Studies on the Exchange and Regulation of Sodium in the Larva of Aedes Aegypti (L.): II. The Net Transport and the Fluxes Associated with it

In an earlier paper (Stobbart, 1959) I showed that the steady-state exchange of sodium between the haemolymph of this larva and the medium can be increased roughly six-fold by feeding. The exchange occurs principally through the anal papillae, and there is evidence which suggests that large parts of the influx and outflux of sodium are mediated by a carrier mechanism in the anal papillae.

In this paper I shall describe experiments on the net transport of sodium by sodium-deficient larvae. As before, the aim was to try to throw more light on the processes of sodium exchange and regulation.

Larvae deficient in sodium were produced by rearing from the egg 700–1000 in 500 ml. of glass-distilled water in a hard-glass beaker. They were given a limited amount of food. In order to prevent some of the larvae from growing more quickly than the rest it was necessary to add the food (total amount about 0·3 g.) in small amounts at roughly daily intervals, taking care that some was always available to the larvae. Despite this the variation in growth rate was greater than in larvae reared as described earlier (Stobbart, 1959). The sodium content of the rearing solution was 0·001–0·002 mM./l. and of the haemolymph of sodium-deficient larvae about 30 mM./l.—roughly one-third of that of normal larvae. The sodium-deficient larvae grew about half as fast as normal ones reared as described earlier, and took about 6 days to reach the 4th instar. Though they were rather smaller than normal ones and had greatly hypertrophied anal papillae they were just as active and seemed quite healthy. Sodium-deficient larvae containing labelled sodium were produced by rearing them in distilled water to which a small amount of practically weightless ²²Na had been added. The specific activity of the rearing solution varied by reason of the addition of the food. In order to reduce variation caused by this the larvae were collected for the experiments as far as possible at the same time.

Both fed and starved 4th-instar larvae were used in the experiments. The fed larvae were used within a few hours of moulting into the 4th instar and were always given a sufficiency or an excess of food. The starved larvae were kept in distilled water without food for 60 hr. before the start of an experiment. In order to study the net transport of sodium and the fluxes associated with it, sodium-deficient larvae were put into what were effectively infinite amounts of unlabelled or labelled media containing 2 mM/1. Na and of the same composition as those described earlier. The increase in sodium level in the haemolymph was found by flame photometry and, where appropriate, the change in radioactivity in the haemolymph was noted. Where necessary the anal papillae were destroyed by Wigglesworth’s method (1933), the larvae being kept in distilled water till scars had formed at the bases of the papillae. All other techniques and symbols were the same as those described earlier (Stobbart, 1959). The lines drawn through the points in some of the figures were drawn by eye unless it is stated to the contrary in the text. The experiments were done at 28° C.

Fig. 1 (which contains data from two experiments) shows the net transport performed by both fed and starved sodium-deficient larvae. In the case of fed larvae the net transport occurs in two phases, both of which are roughly rectilinear. The 1 st phase is rapid, the rate of net transport being 55–65 mM./l./hr. At the end of this phase the sodium level has risen to 85–95 rnM./l. After this there is a slow significant increase in sodium level. For the two experiments the rates of net transport were 0·544 mM./l./hr., standard deviation = 0·182, P = 0·015; and 0·177 mM./l./hr., s.D. = 0·0614, P = 0·02. The transition between the two rates of net transport is rapid. In the case of starved larvae the sodium level rises at a rate of about 10·5 mM./l./hr. and reaches a value of about 110mM./l. in about 8 hr. ; thereafter there is a slight decline to about 105 mM./l. which is the level found in normal larvae (Stobbart, 1959). As the maximum value is approached the rate of net transport decreases rapidly. The net transport by starved larvae in another experiment is shown in Fig. 3 A. The increase in sodium level is largely rectilinear, the rate being 6·96 mM./l./hr., s.D. = 0·463, P = <0·001. These larvae were for various reasons starved for 98 hr. before entry into the medium and this may account for the slower rate of net transport. The results given above show that feeding increases the rate of net transport by a factor of roughly 5 (neglecting the slow phase of fed larvae).

Fig. 2 A shows, for both fed and starved larvae, the outflux of labelled sodium from the haemolymph during net transport. The larvae, which contained labelled sodium, were put into an unlabelled medium at zero time. The flame photometer readings show the initial and final values of the sodium level in the haemolymph. On the basis of evidence presented in Fig. 1 one would expect the rapid phase of net transport in fed larvae to be complete in about 1 hr. and the net transport in starved larvae to be completed in about 8 hr. After these times a steady-state exchange must occur as the sodium level stays effectively constant. The tangents to the radioactivity curves at their points of origin give estimates for the initial outflux of sodium. As the net transport is roughly rectilinear an estimate of the initial influx can be got by adding the initial rate of outflux to the rate of net transport. These estimates are very rough as the initial specifc activity of the sodium in the haemolymph was not found accurately. The estimates are shown in Table 1. Fig. 2B shows the influx of labelled sodium into the unlabelled haemolymph of fed and starved larvae. The straight lines marked 1 and 2 represent the increase in the sodium level in the haemolymph of fed and starved larvae, the vertical extent of these lines being equal to the difference between the initial and final flame photometer readings. Tangents to the radioactivity curves at their points of origin provide estimates for the initial influx and outflux. These estimates must be more accurate than the ones discussed above because the specific activity of the medium was measured accurately. Estimates of the initial fluxes are given in Table 1.

Fig. 4 gives information about the steady-state (or nearly steady-state) exchange which occurs after the net transport of sodium. The larvae were reared in distilled water; when they reached the 4th instar they were put into unlabelled medium from which they took up sodium. At zero time they were put into a labelled medium. The increase in labelled sodium in the haemolymph was noted, and the sodium level in the haemolymph was followed by flame photometry. In the case of starved larvae the sodium level in the haemolymph was constant so that permeability constants could be found (cf. Stobbart, 1959). For these larvae: K_in = 0·474 hr.^–1. K_out = 0·0102 hr. ^–1, [Na_in] =93 mM./l., flux of Na = 0·95 mM./l./hr. These values are all very close to the corresponding values for starved larvae reared under normal conditions (Stobbart, 1959; Treherne, 1954). In the case of fed larvae there was a slow rectilinear increase in haemolymph sodium. The rate of net transport was 0·499 mM./l./hr., S.D. = 0·0479, P ≅ 0·0025. The initial influx was 10·59 mM./l./hr. and the initial outflux therefore 10·09. normal fed larvae the flux in either direction during steady-state exchange is between 7·15 and 8·84 mM./l./hr. It is reasonable to suppose that when the slow net transport is completed the fluxes would fall to this value as the flux in the starved larvae is very close to that in starved normal larvae. But it will be difficult to investigate this point as the fed larvae pupate soon after 60 hr. These results together with those in Table 1 show that the initial values of influx and outflux during net transport are much greater than the fluxes which occur during steady-state exchange.

The net transport of sodium and the fluxes characteristic of it occur almost entirely through the anal papillae. Papilla-less starved sodium-deficient larvae are unable to effect a net transport inwards over a period of 14 hr. and the flux (the same in both directions) is small, about 0·2 mM./l./hr. Papilla-less fed sodium-deficient larvae are however able to effect an appreciable net transport (pre-sumably through the gut) over the period 30–50 hr. (Fig. 5 B, C). The evidence suggests that the larvae become adapted to perform this net transport after 20-40 hr. During the first hour, however, it is clear that the gut makes a negligible contribution to the net transport and its associated fluxes.

During net transport by starved sodium-deficient larvae no change in weight could be detected, nor could any change in weight be detected in starved controls kept in distilled water. The method would have detected changes greater than 10%. It therefore seems reasonable to suppose that during net transport the volume of haemolymph stays constant as any increase in weight due to gain of salts must have been negligible.

It is clear that the larvae can collect sodium from very dilute media. The characteristics of steady-state exchange under sodium-deficient conditions are of interest and were studied in starved larvae reared under sodium-deficient conditions and then equilibrated in a medium diluted to give a sodium concentration of 0·003 mM./l. The exchange was followed only for 16 hr. During this time [Na_in] remained steady at 57·8 mM./l. and the flux of sodium was 0·455 mM./l. in either direction. By comparison with results given earlier (Stobbart, 1959) it can be seen that when [Na_out] is reduced by a factor of 0·0015, [Na_in] and the fluxes are reduced by a factor of about 0·5.

Physostigmine sulphate, when applied to the outside of the larva, was found not to inhibit specifically the net transport of sodium. This is in contrast to the results of Koch (1954) obtained with other arthropod tissues.

Considerable cytological differences exist between the anal papillae of fed and of starved larvae (Stobbart, 1959). Similar differences exist between papillae of fed and of starved sodium-deficient larvae (Fig. 6). In this case the thickness of the cytoplasm of ‘fed papillae’ is more variable, there is no clear-cut striated region, and the cytoplasm contains many elongated bodies which are arranged so as to be roughly normal to the cuticle. The cytoplasm of ‘starved papillae’, however, does show a definite striated region similar to that described earlier. The nuclei of both fed and starved papillae are found inside vesicles in the cytoplasm. The probable significance of cytological differences between papillae of fed and starved larvae was discussed earlier. The papillae were fixed in Camoy’s fluid and were stained with iron haematoxylin and eosin.

It has been clearly shown that this larva can transport sodium from the medium into the haemolymph in the face of steep concentration gradients, and previous work (Wigglesworth, 1938; Ramsay, 1953) has shown that sodium occupies a dominant position among the inorganic constituents of the haemolymph. Because of these facts, and by comparison with the situation in other tissues, I shall assume that sodium is actively pumped into the haemolymph. I shall also assume as a first approximation that the larva consists of only the haemolymph compartment (Treherne, 1954).

With the techniques used in this work a large part of the net transport of sodium from the medium to the haemolymph appears to proceed at a constant rate. Although the techniques would not have detected slight variations in rate of net transport they would certainly have detected marked ones. It seems reasonable therefore to assume that the rate of net transport is largely constant.

The rate of net transport may be increased roughly fivefold by feeding the larvae ; and net transport, together with the fluxes characteristic of it, occur almost entirely through the anal papillae. The steady-state exchange of sodium may be increased roughly sixfold by feeding and it occurs principally through the anal papillae (Stobbart, 1959). These facts suggest that both net transport and exchange are brought about by some carrier mechanism in the papillae which is linked to the metabolism of the larva.

x = concentration of labelled sodium in the haemolymph at any time

k = m_i — m₀

mi influx of sodium (mM./l. of haemolymph/hr.),

m₀= outflux of sodium (mM./l. of haemolymph/hr.),

S_o = concentration of sodium initially present in the haemolymph.

As t increases the term diminishes and the curve of this equation degenerates into a straight line. When m_i/k and m₀/k are large a lot of exchange occurs relative to net transport. The type of curve described by this equation is shown by curves A and B of Fig. 7.

It appears from the foregoing considerations that there is no vast increase in the fluxes during net transport in the case of starved larvae and in the case of fed larvae during the slow phase of net transport. During the rapid phase of net transport by fed larvae, however, there may be a reduction in the fluxes. It should prove possible to obtain more information about the net transport by measuring the fluxes directly at different times during the net transport.

The results which have been presented here are compatible with the sort of sodium pump which was suggested earlier to account for the steady-state exchange (Stobbart, 1959). The pump is supposed to work in conjunction with something similar to an exchange diffusion mechanism (E.D.M.) which is confined to an osmotic barrier in the anal papillae. The pump is supposed to split off from the E.D.M. at the inner surface of the barrier sufficient sodium to balance any passive losses or to bring the sodium level in the haemolymph back to the normal value. The sodium attached to the E.D.M. is free to exchange with that in the haemolymph or medium. An increase in the fluxes could be brought about by (1) an increased rate of move-ment of E.D.M.’S, (ii) an increase in the amount of sodium carried by each E.D.M., (iii) a synthesis of more E.D.M.’S, (iv) an increase in any possible enzymic catalysis of exchange of sodium between E.D.M. and medium of haemolymph. Both the exchange and the pumping of sodium are increased by feeding in sodium-deficient larvae. When sodium-deficient larvae are put into the medium, net transport and the large fluxes associated with it are observed straight away. This suggests (in terms of the proposed sodium pump) that in sodium-deficient larvae in a sodium-deficient medium the capacity of the E.D.M. to carry sodium is increased in an attempt to present the pump with sodium originating from the medium outside. If the E.D.M. has a high affinity for sodium (as is the case in Aëdes) it might act as a mechanism for moving sodium through an osmotic barrier and presenting it to a pump. Sodium not removed by the pump would move back and forth across the barrier and could give rise to an exchange diffusion component in the fluxes (cf. Mitchell & Moyle, 1953). A mechanism of this sort should not cause an out-flux of sodium from the larva when sodium is missing from the medium outside. Seen in this light, exchange diffusion is not necessarily a useless by-product of metabolism. The increase in steady-state exchange brought about by feeding (Stobbart, 1959) may mean that the pump is in a greater state of readiness to deal with emergencies as the influx of sodium is roughly 6 times greater than it is in starved larvae. Exchange diffusions that are apparently useless do occur (Croghan, 1958 a, b). Perhaps exchange diffusion is a general characteristic of biological membranes and is one which in certain cases lends itself to the development of ionic pumps.

It is possible that the increased outflux during net transport is a passive process caused by an increased electrochemical activity of sodium in the haemolymph due to the active influx. Until information about the electrochemical activity is available it is not possible to say whether the increased outflux is more likely to be due to passive diffusion or to be mediated by some sort of carrier.

The net transport of sodium slows down, or slows down and stops, quite abruptly when the level of sodium in the haemolymph approaches that of normal larvae. This rapid control would appear to be worth investigation. In fed sodium-deficient larvae the rate of net transport slows down abruptly, but does not stop, when the sodium level reaches 80 – 90 mM./l. The reasons for this are obscure. A slow net transport cannot be detected in fed normal larvae. Perhaps when the sodium level reaches a certain critical value most of the energy which would have been used for net transport is diverted to other ends.

I should like to thank Dr J. A. Kitching, F.R.S., under whose supervision this work was done, for guidance and encouragement, and Prof. J. E. Harris, F.R.S., for his active interest in the problem. I am grateful to Dr I. M. Glynn for providing the derivations of equations (1) and (2), to Dr K. Zerahn for some helpful discussion, and to Dr K. M. Smith, F.R.S., for letting me use the photographic facilities of the A.R.C. Virus Research Unit, Cambridge.

The isotopes were bought out of a Royal Society grant to Dr Kitching. This work was done while I held a Maintenance allowance from the Department of Scientific and Industrial Research.

During the rectilinear net transport let

influx (assumed constant) = m_i,

outflux (assumed constant) = m₀,

In addition let:

t -time,

amount of Na originally present inside be S₀,

amount of Na present inside at any time be S,

amount of Na* present inside at any time be x.

The medium outside is assumed to stay constant

(2) During the rectilinear net transport : let m_i be the influx at any time ;

let m₀ be the outflux at any time ;

let m_i— m₀= k, where k is a constant.

In addition:

let m₀= k₁S where k₁ is a constant,

and let S₀, S, x, and t be defined as before.