1. The osmotic pressure, sodium, potassium and chloride concentrations have been determined in the haemolymph of normal Drosophila melanogaster larvae and of larvae selected to survive on standard medium containing 7% NaCl (‘selected’ larvae).

  2. The haemolymph composition of normal larvae developing on standard medium shows features characteristic of pterygote insects.

  3. The haemolymph of ‘selected’ larvae developing on standard medium containing 7% NaCl is markedly hypotonic to the medium. There is only a small rise in NaCl concentration.

  4. Both normal larvae and ‘selected’ larvae can survive and develop on standard medium containing 7% KCl.

  5. The haemolymph of ‘selected’ larvae developing on standard medium containing 7% KCl is markedly hypotonic to the medium. There is a decreased Na:K ratio and a very markedly increased Cl:organic anion ratio.

  6. The nature of the mechanisms regulating the haemolymph composition is discussed.

It has been shown by Waddington (1959) that larvae of Drosophila melanogaster (Oregon K) can be selected over several generations to survive and develop on a medium containing 7% NaCl. As few species are known that can survive in such concentrated media, it was considered of interest to study the haemolymph composition of the normal and selected strains. Some previous analyses of the haemolymph of normal D. melanogaster larvae have been given by Gloor & Chen (1950) and Zwicky (1954), but these did not include sodium or potassium estimations.

Cultures were obtained of normal D. melanogaster (Oregon K) larvae and of D. melanogaster (Oregon K) larvae selected to survive on a medium containing 7% NaCl, hereinafter referred to as ‘selected’ larvae. These were grown on standard medium (agar-yeast-maize meal-treacle-water) or on standard medium containing 7% NaCl or 7% KCl.

Full-grown larvae were removed from their medium, rinsed in distilled water, dried on filter-paper and placed under liquid paraffin in a lacquered watch-glass. The body wall was punctured and haemolymph was sucked into a fine pipette. To obtain large samples the haemolymph from 10 to 40 larvae was pooled. A haemolymph sample was stored under liquid paraffin in a lacquered watch-glass. The osmotic pressure, sodium, potassium and chloride concentrations were determined as described previously (Croghan, 1958b). As far as possible a determination was carried out in duplicate or triplicate on a sample, and in many cases all the determinations were carried out on the same pooled sample.

Samples of the media were also analysed. A 1 g. sample was diluted to 5 ml. with distilled water, mixed and centrifuged. Determinations were carried out on the supernatant and the results multiplied by the dilution factor.

Analyses were carried out on normal larvae developing on standard medium and on ‘selected’ larvae developing on standard medium containing 7% NaCl. It was found very surprisingly that both normal larvae and ‘selected’ larvae could survive and develop on standard medium containing 7% KCl. Analyses were carried out on ‘selected ‘larvae developing on standard medium containing 7 % KCl.

The results are summarized in Tables 1 and 2. The derived results are obtained using the means of the measured results. That part of the cation concentration not accounted for by chloride is regarded as organic anion. The osmotic pressure deficit is that part of the osmotic pressure not accounted for by ionized salts, assuming that the organic anion is divalent. As calcium and magnesium were not estimated, the derived results underestimate the organic anion concentration and overestimate the osmotic pressure deficit.

Table 1.

The composition of the haemolymph of Drosophila melanogaster larvae

The composition of the haemolymph of Drosophila melanogaster larvae
The composition of the haemolymph of Drosophila melanogaster larvae
Table 2.

The composition of the media

The composition of the media
The composition of the media

The haemolymph composition of normal larvae developing on standard medium shows features characteristic of pterygote insects. The Na:K ratio in the haemolymph is low. This feature can be associated with a phytophagous diet (Boné, 1944). The chloride concentration is low compared to the cation concentration. This anion deficit is regarded as divalent organic anion such as succinate and malate which occur in high concentration in the haemolymph of Gastrophilus larvae (Levenbook & Wang, 1948; Nossal, 1952). Ionized salts account for only a fairly small proportion of the total osmotic pressure of the haemolymph. This osmotic pressure deficit is probably mainly accounted for by amino acids, and Zwicky (1954) has in fact found a very high concentration of ninhydrin-positive substances in the haemolymph of D. melanogaster larvae.

The haemolymph of ‘selected ‘larvae developing on standard medium containing 7% NaCl was markedly hypotonic to the medium. There is a rise of less than 40 mM./l. in the NaCl concentration. This increases the Na : K and chloride : organic anion ratios.

The haemolymph of ‘selected’ larvae developing on standard medium containing 7 % KCl was also decidedly hypotonic to the medium. A small rise in the potassium concentration is more than compensated for by a fall in sodium concentration. This decreases the Na:K ratio to < 1. The principal difference is that the chloride concentration has risen and the organic anion concentration has markedly decreased. This increases the chloride : organic anion ratio very considerably.

The external cuticle of D. melanogaster larvae is probably highly impermeable, but the larvae were constantly eating the concentrated media, and NaCl or KCl must have been rapidly entering the haemolymph across the gut epithelium. Well-developed mechanisms that can regulate the haemolymph composition must thus be present.

The regulation of body-fluid osmotic pressure in media with a high NaCl concentration is found in a few specialized types such as Aedes detritus larvae (Beadle, 1939) and Artemia salina (Croghan, 1958b). The ability with which these selected D. melanogaster larvae can regulate the haemolymph composition is thus remarkable. Selection for survival on a medium with a high NaCl concentration must have involved selection of a mechanism for the rapid excretion of NaCl. Gloor & Chen (1950) describe anal organs in Drosophila larvae. These consist of two silver-staining ventro-lateral plates with a thin cuticle and large underlying epidermal cells. They present evidence that these are concerned in salt uptake from hypotonic NaCl solutions. Waddington (1959) showed that these anal organs (papillae) were increased in area when the larvae were grown on media containing large amounts of NaCl, and that this increase was most marked in the selected larvae. It seems probable therefore that the anal organs are concerned in NaCl excretion in hypertonic media. The anal organs would function like the branchiae of Artemia salina (Croghan, 1958c). This mechanism would be distinct from that found in Aedes detritus larvae, where the area of the anal papillae is reduced compared to freshwater culicid larvae and where the mechanism is a specialization of the Malpighian tubule-rectal gland system involving the secretion of NaCl into the Malpighian tubule and the concentration of this by uptake of water in the rectum (Ramsay, 1950).

The ability of both normal larvae and ‘selected’ larvae to survive and develop on standard medium containing 7% KCl might seem even more remarkable. This is very different from the case of a type such as Artemia salina where a high KCl concentration is rapidly fatal (Croghan, 1958a). A very high potassium concentration is a feature of the Malpighian tubule fluid of insects. It has been demonstrated by Ramsay (1953a, b, 1955a, b) that the insect Malpighian tubule-rectal gland system operates in a cyclic manner. Potassium is actively transported into the tubule. This must be accompanied by an anion, which probably follows the potassium passively. In the proximal end of the tubule or in the rectum the potassium and anion are mainly reabsorbed. This cycle is associated with the circulation of water in the Malpighian tubule-rectal gland system (Ramsay, 1958). Given such a potassium cycle it seems clear that adaptation to a medium with a high KCl concentration could involve simply a decrease in the uptake side of the cycle. Potassium and accompanying anion would then be very rapidly excreted. Insects would thus be expected to tolerate readily a medium with a high KCl concentration that would be fatal to other groups.

The fall in the sodium concentration in the haemolymph of D. melanogaster larvae on the medium containing 7% KCl suggests that normally the anion is actively reabsorbed, the cations following passively, as a decrease in the active reabsorption of anion in a medium with a high KCl concentration would then result in an increased sodium loss. The considerable fall in the organic anion concentration in the haemolymph of D. melanogaster larvae on the medium containing 7 % KCl suggests that organic anion accompanies the active secretion of potassium into the Malpighian tubule, and that a failure to reabsorb this anion occurs in a medium with a high KCl concentration.

We wish to thank Prof. C. H. Waddington, F.R.S., for permission to use his normal and selected strains of D. melanogaster (Oregon K) and Miss E. Paton for looking after the cultures.

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