Effect of diet on the free amino acid pools of symbiotic and aposymbiotic pea aphids, Acyrthosiphon pisum

J. Insect Physiol. Vol. 41, No. I, pp. 33-40, 1995 Copyright 0 1995 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0022-1910/95 $9.50 + 0.00

0022-1910(94)00085-9

Effect of Diet on the Free Amino Acid Pools of Symbiotic and Aposymbiotic Pea Aphids, Acyrthosiphon pisum I. LIADOUZE,* Received

G. FEBVAY,*t

J. GUILLAUD,*

G. BONNOT*

I1 April 1994; revised 12 July 1994

Free amino acid pools were analysed in Acyrtirosiphon pisum reared on Vicia faba L. and on three artificial diets with different amino acid profiles: diets A and B copying the very unbalanced profiles of phloem saps of alfalfa and broad bean respectively, diet C deriving from aphid carcass analysis. Total free amino acid levels ranged from 46.9 f 2.3 nmol-mg-’ fresh weight for aphids maintained on host plant to 86.0 f 3.6 nmol-mg-’ for those reared on diet B. Whatever the food source was, the free amino acid pools of aphids displayed roughly similar patterns, except for lysine and arginine. The role played by the intracellular symbionts in this homeostasis was investigated with aposymbiotic aphids reared on the same diets. The treated aphids had significantly higher free amino acid content (119.2 f 1.7 to 140.0 f 7.7 nmol-mg-’ fresh weight) than control aphids. In contrast to the symbiotic situation, the well balanced free amino acid pools were not maintained in aposymbiotic aphids: four amino acids were found in higher concentrations in aposymbiotic aphids (asparagine, aspartic acid, glutamine and proline), while isoleucine, leucine, tyrosine, phenylalanine, threonine and glutamic acid were in lower concentrations. These results are consistent with the hypothesis that symbiotic bacteria contribute to the nutrition of aphids by the synthesis of essential amino acids. The observed negative correlation of free amino acid levels with aphid performances suggested a possible use of this parameter as an indirect criteria to measure the quality of a natural food for aphid development, and to test the nutritional fitness of an aphid population to its host plant. Acyrthosiphon

pisum

Homoptera

Aphididae

Amino acids

INTRODUCTION

Symbiosis

Homeostasis

amino acids supply the free amino acid pool of the aphid. Protein degradation during their turnover as well as amino acid neosynthesis provide the main other inputs to this pool. Amino acids from this pool have a number of different functions in insects (Candy, 1985), one of the most important being protein synthesis. All 20 common amino acids are required simultaneously for this synthesis (Bonnot et al., 1976; Horie and Inokuchi, 1978). The ratio of available amino acids can be modified by their synthesis either from precursors by the insect itself (non-essential amino acids) or from the activities of symbiotic micro-organisms. Therefore, the size and composition of this free amino acid pool is a picture of the regulatory mechanisms driving the whole nitrogen metabolism of the insect. Aphids display an obligate association with bacterial endosymbionts harboured by the bacteriocytes, a complex of cells differentiated specifically for this purpose (Buchner, 1965). These symbionts were recently assigned to the new genus Buchnera in the gamma-subdivision of Proteobacteria by Munson et al. (1991). The bacteria are believed to complement the aphid diet by provision of

The phloem-sap, on which aphids (Homoptera: Aphididae) feed, is generally considered as nutritionally poor (Raven, 1983; Slansky and Scriber, 1985). Concerning the nitrogen supply, this food is mainly constituted of amino acids (Ziegler, 1975; Rahb& et al., 1990) that often show an exceptionally unbalanced composition. For example, the sap of Leguminosae, the host plants of the pea aphid Acyrthosiphon pisum (Harris), has one or two amino acids in large excess. In the sap of alfalfa, collected by aphid stylectomy, asparagine amounts to 70% of the total amino acids (Girousse et al., 1990). Asparagine is also the main amino acid (58%) of the sap of broad bean obtained by the EDTA-exudation technique (Douglas, 1993) and, Sasaki et al. (1990) showed that asparagine and glutamine total together approx. 50% of the amino acid content of phloem sap collected by stylectomy. After ingestion and absorption, these *INSA, Laboratoire de Biologie appliquke 406, UA INRA 20 av. A. Einstein, F-69621 Villeurbanne cedex, France. tTo whom correspondence should be addressed.

Artificial diet

227,

33

34

I. LIADOUZE

vitamins, sterols and certain amino acids (Ehrhardt, 1968; Mittler, 1971; Houk and Griffiths, 1980; Douglas, 1988a). The nutritional dependency of aphids upon the symbionts was first studied by maintaining symbiotic and/or aposymbiotic aphids on defined diets from which individual amino acids were omitted (Dadd and Krieger, 1968; Mittler, 1971). In the last years, direct investigations of amino acid synthesis by the symbionts were carried out, as in the case of the incorporation of inorganic sulphate into the sulphur amino acids for Myzus persicae (Douglas, 1988b). Douglas and Prosser (1992) also provided results demonstrating the synthesis of tryptophan by the symbionts of A. pisum. Nitrogen recycling is also hypothesised as a possible function for the aphid symbionts, and it was recently demonstrated that aphids deprived of their symbiotic bacteria produce honeydew containing a high concentration of glutamine (Sasaki et al., 1990; Prosser and Douglas, 1991). Prosser and Douglas (199 1) first suggested that ammonia (nitrogenous waste product) is utilised by the symbiotic bacteria in untreated aphid, but is detoxified in the aposymbiotic aphid primarily by incorporation into glutamine. This suggestion was consistent with the ammonia assimilation capabilities of the symbiotic bacteria of aphids (Whitehead et al., 1992). However, other authors hypothesised that amides are important nitrogen sources for the symbiotic aphids, that may be unused, and therefore excreted, by aposymbiotic aphids (Sasaki and Ishikawa, 1993; Sasaki et al., 1993). Several investigations of the metabolic capabilities of aphids were carried out through honeydew analyses, but only one paper reported results on free amino acid content of aphids (Prosser and Douglas, 1991). This study was however limited to the comparison of symbiotic and aposymbiotic aphids reared on one holidic diet. In the study reported here, the amino acid metabolism of the pea aphid A. pisum is investigated further by analysing the free amino acid composition of symbiotic aphids reared on three different artificial diets and on plant. The influence of symbionts in the regulatory mechanisms governing this pool is examined by a similar analysis of aposymbiotic aphids.

MATERIALS

Symbiotic

AND METHODS

insects

A parthenogenetic clone of A. pisum (LLOl) was established from a field infestation on lucerne in Lusignan, France (October 1986). The stock culture of aphids was maintained in the laboratory on young broad bean plants (Viciu fubu L. var. Aquadulce) in Plexiglas cages (21°C 70% r.h., 16:8 light-dark photoperiod). A limited number of mass-reared winged adults was allowed to lay progeny for 24 h on young Viciu plants. Part of the offspring was kept on seedlings during 7 days and the resulting larvae or young adults were used for amino acid analyses (control sample reared on plant).

et al.

With the remaining offspring, groups of 20 individuals (aged between 0 and 24 h) were directly transferred to artificial diets and reared during the same period of time (7 days) in the same conditions of photoperiod, humidity and temperature. Artificial

diets

Three different diets were used; two of them (diets A and B) copied the amino acid composition of phloemsap of common host-plants of the pea aphid, when diet C derived directly from the total amino acid profile of the whole aphid tissue (Febvay et al., 1988b). The sucrose content of all diets was the same (20% w/v) and the supply of vitamins and oligoelements was identical to the diets previously described (Febvay et al., 1988b). The total amino acid concentration of the three diets was very similar (A: 259 mM, B: 254 mM and C: 260 mM). The amino acid profiles of these diets are shown in Fig. 1. These profiles were derived from phloem sap analysis of alfalfa (Girousse et al., 1990) for diet A and of broad bean (Sasaki et al., 1990) for diet B. The amino acid composition of diet C was identical to the A5 synthetic diet described by Febvay et al. (1988b). The solutions were filtered through a 0.45 pm filter and aseptically enclosed in parafilm sachets stretched over the experimental chamber (polyvinylchloride tubes, 20 mm high and 35 mm i.d.) and stored before use at - 20°C. During aphid rearing, the sachets were changed twice a week. Aposymbiotic

insects

To produce aposymbiotic aphids, neonate larvae obtained on Viciu plants were transferred to sachets of artificial diets containing 50 pg.ml-’ of rifampicine, a concentration below its LC,, at day 7 for A. pisum (Rahbe and Febvay, 1993). After 48 h at 2 1“C, the larvae were transferred back to the corresponding artificial diet or to Viciu plants and grown under standard conditions during 5 more days. For plant grown aposymbiotic aphids, the rifampicine diet was diet C. A full description of this aposymbiosis protocol has been provided elsewhere (Rahbe et al., 1993). Sample

analysis

Aphids were individually weighed immediately after removal from plant or artificial diets (fresh weight). For each treatment, three replicates of five aphids were used for free amino acid analysis. Each sample was crushed in 200 ~1 of water with 50 nmol of glucosaminic acid added as an internal standard. The homogenate was centrifuged at 7000g for 5 min to eliminate cellular fragments and then deproteinised by filtering through a membrane with a 10 kDa cut-off threshold (UltrafreeMC Millipore, St Quentin-Yvelines, France). After having removed lipids by extraction with 100 ~1 of chloroform, the sample was dried by evaporation (Speedvac apparatus) and taken up with 60 ~1 of 0.05 M lithium citrate buffer pH 2.2. The sample was then

Viciu

faba plants

+

2~2

FIGURE 1. Free amino acid composition of symbiotic and aposymbiotic pea aphids grown on different artificial diets or on broad bean plants. Values are expressed in mol% as mean + SD (n = 3). The three graphs (a, b, c) show the amino acid profiles of used artificial diets. (d) symbiotic and (h) aposymbiotic aphids reared on diet A; (e) symbiotic and (i) aposymbiotic aphids reared on diet B; (f) symbiotic and (j) aposymbiotic aphids reared on diet C. (g) symbiotic and (k) aposymbiotic aphids reared on broad bean plants.

submitted to ion-exchange chromatography on an automatic amino acid analyser (Beckman 6300) in which amino acids were detected by ninhydrin reaction, identified by their retention time and wavelength ratio, and quantified by their absorption at 570 nm (440nm for proline). Statistical analysis Weight and free amino acid data were subjected to a 2-ways analysis of variance (ANOVA) and the statistical differences were estimated by Fisher’s PLSD test. The amino acid composition of the free pool was calculated as percent of the total amino acid content; the statistics (mean and standard deviation) on these data were calculated after arcsin-square root transformation. Global interpretation of the differences in amino acid profiles was carried out by a multivariate statistical procedure. A principal component analysis (PCA) was performed on the covariance matrix of the 21 variables (amino acid concentrations). PCA is an exploratory multivariate procedure that determines, by a sequence of linear combinations of variables (eigenvectors) into components explaining the greatest proportion of

variation in the data set (Dagnelie, 1986). This is done without any a priori assignment of individuals (or samples) to categorical groups. The AnaMul software for Macintosh was used for PCA computation (Febvay and Bonnot, 1991).

RESULTS

The weights of symbiotic pea aphids reared on the different diets were significantly different (Table 1). As classically observed, aphids maintained on plant were bigger. When the aphids were raised on artificial diets, their body weights ranged from 34% (diet B) to 50% (diet C) of that of the aphids grown on plant. Rifampicine treatment depressed significantly the aphid body weights (Table 1), which were consistently lower (65575%) than that of their symbiotic counterparts. The free amino acid contents of 7 day-old aphids are shown in Table 2. Expressed as per unit of fresh weight, the free amino acid level was not significantly different in symbiotic aphids between larvae raised on plant and on the balanced diet C, but was approx. 40% lower than that of aphids reared on diets A or B. When compared

I. LIADOUZE

36

TABLE 1. Fresh weight in mg (mean k SE with number of repetitions in parentheses) of symbiotic and aposymbiotic aphids reared on different diets Symbiotic

aphids

Aposymbiotic

aphids

Diet A

1.25 * 0.05b+

0.31 * o.ola~’

Diet B

(51) 0.96 & 0.04”,”

(45) 0.32 + 0.02”,’

Diet C

(50) 1.41 k 0.06c,”

(47) 0.44 + 0.03bJ

Plant

(44) 2.81 & 0.09d+

(43) 0.74 * 0.03’J

(26)

(46)

(Vicia faba)

Means with the same letter (ad: comparisons between lines, u-v: comparisons between columns) are not significantly different (2ways ANOVA and Fisher’s PLSD test, P < 0.05).

to the corresponding control symbiotic aphids, the free amino acid content of aposymbiotic insects was considerably increased. This rise of the free amino acid level following antibiotic treatment was more marked for aphids grown on plant or on optimal artificial diet C (+ 130% or more) than for aphids grown on unbalanced diets A or B (+60%). The body weight of aphids was strongly and negatively correlated with the free amino acid level (Pearson correlation coefficient r = -0.88, 6 d.f., P < 0.01). The free amino acid compositions of symbiotic and aposymbiotic aphids raised on different diets are shown in Fig. 1. We observed a remarkably constant pattern for the symbiotic aphids whatever the diets used. In contrast, rifampicine treatment largely altered these profiles, which were closer to the patterns of the original rearing diets. To quantify such profile alteration, a coefficient of similarity between two patterns of free amino acids was calculated according to the equation shown in Table 3, as described by Tamura and Osawa (1969) and Tamura et al. (1969). The divergence from the control pattern (free amino acid profile of symbiotic aphids grown on plant) was first determined (Table 3). The values obtained with symbiotic aphids on the three diets were all greater than 0.9, that on diet C being very close to 1. On the other hand, these coefficients dropped dramatically for rifampicine treated aphids. In this case the highest value (0.81) was obtained with aphids raised on the balanced diet. With aphids reared on broad bean plant or on diet B that mimics the amino acid composition of broad bean, the coefficients were very close.

TABLE 2. Free amino acid content (nmol mgg’ aphid fresh weight) of symbiotic and aposymbiotic aphids reared on different diets. Data are expressed as mean f SE (n = 3) Symbiotic Diet A Diet B Diet C Plant (Vicia fiba)

77.4 86.0 52.3 46.9

aphids

+ 2.7”,” + 3.6”,” + 4.9bs” & 2.3b+

Aposymbiotic 124.6 140.0 120.5 119.2

+ & + ;

aphids 7.2=+’ 7.7a.’ 6.9b,’ 1.7b,’

Means with the same letter (a-b: comparisons between lines, u-v: comparisons between columns) are not significantly different (2ways ANOVA and Fisher’s PLSD test, P -e 0.05).

et al.

When the aphid patterns were next compared with those of the diets used for aphid rearing (Table 3), high values were obtained for aposymbiotic aphids, indicating that the profiles exhibited great analogy with the profile of food amino acids. Further interpretation of the concentrations of each amino acid measured on symbiotic and aposymbiotic aphids on different diets were carried out using the multivariate principal component analysis. As shown in Fig. 2(A), the two first components (accounting for 82.4 and 13.8% of the variance in the data set, respectively) allowed a complete separation of the four groups of aposymbiotic aphids reared on different foods, the symbiotic group was also completely separated from the latter. However, the first two axes did not allow an accurate separation within the four groups of symbiotic aphids reared on different diets. Within aposymbiotic insects, the aphids reared on the optimal diet C formed the closest group to the symbiotic group and, the two aposymbiotic groups on broad bean plants and on diet copying broad bean sap were neighbouring. The separation of symbiotic and aposymbiotic aphids in five groups is due to strong correlations of some amino acids with the principal components [Fig. 2(B)]. Trivial factors were asparagine, aspartic acid, glutamine and proline (higher in the aposymbiotic aphids) and isoleucine, tyrosine, phenylalanine, leucine, threonine and glutamic acid (higher in symbiotic aphids). The third component (accounting for 2.1%) allowed a limited separation of the four symbiotic groups [Fig. 2(C)]. Only aphids reared on the two unbalanced diets (A or B) could not be distinguished from one another and were clearly separated from aphids on plant or on diet C. Two amino acids (lysine and arginine) were responsible for this separation, displaying higher proportions in symbiotic aphids on diets A or B than in the other symbiotic aphids [Fig. 2(D)]. DISCUSSION

The nitrogen supply from the food source (host plant) have long ago been studied as a possible nutritional mechanism of resistance to aphids. If the total amino acid level in plants was the first factor invoked (Schaefer, 1938; Maltais and Auclair, 1957; Auclair et al., 1957; Van Emden and Bashford, 1969; Jansson et al., 1987), the amino acid balance was early postulated as playing a crucial role in determining resistance to aphid attacks (Van Emden and Bashford, 1971; Havlickova, 1987; Febvay et al., 1988a; Rahbe et al., 1988). The possibility to rear aphids on holidic liquid diets (Auclair and Cartier, 1963; Akey and Beck, 1972) facilitated studies on amino acid quality and its consequences to aphid development. The first experiments concerned mainly the “essentiality” of amino acids and most studies were led by deletion or addition of dietary components and analysing their effects on aphid development (Retnakaran and Beck, 1968; Harrewijn and Noordink, 1971; Leckstein and Llewellyn, 1973, 1974; Srivastava

FREE TABLE

3. Pattern

similarity (symbiotic

AMINO

ACIDS

coefficient* of free amino acid composition and aposymbiotic) reared on different diets

Symbiotic aphids

fiba)

Aposymbiotic aphids

0.95 0.91 0.98 1

S(A,

Symbiotic aphids

0.54 0.68 0.81 0.70

*The following equation was used for the calculation, . a,) and B (b,, b,, b,)

W,

B) = cost7 = v’Cu;JCb:(i

of pea

aphids

Against the free amino acid pattern of the diet used for the aphid rearing

Against the control free amino acid pattern of symbiotic aphids reared on plants

Diet A Diet B Diet C Plant (Vi&

37

IN A. PISUM

0.48 0.71 0.80 0.637 when comparing

=

1

Aposymbiotic aphids 0.96 0.94 0.86 0.91t two patterns,

A (a,,

a,,

n)

It has been thought that the pattern similarity is a cosine of the angle between the two vectors OA and OB in the n dimensional space (Tamura and Osawa, 1969; Tamura et al., 1969). When one pattern is identical with the other, the pattern similarity is 1; if no components exist simultaneously in the two patterns, the pattern similarity is 0. tThese two coefficients were calculated against the free amino acid pattern of diet B that copied the amino acid composition of the broad bean sap.

and Auclair, 1975; Srivastava et al., 1985). However the supply of individual amino acids was not the sole criterion to take into account to analyse optimal development of aphids on artificial diets, and we previously showed on A. pisum that the balance of amino acid composition was an important factor (Febvay et af., 1988b). The growth results presented here confirm this point. On the three diets with an identical total amino acid concentration, that are differentiated only by their amino acid profile, the growth of symbiotic larvae was significantly different. On the well balanced diet, the development of the aphids was much improved, as observed with equivalent diets by Sasaki et al. (1991) on a different clone of A. pisum. The best growth obtained on diet C is not surprising since this diet was elaborated through an optimisation process based on a growth criterion (Febvay et al., 1988b). Independently of this amino acid composition of food, the aphids on artificial diets remained however lighter than on plant. The pea aphids used in this study were parthenogenetic and viviparous and the weight of aged larvae or adults depended highly on the embryo number in the oviducts. The fecundity of adults on artificial diet was markedly depressed (US plant-grown aphids; data not shown here) and this may largely explain the weight differences between aphids on plant and on artificial diets. Furthermore, as shown earlier (Febvay et al., 1988b) the developmental time is increased on artificial diets, resulting in a reduced weight at a given time, as measured in this study. Different methods were used to obtain routinely aposymbiotic aphids, involving either short treatment with antibiotics in artificial diets (Rahbe et al., 1993), antibiotic-infused plants (Douglas and Prosser, 1992) or even heat treatment (Ohtaka and Ishikawa, 1991). Whatever the treatment, it was shown that the effect on aphid performances, such as growth or reproduction,

resulted mainly from aposymbiosis (i.e. not from treatment artefacts). Our results on body weights confirm this point and are in very good concordance with those of Sasaki et al. (1991). The influence of amino acid supply on aphid performances has been largely investigated, but no study has focused up to now on the influence of this factor on the free amino acid pool of aphids. As reviewed by Chen (1985), interrelation between extracellular amino acids, their uptake into the cell and subsequent incorporation into proteins remains obscure (in insects as in other higher organisms). The intracellular amino acid pool, assumed to be the obligatory free precursor pool for protein synthesis, is more or less rapidly in equilibrium with the amino acid pool of haemolymph and analyses of free amino acid pool in insects are generally carried out on the haemolymph compartment. In aphids, however, as haemolymph is difficult to sample routinely, we decided to analyse the total free amino acids. This pool represents an integrated picture of free amino acid content of total tissues and haemolymph but is somewhat altered by dietary amino acids contained in the alimentary tract. Nevertheless, as shown by the results reported here (for example, free amino acid pattern comparison of symbiotic aphids grown on diet A or B and of the corresponding diet), the contribution of the dietary amino acids may be neglected. One major finding in the present study is that the symbiotic pea aphids showed a free amino acid pattern remarkably constant, whatever the diet composition used for larval development. To our knowledge, this result is the first demonstrating a real homeostasis of the internal medium of aphids. The sole difference in free amino acid patterns observed in symbiotic aphids concerned lysine and arginine, that were in higher concentrations in aphids on the two “unbalanced” diets although these diets present lower levels in these amino

38

I. LIADOUZE

er al.

PC1

FIGURE 2. Principal component analysis of the free amino acid concentrations of symbiotic and aposymbiotic pea aphids grown on different artificial diets or on broad bean plants. (A) individual projections on the first two principal components (ellipses contain 95% of the corresponding group); (B) correlation circle showing for the two principal components (PC1 and PC2) the contributions of each variable (amino acids); (C) and (D) same as (A) and (B), with the first and third principal components. Open symbols show the symbiotic aphids and solid symbols show the aposymbiotic ones. (0, +) aphids grown on diet A; (A, A) aphids grown on diet B; (0, n ) aphids grown on diet C; (0, 0) aphids grown on broad bean plants.

acids than diet C. It is important to note that these two amino acids are also the two ones over-excreted in honeydew of aphids reared on similar unbalanced diets, when compared to honeydew from aphids grown on plant or on a well balanced diet (Sasaki et al., 1991). These amino acids, with two and four nitrogen atoms per molecule respectively, are probably concerned with nitrogen excretory metabolism. Nitrogen excretion might be somehow disturbed in aphids reared on diets A or B, as these diets contained a great proportion of amide amino acids. Nitrogen excretory metabolism is also probably modified in aphids reared on artificial diets (US plant reared aphids): lysine and arginine levels were not as high in the free amino acid pool (nor in honeydew; Sasaki et al., 1991) of plant grown aphids, in contrast to what is observed on diet B, although the amino acid balance of diet B was derived from sap analysis. The other important finding of this study is that symbiosis seems to be the key of the observed homeostasis of the free amino acid pool. In aposymbiotic aphids, the pattern of this pool was not constant anymore, and appeared close to that of the ingested diet.

These results demonstrate that the metabolic pathways leading to the maintenance of free amino acid pool equilibrium depend on the activity of symbiotic bacteria. Through the analysis of PCA results, patterns for this bacterial-dependent regulation may be hypothesised. Four amino acids (aspartic acid, asparagine, glutamine and proline) were found in higher concentrations in aposymbiotic aphids, and may thus be regarded as the main nitrogen pool utilised by symbionts for the synthesis of other amino acids. The utilisation of amide molecules by symbiotic aphids was already hypothesised by Sasaki and Ishikawa (1993), but it is the first result showing that aspartic acid and proline may also be concerned. The amino acids potentially synthesised by the symbionts may logically be found among those traced by their lower concentrations in aposymbiotic aphids: isoleucine, leucine, tyrosine, phenylalanine, threonine and glutamic acid. Three of them (isoleucine, phenylalanine and threonine) were found already by Prosser and Douglas (1991) at lower concentrations in chlortetracycline-treated aphids. All these amino acids, except glutamic acid, are so-called essential amino acids

FREE

AMINO

ACIDS

these data are consistent with the hypothesis that the bacteria contribute to the nutrition of aphids by synthesis of essential amino acids (Mittler, 1971). The position of glutamic acid in this group reflects that this amino acid plays a central role in the metabolic pathways of the bacteria. Our hypothesis is that the four amino acids that accumulate in aposymbiotic insects are substrates for glutamate production in symbiotic conditions. As glutamic acid seems to be the major bacterial nutrient and displays peculiar transport features (Whitehead and Douglas, 1993) it is likely that the synthesis of essential amino acids by bacteria depends on glutamic acid metabolism. A work using radiotracers is presently in progress to confirm or to invalidate this hypothesis. The free amino acid pools of aposymbiotic aphids seem clearly deficient in some amino acids, a situation leading to a reduced protein synthesis and to an important growth depression. This reduction in synthesis may in turn explain the observed increase of the free amino acid content in aposymbiotic aphids, reflecting the nonutilisation of the available amino acids. These results are consistent with the view that all 20 protein amino acids are required simultaneously for the protein synthesis as demonstrated early in vertebrates by Munks et al. (1945) and since confirmed in insects as in the Tachinidae, Phryxe cauduta, by Bonnot et al. (1976) or in the silkworm, Bombyx mori, by Horie and Inokuchi (1978). Accordingly, but on a lesser scale, symbiotic aphids reared on artificial diets showed a decreased body weight and a higher free amino acid concentration, when compared to aphids grown on plants. Two effects may be discriminated by these results. The first effect, displayed by the comparison of aphids grown on plant and on artificial diet C, is the “artificial diet effect”. As a consequence, in comparison with plant grown aphids, the artificial diet reared aphids show a lighter body weight but no significantly different level of free amino acid content. This effect results probably from a decrease of the general metabolism, following the failure of artificial diets to supply some “vitamin-like” cofactors that are normally provided by the phloem sap to aphids grown on plant. On the other hand, the comparison of aphids grown on balanced artificial diet C and on unbalanced diets A or B, displays the second effect (“unbalanced diet effect”), which results in an additional and moderate decrease in body weight, and an increase in free amino acid contents, resulting probably again from a limitation of protein synthesis. The free amino acid pool being identically well supplied in all symbiotic aphids, this limitation should here again not be a consequence of a deficiency in particular amino acids. In addition, maintaining a constant composition of the free amino acid pool may represent a metabolic cost that is higher for aphids fed on unbalanced diets than for those fed on balanced diets and could lead to the observed additional growth depression. The free amino acid content of aphids is strongly and negatively correlated with their body weight, and might and

IP41,1*

IN A. PISUM

39

be used as a routine parameter to estimate the quality of a food for aphid development in natural situations. If this was confirmed, particularly in the case of different insect/plant genotype combination, these free amino acid levels and profiles would be interesting instantaneous criteria, easy to measure, for the evaluation of the nutritional origin of aphid fitness to its host plant (ecological variability, host-plant resistance, . . .).

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Acknowledgements-We thank G. Duport who helped with the insect cultures. We are grateful to Y. Rahbe and B. Delobel for helpful discussions and comments on the manuscript. We also thank the Conseil Regional Rhone-Alpes for financial support.