Evidence for Na⁺/H⁺ and Cl⁻/HCO₃⁻ Exchanges During Independent Sodium and Chloride Uptake by the Larva of the Mosquito Aedes Aegypti (L.)

The starved aquatic larva of Aëdes 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 Aëdes larvae resemble Carassius auratus (Garcia Romeu & Maetz, 1964; Maetz & Garcia Romeu, 1964) and Astacus fiuviatilis (Shaw, 1960a–c), the chief difference being the lack of affinity of the sodium pump of Aëdes for the ammonium ion. Although fairly convincing, the evidence for ion exchange in Aëdes 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.

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 60–72 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 Na₂SO₄ at 1 mM/l or KCL at 2 mM/l (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 rearing medium (NaCl 2·0 mM/l; KCl, 0·5 mM/l; CaCl₂, 0·5 mM/l; MgCl₂, 0·2 mM/l) 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 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.

• Cations: Na⁺, K⁺, Ca²⁺ and Mg²⁺ were measured with the Unicam SP₉₀₀ 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.

• 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 NH₄OH (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 NH₄OH.

• 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 0·099 N-HNO3 and an ‘Agla’ micrometer syringe.

• Measurements of pH: A direct-reading Pye pH meter was used for these in conjunction with a specially chosen wick-ended mercury-calomel-saturated KCl electrode which allowed only negligible amounts of KCl 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 25–28°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.

In Fig. 1 the pH changes caused by 1000 depleted larvae in 100 ml of Na₂SO₄ (group A) or KCl (group B) are compared with those caused by 1000 undepleted larvae in 100 ml of unbuffered rearing medium. The results of the analyses performed on the larvae and the solutions are shown in Table 1. It is clear that uptake of Na⁺ from Na₂SO₄ and of Cl⁻ from KCl causes these solutions to become less and more alkaline respectively than the controls’ solution, and that (from this and earlier work Stobbart, 1960, 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 Na₂SO₄ have brought their Na concentration back from 57·44 μ-equiv/kg to normal (73·66). As the mean weight per larva is 1·175 mg (based on 700 larvae) it can be calculated that 19·1 μ-equiv of Na⁺ were taken up. During their stay in Na₂SO₄ they lost 8·79 μ-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 μ-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 μequiv. of Cl⁻ during their stay in KCl, and analysis of this solution shows that 9·8 μequiv. 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) 1000 larvae. The groups were placed for 10–11·25 h into 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 HNO₃ 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 Na₂ SO₄ at 1 mM/l, and group B’ into KCl at 2 mM/l. 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 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 3B 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, 13·05 and 12·3 μ-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 μ-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 Na₂SO₄ is much lower than that of the controls and is probably due to the production of acid by the larvae; conversely group B’ larvae (in KCl) 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.

From Fig. 2 and Table 2 we again see that considerable amounts of Na⁺ have been taken up from Na₂ SO₄ and Cl⁻ from KCl, 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 1·175 mg) are in 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 μ-equiv., and a K⁺ loss of 9·5; and in keeping with the very small loss of Cl⁻ there is no significant drop in internal Cl⁻during the stay in Na_aSO₄. For group B’ larvae we find no significant change in internal Na⁺ during the stay in KCl in keeping with the negligibly small Na⁺ loss, a K⁺ uptake of 1·7 μ-equiv. and a Cl⁻ uptake of 19·2 μ-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 μ-equiv. of Na⁺ are taken up and 6·67 μ-equiv. of K⁺ are lost leaving 13·33 μ-equiv. (67%) to be accounted for electrostatically, of which 9·48μ-equiv. (49%) could have been exchanged for H⁺. This leaves (neglecting the very small losses of Cl⁻, Ca²⁺ and Mg²⁺) 3·49 /-equiv. (17·5%) unaccounted for, an amount which earlier work suggests could have been accompanied by uptake of SO₄^2- (see Discussion). In the case of group B’ larvae 27 μ-equiv. of Cl⁻ are taken up with 6·1 μ-equiv. of K⁺, leaving 20·9 μ-equiv. (77·5%) to be accounted for electrostatically, of which II-I6 (41·4%) could have been exchanged for OH” (or HCO₃”). This leaves (neglecting the very small losses of Ca²⁺ and Mg²⁺)9·74μ-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 KCl or Na⁺ from Na_aSO₄, 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).

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 NH₄HCO₃ (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 KCl). We know from the controls that the normal base production for 950 larvae/95 ml over an 11 h period is 9·84 μM (a), and that the base production associated with Cl uptake is 11·16 μM (b), these are equivalent to concentrations of 0·1035 (a) and 0·1175 (b) m-equiv./l respectively. Let us assume that (a) is NH₄OH and (b) is KHCO₃ (any HCO’₃ exchanged for Cl’ will appear as KHCO₃ in the external solution). If this assumption holds we will have finally a solution of 0·1035 mM/l NH₄OH +0·1175 MIM/1 KHCO₃ in 2 mM/l (approximately) KCl. The pH of such a solution is 9·35 [OH⁻] = 0·228 mM/l) in marked contrast to the observed pH of 6·8 [OH⁻] = 0·0644 μM/1). The ratio predicted [OH⁻]/observed [OH⁻] exceeds 3500/1. But if we assume that (a) is NH₄HCO₃ and (b) is KHCO₃ our final solution will be 0·1035 mM/l NH₄HCO₃+ 0·1175 mM/l KHCO₃ in 2 MM/I (approximately) KCl.The pH of such a solution is 7-3 and the ratio predicted [OH⁻]/observed [OH⁻] 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 NH₄HCO₃ 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 affinity of the chloride pump for HCO₃⁻ (Stobbart, 1967), provide reasonable evidence that about half the Cl⁻ taken up from KCl is exchanged for HCO₃⁻. Nevertheless, in view of the high affinity of the chloride pump for OH⁻ (Stobbart, 1967) we cannot rule out the possibility of a certain amount of C1⁻/OH⁻ exchange giving the same overall result due to diffusion of CO₂ 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 Cl⁻, 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 Na₂SO₄ in exchange for H⁺. The unaccounted 17·5% of the Na⁺ uptake is almost certainly accompanied by a SO₄⁻² uptake. Thus earlier work (Stobbart, 1967) showed that when depleted larvae were placed in Na₂SO₄ at 1 mM/l labelled with ³⁵S the influx of SO₄⁻² over the 11 h period equalled 15% of the Na⁺ uptake. Now the SO₄⁻² concentration of the haemolymph is likely to be very low (Buck, 1953; Prosser et al. 1950) which means that ‘back movement’ of SO₄⁻² may be neglected and that the influx of labelled SO₄⁻² 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.