was cut away. The cut ic le between the ligatures was torn, being careful not to pinch or tear either the rectum or the trachea in the process. The whole preparation was then l i f t e d , using the ends of the thread used in l igaturing and placed in a r t i f i c i a l hemolymph. Small petri 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 wells, the preparations were placed therein such that the t ip 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 in a covered plast ic container the bottom of which was f i l l e d with water to increase the humidity. After 1.5-2 hours the rectal preparations were removed with forceps using the end of the thread ligatures and placed on dry f i l t e r paper. The cut icle 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 needle-tipped 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 td . ) . Two cuts into the cut ic le 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 pul l the posterior half gently using the siphon as a handle. In three cases out of four the cut ic le would break where the two incisions 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 is pulled from a sleeve. The rectum could then be ligatured as required by the experiment in pro-gress, usually at any two of the following three locations; the intest ine, the anal canal or between the anterior and posterior rectal portions. Small petr i dishes were washed thoroughly in detergent, rinsed six times in tap water, six times in d i s t i l l e d water and a i r dried. On the inside of the petri dish top a c i rc le of s i l i c o n grease was smeared such that a c i rcular spot of ungreased glass approximately 5 mm in diameter was le 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 petri 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 in 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 in 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 sl ide 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 ter paper. While holding the rectum with forceps by means of the end of the thread l igature, the rectum could be punctured with a micropipette as described for whole larvae, and the sample handled as with other prepar-ations. Rectal Preparation for E lec t r ica 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 petri dish was f i l l e d with paraffin wax to a depth of 1 cm. A depression was made in the wax 2 mm wide, 5 mm long and 3 mm deep. The siphon of the l igated posterior portion of 166 the larvae was placed in the depression such that the larvae l a / dorsal side down. A glass microscope s l ide coverslip was s l i d into place covering most of the depression but allowing the siphon to remain in place. The edge of the coverslip 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 issue. The entire petri dish was then flooded with paraffin o i l . The larva was there-fore immersed in o i l but the siphon was safely sealed into the socket of a i r under the coversl ip. The cut ic le of the ventral side of the larva was torn and a drop of hemolymph was placed over the larva, there-by bathing the rectum in a solution of choice. The e lec t r ica l potentials generated across the rectum were measured using a Radiometer voltmeter and M 701 Amplifier (W-P Instruments, Hamden, Conn.). The indifferent 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 amplif ier . The electrodes were pulled on a ver t ica l glass electrode puller and f i l l e d by boil ing in 37 MKC1 under vacuum. Unless int racel lu lar potentials were required, the t ip was broken off to reduce t ip potentials. The e lect r ica 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 re-corded. Using this preparation the hemolymph could be changed to follow i t s effect on the e lec t r ica l potential across the rectum. 2) The e lec-trode was used to pierce the rectum and measure the potential in the anterior or posterior portion of the rectum. Intracel lular potentials were obtained by using a f ine-t ipped microelectrode (resistance 10 Meg Ohms) and slowly approaching the rectum unt i l an abrupt change of potential occurred. This potential was recorded, the electrode was advanced unt i l a potential was observed indicating that the rectal lumen had been reached. The electrode was then backed off again so that the t ip was in the c e l l again, and this potential was recorded as wel 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 relat ively dry and were trans-ferred with needle-tipped forceps to a small piece of "Parafilm" on the stage of a microscope. The cut icle 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 in 1 ul Drummond pipettes 167 In most cases, one microliter of hemolymph could be isolated* When a lesser volume was taken up, the length of the f l u i d column in the micropipettes was measured and the ratio of that length to the total length of the pipette was considered to be the ratio of the volume of the sample relative to one microliter* For osmotic concentration readings, the hemolymph on the *Parafilm* was taken up directly into the pipette used for loading the osmometer. For ion concentration measurements, the Drummond microcap pipettes were emptied into the proper solutions for 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 (Fisher) . The drops were placed in half a petri dish which had been l ined 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 dist inct edges i f illuminated from below. The diameters of the drops were measured at 50 times magnification using an eyepiece micrometer in a Wild stereo binocular microscope. This method was calibrated with a solution of radioactive inul in C-carboxy dissolved in 10$ sea water. Drops were placed in o i l and their diameters were measured. These drops were then taken up in drawn-out Pateur pipettes and placed in 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 iqu id s c i n t i l l a t i o n counting system (Nuclear Chicago). Several samples of the identical f l u i d were taken up in 1 microl i ter Drummond s e l f - f i l l i n g micropipettes and these samples were used to calculate the radioactivity of a known volume. Figure 1 shows the results of this ca l ibrat ion. 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 rectal secretion produced from a hemolymph solution of known ionic and osmotic character ist ics. In addit ion, i t was of interest to vary the ionic parameters and monitor the effect of these changes on rectal secretion and components and trans-rectal e lec t r ica l potentials. 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 is ted in Chapter 1. The purpose of this appendix i s the c la r i f i ca t ion of the specif ic order in which the hemolymph components wexe added so as to optimize the match with the measured parameters of real mosquito larva hemolymph. In addition, 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 deficient in one ion are to resemble the complete one in every ionic 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 Cl 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 so l id NaOH pe l le ts , dipped into the solution and removed as a means of gradual t i t r a t ion . The magnesium and potassium concentrations were measured and adjusted to the proper level with MgCl2 a n c ' KC1, respectively. Chloride concentra-tion was then measured and adjusted with NaCl. Next, the sodium concen-tration was' determined and adjusted with Na2Su"4. F ina l l y , the osmotic concentration was measured and adjusted with sucrose. 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 ike the normal a r t i -f i c i a l hemolymphs. When no sucrose was added, the lowest osmotic concen-tration in 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 in Chapters 2 and 3 contained the normal organic components, CaCl2, MgC^ and NaOH to pH 6.8. The sodium levels were adjusted with Na2S04. 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 lectr ica l studies in Chapter 4, therefore CaSO^ and MgSO^ were substituted for the chloride salts of these metals. 17.0 A r t i f i c i a l hemolymphs with variable sodium concentrations were produced using a more restricted 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 in this way, largely because the amount of c i t r i c and malic acid were restr icted. I suggest that the reader, i f he wishes to produce this hemolymph, omit the malic acid when the organics are added, adjust potassium with KOH, and then t i t rate down to pH 6.8 with malic ac id . This gives an uncertain malic acid concentration but a l l the inorganic ions w i l l be at their 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 con-centration levels were brought to the proper level with choline chloride, yielding a hemolymph with a low sodium concentration (5mW), and with unadjusted osmotic concentration. Half of this solution was re-moved and adjusted to a high sodium concentration (200mM) with Na2S04. 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 solut ions. PUBLICATIONS Bradley, T.J. & P e r k i n s , D.L. (1975) I o n i c Antagonism i n Mosquito Larvae, Culex p i p l e n s . Comp. Biochem. P h y s i o l . 52A: 403-407. Bradley, T.J. (1975) R e c t a l S e c r e t i o n i n Saline-water Mosquito Larvae. Am. Zool. 15 (3): 794. Bradley, T.J. & P h i l l i p s , J.E. (1975) The S e c r e t i o n of Hyperosmotic F l u i d by the Rectum of a Saline-water Mosquito Larva, Aedes taeniorhynchus. J_. exp. B i o l . .63: 331-342. P h i l l i p s , J.E. & Bradley, T.J. (1976) Osmotic, and I o n i c Regulation i n Saline-water Mosquito Larvae. In Transport of Ions and Water i n Animal Tissues. (Eds. Gupta, Moreton, Oschman and Wall) Academic Press ( i n press.) 

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