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Role of the Malpighian tubules in acid-base regulation in the desert locust Schistocerca gregaria Stagg, Andrew Peter 1992

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ROLE OF THE MALPIGHIAN TUBULES IN ACID-BASE REGULATION IN THEDESERT LOCUST SCHISTOCERCA GREGARIAbyANDREW PETER STAGGB.Sc., The University of British Columbia, 1988A THESIS SUBMITTED IN PARTIAL FULFILLMENTOF THE REQUIREMENTS FOR THE DEGREE OFMASTER OF SCIENCEinTHE FACULTY OF GRADUATE STUDIES(Department of Zoology)We accept this thesis as conformingto the required standardTHE UNIVERSITY OF BRITISH COLUMBIAOctober 1991© Andrew Peter Stagg, 1991In presenting this thesis in partial fulfilment of the requirements for an advanceddegree at the University of British Columbia, I agree that the Library shall make itfreely available for reference and study. I further agree that permission for extensivecopying of this thesis for scholarly purposes may be granted by the head of mydepartment or by his or her representatives. It is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.Role of the Malpighian tubules in acid—base regulation in thedesert locust Schistocerca gregaria byAndrew Peter Stagg(Signature) Department of Zool ogyThe University of British ColumbiaVancouver, CanadaDate  November 29. 1991DE-6 (2/88)11AbstractMalpighian tubule fluid from Schistocerca gregaria adults, starved for one day, wascollected by cannulation of the gut in situ, both before and after injecting 10 gmol of HC1 orNaC1 into the haemocoel. Haemolymph pH at the neck remained depressed by 0.3 units for atleast 8 hours in HC1- compared with NaCl-injected locusts. Haemolymph pH remained lowercompared with pre-injected haemolymph values for several hours. The pH of tubule fluidremained about 0.5 units more acid than haemolymph under all conditions. Thus, net tubular acidsecretion was proportional to haemolymph acid-base status. The greater acidity of tubular fluidafter acid-injection was associated with lower estimated bicarbonate concentrations and higherPco2 , without any change in total CO 2 when compared with controls. The combined contributionof bicarbonate, phosphate, and urate to total buffer capacity of tubular fluid was estimated to be75%, with bicarbonate responsible for 55% of the total. The maximum rate of acid removal byall Malpighian tubules of starved locusts, including W trapped in ammonium ions, was calculatedto be very small in relation to the acid-load injected into the haemocoel. Ligation of the locusthindgut so as to prevent posterior Malpighian tubule fluid flow significantly lowered haemolymphpH as compared with either anterior or sham ligations after HC1 injection. Injection of anartificial saline into the hindgut restored haemolymph pH after HC1 injection. Thus the hindgutper se directly contributes to haemolymph pH recovery from acidosis. Increasing the temperatureto 37° C from 21° C caused a 4-fold increase in tubular secretion rates. In addition, starvedlocusts maintained a temperature/haemolymph pH ratio that is consistent with the alphastathypothesis under starved conditions, but temperature does not affect haemolymph pH under fediiiconditions. Feeding locusts at 21° C lowered haemolymph pH 0.28 units. At 37° C, feeding hadno effect on haemolymph pH. Feeding did not initiate any changes in tubular fluid acid-basevariables at either 21° C or 37° C. The buffering capacity of tubular fluid was not affected bytemperature or feeding. Micropuncture of the gut indicated feeding initiated a movement ofalkaline midgut contents posteriorly into the hindgut. The maximum rate of acid removal wascalculated to double for starved locusts at 37° C as compared to 21° C, but feeding reduced netacid excretion to zero at both 21° C and 37° C.ivTable of ContentsPageAbstract ^  iiTable of Contents ^  ivList of Tables  viiList of Figures  viiiList of Abbreviations ^Acknowledgements  xiiiChapter 1:^General Introduction ^  1General Mechanisms of pH Homeostasis ^  1Ventilation ^  1Ionic transport  2Buffers  3Temperature ^  3Metabolic Compensation ^  4Insect Acid-Base Regulation  4Preliminary Observations ^  4Anatomy of the Locust Excretory System^  7Acid-Base Transport in the Locust Hindgut  8Ammonia Secretion in Locusts ^  10Chapter 2:^Acid-Base Parameters of the Malpighian Tubules and Response to Acidosis 13Introduction ^  13Materials and Methods ^  14Animals  14Collection of Malpighian Tubule Fluid in situ ^  14Experimental Protocols ^  15Acid-Base Measurements  16Biochemical Measurements  17Statistics ^  17Results ^  18Time Course of Fluid Secretion ^  18Time Course of Tubular Acid-Base Variables in UninjectedLocusts ^  18Effect of HC1 or NaCI Injection on Haemolymph Acid-BaseParameters  23VEffect of Haemolymph Acidosis on the Composition of Malpighian TubuleFluid ^  23Tubular Fluid Buffer Values ^  30Phosphate, Urate, and Total Ammonia Levels in Tubule Fluid ^ 30Discussion ^  31Chapter 3:^Effect of Temperature, Feeding, and Malpighian Tubule Fluid Flow onHaemolymph Acid-Base Regulation ^  38Introduction ^  38Materials and Methods ^  39Animals  39Feeding Protocols  39Hindgut Ligation and Malpighian Tubule Fluid Collection ^ 40Experimental Pertubations ^  41Acid-Base Measurements  41Biochemical Measurements  42Statistics ^  42Results ^  43Effect of Hindgut Ligation on Haemolymph pH Recovery After AcidInjection ^  43Effect of Saline Addition into the Hindgut Lumen^  46Time Course of Fluid Secretion in Fed Locusts: TemperatureEffect ^  48Haemolymph pH Variables ^  51Effect of Feeding and Temperature on Tubular Acid-BaseVariables ^  54Effect of Feeding on Tubular pH as Determined by Micropuncture ^ 56Discussion ^  59Chapter 4: General Discussion ^  63Effects of Cannulation  63Malpighian Tubule Secretions ^  64Comparison of the Locust Excretory System with the VertebrateNephron ^  65References  69viAppendix ^  75viiList of TablesPageTable 2.1^Effect of HC1 and NaC1 injection into the haemocoel on acid-base statusof Malpighian tubule fluid in S. gregaria at 21° C ^ 28Table 2.2^Final pH determinations of haemolymph (neck sample) and tubule fluid atthe end of experiments on three groups of locusts at 21° C ^ 29Table 2.3^Composition (mmol 1- ') of S. gregaria Malpighian tubule fluid collectedin situ at 21° C ^  31Table 2.4Table 3.1Table 3.2Estimated buffer contribution (13) of measured solutes in Malpighian tubulefluid of HC1-injected S. gregaria at 21° C, pH 6.71, and constantPco2 ^  36Food uptake in S. gregaria starved for three days at 21° and 37° C 49Effect of feeding on haemolymph acid-base variables in S. gregaria at 21°and 37° C ^  53Table 3.3^Effect of feeding on Malpighian tubule fluid acid-base variables in S.gregaria at 21° and 37° C ^  55Table 3.4^Composition of Malpighian tubule fluid collected in situ from S. gregariabefore and after feeding at 21° and 37° C ^  57viiiList of FiguresPageFigure 1.1^Anatomy of locust excretory system ^  6Figure 1.2^In vivo pH profile of the locust alimentary canal ^ 9Figure 2.1^Fluid production rates (JO for the full complement of Malpighian tubulesof starved locusts cannulated in situ at 21 ° C ^ 19Figure 2.2Figure 2.3Figure 2.4Figure 2.5Tubule fluid pH in uninjected locusts cannulated in situ at 21° . . . . 20Haemolymph pH relative to the time of injection for different experimentalgroups of locusts starved for one day at 21° C ^ 21Total CO2, Pco2, and HCO3 concentration in Malpighian tubule fluid fromuninjected, starved animals at 21° C as a function of time aftercannulation   22Total CO2, Pco2, and HCO3 concentration in haemolymph collected at theneck from NaCl-injected, starved locusts at 21° C as a function oftime^  24Figure 2.6^Total CO2, Pco2, and HCO3 concentration in haemolymph collected at theneck from HC1-injected, starved locusts as a function of time. . . . . 25Figure 2.7^The pH of Malpighian tubule fluid prior to injection and after injectionwith either NaCl or HC1 ^  26Figure 3.1^Haemolymph pH relative to time of injection for different ligated groupsof locusts starved for one day at 21° C ^  44Figure 3.2^Haemolymph pH as a function of time after injection of HCl ^ 45ixFigure 3.3^Haemolymph total CO2 and HCO3 with time after injection for differentexperimental groups of locusts starved for one day at 21° C ^ 47Figure 3.4^Fluid production rates (JO for the full complement of Malpighian tubulesof starved and fed locusts cannulated in situ at 21° C ^ 47Figure 3.5^Fluid production rates (.4) for the full complement of Malpighian tubulesof starved and fed locusts cannulated in situ at 37° C ^ 50Figure 3.6^Malpighian tubule fluid pH as measured by the micropuncturetechnique ^  58Figure 4.1^Schematic diagram of the locust excretory system ^ 66Figure A.1.^Diagram of pH sensitive microelectrode ^  76List of Abbreviationspequiv^-microequivalent (s)111 - micrcoliter (s)ANOVA^-analysis of varianceATP - adenosine 5'-triphosphateATPase^- adenosine 5'-triphosphataseCa2+ -calcium ioncAMP^- adenosine 3':5'-cyclic monophosphoric acidCC -corpora cardiacaCco,^-complete carbon dioxideCl-^-chloride ioncm2 -square centimetre (s)Cn^-colonCO2^-carbon dioxide gasDH -diuretic hormoneDIDS^-4,4'-Diisothiocyanato-stilbene-2,2'-disulfonic acidg -gram (s)h^- hour (s)Fr -hydrogen ionH2O^-waterHa -haemolymphHC1^-hydrochloric acidxiHCO3^-bicarbonate ionHPO42"^-monobasic phosphate ionIl -ileum- rate of ammonia secretion.111*^ - rate of acidificationJNH,^ -rate of molecular ammonia secretionJrai4+ -rate of ammonium ion secretion-potassium ionKCl^-potassium chloridekg - kilogramKOH^-potassium hydroxide1 - liter (s)M^- moles per liter (molar)mequiv^-milliequivalentsmg2+ -magnesium ionml^- milliliter (s)mm -millimetresmM^- millimolarmmol^- millimole (s)N - numberNa+^-sodium ionNaC1 -sodium chloridexliNaOH^-sodium hydroxideNH3^-molecular ammoniaNH:^-ammonium ionnm - nanometer02^-oxygen gasOH-^-hydroxyl ionPco2^-partial pressure carbon dioxidepH -negative log hydrogen ion concentrationpK^-negative log dissociation constantRe -rectum13^-non-bicarbonate buffer valueJv -rate of fluid secretionS. gregaria^-Schistocerca gregariaS.E.^- standard errorVG -ventral gangliaA^ -change°C -degrees centigradeAcknowledgementsI would like to sincerely thank Dr. John Phillips for his guidance and support throughoutthis project. Dr. Phillips' seemingly limitless enthusiasm was a great help.I would also like to thank Drs. W. Milsom, D. Jones, and D. Randall for their helpfulcomments on the manuscript.I thank Joan Martin for many helpful conversations, technical assistance, and excessivetea breaks.Dr. Jon Harrison was instrumental in getting this project off the ground, and Iacknowledge his patience, enthusiasm, and friendship through many long nights! I would like tosincerely thank Dr. Harrison for his contribution to this project.I would like to thank Jacqui Peach for uncountable hours of fun and sportmanship thatmade graduate work so enjoyable.I would especially like to thank my father, Peter, and sister, Elizabeth, for support overthe years.1CHAPTER 1General IntroductionThe regulation of acid-base status is fundamental to organismal homeostasis. Manyproteins of physiological importance are bathed in blood or haemolymph and are sensitive tochanges in Ir concentration of these fluids. Maintaining blood pH is critical to physiologicalfunction, but surprisingly little is known of the regulatory mechanisms in insects.Vertebrates use the ventilatory and renal systems to maintain blood Pco 2, HCO3, and pHat constant levels and they have elaborate systems of blood buffers that contribute to maintainingpH within tolerable boundaries. Temperature and metabolism also influence these regulatoryprocesses. I will first review some of the general mechanisms of acid-base homeostasis in air andwater breathing vertebrates and crustaceans, and then outline available information on acid-baseregulation in insects.General Mechanisms of pH Homeostasisa) VentilationMetabolic CO2 dissolves in water to form carbonic acid, which further dissociates intoFr and HCO3. Although the primary function of the respiratory system is gas exchange, the ratioof ventilation to CO2 production influences the tissue Pco 2 . Therefore, because CO2 produced inactively metabolizing tissues will tend to acidify the local tissue and blood, the rate of ventilationaffects the acid-base status of tissue and blood in air breathing animals (Heisler, 1986).2b) Ionic transportIn the vertebrate nephron, active proton secretion is a secondary transport process in theproximal tubule, where 11+ is exchanged for Na+ (Sullivan, 1986). Extrusion of Na+ from the cellsinto the interstitial space via the Na+-K-f ATPase sets up a favourable gradient for Na to enterthe cells from the lumen (Sullivan, 1986). Reabsorption of HCO3 takes place in the proximaltubule primarily as a result of Natfr - secondary transport (see below). There is also a C1 --HCO3exchanger found in other segments of the nephron (Sullivan, 1986). Renal absorption of HCO3shifts the blood bicarbonate buffer system to the alkaline side.The blood protons translocated to the nephron lumen may have several effects, dependentupon the composition of the primary urine formed in the glomerulus (Pitts, 1968). Firstly, if theprimary urine HCO3 levels are high, the extruded protons will titrate HCO3 to carbonic acid andthe resulting CO2 diffuses into the cell and dissociates back into HCO3 and W. Secondly, ifphosphate is present in the alkaline dibasic form, protons will form the titratible monobasic acidwhich can be excreted as a neutral salt. Finally, if tubular buffers such as HCO3 and HPO 42- arelow, then ammonia derived from glutamine and other amino acids in kidney epithelia will trapprotons to form NH4+ in the lumen. Ammonium is relatively lipid insoluble and therefore remainsin the tubule lumen, thereby enhancing Fr removal from the body (Pitts, 1973).Fish regulate blood Pco2 mostly by ionic movements of HCO3, because the differencebetween inspired and expired Pco 2 is low (Heisler, 1986). Diffusional gas exchange of CO 2 takesplace across the gill epithelium; however transport of CO 2 to the gill site is via fr and HCO3.Transepithelial transfer of Fr for Na, NH4+ for Na, and HCO3 for CF also occurs across the fishgill (Heisler, 1986).3Aquatic crustaceans have the same limitations for eliminating CO 2 as fish; i.e. ventilationis largely dependent on 0 2 requirements (Cameron, 1986). The gills again appear to be the majorsite of acid-base regulation. Gill CI1HCO3 and Na+/H+ exchange are the dominant mechanisms.c) BuffersChanges in pH from an acid or alkaline load will be greatest when blood non-bicarbonatebuffer value is lowest (Heisler, 1986). The higher the concentration of blood buffers, the greaterthe resistance to change in pH. Therefore, mobilization of blood buffers in response to a changein blood pH is a mechanism of pH homeostasis. In vertebrates, the CO2/HCO3 buffer systempredominates as the major extracellular blood buffer (Truchot, 1987). In addition, major non-bicarbonate buffers include residues on the polypeptide chain of proteins, and inorganic andvarious organic phosphates (Heisler, 1986).d) TemperatureRegulation of blood acid-base status is affected by temperature, and models of howectothermic animals maintain acid-base balance in the face of a change in temperature iscurrently a matter of considerable debate. One model, the alphastat hypothesis, predicts thatchanges in blood pH reflect an attempt to maintain fractional dissociation state of proteins whenthe temperature changes (Reeves, 1977). Reeves has argued that the imidazole moieties ofhistidine residues of proteins, with a pK near 7 and a heat of enthalpy around 7 kcal mor i ,produce a LpK/AT relationship in the range of -0.017 to -0.023, which fits many observedApH/AT relationships (Cameron, 1988). However, many animals show changes in pH with4temperature that are not consistent with the alphastat model (Heisler, 1984). A second modelpredicts that blood pH should change with temperature to maintain a constant relative alkalinity•i.e. the difference between blood pH and the pH of neutral water is maintained constant (Heisler,1984).e) Metabolic compensationMetabolic reactions can influence the pH of blood (Hochachka and Mommsen, 1983;Portner, 1987). For example, Atkinson and Bourke (1987) maintain protein metabolism resultsin large amounts of released bicarbonate, about 1 mole/100 g protein. However, the traditionalview of renal physiologists is that the acid formed from sulphur and phosphate groups associatedwith protein lead to net acid production.Insect Acid-Base RegulationPreliminary ObservationsInsects have recently been shown to also maintain acid-base status, and attention is nowfocused on mechanisms of this regulation. There are a few early studies of haemolymph acid-baseparameters (Craig and Clark, 1938; Levenbrook, 1959); however CO 2 loss during measurementsin these studies may have led to questionable values for haemolymph pH. Strange (1982)conducted the first detailed study of insect haemolymph pH regulation using saltwater mosquitolarvae.More recently, acid-base regulatory mechanisms have been investigated in a terrestrialinsect, the locust, by Harrison (1988, 1989) at different environmental Pco 2 's and temperatures.5Locusts regulate ventilation in response to environmental hypercapnia, thus controllinghaemolymph pH changes due to changes in blood Pco2. Harrison (1989) also showed thathaemolymph pH is regulated with changes in temperature. In two locusts, M. bivittatus and S.nitens, a pattern of haemolymph pH regulation consistent with the alphastat hypothesis wasobserved over the temperature range 25° to 35° C (Harrison, 1989). From these studies it is clearlocusts regulate haemolymph pH in response to temperature and changing blood Pco 2 levels.Haemolymph buffer systems have also been investigated in the desert locust. Harrison etal., (1990) found that, as in vertebrates, the major buffers of physiological importance arebicarbonate and protein, with HCO3 accounting for 60% of the total haemolymph buffer value.Haemolymph acid-base regulation has also been investigated in response to exercise andacid injection. Harrison et al. (1991a) found that regulation of pH back to resting levels aftervigorous hopping can be largely explained by increased tracheal ventilation.Haemolymph recovery from severe acidosis initiated by HC1 injection (blood pH reducedby 0.5 units) has also been investigated in locusts by Harrison et al., (submitted). HaemolymphpH values returned to pre-injection levels within 8 hours primarily by transfer of additionalHCO3 into the haemocoel while Pco 2 remained constant. This recovery of haemolymph pH afterinjection of acid (10 [mop was accompanied by significant lowering of luminal pH in the crop,midgut, and at the point of Malpighian tubule entry to the gut (see Fig. 1.1). Haemolymph buffervalues are not altered according to acid-base status (Harrison et al., 1990b). Harrison (1989) hascalculated that the alimentary canal contains greater than 30% of total body water and a largefraction of bicarbonate. Therefore, it seems the lumen of the alimentary canal is an importantpotential sink for net acid equivalents during regulation. A great deal is now known about the6MIDGUTTUBULESMALPIGHIANILEUM^COLONc 11 11 KC1, Na' andWater secretion 4••••••^•11 Water and ion, reabsorption•••Water, ion andmetabolite reabsorptionRECTUMANUSStrongly hyperosmoticor hyposmotic excretaFigure 1.1. Anatomy of the locust excretory system. The flow of urine is indicated by the thinarrows and transfer across the epithelia is indicated by the thick arrows. (From Audsley, 1991).7acid-base transport mechanisms in locust hindgut epithelia but the anatomy of the locustexcretory system should first be described.Anatomy of the Locust Excretory SystemThe locust excretory system consists of approximately 250 Malpighian tubules that insertbetween the midgut and hindgut complex (see Fig. 1.1; Garrett et al., 1988). The hindgut isdivided into 3 segments: ileum, colon, and rectum. The ileum and rectum are the main sites ofselective ionic transport and water reabsorption with the ileum reabsorbing the bulk of fluidisosmotically, while the rectum adjusts the final osmolarity of the faeces (Phillips et al., 1986).The Malpighian tubules consist of a simple epithelium, are 10-23 mm in length, and aredistally blind-ended (Bradley, 1985; Garret et al., 1988). Approximately 30% of the tubules aredirected anteriorly and anchored to trachea and caecae. The rest of the tubules run posteriorlyalong the alimentary canal and rest on rectal pads with their associated tracheal connections orfloat freely in the haemocoel cavity. Malpighian tubules can be categorized into 3 sections onthe basis of histology but there is no evidence for physiological differentiation (Garrett et al., 1988).Insect Malpighian tubules are functionally analogous to the vertebrate glomerulus in thatboth produce a primary isosmotic urine (Phillips, 1981). Locust Malpighian tubules drive fluidsecretion by active KC1 transport (Maddrell and Klunsuwan, 1973) rather than by pressure-drivenfiltration at the glomeruli. This IC+ rich isosmotic primary urine flows into the hindgut (andmidgut; Dow, 1986) where selective reabsorption occurs in the ileum and rectum. The rate offluid secretion is usually proportional to the concentration of IC+ in the external medium bathingin vitro preparations. This is true for most insects studied except in the case of Rhodnius (and8other blood-sucking insects), where Na+ ingestion is high and therefore fluid secretion is both Na+and IC-1- driven (Phillips, 1981).Acid-Base Transport in the Locust HindgutThe pH of the gut content differs from that of haemolymph over much of the gut length(see Fig. 1.2; Phillips et al., 1986; Thomson et al., 1988). Earlier in situ studies showed that thelocust rectum was capable of actively acidifying the luminal contents even after rinsing severaltimes with an alkaline buffer (Phillips, 1961). Thompson et al., (1988a) showed conclusively thatprotons are actively secreted across the apical membrane of the rectum against a largeelectrochemical gradient. This active proton extrusion is Na+, Cl- , Me+, and Ca2+ independentand can be inhibited by azide. This locust proton pump is similar in properties to that describedin the turtle bladder (Thomson, 1990).Both the ileum and rectum have been identified as sites of HCO3 reabsorption. Thompsonand Phillips (1985) showed that HCO3 reabsorption is 100% inhibited by DIDS. Thompson(1990) has proposed that the powerful proton pump in the apical membrane acidifies the rectallumen, thereby titrating HCO3 to CO2, which diffuses across the apical membrane and combinesin the cell with OH- formed behind the 1-14 pump. The resulting HCO3 moves to the haemocoel.Lechleitner et al., (1989) showed that HCO3 reabsorption occurs in everted ileal sacs andthis absorption is inhibited by extracts of ventral ganglia (VG) and corpora cardiacum (CC). Thelocust ileum also secretes acid equivalents (Audsley, 1991), and although the mechanism has notbeen described, it seems probable that this is what generates the high rates of HCO3 reabsorptionobserved in this segment. Acid secretion in the ileum is inhibited by extracts of CC, VG, and byaddition of cAMP (Audsley, 1991). HCO3 reabsorption can also be inhibited by addition of these8I 7 -a6i9I^1Ha Midgut II Cn ReFigure 1.2. In vivo profile of the locust alimentary canal. Ha, haemolymph; Cn, colon; Re,rectum. N= 10 for each value; Mean ± S.E. (From Thomson, 1990).10factors to the serosal side of flat sheet preparations. Acid secretion rates are significantly reducedin the rectum by serosal addition of cAMP (Thomson et al., 1988a). Therefore, both the rectumand ileum are sites where titratible acidity can be modified via putative hormonal mechanisms.Hence these sites could be implicated in haemolymph pH homeostasis. In support, the pH ofrectal contents increases to 6.2 after feeding from below 5.0 in starved individuals (Speight, 1967;Harrison, et al., submitted).Ammonia Secretion in LocustsThe Malpighian tubules secrete large amounts of amino acids (50mM), particularlyproline, into the hindgut (Chamberlin, 1981). Proline and other amino acids have been shown toact as respiratory substrates to support active Cl - transport and hence fluid reabsorption in boththe rectum and ileum (Chamberlin and Phillips, 1982; Lechleitner and Phillips, 1989; Peach andPhillips, 1991). Metabolism of these amino acids leads to the production of ammonia.' Thomsonet al., (1988b) have demonstrated that the rectum secretes significant quantities of ammonia intothe rectal lumen as NW (rather than NH 3) primarily by apical, amiloride-sensitive NaINH 4+exchange. More recently, Peach (personal communication) has characterized ammonia secretionin the ileum and found even higher rates of ammonia secretion which is independent of luminalNa+ levels. If the diffusion-trapping mechanism proposed for the vertebrate kidney (Good andKnepper, 1985) were present in the excretory system of the locust, one would expect the rate ofammonium secretion (JNTH,+) to increase as the hindgut lumen becomes more acidic. This is notthe case for either the rectum (Thompson et al., 1988b) or the ileum (Peach, personalAmmonia refers to total ammonia: i.e. molecular ammonia (NH 3) plus ammonium ion (NH4').11communication). Therefore an alternative model to diffusion trapping must be proposed for bothlocust ileum and rectum.The acidic rectal contents, coupled with the large amounts of ammonia production, againsuggest the rectal epithelium is involved in pH regulation. Moreover, a low luminal pH couldincrease phosphate reabsorption and precipitate urate and ammonium salts (Harrison and Phillips,submitted). The discovery of high ammonia secretion rates in locust hindgut also raises thequestion of the relative importance of ammonia and uric acid as excretory end-products (Phillips1986). The traditional dogma is that NH 4+ excretion is associated with copious water loss whereasurates are the major nitrogenous end-product in dry environments where water conservation iscritical to survival (Cochran, 1985). Harrison et al. (submitted) report, however, that ammoniumurate is in fact less soluble than uric acid and excess NH4+ is precipitated apparently with organicanions in locust excreta, which can be quite dry. It is not known whether insect Malpighiantubules might secrete NH3 , and if so, how important this might be compared with ammoniasecretion demonstrated in hindgut.Despite the recent research into acid-base regulation in terrestrial insects, no research hasbeen done on the involvement of Malpighian tubule secretion in haemolymph acid-baseregulation. In Chapter 2, I measure major acid-base parameters in Malpighian tubule secretioncollected by cannulation in vivo. The response of these tubules to acid injection into thehaemocoel was assessed by measuring changes in pH, CO 2, calculated Pco2 and HCO3 -, and totalbuffer value in the secretion. Ammonia and urate values for tubular fluid were also measured toassess their relative contribution to nitrogenous excretion and acid-base balance. In Chapter 3,I describe the effect of feeding and temperature on acid-base physiology of Malpighian tubules12and implications for haemolymph pH regulation. Finally, in Chapter 4, I discuss the similaritiesand differences between the vertebrate nephron and the locust excretory system in whole bodyacid-base regulation.13CHAPTER 2Acid-Base Parameters of the Malpighian Tubules and Response to AcidosisIntroductionRegulation of acid-base balance in terrestrial insects has been a neglected area (Chapter1). Phillips et al. (1986) proposed that the process is probably similar to that in vertebrates: ie.the respiratory (tracheal) system regulates haemolymph Pco2 and the excretory system (consistingof Malpighian tubules and hindgut) regulates haemolymph bicarbonate levels. As a result,haemolymph pH is maintained within narrow limits as recently reported for locusts during rest,temperature change and activity (Harrison, 1988; 1989; Harrison et al., 1991a).Indeed, during hopping and recovery at 35° C, regulation of pH back to resting valuescan be largely explained by increased tracheal ventilation (Harrison et al., 1991a). In contrast,recovery from acidosis caused by injection of HCl into desert locusts is almost entirely due toincrease in haemolymph bicarbonate levels (Harrison et al., submitted). Although the source ofthis additional haemolymph bicarbonate has not been firmly established, the excretory system ofthe locust seems likely. A significant fraction of the acid removed from the haemocoel withina few hours of acid injection appears (temporarily at least) in the midgut contents (Harrison etal., submitted). This transfer may be partially mediated by the Malpighian tubules.It is now firmly established that locust ileum and rectum secrete H+ and NH4+, and absorbOH- and HCO3; moreover, these processes can be substantially modified by cAMP, and bycorpora cardiaca and ventral ganglia factors (Thomson et al., 1988a, 1988b, 1991; Lechleitneret al., 1989; Audsley, 1991; Chapter 1). However, the potential contribution of Malpighiantubules to acid-base regulation has not been investigated in any terrestrial insect. Even simple14measurements of the primary determinants of acid-base excretion (ie the concentrations of CO 2 ,levels of bicarbonate, phosphate, ammonia, organic acids, and total buffer capacity) have neverbeen measured in tubule secretion. The exception is some in vitro values for tubular bicarbonatein corixids living in alkaline lakes, an unusual situation for most insects (Cooper et al., 1987).Previous relevant information for terrestrial insects is restricted to some isolated estimates of pH,urate or phosphate levels in fluid collected from Malpighian tubules isolated in vitro (reviewedby Phillips, 1981; also O'Donnell et al., 1983; Andrusiak et al., 1980; Hanrahan et al., 1984).In no insect has the response of tubular acid-base parameters to changes in haemolymph pH beeninvestigated. In this chapter I describe such a study, using tubules of the desert locust cannulatedin situ and with acidosis initiated by HC1 injection into the haemocoel.Materials and MethodsAnimalsAdult female Schistocerca gregaria, 2-3 weeks past final moult, were used in allexperiments. Locusts were maintained as described previously (Thomson et al., 1988a).Collection of Malpighian Tubule Fluid in situAnimals were restrained under dry cotton to prevent any visual stimulus. An incision wasmade in the third and fourth abdominal segments, and the gut was gently lifted with glass hooks.The gut was ligated just anterior to the point of tubule entry and at the posterior end of the ileum.The ileum was then partially severed near the tubule junction and the gut contents were gentlyteased out with a glass hook. A glass cannula (1.0 mm wide) was inserted into the anterior ileum15and secured with surgical thread. (The end of the cannula was thinly coated with wax to ensurea tight seal). The gut and cannula were then gently inserted back into the abdomen and theincision sealed with wax. The cannula and gut were then rinsed with 100mM KC1 to ensure noblockage was present and the rinse solution was removed with a syringe. Rates of tubule fluidproduction were calculated by measuring the fluid advancement through the glass cannula atintervals and calculating volume from the inside diameter of the tubing.Experimental ProtocolsLocusts were placed in individual containers and fed lettuce ad libitum for 2 hours at 35°C the day before cannulation. Animals were then starved for 18-24 hours at 21° C (theexperimental temperature) with access to cotton soaked with distilled water, and then cannulatedas above.The influence of haemolymph acidosis on composition of Malpighian tubule fluid wasassessed as follows: When 20 pl of tubular fluid had been collected in the cannula (usually after2-3 hours), this fluid was drawn into a length of fine PE 20 tubing by a Hamilton syringe. Thisconstituted the pre- injection sample. Then acidification was initiated by injecting 25 pl of 0.4M HCl into the abdominal haemocoel through the 7th or 8th intersegmental membrane. A further20 pl of tubular secretion was collected after another 2-3 hours. This constituted the post-injection sample on the same locust. A second 20 pl post injection aliquot was often collected, but no significant difference in solute composition were observed as compared to the first post-injection aliquot. Only data for the first period is therefore included in this report. Since mymethod precluded reabsorption of tubule fluid in the midgut and hindgut, the resulting reduction16in haemolymph volume must be considered. In most later experiments, 20 pl was collected beforeand another 20 pl of tubule fluid collected after injection of acid or NaC1 into the haemocoel.This represents roughly 10% of haemolymph volume. No more than 20% of haemolymph volumewas estimated to be secreted in any of the long experiments lasting up to 8 hours. Preparationscontinued to secrete at low rates for 1-2 days.As a control for possible diuresis resulting from fluid injection, other locusts were treatedin the same way, but they were injected with 25 pi of 0.4 M NaCl. Finally a third group werenot injected but otherwise treated identically to assess changes in composition of tubular secretionand haemolymph with time due to hindgut cannulation.Acid-Base MeasurementsTubular fluid pH was measured with glass-microelectrodes on 1-2 IA samples aspreviously described (Harrison et al., 1990b). Total CO2 was measured with 4 IA aliquots usingthe technique of Boutilier et aL (1985). Bicarbonate and Pco2 were calculated from total CO2and pH assuming carbonic acid pK values and CO2 solubility coefficients were identical to thosein locust haemolymph. (Harrison, 1988).The method of Chamberlin and Phillips (1982) was used to verify that collection of fluidfrom a cannula did not result in CO2 loss. As before, an incision was made in the third and fourthabdominal segments. Instead of placing a cannula in the gut, ligatures were made just anteriorand posterior to the point of tubule entry. Fluid was allowed to collect in the distended sac, andpH and total CO2 determinations were made on fluid collected by puncturing the sac with aHamilton syringe. In preliminary experiments, the pH and total CO 2 values estimated in this way17were similar to values determined by cannulation.Biochemical MeasurementsTotal urate and inorganic phosphate were determined on 5 pl samples of tubular fluiddiluted 100-fold with distilled water. Urate concentrations were determined spectrophotometricallyat 520 nm with a commercial kit (Sigma) and uric acid stock solutions as standards. Inorganicphosphate was determined spectrophotometrically at 820 nm by the technique of Chen et al.(1956). Total ammonia (ammonia + ammonium) concentrations were determined by theenzymatic assay of Kun and Kearny (1974). The non-bicarbonate buffer value (13) for tubularsecretion was determined using samples diluted 100-fold with 100 mM KC1. Dilute samples wereacidified with 20 mM HC1 until the pH was below 4.0. Samples were then stirred for 2 hours todrive off CO2 and HCO3. Buffer value was then determined from pH 6.4 to 7.0 by titrating with20 mM NaOH using a PHM 84 research pH meter, TIT 80 titrator, and ABU 80 autoburrette(Radiometer, Copenhagen, Denmark).StatisticsAll data are presented as mean ± standard error (S.E.) with N indicating the number of locusts.Paired t-tests were used to determine significant differences in composition of secretion causedby acid injection. Where paired t-tests were not appropriate, significance of difference betweenmeans was determined by one way ANOVA, with p<0.05 accepted as significant.18ResultsTime course of fluid secretionThe rate of fluid secretion (4) by insect Malpighian tubules isolated in vitro generallyfalls with time (reviewed by Maddrell, 1980). I therefore first followed Jv with time for the fullcomplement of locust tubules in starved animals, as determined by hindgut cannulation in situ(Fig. 2.1). The initial mean A, over the first 2 hours varied between 8 and 15 p1 If' in differentexperiments and gradually fell by 20-40% over 9 hours. These rates are within the range ofvalues previously reported for the full complement of Schistocerca tubules using in vitro andother in vivo methods (reviewed by Phillips, 1981).The injections of 25 IA of 0.4 M NaC1 or HCl into locusts between the 2nd and 3rd hourafter cannulation had no measurable effects on J v as compared to uninjected controls (Fig. 2.1).There was no statistical difference between Jv values for the three treatment groups except forHC1 injected locusts at the third hour.Time Course of Tubular Acid-Base Parameters in Uninjected LocustsThe pH of tubular fluid from starved, uninjected locusts did not change significantly over7 hours and averaged 6.63 (Fig. 2.2). This is 0.61 pH units below the initial haemolymph valueof 7.24, which fell slightly to 7.11 over 6 hours of cannulation (Fig. 2.3). Initial values in tubularfluid over the first 2 hours were: total CO 2 (8.9mM), Pco2 (55 toff), and HCO3 - (6.5 mM), andthese did not change significantly over 8 hours (Fig. 2.4).15—T40 1 0*4as1.400.•.;000  5$4ta,ny0 190Io4 I^I^i^I^12 4 6Time (hours)I^48110Figure 2.1. Fluid production rates (4) for the full complement of Malpighian tubules of starvedlocusts cannulated in situ at 21° C. Mean ± S.E. (N=10-20) for elv over an equal period beforeand after the point of injection (ie: 1 or 2 hour total period). Values for the three treatmentgroups [uninjected (•), HC1-injected (s), and NaCl-injected (A)) are not significantly differentexcept for the HC1 group at 3 hours after cannulation.207.0-6.8-6.6—C).54oE6.4 -6.2 —6.00^2^4^6^8Time (hours)Figure 2.2. Tubule pH in uninjected locusts cannulated in situ at 21° C (Mean ± S.E., N=5-9).The apparent decrease in pH over 8 hours is not significant (p>0.5).210^2^4^67.4—7.2: 47.0—c) 6.6—=6.4—6.2 4—2Time (hours)Figure 2.3. Haemolymph pH relative to the time of injection (0 h) for different experimentalgroups of locusts starved one day at 21° C. Mean ±S.E. (N=4-9). Values are for haemolymphcollected at the neck (all symbols except A), or near the abdominal injection site (A), of locustsinjected with 25 Al of 0.4 M NaCI (0) or 0.4 M HCl (+) or uninjected (•). The pH ofhaemolymph sampled from the neck prior to cannulation is also given (R). HC1 injection causeda significant decrease (p=0.000) in haemolymph pH within 15 min (first sample) of injection.Haemolymph pH remained depressed after HC1 injection for 7 hours.2212— — 8010-— 608— c.0 6— — 40 owA4—— 202—0^I I 0I0 2^4^6^8Time (hours)Figure 2.4. Total CO2 (0), Pco2(■), and HCO3- (A) concentrations in haemolymph collected atthe neck from uninjected, starved animals at 21° C as a function of time after cannulation. (MeanS.E. N=4-5). There was no significant change in any of these variables due to injection, or withtime after injection.23Effect of HCl or NaCl injection on Haemolymph Acid-Base ParametersBefore cannulation, locust haemolymph pH averaged 7.28. Injection of 25 p1 of 0.4 MNaC1 into the abdomen of locusts (control) did not significantly change haemolymph pH, whichremained above 7.2 for at least 5 h after injection (Fig. 2.3). Likewise haemolymph total CO 2 ,Pco2 and HCO3 were not significantly changed by NaC1 injection and remained close to 8.0 mM,18 toff, and 7.5 mM respectively (Fig. 2.5). Within 15 min of injecting 25 p1 of 0.4 M HC1 intothe abdomen, haemolymph pH fell by 0.34 units to 6.87, as measured at the neck (30-50 1..ilsample) and to 6.4 as measured by abdominal sample near the site of injection. Apparently,complete mixing in the haemolymph compartment is not attained over several hours.Abdominal injection of HC1 had a marked effect on acid-base parameters of haemolymphcollected at the neck (Fig. 2.6). Initial values before injection were similar to those for the NaClinjected group (Fig. 2.5). Over the first 15 min after HC1 injection, total CO2 fell significantlyby over 30% to 5.2 mM, Pco 2 increased temporarily but not significantly, and calculated HCO3fell by 40% to 4.2 mM. Total CO 2 and HCO3 remained significantly depressed 7 hours afterinjection (Fig. 2.6).In summary, changes in haemolymph acid-base parameters (as measured at the neck)caused by acid injection were sustained for at least 7 hours. Subsequent studies on compositionof tubule fluid were normally made within 4 hours of acid injection.Effect of Haemolymph Acidosis on Composition of Malpighian Tubule FluidAcid injection into the haemocoel initially caused a significant decrease in the pH oftubular fluid by 0.46 pH units as compared to NaC1 injected controls (Fig. 2.7). However tubular— 25—15 ti04.,.......cv—10  c.0)a.524I10—T^T• 4•^T• •^T^•8 — 1^1 \410IIIP. ------____ti ,....--------? co 600=4—2rt0U0^•^I^•^I^•^I^t^I^1^I^1^I^•^0—3^—1 1 3 5 7 9Time from saline injection (hours)Figure 2.5. Total CO2 (•), Pco2 (■), and HCO3- (A) concentrations in haemolymph collected atthe neck from NaCl-injected, starved locusts at 21° C as a function of time (injection at 0 hour).Mean ± S.E. (N=5-9). There was no significant change in any of these parameters due toinjection, or with time after injection.EI el04610 —225— 30^T^ -25•T^• ^— 20•• • — 15•••^46:1'..^•^•1—10—50C‘)—3^—1^1^3^5^7Time from acid injection (hours)Figure 2.6. Total CO2 (•), Pco2 (M), and HCO3- (A) concentrations in haemolymph collected atthe neck from HCl-injected starved animals at 21° C, as a function of time (injection at 0 hour).Mean ± S.E. (N=5-8). Pco2 did not change significantly after acid-injection (p=0.13); but bothtotal CO2 and HCO3- levels significantly decreased (p=0.028, 0.007 respectively) after injectionand remained depressed for the next 7 hours.TA1 TAIiiTA1T0I 1^I^I^I^1^I^1^I^I267.2 —7.0 — TI6.8 —=041 6.6 —4E-I6.4 —6.2 —6.0 1 I0^2^4 6 8 10 12Time (hours)Figure 2.7. The pH of Malpighian tubule fluid prior to injection (filled symbols) and afterinjection (open symbols) with either NaCl (A) or HCl (0). Mean ± S.E. (N=10-14). The pH wasdecreased significantly (p=-0.001) immediately after HC1 injection relative to NaCl injectedcontrols.27fluid pH in the NaC1 injected controls also gradually fell with time so that after 6-8 hours therewas no difference between the acid and salt injected groups. Thus the cannulation/injectionprocedure itself caused a slow increase in tubule acidity. Therefore, in the remaining series ofexperiments, I compared the composition of Malpighian tubule fluid pooled over approximately2.0-2.5 hours before injection with tubular fluid collected within 3 hours after injection from thesame locust.The effect of injecting either 10 gmol of NaCl or HC1 on tubular acid-base parametersis shown in Table 2.1. Before injection, the tubule fluid pH of 6.9 was similar in acid and NaC1injected groups. Acid but not NaC1 injection caused a significant decrease in tubule fluid pH by0.46 units to pH 6.48. Total CO2 did not change significantly in the acid injected group, so thatthe effect of increased tubular acidification was to titrate HCO3 (which fell significantly from5.5 to 3.2 mM), to CO 2 . As a result, tubule fluid Pco 2 doubled after acid injection. There is noindication from these results that Malpighian tubules contributed additional HCO3 to thehaemolymph after acid injection.By comparison, the NaC1 injected controls showed no change in Pco 2 after injection.However total CO2 and HCO3 in tubule fluid were initially higher in this group and bothdeclined slightly but significantly after injection.There is no indication that acid injection stimulated long- term active acid secretion byMalpighian tubules, because the final pH difference between haemolymph and tubular fluid wasnot significantly different in uninjected, NaC1 injected and HC1 injected groups (Table 2.2).After 4 hours, tubule fluid was on average 0.56 to 0.75 units acid to haemolymphregardless of treatment. The lower tubule fluid pH initially observed (2 hours) after acid injectionTable 2.1. Effect of HCl and NaC1 injection into the haemocoel on acid-base status of Malpighian tubule fluid in Schistocercagregaria at 21° C. Pre samples were secreted over a 2-3 hour period before injection, post samples were secreted over a 2-3 hourperiod after injection.HCI injected NaC1 injectedpre-inj post-inj pre-inj post-injpH 6.89 6.42* 6.91 6.81± 0.07 ± 0.1 ± 0.08 ± 0.07Total CO2 (mM) 6.2 5.5 11.8 9.1'± 0.8 ±0.7 ± 1.3 ±0.9Pco2 (ton) 24 51' 45 42± 2.8 ± 11.3 ± 5.0 ± 3.8HCO3 - (mM) 5.1 3.2' 9.8 7.3*± 0.7 ±0.4 ± 1.3 ±0.9Non bicarbonate buffer value 9.5 10.4 10.4 15.5'(B)(mmol 1 -1 pH unit')± 1.0 ± 1.2 ± 1.1 ± 2.6Mean ± S.E. (N=9-14). Significantly different from pre-injection value, paired t-test, p<0.05.Non-bicarbonate buffer value is for the pH range 6.4-7.0.Table 2.2. Final pH determinations of haemolymph (neck sample) and tubule fluid at the end ofexperiments** on three groups of locusts at 21° C.Uninjected HC1 injected NaCl injected1) Hemolymph pH 7.15 ± 0.04 6.95 ± 0.01 7.12 ± 0.032) Tubule pH 6.39 ± 0.21 6.40 ± 0.11 6.51 ± 0.113) Difference 0.75 ± 0.18 0.56 ± 0.11 0.60 ± 0.13(1-2)Mean ± S.E. (N=9-14). The pH difference across the tubule wall (3) was not significantlydifferent between treatments.*Values were made on fluids collected between 3 and 6 hours after injection time and 6 to 9hours after cannulation.30(reported in Table 2.1 and Fig. 2.7) may simply reflect greater haemolymph acidity of the formergroup rather than enhanced acid secretory activity. In particular, different pH values ofhaemolymph collected from the abdomen and neck (Fig. 2.3) make it difficult to decide theeffective pH experienced by tubules after acid injection (see Table 2.3).Tubular Fluid Buffer ValuesThe rate of acid secretion is determined not only by tubular pH, but by the buffer capacity(13) which might change following acid injection. I measured the non-bicarbonate buffer value (13)for Malpighian tubule fluid over the pH range 6.4-7.0 observed in earlier experiments (Table 2.1).Titration indicated a constant value for 13 between pH 6.4 and 7.0 (data not shown). The valueof 13 was 9.5 mequiv r' pH unit' before injection, and this was unchanged after HC1 injection.However, NaC1 injection caused a significant increase in 13 to 15.5 mequiv 1 -1 pH unit'. Thenature of this increased buffer capacity was investigated by measuring the levels of majorpotential buffers previously reported in tubule fluid, namely phosphates and urates. Moreover,ammonia will trap secreted Fr to form NH:. Indeed enhanced ammonia production by vertebrateproximal tubules is a major mechanism of removing excess acid (Pitts, 1973). I therefore alsomeasured total ammonia concentrations in locust tubular fluid.Phosphate, Urate, and Total Ammonia Levels in Tubule FluidAs shown in Table 2.3, phosphate levels in tubule fluid increased with time in uninjectedlocusts, and this was significantly enhanced (4-fold increase) by NaCl injection but not HC1injection. This difference in phosphate secretion may account for the greater 13 value of tubularTable 2.3. Composition (mM) of Schistocerca gregaria Malpighian tubule fluid collected in situat 21° C.Uninjectedpre postHC1 injectedpre postNaCI injectedpre postUrate 1.6 1.6 5.3 2.2' 3.6 3.8± 0.4 ± 0.5 ± 0.8 ± 0.2 ± 0.4 ± 1.3Phosphate 2.7 4.8' 4.5 6.7' 4.5 16'± 0.7 ± 1.2 ± 1.0 ± 1.0 ± 1.2 ± 3.9Ammonia 5.2 8.1' 4.6 6.7' 5.3 6.2± 1.0 ± 1.8 ± 0.7 ± 0.9 ± 0.9 ± 1.3Mean ± S.E. (N=9-21 locusts) for fluid collected by cannulation over 2-3 hours before and 2-3hours after HC1 or NaC1 injection, and during same time periods for uninjected locusts." Indicates were significantly different from pre- injection values on the same animals.32fluid from NaC1- as opposed to HC1-injected locusts (Table 2.1). However the contribution ofphosphate to acid removal depends on the form of this anion that is actively secreted by tubules,and this is unknown (reviewed by Phillips, 1981). Average urate concentrations varied from 1.6to 5.3 mmol 1 - ' in the three treatment groups prior to injection. Urate levels decreasedsignificantly from 5.3 to 2.2 mmol -1 following HC1 injection (Table 2.3).Total ammonia was 4 to 5 mM in tubular fluid prior to injection in all three groups. Whilethere was a small but significant increase in total ammonia to 6.7 mM following acid injection,this could reflect a change with time because a similar significant change with time was observedin uninjected locusts (Table 2.3).DiscussionThis study provides the first comprehensive measurements of the major acid-baseparameters for Malpighian tubule secretion in a terrestrial insect in situ. In control locusts starvedfor one day, both total CO2 and HCO3- levels are similar in haemolymph and tubular fluids at 8-9and 7-8 mM respectively. In contrast, calculated Pco 2 levels (55 ton) are nearly 3 times higherin tubular fluid than haemolymph. Tubule fluid, at pH 6.63 is 0.5 pH units more acid thanhaemolymph in the control group, suggesting that locusts already may experience an acidoticstate within one day of starvation. In support, Harrison et al. (submitted) found that the pH offaecal pellets from similarly starved locusts averaged 4.62, compared to 6.2 in feeding animals.Thus control locusts used in my study may have already been responding to a natural acid loadprior to acid injection, and this may have diminished the response to my experimental acidoticchallenge. Regardless, my results permit an estimate of the maximum capacity of the full33complement of locust tubules to eliminate excess acid equivalents.Using measured J.,, (initial average of 10 gl hour'; Fig. 2.1), a median value for themeasured pH difference between haemolymph and tubular fluid under the three conditions studied(0.6 pH units), and the measured highest buffer values for tubular fluid (15 mmol 1-' pH unit')for NaCl injected locusts (Table 2.1), the estimated maximum rate of excess acid removal bylocust Malpighian tubules (JO is 0.09 iirnoles If'. This value could be considerably less if moreconservative values are used in these calculations. While tubule fluid was initially much moreacid after HC1 injection (Table 2.1), so was the haemolymph. It is therefore not possible toconclude that active tubular acid secretion rate was stimulated by HCl injection, especially giventhe heterogeneity in haemolymph pH observed between the neck and abdomen (Fig. 2.3). Clearlymechanisms and possible control of tubular acid secretion will require in vitro studies, where thecomposition of the fluid bathing isolated tubules can be precisely controlled. Nevertheless myin vivo studies do provide the necessary groundwork for such future studies.Given that 10 moles of acid was injected into locusts, the Malpighian tubules clearly donot have the capacity (at a maximum fir of 0.09 mmoles 11 -1 ) to return haemolymph pH to normalvalues within an 8 hour period, as observed by Harrison et al. (submitted) in a parallel study onuncannulated animals. The potential regulatory capacity of the hindgut is many times greater.Both locust ileum and rectum actively secrete acid in vivo at 1.5 limoles hr' cm2 in the absenceof a pH difference (Thomson et al., 1988a, 1988b, 1991). Correcting for surface area of thesehindgut segments, the locust rectum (0.62 cm 2) and ilea (0.4 cm 2) can still secrete IT at 0.6 and0.3 moles hour' respectively against a gradient (0.6 pH units) comparable to the maximumdeveloped by the tubule epithelium. Clearly all three segments of the locust excretory system34(tubules, ileum, and rectum) contribute to acid excretion, with W secretory capacity increasingand luminal pH decreasing posteriorly. Fall in luminal pH during passage through the hindguthas been observed by Thomson et al., (1988a) and Harrison et al. (submitted).Haemolymph pH measured either from the neck or abdomen did not recover substantiallyto control values even after 8 hours (Fig. 2.3). This lack of recovery in the cannulated animalsis evidence for a role of the locust hindgut in haemolymph pH regulation, suggesting interruptionof tubule fluid reabsorption in the midgut and hindgut prevents bicarbonate reabsorptionnecessary for haemolymph pH recovery. In a parallel study using uncannulated desert locusts,haemolymph pH measured at the neck recovered to control values within 8 hours of a similaracid injection, accounted for by a rise in haemolymph HCO3 (Harrison et al., submitted).Calculations of Ai' presented above using buffer values and pH differences do not evaluatethe additional acid removal which may be associated with total ammonia excretion. I did notobserve increased tubular ammonium secretion attributable specifically to HCl injection; however,using the typical total ammonium concentration in tubular fluid of 5.5 mM (Table 2.3), themaximum rate of ammonium secretion (J ), assuming Jv of 10 111 If% is 0.06 p.tmoles 11 - '. ThusJam, could potentially increase net acid secretion by locust tubules to a maximum of 0.15 !moles11- ', assuming all secreted ammonium trapped W which came from the haemolymph. Again, thelocust hindgut is a much more important site for potential acid removal in the form of ammonia.Oxidation of amino acids by the locust rectum supports a J to the lumen of 0.4 gimoles hour'(Thomson et al., 1988b: corrected for surface area). However the locust ileum is by far the majorsource of excreted ammonia, with an unstimulated J of 0.6 !moles h4 , and this can bestimulated to 1.4 grnoles W I by adding 5 mM cAMP. (Lechleitner, 1988; Audsley, 1991).35A surprising observation is that ammonia concentrations equal or exceed those of uratein locust tubular fluid (Table 2.3). As a result of the additional and much larger J in locusthindgut, the final excreta in locusts would be expected to contain ammonia rather than urate asthe predominant nitrogenous end-product, contrary to previous dogma that urate is the major endproduct in locusts (reviewed by Cochran, 1975). (There is no evidence for urate production orsecretion by locust hindgut). In a companion study (Harrison et al.,1990a; Harrison and Phillips,submitted), the high ammonia content prediction is confirmed. The faecal concentrations ofammonia and urate are 270 and 68 (mM Kg' H 2O) respectively in locusts starved for a day,when care is taken to prevent ammonia loss on exposure of excreta to air. A comparison of thesefaecal concentrations with those of Malpighian tubule fluid (Table 2.3) provide interesting newinformation. Assuming no urate secretion in hindgut, the 15-fold increase in urate levels duringpassage through the hindgut suggests that about 95% of tubular fluid is reabsorbed in the hindgut,in agreement with previous estimates (reviewed by Phillips, 1981). The increase in ammonia-to-urate ratio from near 1:1 at the tubules (Table 2.3) to about 4:1 in the faeces, confirms thathindgut contributes over 70% of total ammonia excreted.Finally, I have assessed the contribution of individual potential buffers to the total buffercapacity of locust Malpighian tubule fluid (Table 2.4). Using average measured values of pH andsolute concentrations in tubule fluid, and pK values from the literature (Robinson and Stokes,1959), phosphate (3.2 mequiv pH unit"') is the major non-bicarbonate buffer in Malpighiantubule fluid, while urate (0.84 mequiv pH unit -1) plays a minor role. The contribution ofphosphate to non-bicarbonate buffer value after NaCl injection is 8.4 mequiv r' pH unit " 1 , whichaccounts for the increase in non-bicarbonate buffer value shown in Table 2.1. However,Table 2.4. Estimated buffer contribution (13) of measured solutes in Malpighian tubule fluid ofHC1-injected Shistocerca gregaria at 21° C, pH 6.71, and constant Pco2 .13 (mequiv/l/pH unit)a) Bicarbonate^ 9.90b) Phosphate 3.16c) Urate^ 0.84d) Total value of a+b+c^13.9Component 13 values calculated according to Heisler (1986), using pK values from Robinson andStokes (1959) and measured solute concentrations in tubule fluid post injection (Table 2.3).37bicarbonate ion is overall the major buffer (9.9 mequiv 1- ' pH unit- ') in tubule fluid, accountingfor greater than 50% of total buffer capacity (Table 2.4). Only about 40% of non-bicarbonatebuffer value is represented by total inorganic phosphate and urate. Despite the variability in thetubule fluid concentrations, there must be some additional unmeasured ionic species that accountfor the remaining 20-30% of total buffer capacity of tubule fluid.38CHAPTER 3Effect of Temperature, Feeding, and Malpighian Tubule Fluid Flow onHaemolymph Acid-Base RegulationIntroductionIn Chapter 2 I observed that prevention (by cannulation) of Malpighian tubule fluid flowinto the hindgut reduces the ability of locusts to regulate haemolymph pH. After 8 hours,haemolymph pH of cannulated locusts still had not recovered to pre-HC1 injection values. In acompanion study, Harrison et. a/. (submitted) report haemolymph pH recovery from HC1 inducedacidosis in uncannulated locusts within 8 hours. Harrison suggests haemolymph recovery is dueto a transfer of net acid equivalents to the alimentary canal, associated with movement of HCO3into the haemocoel. I suggested (Chapter 2) that the lack of haemolymph recovery from acidosisin cannulated animals may be due to reduced HCO3 - reabsorption in the hindgut. Previous studieshave shown high rates of HCO3 transport in locust hindgut (Thomson and Phillips, 1985;Lechleitner et al., 1989) and cannulation would deprive the hindgut of respiratory substratessecreted by Malpighian tubules into the gut lumen.Temperature affects the acid-base status of blood and haemolymph (Chapter 1). Harrison(1988) has shown haemolymph pH of locusts decreases with a slope of -0.017 pHU/ 'C,consistent with temperature/blood pH relationships observed for other taxa. Since locustsnormally experience high temperatures (35-40° C) during the day, acid secretion by tubules maybe much more important than suggested by my previous study at 21° C (Chapter 2).The state of feeding can also affect the acid-base status. Possibly hemolymph pH can beregulated faster when food is passing rapidly through the gut after feeding. Harrison et al.39(submitted) have suggested that the alimentary canal might be utilized as a temporary storage sitefor acid equivalents. Speight (1967) showed that feeding state of locusts affects the pH of rectalluminal contents, which has since been implicated circumstantially in haemolymph pH regulation.In this chapter, I examine the importance of respiratory substrates and HCO3 content inthe hindgut lumen in haemolymph pH regulation. In addition, since temperature and feeding arelikely to affect acid-base status, I examine the effects of feeding at two different temperatureson acid-base parameters in Malpighian tubule fluid and on haemolymph acid-base status.Materials and MethodsAnimalsAdult female S. gregaria, 2-4 weeks past their final molt, were used in all experiments.Locusts were maintained and their Malpighian tubules cannulated in situ as previously described(Chapter 2; Thomson et al., 1988a).Feeding ProtocolsGroups of locusts were isolated in a container for 2 days with access to cotton woolsoaked with distilled water. The day before the experiment, animals were placed in individualcontainers at the experimental temperatures (21° C or 37° C), again with access to distilled water.Half of these animals were fed lettuce and bran ad libitum in individual containers for at least2 hours prior to experiments. Net  food uptake was determined by weighing individual locustsbefore and after feeding.40Hindgut Ligation and Malpighian Tubule Fluid CollectionLIGATION: Animals were restrained under dry cotton wool to prevent any visual stimulus. Anincision was made in the third and fourth abdominal segments, and the gut gently lifted withglass hooks. A curved glass rod was carefully dragged from the midgut posteriorly along thealimentary canal, allowing clear access anteriorly to the point of tubule entry, without anyMalpighian tubules lying close to the gut. This procedure does not damage the tubules. A surgicalligature was tied immediately anterior to the point of tubule entry. A similar procedure wasperformed posterior to the point of tubule entry. Loops of surgical thread, remaining loosely tiedaround the gut just anterior or posterior to the tubule entry point, constituted a sham operation.The ligated gut was then gently inserted back into the haemocoel cavity, and the incision sealedwith sealing wax. Animals remained restrained throughout the experiment.TUBULE FLUID COLLECTION: Locusts were cannulated as previously described (Chapter 2). Fluidproduction rates were measured by the advancement of fluid through the cannula. When 20-30ul of tubular fluid had collected in the cannula, the sample was mixed by withdrawal into alength of PE tubing attached to a Hamilton syringe. Total CO2 and pH were measuredimmediately on the pooled sample and biochemical measurements were made as outlined below.41Experimental PertubationsHaemolymph acidosis was initiated by injection of 25 pl of 0.4 M HC1 (10 'mop throughthe 7th or 8th intersegmental membrane. Haemolymph acid-base parameters were measured onsamples collected from the neck membrane as described in Chapter 2.In some experiments, a saline mimicking Malpighian tubule fluid was injected into theposterior ligated hindgut complex by injecting the saline through PESO tubing wedged into theanus. The volume of saline injected into the hindgut equalled the average initial rate ofMalpighian tubule fluid production of 10 pl/hr (Chapter 2) over 6 hours (=60 pl). The saline hadthe following composition (mM): NaH 2PO4 .H 20 (6), NaC1 (35), K2SO4 (126), CaC12 (7),Mg(C2H302)2 4H20 (4), MgSO4 .7H20 (16), KCl (39), glucose (4.6), alanine (1.0), aspartate(0.5), glutamate (0.8), glutamine (0.5), glycine (4.0), proline (38.0), serine (1.0), NaHCO 3 (6),pH 6.63.Acid-Base MeasurementsCANNULATION AND LIGATION EXPERIMENTS: Haemolymph pH was measured on 2 pl samplescollected from the neck as previously described (Harrison et al., 1990b). Total CO2 was measuredon 8 pi aliquots using the technique of Boutilier et al., (1985). Tubular fluid values weredetermined on samples collected as described above. Bicarbonate and Pco 2 were calculated fromtotal CO2 and pH assuming that carbonic acid pK values and CO 2 solubility coefficients wereidentical to those for locust haemolymph (Harrison, 1988).MICROPUNCTURE EXPERIMENTS: Tubule fluid pH was also determined in situ. The abdominalcavity of animals was quickly dissected open and a small incision was made in the gut at the42point of tubule entry into the gut. The glass pH microelectrode was inserted directly into the gutlumen, along with a Ka/agar reference electrode. Similar measurements were made on theposterior midgut contents. These measurements were completed within 2 minutes of capture.Biochemical MeasurementsTotal ammonia (ammonia + ammonium) concentration was determined on 10 gl of thepooled fluid sample that was transferred directly into 40 Ill of 5% tricarboxylic acid (TCA). Theenzymatic assay used was that of Kun and Kearney (1974). Total urate and inorganic phosphatewere determined on 5 gl samples of tubular fluid diluted 100-fold with distilled water. Urateconcentrations were determined spectrophotmetrically at 520 nm with a commercial kit (Sigma)using uric acid stock solutions as standards. Inorganic phosphate was determinedspectrophotometrically at 820 nm by the technique of Chen et al. (1956).The non-bicarbonate buffer value (B) for tubular secretion was determined using samplesdiluted 100-fold with 100 mM KC1. Dilute samples were acidified with 20 mM HC1 until the pHwas below 4.0. Samples were then stirred for 2 hours to drive off CO 2 and HCO3. Buffer valuewas then determined from pH 6.4 to 7.0 by titrating with 2 mM KOH using a PHM 84 researchpH meter, TTT 80 titrator and ABU 80 autoburrette (Radiometer, Copenhagen, Denmark).StatisticsAll data are presented as mean ± standard error (S.E.) with N indicating the number oflocusts. Paired t-tests were used to determine significant differences in composition of secretion43caused by acid injection. Where paired t-tests were not appropriate, significance of differencebetween means was determined by one-way ANOVA, with p< 0.05 accepted as significant.ResultsEffect of Hindgut Ligation on Recovery of Haemolymph pH After Acid InjectionHaemolymph pH is closely regulated under starved conditions in locusts and returns tonormal within 8 hours of acid injection (Harrison et al., submitted). In my initial study (Chapter2) it was observed that cannulation of the gut to collect Malpighian tubule secretions reduced theability of locusts to recover haemolymph pH after injection of HC1. I first repeated an earlierexperiment by Harrison et al. (submitted) to confirm their observation on pH recovery after acidinjection. Unmanipulated animals injected with 10 pmol HCI did indeed recover from the acidosisto pre-injection haemolymph pH values within 8 hours (Fig. 3.1).The reduced regulatory ability after cannulation was attributed to reduced bicarbonatereabsorption in the hindgut complex (Chapter 2). Alternatively, the operation to cannulateMalpighian tubules might itself interfere with haemolymph pH regulation by unknown feedbackmechanisms. I therefore first studied haemolymph pH recovery in sham operated locusts (Fig.3.2). Before HC1 injection, haemolymph pH was 7.31, which is similar to pre-cannulated valuesin Chapter 2. After 6 hours, haemolymph pH from uninjected sham operated animals was slightlylower at 7.22. Within 20 minutes of HC1 injection haemolymph pH had fallen 0.36 pH units to6.95. Total CO2 was reduced by over 30% to 5.8 mM and Pco2 temporarily increased at thattime. These results are very similar to those observed previously after HCI injection intounoperated animals (Chapter 2; Harrison et al., submitted).447.5a.7.0E6.50^3^6Time from injectionFigure 3.1 Haemolymph pH as a function of time after injection (0 h) of 25 111 0.4 M HCI. MeanS.E. (N = 5-9). Locusts were starved for 1 day at 21° C.7.16.9.7.345iT1T■10 3 6Figure 3.2. Haemolymph pH with time after injection (0 h) for different experimental groups oflocusts starved for 1 day at 21° C. Mean ± S.E. (N = 6-12). Locusts were sham operated (•),anterior ligated (A), posterior ligated (•) and then injected with 25 Al of 0.4 M HCI. A fourthgroup were sham operated and uninjected (0). The pH of haemolymph from posterior ligatedlocusts that were first injected with HCI and then injected with a saline mimicking Malpighiantubule fluid through the anus is also shown (•).46Haemolymph from the sham operated group had recovered to a pH of 7.10 within 6 hoursafter injection of HC1, which is significantly higher than 20 minutes post-injection, buthaemolymph pH did not recover to pre-injection values (Fig. 3.2), as shown by Harrison et al.(1991) and this study (Fig. 3.1). No further significant recovery of haemolymph pH was observedbetween 6 and 24 hours after acid injection into sham operated locusts (data not shown).Therefore, in the remaining experiments, I compared haemolymph values 6 hours post injection,using the partial (61%) recovery of haemolymph pH on sham operated locusts as the control.A posterior ligation, which prevents fluid flow posteriorly into the hindgut complex,results in a haemolymph pH of 6.95 at six hours after injection of 10 limol HC1, which issignificantly lower than the similar means for locusts with either anterior or sham ligations. Therewere no significant differences in total CO 2, or calculated Pco2 and HCO3 of haemolymphbetween any experimental group 6 hours after HC1 injection (Fig. 3.3).Effect of Saline Addition Into the Hindgut LumenThe prevention, by ligation, of Malpighian tubule fluid flow into the hindgut resulted inreduced recovery of haemolymph pH (Fig. 3.2) compared with sham controls. Since the hindgutobtains respiratory substrates from the lumen side (Chamberlin, 1981) to sustain ion transportactivities, hindgut ligation may inhibit metabolism and hence HCO3 reabsorption to restorehaemolymph pH. To test this possibility, I injected 60 gl of an artificial saline resemblingMalpighian tubule fluid into the posteriorly ligated hindgut complex 3 hours after HC1 injectioninto the abdomen. This caused a significant recovery of haemolymph at 6 hours to values equalto the sham operated locusts (Fig. 3.2). There was no change in total CO 2 or HCO3 of^10—^47• T0—, 1 ■______________I1 \ cv^ .^•o 5— ac.)0El01Cr,I0Ux3^6Time (hours)Figure 3.3. Haemolymph total CO2 and HCO3 with time after injection (0 h) for differentexperimental groups of locusts starved for 1 day at 21° C. Mean ± S.E. (N=6-12). Locusts weresham operated (•), anterior ligated (A), posterior ligated (■) and then injected with 25 ill of 0.4M HC1. A fourth group were sham operated and uninjected (0). Values for haemolymph fromposterior ligated locusts that were first injected with HCl and then injected with a salinemimicking Malpighian tubule fluid through the anus is also given (+).48haemolymph (Fig. 3.3). This provides the first direct evidence that hindgut transport activitiescontribute to haemolymph pH recovery from acidosis in locusts, or indeed any terrestrial insect.Anterior ligation did not prevent recovery of haemolymph pH as compared to sham operatedcontrols, suggesting that midgut reabsorption was not essential to regulation.Time Course of Fluid Secretion in Fed Locusts: Temperature EffectAll previous studies have been on starved locusts. Feeding initiates the release of diuretichormones (DH) which stimulated fluid secretion in isolated insect tubules by several fold(Maddrell, 1980; Phillips, 1981). The effect of DH on acid-base transport by insect Malpighiantubules has not been studied. Conceivably DH stimulation could initiate a much greater capacityof tubules to eliminate acid. I therefore cannulated locusts that had been starved for 3 days andthen recently fed lettuce and bran for 2 hours to ascertain the effect of feeding on tubular fluidparameters. Secretion rates were measured between 15 and 120 minutes after feeding.Since all previous studies were done at 21° C (i.e. typical night-time temperature),possibly acid-base elimination by Malpighian tubules is much faster at typical daytimetemperatures (e.g. 37° C). I therefore followed J , for the full complement of tubules in starvedand fed animals at both 21° and 37° C. Temperature affects the amount of food taken in (Table3.1) and hence the rate of passage through the gut which could also influence pH regulation ifthe hindgut is involved. At 21° C, locusts ingest 0.29 g of lettuce and bran and at 37° C theyingest 0.45 g in 2 hours, representing 12% and 20% of total body weight respectively (Table 3.1).Initial fluid production rates for fed locusts at 21° C varied between 9 and 10 tl h .% andgradually decreased by up to 50% in 3 hours (Fig. 3.4). The J, for fed animals gradually49Table 3.1. Food uptake in 2 hours by S. gregaria starved for 3 days at 21° C and 37° C.Locust Weight (g)^Food Uptake (g)^% Body Weight21° C 2.42 ± 0.1 0.29 ± 0.03 12.3%37° C 2.26 ± 0.07 0.45 ± 0.03 20.3%Mean ± S.E. (N=15-16)T•1aTT•115—50......i..430 104.,0r40o.400V0 5Iia...tiFT:1o  I 0 1 2^3^4^5Time (hours)Figure 3.4. Fluid production rates (JO for the full complement of Malpighian tubules of starved(0) and fed (•) locusts cannulated in situ at 21° C. Mean ± S.E. (N =7-8).51increased but remained statistically unchanged from initial values. These values wereinsignificantly different from those for starved locusts in Chapter 2. At 37° C, initial fluidproduction rates are almost 4x higher than those at 21° C (Fig. 3.5). J v for unfed locusts fell byup to 50% over 3 hours but fed animals exhibited no change in initial fluid production over thefirst hour as compared to starved animals. Again feeding did not significantly increaseMalpighian tubule secretion rate, at least over the first 1.25 hours following a meal equal to 20%of body weight.Haemolymph pH VariablesBefore considering the effect of temperature and feeding on acid-base parameters intubular secretion, it is first necessary to consider changes in the haemolymph bathing the tubules.Haemolymph pH in locusts decreases with increasing temperature (Harrison, 1988), but theeffects of feeding state on blood pH is not known. I therefore compared blood pH in fed andstarved locusts at two different temperatures. The different amounts of food uptake were asshown in Table 3.1.Locusts starved for 3 days had a haemolymph pH of 7.23 at 21° C (Table 3.2), which issimilar to the pH of haemolymph from locusts starved for one day at 21° C (Chapter 2). Afterfeeding at 21° C, haemolymph pH decreased significantly to 6.95 (Table 3.2). Total CO 2 andHCO3 of haemolymph also decreased after feeding from 10.9 mM to 6.8 mM and from 9.9 mMto 5.6 mM respectively. Haemolymph Pco 2 remained statistically unchanged (Table 3.2). At 37°C, haemolymph pH in starved locusts is 6.87, which is 0.36 units lower than haemolymph pHat 21° C (Table 3.2). These values give a ApH/AT ratio of -0.023, which is similar to the valueT0I04020520 1 2Time from cannulationFigure 3.5. Fluid production rates (J,,) for the full complement of Malpighian tubules of starved(0) and fed (•) locusts cannulated in situ at 37° C. Mean ± S.E. (N =7-8).Table 3.2. Effect of feeding and temperature on acid-base parameters of Schistocerca gregaria haemolymph.21° C 37° Cstarved fed starved fedpH 7.23 6.95* 6.87* 6.88± 0.01 ± 0.11 ± 0.08 ± 0.04Total CO2 (mM) 10.9 6.8* 6.6* 5.4± 0.3 ± 0.13 ± 0.5 ± 0.4Pco2 (toff) 23.0 27.4 32.8 24.4±1.4 ± 6.0 ± 5.8 ± 2.4HCO3 (mM) 9.9 5.6* 5.5* 4.6± 0.3 ± 0.2 ± 0.4 ± 0.4Mean ± S.E. (N=4-6)* Indicates significant difference of fed from starved group at same temperature.§ Indicates significant difference of starved group at different temperatures.54reported in a previous study of temperature/pH relationship (Harrison 1988). After feeding at 37°C, haemolymph pH was statistically unchanged at 6.88. Total CO2 was depressed compared withstarved conditions at 21° C, and feeding caused a slight decrease to 5.4 mM. Haemolymph Pco 2(32.8 toff) and HCO3 (5.5 mM) both decreased to 24.4 (toff) and 4.6 mM respectively (Table3.2), but these changes were not significant. Therefore, feeding caused changes in the acid-basestatus of locust haemolymph at 21° C, but not at 37° C.Effect of Feeding and Temperature on Tubular Acid-Base VariablesThe effect of feeding locusts at 21° C was to significantly lower haemolymph pH, but thisdid not occur at 37° C. In this series of experiments, I compared the composition of Malpighiantubule fluid pooled over 2-3 hours from starved and fed locusts at 21° and 37° C.At 21° C, tubule fluid pH from starved locusts was 7.01 (Table 3.3), which is similar tovalues from locusts starved for one day (Chapter 2). After feeding, mean tubular pH wasstatistically unchanged at 6.86. Initial total CO 2 was 10.4 mM which was slightly, but notsignificantly depressed to 7.6 mM after feeding (Table 3.3).At 37° C, haemolymph from starved animals was 6.87. Tubular pH was 6.71 beforefeeding, but increased to 6.89 after feeding, while blood pH remained unchanged at a pH of 6.88.Haemolymph total CO2 decreased after feeding but tubular total CO 2 increased from 6.5 mM to8.8 mM (Table 3.3).Changes in acid secretion may be masked by changes in the buffer capacity (B) of tubularfluid. I measured the non-bicarbonate buffer value for Malpighian tubule fluid for starved andfed animals at 21° C and 37 ° C. The non-bicarbonate buffer value for animals starved for 3 daysTable 3.3. Effect of feeding and temperature on acid-base parameters of Malpighian tubule fluidin Schistocerca gregaria.21° C 37° Cstarved fed starved fedpH 7.01 6.86 6.71* 6.89± 0.01 ± 0.13 ± 0.07 ± 0.14Total CO2 (mM) 10.4 7.6 6.5 8.8± 1.5 ± 1.2 ± 1.1 ± 1.6Pc 02 (ton) 34.5 33.0 38.5 37.4± 5.9 ± 6.3 ± 5.1 ± 6.2HCO3- (mM) 8.9 6.2 5.3 7.6± 1.4 ± 1.1 ± 1.0 ± 1.7Non bicarbonate buffer 19.4 17.3 15.8 17.4value (B)(mmol 1- ' pH unit')± 0.9 ± 1.1 ± 0.8 ± 3.1Mean ± S.E. (N=9-14)Non-bicarbonate buffer value is for the pH range 6.4-7.0.§ Indicates significant difference of starved group at different temperatures.56is 19.4 pequiv 14 at 21° C, which is almost double the value for locusts starved for 1 day. Afterfeeding at 21° C, buffer value remained unchanged at 17.3 pequiv 1- '. At 37° C, tubular 13 valuewas 15.8 mequiv 1-1 before feeding, and remained unchanged at 17.4 mequiv 1-' after feeding(Table 3.3).PHOSPHATE, URATE AND TOTAL AMMONIA LEVELS IN TUBULE FLUID: The levels of these threeinorganic solutes were not statistically different before and after feeding except for ammonia at37° C (Table 3.4). Urate was elevated significantly at 37° C as compared to 21° in each feedingstate. Total ammonia, phosphate and urate levels were all elevated after 3 days starvationcompared with animals starved for one day at 21° C. This is probably due to desiccation.Chamberlin (1981) has shown that locusts can loose up to 50% of total body water under starvedconditions.Effect of feeding on Tubular pH as Determined by MicropunctureIt appears that feeding at 21° or 37° C has only small affects on the acid-base status oftubular fluid collected by cannulation in situ. However, the pH of gut contents at the tubule entrypoint was earlier reported to increase substantially after feeding as determined by micropunctureof the gut (Speight, 1967). This is contrary to my observation by cannulation in situ. I thereforemeasured the pH of gut contents at the point of Malpighian tubule entry without prior surgicalintervention to check Speight's observation (Fig. 3.6). Animals starved for 3 days have a luminalpH of 6.9 as determined by micropuncture. After feeding for 1 hour, gut pH at the point ofMalpighian tubule entry increases by over 0.5 pH units, suggesting a dramatic shift in pH oftubular fluid after feeding. This elevated luminal pH is maintained above pH 7.4 as long asTable 3.4. Composition of Schistocerca gregaria Malpighian tubule fluid collected in situ beforeand after feeding at 21° and 37° C.21° C 37° Cstarved fed starved fedAmmonia (mM) 7.1 13.7 10.1 5.0**± 2.2 ± 2.8 ± 1.8 ± 0.9Phosphate (mM) 7.8 8.9 8.2 5.5± 1.7 ± 1.2 ± 2.0 ± 0.9Urate (mM) 3.1 2.9 7.8§ 6.6*± 0.5 ± 0.34 ± 0.8 ± 1.4Mean ± S.E. (N=7-9 locusts)Indicates significant difference of fed from starved group at same temperature.Indicates significant difference of fed or starved group at different temperatures.588.0=040^7.50-I-,c)S'5^7.0A0i -16.5 Time (hours)Figure 3.6. The pH of the gut contents as measured by the micropuncture technique. Mean ± S.E.(N=8-9). Values are for starved (0 h) and fed locusts with time from the start of feeding, for themidgut contents (A), gut contents at the point of tubule entry (0), and from continuously fedanimals at the point of tubule entry (•).59locusts are continuously fed. A clue to the cause of this alkaline lumen content at the Malpighiantubule entry point after feeding came from studying the midgut. The pH of midgut contents doesnot change after feeding. Contrary to previous studies, the midgut pH was found to be alkalineanterior to the point of tubule entry. Thus feeding induced substantial movement of alkalinemidgut contents posteriourly to dilute the influence of more acidic Malpighian tubule fluidentering the gut.DiscussionThis study provides evidence that fluid flow from the Malpighian tubules into the hindgutis necessary for haemolymph acid-base regulation. While the Malpighian tubules per se havebeen shown to contribute very little to net acid removed from the haemolymph in starved animalsat 21° C (Chapter 2) it seems that regulation of non-respiratory acid-base disturbances by thehindgut indirectly requires tubule secretions because bicarbonate reabsorption and Ir secretionin the hindgut depends on Malpighian tubule fluid for metabolic substrates. The rectal tissueutilizes amino acids and glucose as metabolic fuels (Chamberlin, 1981), which are suppliedluminally from the Malpighian tubule secretions. Thomson (1990) has shown high rates of acidand ammonia secretion by locust recta in vitro, and these transport processes require high energyinput. I have shown that readdition of a saline that mimics Malpighian tubule fluid into theligated hindgut complex restores haemolymph pH to values not significantly different from eitheran anterior or sham ligation. This is good evidence for the involvement of the hindgut inhaemolymph pH regulation in locusts.60Malpighian tubule fluid secretion rates for locusts starved for 3 days at 21° C are similarto those reported previously at 21° C in vivo after one day starvation and are comparable toprevious in vitro rates (see Chapter 2). There was not a dramatic increase in fluid production afterfeeding as has been reported for many insects taking large meals (reviewed by Maddrell, 1980).Apparently the smaller meals (20%) taken by locusts in this study do not release much DH, orthe fluid secretion rate has already fallen 1 hour after feeding. Alternatively, handling animalsmay have stimulated DH release in all locusts used in this study. Under starved conditions, thesecretion rate drops off rapidly after cannulation, possibly a result of decreasing haemolymphvolume and the stressful effects of the cannulation operation. These results are consistent withobservations in Chapter 2. After feeding the fluid production does not decrease, an indicationfeeding is causing some affect on fluid secretion. Fluid secretion is 4x higher at 37° C understarved conditions as compared to starved animals at 21° C. Also, food intake after 3 daysstarvation is more than 50% greater at 37° C. Even animals starved for one day ingest more foodat these temperatures (unpublished observations).Comparison of unfed haemolymph pH values at two different temperatures gives the samepattern of pH adjustment as reported for S. nitens over a greater temperature range (Harrison,1989). In S. gregaria, I found pH to decrease by 0.017 pH units/ °C between 21° C and 37° Cunder starved conditions. This change in haemolymph pH is consistent with the alphastathypothesis, but the temperature affects below 21° C have not been studied in S. gregaria. Underfed conditions, the ApH/AT relationship is near zero. Clearly, these results show the alphastatmodel, as proposed by Reeves (1977), does not account for changes in pH with temperature inlocusts under all physiological conditions.61Before feeding, the 11+ gradient across the Malpighian tubule epithelium after 3 daysstarvation was similar to values previously reported in cannulated locusts starved for one day(Chapter 2), the tubule fluid being 0.17-0.5 pH units more acid as compared to the haemolymph.At both 21° C and 37° C, feeding abolishes the transepithelial pH difference. Using the initial(one hour) .4, the calculated pH difference between hemolymph and tubular fluid, and themeasured buffer values for tubule fluid, I have estimated the maximum rate of excess acidremoval (fir) from the Malpighian tubules under each of these conditions. At 21° C, starvedlocusts have a maximal Ai' of 0.05 pmol h', which is almost half the rate of acid secretion forHC1-loaded locusts under similar conditions, but starved for one day (Chapter 2). After feedingat 21° C, the tubules no longer eliminate acid equivalents as the pH gradient is almost completelyabolished. At 37° C, starved locusts eliminate excess Fr at 0.09 pmol If', which is almost twicethe rate at 21° C, and equivalent to acid-loaded locusts at 21° C (Chapter 2). At 37° C, as at 21°C, tubules no longer eliminate acid equivalents after feeding.Thus tubules do not contribute substantially to acid removal from locusts after feeding,unlike starved locusts where they could make a small contribution (Chapter 2). Changes in Frsecretion activities are not masked by changes in tubule fluid buffer value (B) (Table 3.3) beforeand after feeding at both 21° C and 37° C. Furthermore, the major inorganic solutes that couldcontribute to tubular buffering (phosphate and urate) remain statistically unchanged after feeding(Table 3.4). Total ammonia, which includes protons trapped as ammonium ions, changessignificantly only after feeding at 37° C. This is consistent with a transepithelial ammonium ratiofor tubules dependent on the proton gradient across the Malpighian tubule epithelium. If theepithelium moves ammonia via diffusion trapping (Good and Knepper, 1985), then alkalinization62of the tubule fluid and acidification of haemolymph would indeed result in a lower ammoniumconcentration in the tubular fluid.There is no doubt that the pH of the gut lumen at the point of Malpighian tubule entryincreases after feeding, as first reported by Speight (1967). From Figure 3.5, it is clear that theluminal pH increases after feeding, but the alkaline midgut would suggest this area becomescontaminated with midgut fluid after feeding and therefore doesn't reflect a change in tubularacid-base variables. Collection of Malpighian tubule fluid by cannulation indicates that tubulefluid is still acid.63CHAPTER 4General DiscussionThe overall objectives of this thesis were to 1) characterize the major acid-base parametersof Malpighian tubule fluid collected by cannulation, 2) determine the importance of Malpighiantubule secretions in the regulation of haemolymph acid-base status, and 3) study the effect offeeding state and temperature on the acid-base parameters of these fluids. Measurement of pH,total CO2, and pertinent solute concentrations showed that the tubules do not play as major a rolein haemolymph pH regulation as previously anticipated. While the regulatory response to acidloading exhibited by Malpighian tubules was not great, the tubules were clearly shown to playa secondary role in haemolymph pH regulation because of tubule fluid flow into the hindgut isessential for acid-base transport in that segment, leading to haemolymph pH homeostasis.Effects of CannulationCannulation of the gut so as to collect Malpighian tubule secretions provides a way ofcollecting uncontaminated urine. The fluid secretion rates observed were similar to thoseestimated using in vitro preparations or amaranth injection. The latter dye was found to bequickly concentrated in the tubule lumen without any leakage back into the haemocoel cavity.However, the effects of cannulation lead to a major observation in this thesis: i.e. the preventionof tubule fluid flow into the gut prevented normal recovery of haemolymph acid-base status afteran acid challenge. It is conceivable that the operation and subsequent cannulation has unknownphysiological effects, mediated through feedback mechanisms and stress, that prevent normal64recovery. However, cannulation does not prevent tubule fluid flow even one day after theoperation, and locusts are always able to hop and walk when released from the restraining cage.Malpighian Tubule SecretionsThere is evidence that alkali metal pumps that are present in many insect epithelia,including Malpighian tubules, may be the active mechanism for IC 4- transport. Wieczorek et al.(1986) partially purified an ATPase from a purified membrane preparation of tobacco hornwormmidgut that has typical ion transport properties and inhibition of a vacuolar type ATPase. ThisATPase activity was stimulated 2-fold in the presence of K. More recently, Wieczorek et al.(1989) proposed the ATPase activity in the midgut drives proton extrusion to the lumen, creatinga 3 unit pH gradient. The resulting 11+ ion concentration gradient is then utilized to actively pumpIC' into the lumen through a 1-14--K± antiport. This model is consistent with fluid transport beingIC' dependent. The pH of Malpighian tubule fluid is equal to or more acidic than haemolymphunder all conditions tested. It is conceivable that Malpighian tubule secretions, which are ICdependent (Maddrell and Klunsuwan, 1973), are necessarily acid to haemolymph in order to driveIC+ extrusion for fluid transport.Feeding initiates many metabolic and physiological changes, including well documentedincreases in Malpighian tubule secretion rate (Maddrell, 1980). Despite the lack of a dramaticincrease in fluid production after feeding in my study, the cannulated locusts do show changesin fluid production and acid secretion rates. After feeding, the pH gradient that was observed instarved animals in Chapter 2 and Chapter 3 is abolished. If feeding is changing the rate of IC'transport and hence fluid secretion in the tubules, this again fits well with the proposal of65Wieczorek et al., (1989) that active 1C+ extrusion is driven by a 1C+-H+ antiport. Therefore, underconditions that stimulate fluid secretion, one would expect the pH gradient to approach zero, asobserved after feeding at both 21° C and 37° C.In terms of acid equivalents transferred out of the haemocoel, Malpighian tubules clearlydo not have the regulatory capacity of the hindgut in regulating haemolymph pH. The maximalrate of acid extrusion by Malpighian tubules is 0.09 pmol W I for starved, acid-loaded locusts andstarved locusts at 37° C (Chapter 2; Chapter 3). As discussed in Chapter 2, this is not enoughto account for the pH recovery observed within 8 hours in Chapter 3 and by Harrison et al.,(submitted). However, the effect of temperature is interesting. The maximal rate of acid clearanceis temperature dependent in starved locusts and doubles between 21° C and 37° C. This isprobably the result of a higher metabolic rate, leading to greater production of fr in the cell, andgreater H+-ATPase activity.Comparison of Locust Excretory System with the Vertebrate NephronThe Malpighian tubules of the locust form a primary urine by active ionic secretioncausing osmotic movement of water (summarized in Figure 4.1). This contrasts with mostvertebrates which have a pressure-driven filtration system (Phillips, 1981). This secretorymechanism in insect tubules might prohibit much fr elimination by the tubules because protongradients must be used to drive IC' secretion. The tubular secretions of locusts do transfer a smallfraction of acid equivalents out of the haemocoel to the lumen under starved conditions, but thisis reduced to insignificant values when 1C - secretion must be enhanced (by DH) after feeding.Malpiahian TubulesPco2 = 55 toffHCO3- = 6.5 mMNH4+ = 5mMUrate = 2-5 mMHaemolvmuh Pco2 = 15 torrHCO3- = 8 mMNH4+ = 1 mMUrate = 0.2 mMpH 7.3Colon66Midgut ,........rtr0.09 prnol li0.3 lunol 11 1 H+IleumpH 6.80.6 umol 11-1 NH4I0.6 pmol ti 1 H+HCO3- ^ HCO3Rectum 0.4 pmol If' NH4+ ^ Na+NH4+ = 270 mMUrate = 68 mMCco, = 0.9-2.5 mmol kgpH 4.6-Ali-Figure 4.1. Schematic diagram of the locust excretory system. Fluid flow indicated with thickarrows and ionic and molecular transport indicated with thin arrows.67Haemolymph buffer composition in locusts is similar to that reported in many vertebratesystems (Harrison et al., 1990b), and likewise, the solute concentration of the Malpighian tubulefluid I measured resembles the primary filtrate of the glomerulus (Pitts, 1968). The primary urinefrom Malpighian tubules flows posteriorly into the ileum where large quantities of water arereabsorbed concomitantly with Nat, C1, and HCO3. Almost 100% of filtered HCO3 isreabsorbed in the nephron from most normal or acidotic vertebrates. Similarly, HCO3reabsorption rates are high in the hindgut, reduced from an average 6.5 mM in the Malpighiantubules to near zero in the rectum. Each segment of the vertebrate nephron is capable ofmaintaining luminal pH below that of the filtered blood (Pitts, 1968). Accordingly, urine Pco 2is elevated above plasma levels in the kidney tubules, thus enhancing HCO3 reabsorption.Similarly, Malpighian tubule fluid Pco2 is 55 toff, some 4-fold greater than haemolymph(Summarized in Fig. 4.1).As discussed in Chapter 1, the hindgut has been identified as a site of active protonsecretion. Acid equivalents are thought to be transported in the rectum by an apical 11+-ATPase land by an apical Na7/NH4+ exchanger. The ileum is also capable of transporting acid equivalents.The locust hindgut exchanges NH 4+ for luminal Na+ and secretes Fr in exchange for Na, andtherefore acts much in the same manner as the vertebrate proximal tubule in eliminating acid-baseequivalents.In summary, the overall locust excretory system functions as a renal system. As outlinedin Figure 4.1, the Malpighian tubules form a fluid that flows into the hindgut, where selectivesolute and water uptake occur. The ileum and rectum actively maintain haemolymph acid-base1 A small component (16%) of W secretion is Na + dependent (Thomson, 1990).68status by utilizing Malpighian tubule fluid for metabolic substrates and a source of titratible acid.Depending upon the environmental conditions and physiological state, a very hyperosmotic orhyposmotic urine may be excreted, along with varying amounts of acid equivalents.69ReferencesANDRUSIAK, E.W., PHILLIPS, J.E., AND SPEIGHT, J. (1980). Phosphate transport by locust rectumin vitro. Can. J. Zool. 58, 1518-1523.AUDSLEY, N. (1991). Purification of a neuropeptide from the corpus cardiacum of the desertlocust which influences ileal transport. Ph. D. thesis. University of British Columbia,Vancouver, Canada.ATKINSON, D.E., AND BOURKE, E. (1987). Metabolic aspects of the regulation of systemic pH.Am. J. Physiol. 252, F947-F956.BRADLEY, T.J. (1985). The Excretory System: Structure and Physiology. In Comparative InsectPhysiology, Biochemistry, and Pharmacology. Vol. 4, (eds. G.A. Kerkut and L.I. Gilbert),pp. 421-465.BOUTILIER, R.G., IwAMA, G.K., HEMING, T.A., AND RANDALL, D.J. (1985). The apparent pK ofcarbonic acid in rainbow trout blood plasma between 5 and 15° C. Respir. Physiol. 61,237-254.CAMERON, J.N. (1986). Acid-base equilbria in invertebrates. In Acid-Base Regulation in Animals.(ed. N. Heisler), pp. 357-394. New York: Elsevier.CAMERON, J.N. (1988). Acid-base homeostasis: Past and present perspectives. Phys. Zool. 62(4),845-865.CHAMBERLIN, M.E. (1981). Metabolic studies on the locust rectum. Ph. D. thesis. University ofBritish Columbia, Vancouver, Canada.CHAMBERLIN, M.E., AND PHILLIPS, J.E. (1982). Regulation of hemolymph amino acid levels andactive secretion of proline by Malpighian tubules of locusts. Can. J. Zool. 60(11), 2745-2752.70CHEN, P.S., TORIBARA, T.Y., AND WARNER, H. (1956). Microdetermination of phosphorus. Anal.Chem. 28(11), 1756-1758.COCHRAN, D.G. (1975). Excretion in insects. In Insect Biochemistry and Function. (eds. D.J.Candy and B.A. Kilby), pp. 177-281. London: Chapman and Hill.COOPER, P.D., SCUDDER, G.G.E., AND QUAMME, G.A. (1987). Ion and CO 2 regulation in thefreshwater water boatman, Cenocorixa blaisdelli (Hung.) (Hemiptera, Corixidae). Physiol.Zool. 60(4), 465-471.CRAIG, R., AND CLARK, J.R. (1938). The hydrogen ion concentration and buffer value of theblood of larvae of Pieris rapoe (L.) and Heliothis obsoleta (F.). J. Econ. Ent. 31, 51-54.Dow, J.A.T. (1986). Insect Midgut Function. In Advances in Insect Physiology Vol. 19. (eds.P.D. Evans and V.B. Wigglesworth), pp. 187-328. London: Academic.GARRETT, M.A., BRADLEY, T.J., MEREDITH, J., AND PHILLIPS, J.E. (1988). Ultrastructure ofthe Malpighian tubules of Schistocerca gregaria. J. Morph. 195, 315-325.GOOD, D.W., AND KNEPPER, M.A. (1985). Ammonia transport in the mammalian kidney. Am.J. Physiol. 248, F459-F471.HANRAHAN,^MEREDITH, J., PHILLIPS, J.E., AND BRANDYS, D. (1984). Methods for the studyof transport and control in insect hindgut. In Measurement of Ion Transport andMetabolic Rate in Insects. (eds. T.J. Bradley and T.A. Miller), pp. 19-68. New York:Springer-Verlag.HARRISON, J.M. (1988). Temperature effects on haemolymph acid-base status in vivo and in vitroin the two-striped grasshopper Melanoplus bivittatus. J. Exp. Biol. 140, 421-435.HARRISON, J.M. (1989). Temperature effects on infra- and extracellular acid-base status in theAmerican locust, Schistocerca nitens. J. Comp. Physiol. B 158, 763-770.71HARRISON, J.F., STAGG, A.P., AND PHILLIPS, J.E. (1990a). Ammonium, total urate, and acidexcretion in acid-loaded locusts. The Physiologist 33(4), A65.HARRISON, J.F., WONG, C.J.H., AND PHILLIPS, J.E. (1990b). Haemolymph buffering in the locustSchistocerca gregaria. J. Exp. Biol. 154, 573-579.HARRISON, J.F., PHILLIPS, J.E., AND GLEESON, T.T. (1991a). Activity physiology of the two-striped grasshopper, Melanoplus bivittatus: gas exchange, hemolymph acid-base status,lactate production, and the effect of temperature. Physiol. Zool., 64(2), 451-472.HARRISON, J.F., WONG, C.J.H., AND PHILLIPS, J.E. (1991b). Recovery from acute haemolymphacidosis in unfed locusts: 1. Transfer to the alimentary lumen is the dominant mechanism.J. Exp. Biol. (submitted).HARRISON, J.F., AND PHILLIPS, J.E. (1991c). Recovery from acute haemolymph acidosis in unfedlocusts: 2. The role of renal acid and nitrogen excretion. (submitted).HARRISON, D.K., AND WALKER, W.F. (1977). A new design of glass micro-electrode forextracellular pH measurement. J. PhysioL 269, 23p-25p.HEISLER, N. (1984). Acid-Base regulation in Fishes. In Fish Physiology Vol. XA, (eds. W.S.Hoar and D.J. Randall), pp. 315-393.HEISLER, N. (1986). Buffering and transmembrane ion transfer processes. In Acid-base Regulationin Animals. (ed. N. Heisler), pp. 3-47. New York: Elsevier.HOCHACHKA, P.W., AND MOMMSEN, T.P. (1983). Protons and anaerobiosis. Science, 219, 1391-1397.KUN, E., AND KEARNY, E.B. (1974). Ammonia. In Methods of Enzymatic Analysis Vol. 1, (ed.H.U. Bergmeyer), pp.1802-1806. New York: Academic.72LECHLEITNER, R.A. (1988). Properties of ion and fluid transport and control in hindgut of thedesert locust Schistocerca gregaria. Ph. D. thesis, University of British Columbia,Vancouver, Canada.LECHLEITNER, R.A. AND PHILLIPS, J.E. (1989). Effects of corpus cardiacum, ventral ganglia, andproline on absorbate composition and fluid transport by locust hindgut. Can. J. Zool. 67,2669-2675.LECHLEITNER, R.A., AUDSLEY, N., AND PHILLIPS, J.E. (1989). Composition of fluid transportedby locust ileum: influence of natural stimulants and luminal ion ratios. Can. J. Physiol.67, 2662-2668.LEVENBROOK, L. (1950). The physiology of carbon dioxide transport in insect blood. Part I. Theform of carbon dioxide present in Gastrophilus larva blood. J. Exp. Biol. 27, 158-174.MADDRELL, S.H.P. (1980). Characteristics of epithelial transport in insect Malpighian tubules.In Current topics in membranes and transport Vol. 14, (eds. F. Bonner and A.Kleinzeller), pp. 427-463. New York: Academic.MADDRELL, S.H.P., AND KLUNSUWAN. (1973). Fluid secretion by in vitro preparations of theMalpighian tubules of the desert locust Schistocerca gregaria. J. Insect. Physiol., 19,1369-1376.O'DONNELL, M.J., MADDRELL, S.H.P., AND GARDINER, B.O.C. (1983). Transport of uric acid bythe Malpighian tubules of Rhodnius prolixus and other insects. J. Exp. Biol., 103, 169-184.PEACH, J.L., AND PHILLIPS, J.E. (1991). Metabolic support of chloride-dependent short-circuitcurrent across the locust (Schistocerca gregaria) ileum. J. Insect Physiol., 37(4), 255-260.PrrTs, R.F. (1968). "Physiology of the Kidney and Body Fluids." 2nd Edn. Year Book MedicalPublications, Chicago.73PrrTs, R.F. (1973). Production and excretion of ammonia in relation to acid/base regulation. InHandbook of Physiology. Renal Physiology. Washington, D.C.: Am. Physiol. Soc., sect.8, chapt. 15, pp. 455-496.PHILLIPS, J.E. (1961). Studies on the rectal reabsorption of water and salts in the locust,Schistocerca gregaria, and the blowfly, Callifora erythrocephala. Ph. D. thesis. Universityof Cambridge, England.PHILLIPS, J. (1981). Comparative physiology of insect renal function. Am. J. Physiol. 241(10),R241-R257.PHILLIPS, J.E., HANRAHAN, J., CHAMBERLIN, M. AND THOMSON, B. (1986). Mechanisms andcontrol of reabsorption in insect hindgut. In Advances in Insect Physiology Vol. 19, (eds.P.D. Evans and V.B. Wigglesworth), pp. 329-422. London: Academic.PORTNER, H.O. (1987). Contributions of anaerobic metabolism to pH regulation in animal tissues:theory. J. Exp. Biol., 131, 69-88.REEVES, R.B. (1977). The interaction of body temperature and acid-base balance in ectothermicvertebrates. Ann. Rev. Physiol. 39, 559-586.ROBINSON, R.A., AND STOKES, R.H. (1959). Electrolyte Solutions. London: Butterworths.SULLIVAN, L.P. (1986). Renal mechanisms involved in acid-base regulation. In Acid-BaseRegulation in Animals. (ed. N. Heisler), pp. 83-137. New York: Elsevier.SPEIGHT, J. (1967). Acidification of rectal fluid in the locust, Schistocerca gregaria. M. Sc.thesis. University of British Columbia, Vancouver, Canada.STRANGE, K. (1982). Cellular mechanism of bicarbonate regulation and excretion in in an insectinhabiting extremes of alkalinity. Ph. D. thesis. University of British Columbia,Vancouver, Canada.74THOMSON, R.B. (1990). Cellular mechanisms of acid-base transport in an insect excretoryepithelium. Ph. D. thesis, University of British Columbia, Vancouver, Canada.THOMSON, R.B. AND PHILLIPS, J.E. (1985). Characterization of acid-base transport in an insectepithelium. Federation Proc., 44(3), 643.THOMSON, R.B., SPEIGHT, J.D., AND PHILLIPS, J.E. (1988a). Rectal acid secretion in the desertlocust, Schistocerca gregaria. J. Insect Physiol. 34, 829-837.THOMSON, R.B., THOMSON, J.M., AND PHILLIPS, J.E. (1988b). NH 4+ transport in acid-secretinginsect epithelium. Am. J. Physiol. 254(23), R348-R356.THOMSON, R.B., AUDSLEY, N., AND PHILLIPS, J.E. (1991). Acid-base transport and control inlocust hindgut: artifacts caused by short-circuit current. J.Exp. Biol., 155, 455-467.TRUCHOT, J.P. (1973). "Comparative aspects of Extracellular Acid-Base Balance." Springer-Verlag, Berlin.WIECZOREK, H., WOLFSBERGER. M.G., CIOFFI, M., AND HARVEY, W.R. (1986). Cation-stimulatedATPase activity in purified plasma membranes from tobacco homworm midgut. Biochem.Biophys. Acta 857, 271-281.WIECZOREK, H., WEERTH, S., SCHINDLBECK, M., AND KLEIN, U. (1989). A vacuolar-type protonpump in a vesicle fraction enriched with potassium transporting plasma membranes fromtobacco homworm midgut. J. Biol. Chem., 264(19), 11143-11148.75AppendixThe microelectrode to measure extracellular fluid pH was modified from that describedin Harrison and Walker (1977).Glass tubing sensitive to Ir ion concentration (pH 100-15; Clark ElectromedicalInstruments, Pangbourne, Reading, England) was pulled on a Kopf 700C vertical pipette puller(David Kopf Instruments, Tujunga, California, U.S.A.). Thermally matched, pH insensitive,alumina-silicate glass tubing (SM 100E-15; Clark Electromedical Instruments, as above) waspulled such that the shank angle of the pipette tip matched that of the pH sensitive glass (Fig.A.1). The tip of the pH-glass was cut near the wide end of the shank, and placed over the pH-insensitive glass. A glass-glass seal was made using a platinum microforge. The thin end of thepH-glass was then trimmed and melted closed with the microforge. A length of tygon tubingattached to a 50 ml syringe was used to blow a small bubble in the pH glass while gently heatednear the tip of the microforge. Epoxy glue was applied to the edge of the glass-glass seal forstrength.The completed electrodes were backfilled and the gently boiled for 10 minutes in a pHelectrode backfill solution containing (mM): KH2PO4 (40), NaOH (23), and NaCl (15), pH 7.0.Electrodes were stored indefinitely in the backfill solution.Samples were measured in a small chamber made from pulled out PE 50 tubing,connected to a 3% agar bridge, which in turn was connected to a calomel electrode (Fig. A.1).Potential difference was measured with either a Keithly 602 digital electrometer or a Dagan 8800total patch clamp. Typical electrode resistance is 10 14-10 1552. Electrode slope criteria are givenin Chapter 2......1.5 mm pH sensitive glassa)4(11 pH insensitive glass(_ epoxy glue(_ ^glass/glass sealb)« a+ electrode\.... 80-100 pmPE SO simple ebsmber4 PE 10 31I KO's.' bridse76Figure A.1. a) Diagram of pH sensitive microelectrode. b) Sample chamber.

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