ABSTRACT
Non-protein and protein nitrogen fractions of the isopod Sphaeroma rugicauda were measured in animals adapted to 100 and 2% sea water.
The non-protein nitrogen component was reduced in animals acclimatized to the lower salinity.
Free amino acids accounted for 88 and 74% respectively of the non-protein nitrogen in the two salinities.
In 2 % sea water taurine, proline, glycine, alanine and glutamic acid showed the greatest decreases in concentration compared to the levels measured in animals adapted to 100% sea water.
The decrease in total free amino acids of animals acclimatized to 100% sea water and transferred to 2% sea water was measured.
The total free amino acid concentration is reduced to the 2% sea water level within 12 hr. after transfer.
Free amino acid, haemolymph sodium and total body sodium levels after transfer to 2% sea water were compared.
The asymmetry between the fall in haemolymph sodium concentration and the decrease in total body sodium under these conditions is thought to be due to a water shift from the haemolymph into the tissues.
It is suggested that the osmotic pressure of the cells falls at a slower rate than that of the haemolymph.
INTRODUCTION
Euryhaline animals have been shown to prevent cellular swelling as a result of blood dilution by regulating the osmotic pressure of their cells. The amino acid concentration in the cells has been found to be reduced in a wide variety of forms in response to a reduction of blood concentration. It has been suggested that amino acids play an important role in this adjustment of cellular osmotic pressure. Previous investigations have tended to consider the levels of amino acids present in the cells of animals acclimatized to media of different concentration (see Florkin & Schoffeniels (1965) for review). Little is known of the changes in amino acid concentration taking place when the blood concentration is falling or rising immediately after transfer into a lower or higher salinity. Increased nitrogen excretion in Carcinus maenas (Needham, 1957), Eriocheir sinensis (Jeuniaux & Florkin, 1961), Neomysis integer (Austin, 1965), transferred from high to low salinities indicates possible break-down of excess amino acids. However, the relationship between the concentration of cellular amino acids and that of the blood under these conditions is not known.
In the present investigation the concentration of free amino acids in Sphaeroma rugicauda Leach acclimatized to a high and a low salinity was measured. An attempt was made to relate the changes in tissue amino acid concentration to those of the haemolymph when the animal is transferred from a high to a low salinity.
MATERIALS AND METHODS
Sphaeroma rugicauda was obtained from tidal creeks and salting pools at Totton, an area at the head of Southampton water. They occur here in large populations, notably in spring and summer. They were caught by hand-net and maintained in the laboratory at room temperature (16−20° C.).
Total nitrogen was measured by the Kjeldahl method using the ‘Quickfit’ Leurquin and Delville semi-micro apparatus. About 100 mg. of whole specimens of Sphaeroma rugicauda were digested with 2 ml. of concentrated H2SO4 and one B.D.H. ‘copper catalyst’ pellet. The digest was neutralized with 50% (w/v) NaOH and the liberated ammonia was drawn by an air current through 2% boric acid containing methyl red/ bromocresol green indicator. The ammonia was determined by titration against N/20 HCl. Recovery was estimated using a known weight of ‘Analar’ Glycine (mean = 97%).
The distribution of nitrogen between protein and non-protein fractions was determined using about 700 mg. wet weight of Sphaeroma. These animals had been starved for 6 days and subsequent micro-dissection showed that little food material remained in the gut. The animals were homogenized with 3 ml. of 20% (w/v) trichloracetic acid. The precipitated proteins were spun down to a pellet, the supernatant was removed and the precipitate was washed with de-ionized water. After recentrifuging, the washings were added to the supernatant. The nitrogen content of the supernatant (non-protein nitrogen) and of the pellet (protein nitrogen) were estimated as above.
The amino acid component of the non-protein nitrogen fraction was separated by two-dimensional, ascending paper chromatography using Whatman no. 1 paper. An extract was prepared by homogenizing 50 mg. of Sphaeroma in 3 ml. of 90% methanol. The homogenate was centrifuged and the precipitated proteins were discarded. The supernatant was reduced by evaporation to 1 ml. Butanol, pyridine and water (60 ml. of each) followed by phenol, water, ammonia 0·880 (160 g.; 40 ml.; 1 ml.) were the solvents used. The papers were developed with 0·2% ninhydrin in acetone. The twodimensional maps were interpreted by comparison with standards run under identical conditions. Quantitative estimations using a colorimetric method were carried out after elution of the amino acids from the chromatography paper. Separate papers were prepared and very lightly sprayed with ninhydrin. The spots were cut out and placed in separate test tubes. They were treated with the modified Yemm and Cocking reagent of Mathieson & Tattrie (1964). All traces of colour were removed by washing with de-ionized water. The solutions were made up to 25 ml. with de-ionized water and their optical densities were measured on a Unicam S.P. 500 spectrophotometer at 570 mµ (proline 440 mµ). An extraction of 90% was found using known amounts of glycine. This was measured by comparing optical densities of identical aliquots of the amino acid which had been either treated with the reagent directly or treated after extraction from chromatography paper. Standard curves of all the identified amino acids were prepared by running the standards in the same chromatography tank as the animal extracts.
To estimate the change with time of the free amino acid concentration after Sphaeroma had been transferred from 100% sea water into 2% sea water, it was found that chromatographic separation and subsequent estimation of individual amino acids was impracticable owing to the length of time taken and the number of animals required for each estimation. The total free amino acids were also estimated using the method of Mathieson & Tattrie (1964). An extract was prepared by homogenizing about 30 mg. wet weight of Sphaeroma in 1 ml. of 10% (w/v) sodium tungstate and 2 ml. of de-ionized water. After centrifuging 1 ml. of the supernatant was added to the reagent. A standard curve for glycine was prepared. A group of 200 Sphaeroma were acclimatized for 6 days in full strength sea water at 160 C. Three lots of animals each with a wet weight of 30 mg. were removed, weighed, and an extract was prepared. The remaining animals were transferred to 2% sea water, and similar groups of animals were removed at intervals for the same treatment. The extracts were sealed in glass vials and frozen for estimations to be carried out at the completion of the experiment. Samples were taken over a period of 48 hr. A knowledge of the free amino acid composition, obtained previously by chromatography, facilitated correction of the sample optical densities using the data of Cramer (1954) for molar extinction coefficients of all amino acids related to the standard, glycine, as unity. The molarity of the total free amino acids could therefore be estimated. However, as results will show, the concentrations of individual amino acids differ in animals acclimatized to the two salinities and vary also as a proportion of the total amino-acid concentration. The correction to be applied to the sample optical densities will therefore vary. The average of the free amino acid concentrations in the two salinities was used and, applying Cramer’s data, a correction to be applied to the sample optical densities was found.
The sodium concentration of the haemolymph was measured by flame photometry on a Unicam SP 900 flame spectrophotometer. Total sodium content was determined with the isotope 22Na. The activity in whole animals of this radioisotope was counted in a well-type EKCO scintillation counter. The counts were recorded on an EKCO automatic scaler. Dry weights were determined by drying wet-weighed animals in an oven at 70° C. to constant weight, cooling being carried out in a desiccator.
RESULTS
The total nitrogen values and the protein and non-protein fractions of the total nitrogen in Sphaeroma acclimatized to 100% and 2% sea water are shown in Table 1. A t-test on the mean values for non-protein nitrogen in the two salinities gave a value for t of 5·35. The difference between the means is therefore significant at the 5% confidence level. The corresponding difference for the protein nitrogen fraction was not significant (t =1 · 11).
Methanolic extracts of Sphaeroma were found to contain varying amounts of 11 identifiable amino acids. From the concentrations of individual amino acids it was possible to calculate the nitrogen content of the free amino acid component of the non-protein nitrogen (N.P.N.) (Table 2). Free amino acids account for 88% of the N.P.N. in animals acclimatized to 100% sea water as opposed to 74% in animals adapted to the lower salinity. These values represent the sum of amino acids occurring throughout the body, of which muscle is probably the most important source.
In Sphaeroma the concentration of free amino acids in animals acclimatized to 100% sea water falls when they are transferred to 2% sea water. The amino acid concentration previously measured in animals adapted to 2% s.w. is attained within 12 hr. after transfer to the dilute medium (Fig. 1).
The total body sodium of Sphaeroma was measured in animals similarly transferred from the concentrated to the dilute medium. Animals previously fully loaded in 100% sea water were washed in a current of inactive 2% sea water. The loss of 22Na was exponential. The fall in the sodium concentration of the haemolymph of Sphaeroma under the same conditions was non-exponential. The decrease in concentration of haemolymph sodium, total sodium and free amino acids are shown as percentages of their initial values plotted against time in Fig. 2.
DISCUSSION
The mean value for total nitrogen in animals acclimatized to 100% sea water is 5 93 mg./100 mg. dry weight for Sphaeroma, slightly lower than the values obtained for other aquatic isopods. Meyer (1914) gives values of 7·86 and 6·72% nitrogen of dry matter for Asellus aquaticus and Oniscus murarus respectively. Delff (1912) found that Glyptonotus (Mesidoted) entoman contained 7·59% nitrogen of dry matter.
Non-protein nitrogen represents 24·2 % of the total nitrogen in Sphaeroma in 100 % sea water. Comparable values have been found for Calanus finmarchicus (24·3 %; Cowey & Corner, 1963) and Neomysis integer (23% in 90% sea water; Austin, 1965). The non-protein nitrogen of Sphaeroma varied in the two salinities to which it was acclimatized.
Previous workers have found that the largest component of the non-protein nitrogen fraction is free amino acids. Values of 58% for Calanus (Cowey & Corner, 1963) and 80% for Neomysis (Austin, 1965) of the N.P.N. have been found. In Sphaeroma 88% of the non-protein nitrogen (1·27 mg. N/100 mg. dry weight) is present as free amino acids in animals acclimatized to 100% sea water. Animals acclimatized to 2% sea water show a considerable reduction in their free amino acids with a mean concentration of 0·69 mg. N/100 mg. dry weight. This is 74% of the non-protein nitrogen.
A high concentration of free amino acids has been found in the muscle fibres of a number of marine animals, and variation of these amino acids is known to occur in forms capable of withstanding changes in the concentration of the external medium, e.g. Carcinus maenas (Duchateau & Florkin, 1955; Shaw, 1958); Leander serratus and L. squilla (Jeuniaux, Bricteux-Gregoire & Florkin, 1961); Nereis diversicolor and Perinereis cultrifera (Jeuniaux, Duchateau & Florkin, 1961). Without exception these forms exhibit a decrease in the free amino acid concentration of their muscles when acclimatized to dilute salinities.
In Sphaeroma the amino acids that show the most marked fall in concentration are proline, glycine, alanine, glutamic acid and taurine. With the exception of taurine these amino acids also show the greatest decreases in those euryhaline crustaceans studied when the total free amino acid concentration falls. Jeuniaux, Bricteux-Gregoire & Florkin (1961) showed that when Leander squilla and L. serratus were transferred from 100% to 30% sea water the greatest changes in concentration of individual amino acids were those of proline, glutamine, alanine and glycine. Duchateau & Florkin (1955) also found notable falls in proline, glycine and argenine in the tissues of Eriocheir sinensis acclimatized to fresh water when compared to the level present in sea water.
One of the effects of acclimatization to a dilute medium in the majority of euryhaline forms is a fall in the blood concentration and consequently a dilution of the cell contents by osmotic intake of water. Shaw (1955, 1958) found that the water content of Carcinus muscle rose from 74·0 to 77·8% when the animal was transferred from 100 to 40% sea water. This is a smaller increase, however, than would be expected if there were no change in the concentration of the osmotically active constituents of the cells. It is presumed that adjustment of amino acid levels is involved in the regulation of cellular water content. The ability of Sphaeroma to vary the free amino acid concentration of its tissues may similarly be interpreted as being an aid to cell volume regulation.
All the values of free amino acids from various euryhaline animals have been obtained from animals adapted to the salinities involved. Little is known of the changes taking place when the concentration of the blood is falling or of the relationship between tissue levels of free amino acids and blood concentration in these circumstances. In Sphaeroma this relationship was investigated by measuring changes in the concentration of sodium in the haemolymph. The major inorganic ions in the haemolymph of all the crustaceans that have been studied have been found to be sodium and chloride. In the isopod Ligia oceanica these ions contribute to 90% of the total haemolymph osmotic pressure (Parry, 1953). It is reasonable to assume therefore that measurement of one of these ions will indicate the extent of the gross haemolymph changes that occur. Measurement of the changes in the total sodium content of the animal will include sodium in both the tissues and haemolymph.
Initially, in Sphaeroma transferred from 100% sea water to the dilute medium, the haemolymph sodium concentration falls more rapidly than the total sodium owing perhaps to invasion of water into the animal as a result of the large concentration gradient between haemolymph and medium. This may be the result of an inability of the excretory organs to maintain water balance initially. Preliminary experiments with tritiated water have shown that Sphaeroma is fairly permeable to water (Harris, 1967). The rate of fall of haemolymph concentration is matched by a fall in the free amino acid concentration. After about 2 hr. a decrease in the rate of fall of free amino acid concentration occurs. The reason for this is not known. If, however, the cellular osmotic pressure falls at a slower rate than that of the haemolymph, water may move into the cells causing a reduction in the rate of decrease of haemolymph osmotic pressure as a result of a decreasing haemolymph volume. A slowing of the decrease of haemolymph concentration does occur in Sphaeroma. The total sodium content of the animals however continues to fall. The characteristic non-exponential fall in haemolymph concentration with time cannot therefore be due to a change in permeability to sodium at the body surface. A similar faster rate of fall of total sodium compared with haemolymph sodium concentration has been found by Lockwood (1964) in Gammarus duebeni.
Movement of water into the cells will result in dilution of the cell contents and cause cellular swelling. Shaw (1955) has shown that muscle cells of Carcinus maenas swell as a result of a fall in haemolymph concentration. Gross & Marshall (1960) have shown that the muscle of Pachygrapsus gains water when the animal is immersed in 50% sea water.
If no reduction in cellular osmotic pressure occurs other than by dilution resulting from water uptake from the haemolymph, the haemolymph volume could be reduced to a point where circulatory crisis occurs. However, in Sphaeroma this does not occur and the haemolymph sodium starts to fall at a rate similar to that recorded initially. The free amino acids, however, are maintained at a high level and over-all do not show as great a fall as haemolymph and total sodium values. This may be suggestive of some other component of cellular osmotic pressure being involved in changes to maintain isosmocity with the blood.
ACKNOWLEDGEMENT
I wish to thank Dr A. P. M. Lockwood, under whose supervision this work was carried out, for his advice and criticism. I am indebted to the Science Research Council for a maintenance grant.