The Regulation of Sodium, Potassium and Chloride in an Aphid Subjected to Ionic Stress

Aphids lack Malpighian tubules, thought to be one of the most important parts of the insect excretory apparatus, and present in all but a few groups (Wigglesworth, 1972). Despite this, the osmotic and ionic regulation of these small terrestrial animals has not until recently been studied, nor barely considered, even though the feeding and nutrition of aphids have been the subject of much investigation (see reviews by: Kennedy & Stoyan, 1959; Auclair, 1963; van Emden et al., 1969; Pollard, 1973).

The majority of aphids feed on phloem sap. This fluid is best obtained for analysis from the severed mouthparts of a feeding aphid. Although the cutting of the mouthparts and collection of the sap is not an easy technique, several authors have reported success (Kennedy & Mittler, 1953; van Soest, 1955; Weatherley, Peel & Hill, 1959; von Dehn, 1961 ; Ehrhardt, 1962; Barlow & McCully, 1972), but they have used the technique to concentrate largely on the carbohydrate and amino acid composition of the fluid, or else aspects of plant virus transfer (van Soest & van de Meesters-Manger Cats, 1956). More recently, reliable techniques have been described which enable the collection of stylet sap, haemolymph and honeydew to be made from aphids, in such a way as to avoid the contamination of the fluids or the concentration of their solutes by evaporative loss (Downing & Unwin, 1977; Downing, 1978).

The quantities of fluids collected by these methods are extremely small, of the order of 1 ni in volume, but the analytical methods developed by J. A. Ramsay have allowed a comparison of the ionic levels of these three fluids to be made. Furthermore, the aphids have been successfully cultured on a salt marsh plant, Aster tripolium. By growing the plants in nutrient media made up with fresh or sea water (Downing, 1978) it has been possible to stress the plant and, in turn, the aphids both osmotically and ionically.

In the following paper are described the changes that occur in the sodium, potassium and chloride content of the aphid’s diet, blood and excreta under such conditions. It also highlights an important finding : in this aphid the blood sodium level is maintained at an exceptionally low concentration.

The methods used to collect stylet sap, haemolymph and honeydew were those reported in previous papers. In short, sap was obtained by severing the mouthparts of feeding aphids using a radio frequency probe (Downing & Unwin, 1977). The fluid was allowed to exude from the cut ends of the stylets into a small drop of oil, placed over the stylets on the leaf surface, and then drawn into an oil-filled micropipette. Honeydew was sampled by direct collection (Downing, 1978). Blood was collected by cutting the front leg of an aphid submerged in an oil bath.

Because of the very low sodium levels found in some of the fluids, safeguards in addition to those described in the above papers had to be taken to prevent contamination, particularly from the glassware. All glass micropipettes used in the collection, manipulation and storage of the fluids were made from thin-walled ‘Vitreosil’ silica tubing (Ramsay, 1949) and were carefully cleaned prior to use.

Sodium and potassium concentrations were measured using a highly sensitive integrating flame photometer (Ramsay, 1955; 1976). For sodium, the instrument can reliably measure samples containing of the order of 10⁻¹¹ mol sodium, but for the very low levels of the ion found in the fluids from fresh water plants this required relatively large volumes to be collected, about 30 nl for each determination.

Ramsay (1955) has drawn attention to the problem of interference errors in flame photometry. The possible interference of the large quantities of carbohydrate present in phloem sap and honeydew was investigated, but none was detected at the concentrations known to occur in these fluids. To calibrate the instrument standards were used that contained both sodium and potassium in the ratios expected in the samples.

Fluids were collected from apterous Myzus persicae feeding on sea astep grown from seed in either fresh or sea water nutrient medium (Downing, 1978). On a given day’s sampling all three fluids were obtained from a group of aphids feeding on a selected leaf. Samples of each fluid were pooled, so that sodium, potassium and chloride analyses could be performed on that same fluid. For the analysis of sodium in the fluids sampled under the fresh water condition, a separate collection was required because of the large quantities of fluid needed. Sodium determinations were made from the pooled sample of each fluid: stylet sap from six cut stylets, honeydew from 22 aphids, and haemolymph from 60 aphids.

In a further experiment, a fresh water aster with its attendant aphid colony was subjected to a watering medium of 10 parts 10⁻³ salinity. After the initial wilting and recovery of the plant, the salinity of the medium was progressively increased to that of full strength sea water. Changes in the sodium levels of the fluids were monitored over a period of 62 days.

All fluids were stored in silica glass tubes (Ramsay, 1949) under oil, and frozen until required for analysis. Null hypotheses were set before the experiment (Ridgman, 1975); th^e statistical tests used and their outcome are given in the text.

The results of the analyses are given in Table 1 and Fig. 1. In Fig. 2 are illustrated the changes in the sodium levels of the stylet sap, haemolymph and honeydew under conditions of changing salinity.

As mentioned earlier, in order to analyse sodium relatively large volumes of fluid had to be collected from aphids feeding on fresh water plants, and it is for this reason that only three determinations were made on each fluid (Table 1). Referring in particular to these results it should be noted that while the possibility of contamination of the samples was extremely low, because of the precautions taken, it cannot be dismissed. However, the effect of contamination could only be to add to the sodium present in the samples. Thus, the true values can be no higher than the ones shown, and may be less.

The results indicate that on a fresh water plant the aphids ingest sap with an extremely low concentration of sodium in it. They evidently make no attempt to concentrate the ion from the sap, as the haemolymph contains equally little sodium. Furthermore, when cultured on a sea water plant sodium is excluded from the blood. This is particularly clearly shown in Fig. 2. A stylet was cut and fluid collected within 3 h of initially watering the plant with 10 parts 10⁻³ saline medium. The increase in sodium concentration was sudden, yet the aphids did not leave the plant and continued to feed. The honeydew sample was then collected, followed by the haemolymph. As expected, the sodium level of the honeydew was also high, but that of the haemolymph shows no detectable increase. Over the remainder of the experiment the sodium values of all three fluids fluctuate within the limits observed on an established sea water plant (see Fig. 1).

The results for potassium and chloride (Fig. 1) show that the aphids ingest a diet that is highly variable in the concentration of these ions. This is reflected in the variability of the ionic concentrations in the honeydew. In either case, whether on a sea water or fresh water plant, the concentration of the potassium or chloride ion in the sap is not significantly different from that in the honeydew (F-test). Comparing the fresh water to the sea water condition, the levels of both ions in the blood increase slightly under the sea water condition, (for chloride: Fischer-Behren’s test, P < 0·01; for potassium: Student’s t-test, P < 0·001). The increase in blood potassium level might indicate that the apparent rise in potassium concentration in the diet is a real one, although this is not detectable by statistical analysis (Student’s t-test). The difference in sap chloride concentration between the fresh water and sea water plant is significant (Fischer-Behren’s test, P < 0·01).

The most important finding of the present work is that the haemolymph sodium level, at 0·2 mmol 1⁻¹, in this aphid feeding on fresh water sea aster is the lowest yet to be found in the blood of any animal, by about one order of magnitude. Furthermore, the exclusion of the sodium ion from the blood, under sea water conditions, supports the result in that it indicates that low sodium levels in the blood are not only tolerated, but also required.

This is an extraordinary finding when compared with other herbivorous insects feeding on diets containing low levels of sodium. For example, in the cercopid, Cosmoscarta abdominalis, a Homopteran which feeds on xylem sap, the sodium concentration of the blood is 12·8 mmol 1⁻¹, whereas that of the excreta is only 0·44 mmol 1⁻¹ (Marshall & Cheung, 1975). A similar result was obtained, by these authors, from the fulgorid Pyrops candelaria, also a xylem sap feeder. In Myzus persicae this pattern is exactly reversed.

As far as is known virtually all insect axons use sodium in the propagation of action potentials (Pichon, 1974). Insects possess an effective ‘blood-brain barrier’ system, in the form of a perineurium and glial cells, which together regulate the ionic environment of the nerve cells in the central nervous system (cf. Treherne & Schofield, 1979). If such a system functions in Myzus persicae, to maintain a suitably high concentration of sodium around the nerve cells, it must be extremely effective. However, it does then seem remarkable that when the sodium ion is available to the aphid in higher concentrations, it is still maintained at very low levels in the blood.

Chloride, and possibly potassium increase in the aphid’s diet, when fed on a sea water plant. In the face of this ionic challenge the aphid continues to regulate these ions in the blood, at levels below those of the ingested fluid. No biological membrane is completely impermeable to sodium, potassium and chloride, so that there must be a method by which the ions that will inevitably enter the haemolymph are actively transported out.

In most insects where they have been studied, the Malpighian tubules are the organ reesponsible for the active removal of potassium from the haemolymph (Maddrell, 1977).

However, the midgut of some insects, particularly phytophagous species has been found to be an important site for the excretion of potassium (Harvey & Nedergaard, 1964). In Hyalophora cecropia, the species that has been most studied in this respect, the potassium pump is not an exchange pump and it is potassium specific (Zerahn, 1977). Sodium can, however, be transported in the same direction by the pump, but in the Hyalophora cecropia midgut under normal conditions it is apparently a poor competitor. In Myzus persicae this might not be the case, and the sodium ion might be excluded from the haemolymph at the cost of some increase in the potassium level, which would be one explanation for the observed rise in blood potassium under the sea water condition. Thus, it can be tentatively proposed that the main site of potassium and sodium transport in the gut of aphids, such as Myzus persicae might also be the midgut. There is, of course, no question of their being moved by the Malpighian tubules, as aphids do not have any.

The author would like to thank the N.E.R.C. and the Cambridge Philosophical Society for supporting this work, Professor J. Arthur Ramsay for his advice on the use of the microanalytical apparatus, and Dr S. H. P. Maddrell for his criticism of the manuscript.