EVIDENCE FOR Na + /H + AND Cr/HC<V EXCHANGES DURING INDEPENDENT SODIUM AND CHLORIDE UPTAKE BY THE LARVA OF THE MOSQUITO AEDES AEGYPTI (L.

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1 J. Exp. Biol. (1971), With 3 text-figures Printed in Great Britain EVIDENCE FOR Na + /H + AND Cr/HC<V EXCHANGES DURING INDEPENDENT SODIUM AND CHLORIDE UPTAKE BY THE LARVA OF THE MOSQUITO AEDES AEGYPTI (L.) BY R. H. STOBBART Department of Zoology, University of Newcastle upon Tyne, Newcastle upon Tyne, NEx 7RU (Received 6 May 1970) INTRODUCTION The starved aquatic larva of Aedes aegypti is capable, when deficient in salts, of actively taking up sodium and chloride independently of each other through the anal papillae; and I have suggested, largely on the basis of the competitive effects between various ions for the sodium and chloride pumps, that during independent uptake sodium is exchanged for hydrogen ions, and chloride for hydroxyl or bicarbonate ions (Stobbart, 1965, 1967). According to this suggestion Aides larvae resemble Carassius auratus (Garcia Romeu & Maetz, 1964; Maetz & Garcia Romeu, 1964) and Astacus fiuviatilis (Shaw, ), the chief difference being the lack of affinity of the sodium pump of Aedes for the ammonium ion. Although fairly convincing, the evidence for ion exchange in Aedes larvae was largely circumstantial, and so I undertook work, now to be described, which was designed to characterize more closely the ions exchanged for sodium and chloride. Following earlier practice I shall use the terms ' influx' and 'outflux' to describe ionic fluxes found with radioactive tracers, and the terms 'net loss', 'loss', 'net uptake' and 'uptake', to describe net movements of ions. MATERIALS AND METHODS The general design of the experiments (which were performed at 28 C) was such as to allow the construction of a fairly complete balance sheet of ionic movements into and out of the larvae, and the experiments were rather similar to those of Conway & O'Malley (1946) on yeast. Fourth-instar larvae (stock L, Stobbart, 1967) were reared as described earlier (Stobbart, 1959) and used after h of starvation. They were depleted of salts by treating them with 5-6 changes of de-ionized water at a population density of 1 larva/ml during the period of starvation. The larvae were then sampled to assess the extent of salt depletion, and were then placed (at a density of 10 larvae/ml) in either Na^SO^ at 1 HIM/I or KC1 at 2 mm/1 (both unbuffered) for about 11 h. During this period the ph values of the solutions were measured while net uptake of sodium or chloride was occurring; at the end of the period the larvae were analysed for sodium, potassium and chloride, and various analyses were performed on the solutions. The metabolic activity of the larvae causes the ph of the external medium to rise (Stobbart, 1959) and so a group of undepleted larvae at the same density but kept in unbuffered

2 20 R. H. STOBBART rearing medium (NaCl 2-o mm/1; KC1, 0-5 mm/1; CaCl a, 0-5 ITIM/1; MgCl,, 0-2 ITIM/1) acted as controls. Approximately 1000 larvae in 100 ml of solution were used for each treatment. The solutions and larvae were placed in 250 ml polypropylene beakers which were loosely covered with aluminium foil. The lower halves of Polythene bottles (perforated with numerous fine holes, and of such a diameter as just to fit inside the beakers) were used as sieves, and allowed the larvae to be removed from one solution, rinsed with de-ionized water and transferred to another, in a matter of seconds. Analyses of the larvae Analyses of total sodium, potassium and chloride were carried out as described earlier (Stobbart, 1967) using the E.E.L. flame photometer and the Aminco-Cotlove chloridometer. For each analysis five groups of ten larvae were used, the mean and its standard deviation (s.d.) being calculated and expressed in terms of m-equiv./kg wet weight. Analyses of the solutions (i) Cations: Na +, K +, Ca 2+ and Mg 2 " 1 " were measured with the Unicam SP 900 flame spectrophotometer, though in some cases the E.E.L. flame photometer was used for Na +. Allowance was made where necessary for interference effects between different ions by having the interfering ions present at the same concentration in both the standard and test solutions. (ii) Anions: In some cases the solutions were analysed for Cl~ which had originated from the larvae. To do this the solution (100 ml in volume) was made alkaline with a small quantity of NH 4 0H (to ensure capture of all Cl~) and it was then dried completely (at 100 C) before being taken up in a small amount of water for analysis with the chloridometer. As only small amounts of Cl~ were being measured, a correction had to be applied to compensate for Cl~ added to the solution as a contaminant in the NH 4 0H. (iii) Estimation of the base produced by the larvae: This was not identified chemically, but the amount of base produced by a group of larvae during their stay in a given solution was estimated by titrating the solution back to its initial ph using 0-099N- HNO3 and an 'Agla' micrometer syringe. (iv) Measurements of ph: A direct-reading Pye ph meter was used for these in conjunction with a specially chosen wick-ended mercury-calomel-saturated KC1 electrode which allowed only negligible amounts of KC1 to diffuse into the test solutions during the period of measurement. The ph changes associated with net uptake of sodium and chloride were measured at 28 C, while those associated with the titration of base were measured at C. The lines through the points in the figures were drawn in by eye and the usual convention of significance was employed in the statistical tests. 'Analar' grade chemicals were used throughout. RESULTS In Fig. 1 the ph changes caused by 1000 depleted larvae in 100 ml of NajSOa (group A) or KC1 (group B) are compared with those caused by 1000 undepleted larvae in 100 ml of unbuffered rearing medium. The results of the analyses performed

3 Exchanges during independent sodium and chloride uptake 21 on the larvae and the solutions are shown in Table 1. It is clear that uptake of Na + from NajSO,, and of Cl~ from KC1 causes these solutions to become less and more alkaline respectively than the controls' solution, and that (from this and earlier work Stobbart, i960, 1965, 1967) the uptake of the ions and the ph changes are contemporaneous. However, it is quite obvious that the changes in H + and OH~ concentrations as between the experimental and control solutions are far too small to account for the amounts of Na + and Cl~ taken up. From Table 1 we see that depleted larvae (group A) after their stay in Naj,SO 4 have brought their Na concentration back from /i-equiv/kg to normal (73-66). As the mean weight per larva is mg (based a -o 10 z Hours Fig. I. The ph changes brought about by the presence of iooo larvae in ioo ml of solution. O O, Controls, undepleted larvae in unbuffered rearing medium;, depleted larvae in i mm/1 Na,SO 4 (group A); O, depleted larvae in 2 mm/1 KC1 (group B). Table 1. The bsses of terns by 1000 depleted larvae, and the gains and losses which occurred after 11 h in NaaSC^ or KC1. (The ph of these solutions is shown in Fig. 1.) Treatment Na K Cl Normal Depleted Na,SO 4 1 mm/1 after depletion group A KC1 2 mm/1 after depletion group B ± ± ± ± ± ± [-8-79] [ + 9-8] ± ±04822 [-0013] Ionic concentration in larvae; m-equiv./kg wet wt.±s.d., ( ) = degrees of freedom. [ ] = Uptake ( + ) or loss ( ) of ions by larvae in /J-equiv. found by analysis of solutions. on 700 larvae) it can be calculated that 19-1 /i-equiv of Na + were taken up. During their stay in NagSO 4 they lost 8-79 /i-equiv. of K + (by analysis of solution) additional to the loss brought about by depletion. This figure agrees well with an additional K + loss of 8-7 /i-equiv. calculated from the change in internal K + concentration. From the change in internal Cl~ concentration of group B larvae we can calculate that they took up 22-7 /i-equiv. of Cl~ during their stay in KC1, and analysis of this solution shows

4 22 R. H. STOBBART that 9/8 /i-equiv. of K + were also taken up, though in this case no measurements of internal K + concentration were made. Fig. 2 partially illustrates a more comprehensive experiment again with three groups of (approximately) iooo larvae. The groups were placed for n mt0 initial lots of solution (density 10 larvae/ml) of known ph; undepleted controls were placed into unbuffered rearing medium and two depleted groups of 950 larvae each (groups A', B') into de-ionized water. The final ph of these solutions was measured and the solutions were then titrated back to their initial ph with dilute HN0 3 to give an estimate of the amount of base produced by the larvae. Meanwhile the three groups had been placed into fresh solutions of known ph, the controls into unbuffered rearing medium, group A' into Na^C^ at 1 ntm/1, and group B' into KC1 at 2 mm/1. The ph of these solutions was now measured over an 11 h period, and at the end of this time the base produced was estimated by titration; finally the solutions of groups A' and B' were analysed for the various ions under consideration. Comparison between the 0-1 T Fig. 2. (A) The ph changes brought about by the presence of iooo larvae in ioo ml of solution (controls) or 930 in 95 ml (experimentals). O, Controls, undepleted larvae in unbuffered rearing medium;, depleted larvae in de-ionized water (group AO;, depleted larvae in deionized water (group B0. (B) The ph changes brought about by the above groups of larvae after transference to new solutions. O O, Controls, undepleted larvae in unbuffered rearing medium;, group A' larvae, depleted, in 1 mm/1 Na,SO 4 ; III D, group B' larvae, depleted, in 2 mm/1 KC1. performance of the depleted groups and of the control group in their initial solutions will now show whether the amount of base produced is affected by depletion and the absence of salts in the external medium, while the control group in its second lot of solution serves as a control for the main part of the experiment. Fig. 3 and Table 3 B in fact show that depletion and absence of salts have no important effect on base production which is, per 950 larvae per 10 h, 14-35, I 3" 5 an< i I2 '3 /*-equiv. respectively for the controls and groups A' and B' (mean 13-2). But when the control group is placed in a second lot of rearing medium the base production is substantially lower (9-84/i-equiv./95O larvae/11 h) over the second 11 h period, presumably due to the progressive effects of starvation. The differences between the base production of the control group in this second period and that of groups A' and B' must clearly be associated with the uptake of Na + and Cl~. The base production of the group A' larvae (in NajSOj) is much lower than that of the controls and is probably due to the produc-

5 Exchanges during independent sodium and chloride uptake 23 tion of acid by the larvae; conversely group B' larvae (in KC1) show a much higher base production than the controls. These differences expressed as acid or base production are set out in Table 3 B, and, together with the results of the analyses on the larvae and the solutions, in Table 2. H» HNO, Fig. 3. The amounts of HNO t required to bring back to their original values the ph of solutions in which larvae have been kept at a density of 10 larvae/ml for approximately 10 h. For each solution an arrow marks the point where the titration curve reaches the original ph value. The ph changes which occurred in these solutions are shown in Fig , Controls, undepleted larvae in first lot of unbuffered rearing medium; p D, depleted larvae in deionized water (group AO; O O, depleted larvae in de-ionired water (group BO;, depleted larvae in KCL 2 mm/1 (group BO; 0, controls undepleted larvae in second lot of unbuffered rearing medium; O> depleted larvae in Na,SO 4 1 mm/1 (group AO, no titration was necessary here. From Fig. 2 and Table 2 we again see that considerable amounts of Na + have been taken up from Na^O^, and CF from KC1, and that the changes in H + and OH~ concentrations as between the experimental and control solutions are (in contrast to the amounts of acid or base produced) far too small to account for these uptakes. Again the estimates of ionic movements made from the measurements of their concentrations in the larvae (using a mean weight per larva of m S) are m reasonable agreement with the analyses of the solutions. Thus from these measurements we can calculate for group A' larvae an Na + uptake of 17-9 /^-equiv., and a K + loss of 9-5; and in keeping with the very small loss of CF there is no significant drop in internal CF during the stay in Na a SO i. For group B' larvae we find no significant change in internal Na + during the stay in KC1 in keeping with the negligibly small Na + loss, a K + uptake of 1*7 /t-equiv. and a CF uptake of 19-2 /i-equiv. It must be stressed, however, that, based as they are on relatively small samples of larvae, these estimates are bound to be less accurate than the measurements made on the solutions. Dealing now with the latter measurements, we see that in group A' larvae 20 /i-equiv. of Na + are taken up and 6-67 /t-equiv. of K + are lost leaving A-equiv. (67%) to be accounted for electrostatically, of which 9-48 /4-equiv. (49 %) could have been

6 2 4 R. H. STOBBART Table 2. The losses of ions by 950 depleted larvae, and the gains and losses which occurred after 11 h in NajSO 4 or KCl. (The ph of these solutions is shown in Fig. a.) Treatment Normal Depleted Na,SO«1 mm/1 after depletion group A' KCl 2 mm/1 after depletion group B' Na T [ + 200] [0] K 1 " [-6-67] [+6-i] cr [ ] [ ] Ca»+ [- < 0038] [ < 0-019] Treatment Mg«+ Acid Base Normal Depleted Na,SO 4 1 mm/1 after depletion group A' KCl 2 mm/1 after depletion group B' [ < 0-019] [ < 0019] {-984} Ionic concentration in larvae; m-equiv./kg wet wt.is.d., ( ) = degrees of freedom. [ ] = Uptake ( + ) or loss ( ) of ions by larvae in /i-equiv. found by analysis of solutions. { } = Acid or base production in /i-equiv. found by titration of solutions. Acid and base production are assigned negative values in keeping with the sign convention, used here and in Table i, that ionic uptake is positive and loss is negative. Table 3. Base production by normal and depleted larvae over h periods and by depleted larvae over an 11 h period after entry into NajSC^ or KCl. (The data are derived from Fig. 3, and the ph changes which occurred in the solutions are shown in Fig. 2.) (A) Base production in initial period Treatment Unbuffered rearing-medium De-ionized water after depletion De-ionized water after depletion Group Control A' B' Base production /t-equiv./95o larvae/io h Treatment Unbuffered rearingmedium Na,SO 4 1 mm/1 after depletion KCl 2 mm/1 after depletion (B) In second period Group Control A' B' oduction >larvae/ii h I'O Difference between controls and experimentals expressed as base or acid* production; /*-equiv./95o larvae/i 1 h 9-84*

7 Exchanges during independent sodium and chloride uptake 25 exchanged for H +. This leaves (neglecting the very small losses of Cl~, Ca 2+ and Mg a+ ) 3-49 /i-equiv. (17-5 %) unaccounted for, an amount which earlier work suggests could have been accompanied by uptake of SO 2 4 ~ (see Discussion). In the case of group B' larvae 27 /i-equiv. of CV are taken up with 6-i /i-equiv. of K +, leaving 20-9 /i-equiv. (77-5 %) to be accounted for electrostatically, of which (41 '4%) could have been exchanged for OH~ (or HCO 3 ~). This leaves (neglecting the very small losses of Ca 2+ and Mg 2+ ) 9-74/i-equiv. (36%) unaccounted for, an amount large enough to suggest that an appreciable fraction of the Cl~ taken up is exchanged for some other anion. Tables 1 and 2 also show that depleted larvae retain Na + or Cl~ very effectively when taking up Cl~ from KC1 or Na + from NagSO 4, but that some K + is lost during Na + uptake, and some K + is taken up during Cl~ uptake. These results are in complete agreement with earlier observations (Stobbart, 1967). DISCUSSION It is clear from the foregoing that solutions become appreciably buffered when large numbers of larvae are kept in them. I have made no attempt to identify this buffering factor other than by measurements of ph, as the situation is likely to be complex. Thus the larvae probably excrete uric acid (Wigglesworth, 1933) which is likely to be degraded by micro-organisms, and in common with other aquatic insects they probably also excrete NH 4 HCO 3 (Staddon, 1955, 1959, 1963, 1964; Shaw, 1955). However a few simple measurements suggest that, with the fairly severely starved larvae used here, the buffering effect may be to a considerable extent due to bicarbonates. Consider the group B' larvae (depleted, then placed for 11 h in KC1). We know from the controls that the normal base production for 950 larvae/95 ^ over an 11 h period is 9-84 /IM (a), and that the base production associated with Cl uptake is u-i6/im (b), these are equivalent to concentrations of ( a ) an d (P) m-equiv./l respectively. Let us assume that (a) is NH 4 OH and (b) is KHCO 3 (any HCO3 exchanged for Cl' will appear as KHCO 3 in the external solution). If this assumption holds we will havefinallya solution of o mm/l NH 4 OH mm/l KHCO3 in 2 mm/l (approximately) KC1. The ph of such a solution is 9-35 ([OH^ = mm/l) in marked contrast to the observed ph of 6-8 ([OH~] = /iM/1). The ratio predicted [OH~]/observed [OH~] exceeds 3500/1. But if we assume that (a) is NH 4 HCO 8 and (b) is KHCO 3 our final solution will beo-io35 mm/l NH 4 HCO mm/l KHCO 3 in 2 mm/l (approximately) KCl.The ph of such a solution is 7-3 and the ratio predicted [OH~]/observed [OH^j is now only about 3:1. However, the observed [OH~] is lower than that predicted which suggests that buffers more effective than bicarbonate may be produced, a suggestion supported by the fact that the ph increase in the control medium is less than expected on the basis of NH 4 HCO 3 production the ph would have been expected to rise to approximately 7 instead of 6-2 (see Fig. 2). Such arguments of course merely show that the ph changes observed are compatible with a certain amount of bicarbonate production. What the present work does show is that rather less than half the Cl~ taken up can be accounted for by exchange with base produced by the larvae. However, these considerations, together with the high

8 26 R. H. STOBBART affinity of the chloride pump for HCO 3 ~ (Stobbart, 1967), provide reasonable evidence that about half the CT taken up from KC1 is exchanged for HCO 3 ~. Nevertheless, in view of the high affinity of the chloride pump for 0H~ (Stobbart, 1967) we cannot rule out the possibility of a certain amount of CF/OH" exchange giving the same overall result due to diffusion of CO a into the solution. No direct evidence is available at present concerning other ions which might participate in an exchange for the unaccounted 36% of the d~, but Wigglesworth (1938) demonstrated that the Cl~ concentration in the haemolymph drops at very low external concentrations while the osmotic pressure of the haemolymph, and its Na + concentration (Ramsay, 1950, 1951, 1953), are kept constant. This suggests that organic ions may be mobilized to maintain the osmotic pressure, and that some of these may be exchanged against Cl~ during any subsequent uptake. With respect to the uptake of Na + the situation is more definite. Nearly 50 % of the Na + taken up is balanced by a decline in base production by the larvae, and this decline must be due to their producing H +, unless we make the highly improbable assumption that the metabolic processes which generate the base in the control larvae are completely inhibited when Na + is taken up. Taken in conjunction with the very high affinity of the sodium pump for H + (Stobbart, 1967) the present results indicate that Na + is taken up from NajSO^ in exchange for H +. The unaccounted 17-5% of the Na + uptake is almost certainly accompanied by a SO 4 ~ 2 uptake. Thus earlier work (Stobbart, 1967) showed that when depleted larvae were placed in Na a SO i at 1 ITIM/I labelled with M S the influx of SO 4-2 over the 11 h period equalled 15% of the Na + uptake. Now the SO 4 ~ 2 concentration of the haemolymph is likely to be very low (Buck, 1953; Prosser et al. 1950) which means that 'back movement' of SO 4 ~ a may be neglected and that the influx of labelled SO 4 ~ 2 is likely to be an accurate estimate of its net uptake, which is thus seen to account for the outstanding Na + uptake demonstrated by the present work. In conclusion we may note that the present findings are quite compatible with the model proposed earlier (Stobbart, 1967) to account for the fluxes and net uptake of sodium and chloride. SUMMARY 1. Ionic movements into and out of the starved salt-depleted fourth instar larvae of Aides aegypti have been studied during independent uptake of Na + and Cl~. 2. About 33 % of the Na + taken up from NajSC^ is balanced electrically by a loss of K +, about 49% is exchanged for H +, and about 17-5 % is accompanied by SO 4 ~ About 36% of the CF taken up from KC1 is presumably balanced electrically by a loss of unknown and possibly organic ions, about 41 % is exchanged for HCO 3 ~ (and possibly OH~) and about 23 % is accompanied by K +. REFERENCES CONWAY, E. J. & O'MALLEY, E. (1946). The nature of the cation exchange during yeast fermentation, with formation of 0-02 NH ion. Biochem. J. 40, BUCK, J. B. (1953). Physical properties and chemical composition of insect blood. In Insect Physiology. Ed. K. D. Roeder. New York: John Wiley and Sons. GARCIA ROMEU, R. & MAETZ, J. (1964). The mechanism of sodium and chloride uptake by the gills of a fresh-water fish, Carasrius auratus. I. Evidence for an independent uptake of sodium and chloride ions. J. gen. Physiol. 47,

9 Exchanges during independent sodium and chloride uptake 27 MAETZ, J. & GARCIA ROMEU, R. (1964). The mechanism of sodium and chloride uptake by the gilli of a fresh-water fish, Carassius auratus. II. Evidence for NH 4 + /Na + and HCO,~/Cr exchanges. J. gen. Pkytiol. 47, PROSSBR, C. L., BISHOP, D. W., BROWN, F. A., JAHN, T. L. & WULFF, V. J. (1950). Comparative Animal Physiology, chapter 3. Philadelphia: W. B. Saunders Company. RAMSAY, J. A. (1950). Osmotic regulation in mosquito larvae. J. exp. Biol. 27, RAMSAY, J. A. (1951). Osmotic regulation in mosquito larvae: the role of the Malpighian tubules. J. exp. Biol. 38, RAMSAY, J. A. (1953). Exchanges of sodium and potassium in mosquito larvae. J. exp. Biol. 30, SHAW, J. (1955). Ionic regulation and water balance in the aquatic larva of Sialis lutaria. J. exp. Biol. 3* SHAW, J. (1960a). The absorption of sodium ions by the crayfish Astacus pallipet Lereboullet. II. The effect of the external anion. J. exp. Biol. 37, SHAW, J. (19606). The absorption of sodium ions by the crayfish Astacus pallipet Lereboullet. III. The effect of other cations in the external solution. J. exp. Biol. 37, SHAW, J. (1960c). The absorption of chloride iona by the crayfish Astacus pallipes Lereboullet. J. exp. Biol. 37, STADDON, B. W. (1955). The excretion and storage of ammonia by the aquatic larva of Sialis lutaris (Neuroptera). J. exp. Biol. 33, STADDON, B. W. (1959). Nitrogen excretion in nymphs of Aeschna cyanea (Mull.) (Odonata, Anisoptera). J. exp. Biol. 36, STADDON, B. W. (1963) Water balance in the aquatic bugs Notonecta glauca L. and Notonecta marmorea (Fabr.) (Hemiptera, Heteroptera). J. exp. Biol. 40, STADDON, B. W. (1964). Water balance in Corixa dentipes (Thorns.) (Hemiptera, Heteroptera). J'. exp. Biol. 41, STOBBART, R. H. (1959). Studies on the exchange and regulation of sodium in the larva of Aides aegypti (L.). I. The steady-state exchange. J. exp. Biol. 36, STOBBART, R. H. (i960). Studies on the exchange and regulation of sodium in the larva of Aides aegypti (L.). II. The net transport and the fluxes associated with it. J. exp. Biol. 37, STOBBART, R. H. (1965). The effect of some anions and cations upon the fluxes and net uptake of sodium in the larva of Aides aegypti (L.). J. exp. Biol. 43, STOBBART, R. H. (1967). The effect of some anions and cations upon the fluxes and net uptake of chloride in the larva of Aides aegypti (L.), and the nature of the uptake mechanisms for sodium and chloride. J. exp. Biol. 47, WiCGLESWORTH, V. B. (1933). The adaptation of mosquito larvae to salt water. 3- «* Biol. 10, WICCLESWORTH, V. B. (1938). The regulation of osmotic pressure and chloride concentration in the haemolymph of mosquito larvae. 3- txp- Biol. 15,

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