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Synthesis of the essential amino acid tryptophan in the pea aphid (Acyrthosiphon pisum) symbiosis
J. Insect Physiol.
Vol. 38, No. 8, pp. 565-568, Printed in Great Britain. All rights reserved
1992
0022-1910/92 $5.00 + 0.00
Pergamon Press Ltd
SYNTHESIS OF THE ESSENTIAL AMINO ACID TRYPTOPHAN IN THE PEA APHID (ACYRTHOSIPHON PISUA4) SYMBIOSIS A. E. DOUGLAS and W. A. PROSSER Department of Zoology, University of Oxford, South Parks Road, Oxford OX1 3PS, U.K. (Received 17 December 1991; revised 4 February 1992)
aphids (Acyrthosiphonpisum) containing their symbiotic bacteria do not require a dietary supply of the essential amino acid tryptophan over two generations on chemicallydefined diets; but aphids, whose bacteria are disrupted by chlortetracycline, die as firstgeneration larvae on tryptophan-free diet. Consistent with the interpretation that the symbiotic bacteria in the aphids synthesize tryptophan, tryptophan synthetase activity was detected in both symbiont-bearing aphids and isolated bacteria, but not in chlortetracyclinetreated aphids. Aphids reared on tryptophan-free diet produced honeydew containing 0.32 mM tryptophan, from which it can be calculated that each bacterial cell in the aphid releases at least 0.04 fmol tryptophan d-‘. Abstract-Pea
Key Word Index: Symbiosis; aphids; essential amino acids; tryptophan
INTRODUCTION
Symbiotic microorganisms can be regarded as “vehicles” by which animals gain access to complex metabolic capabilities (Douglas, 1992). For example, some xylophagous insects (e.g. lower termites) utilize the cellulolytic capabilities of the protists in their guts (reviewed by Breznak, 1982); and many insects use the ability of intracellular microorganisms to synthesize “high value” nutrients, such as essential amino acids, sterols and vitamins (Douglas, 1989). The latter associations are commonly described as mycetocyte symbioses because the microorganisms are located in the cytoplasm of specialized insect cells called mycetocytes. A relatively well-characterized mycetocyte symbiosis is in aphids. The microorganisms are bacteria, which have recently been assigned to a new genus, Buchneru, in the yproteobacteria (Munson et al., 1991). Evidence is gradually accumulating that these bacteria provide essential amino acids to the aphid tissues, and that this contributes to the aphids’ ability to utilize phloem sap, a diet of low essential amino acid content. In particular, Douglas (1988a, 1990) has demonstrated that the bacteria in one clone of the green peach aphid Myzus persicae can synthesize the essential amino acid methionine, which is released to the insect; this clone can persist for at least five generations on methionine-free diets. More generally,
aphids, unlike most insects, appear not to require a dietary supply of the full complement of essential amino acids [see review of Dadd (1985)], suggesting that the bacteria may contribute to the aphids’ requirements of most, perhaps all, essential amino acids. In a recent study of honeydew production by the pea aphid Acyrthosiphon pisum, Prosser et al. (1992) found that aphids reared on diets of low nitrogen content produce honeydew with a higher concentration of tryptophan than in the diet. They hypothesized that the bacterial symbionts may have synthesized the “excess” tryptophan. (A. pisum bears two morphologically-distinct bacteria, the primary symbionts, Buchnera, and small numbers of secondary symbionts, either or both of which could contribute to tryptophan synthesis.) This paper describes dietary and metabolic investigations which confirm the interpretation of Prosser et al. (1992), specifically that the bacteria are capable of tryptophan synthesis and make this amino acid available to the aphid tissues in A. pisum.
MATERIALS
AND METHODS
The aphids were a clonal culture of A. pisum, known as “clone OX-~“, and maintained on Viciu faba var. The Sutton at 20°C and 18 h light-6 h dark 565
566
A. E. DOUGLAS and W. A. PROSSER
regime. For some experiments, the aphids were transferred to chemically-defined diets within 24 h of birth. The diets were prepared as in Douglas (1988b) and diet composition was that of Akey and Beck (1972), as modified by Prosser and Douglas (1991). The symbiotic bacteria were routinely disrupted by allowing the aphids to feed for 5 days on the chemically-defined diet supplemented with the antibiotic chlortetracycline at 50 pg ml-’ (Douglas, 1988b). For aphids used in enzyme assays (see below), chlortetracycline was administered via the plant. V. f&a were raised to the three-leaf stage in a substratum of expanded clay (“Hydroleca”: Sinclair Horticulture and Leisure Ltd, Lincoln, U.K.) (Gange and Brown, 1991). The roots of these plants were separated from the Hydroleca particles, and immersed in nutrient solution comprising: 0.05 mM (NH,), S04, 2 mM 0.1 mM CaCl,. 2H,O, 50pM MgSO,. 7H,O, Fe,Na.EDTA, 2mM K,HP04/KH2P0, buffer pH 7, and 250 pg chlortetracycline ml-‘. Preliminary experiments showed that the concentration of chlortetracycline in the leaf tissues [assayed as in Douglas (1988c)] rose to 50-7Opg g-’ leaf tissue within 24 h, and remained stable at this level for a further 4 days. The following protocol was therefore adopted; day 1, first group of plants transferred to nutrient solution containing chlortetracycline; day 2, aphids introduced to chlortetracycline-treated plants; day 4, second group of plants transferred to nutrient solution with chlortetracycline; day 5, aphids transferred from first to second group of chlortetracyclinetreated plants; day 7, chlortetracycline-treated aphids transferred to antibiotic-free plants. The aphids were assayed on day 9. For studies of the dietary requirement of A. pisum for tryptophan, the aphids were raised on chemicallydefined diets either containing or lacking the standard concentration of tryptophan (2.5 mM), such that the total concentration of amino acids in the diet were 135 mM and 132.5 mM, respectively. Within 24 h of reaching adulthood, each aphid was weighed on an electrobalance (Elmer-Parker-AT4) to the nearest pg, and the relative growth rate of apterae over larval development was determined from the equation: [log(teneral weight/birth weight)J/[development
time].
The offspring produced over 5 days by apterae were retained; and the relative growth rate of these second generation aphids was assessed on diets of the same formulation as used for their mothers. The tryptophan synthetase activity of aphids and their symbiotic bacteria was determined by the spectrophotometric assay of Smith and Yanofsky (1962). Each sample of 0.5-1.0 g aphids was ground in a pestle and mortar with ice-cold 50 mM Tris-HCl
pH 7.8, and passed through two layers of muslin. For total aphid enzyme activity, the filtrate was sonicated at 4°C in six 10 s bursts at full power over 3 min, with a W-375 sonicator (Heat-Systems, Ultrasonics Inc.); the sonicate was centrifuged at 13,OOOgfor 15 min; and the supernatant was assayed. For assays of enzyme activity in the bacteria, the bacteria were purified by a method modified after Harrison et al. (1989), as follows: the filtrate was combined with an equal volume of ice-cold 0.01% (v/v) Nonidet P-40 in 50 mM Tris-HCl, pH 7.8, and washed five times, by centrifugation at 6000g for 5 min and resuspension of the pellet in fresh ice-cold buffer. The final pellet, which, by phase contrast microscopy, comprised predominantly intact bacterial cells, was sonicated and centrifuged as for the aphid material. Honeydew produced over 24 h by aphids raised to the fourth larval instar on the complete and tryptophan-free diets was allowed to collect on tinfoil strips placed below each diet cage. The amino acids in the honeydew were quantified by reversed phase high performance liquid chromatography, after derivatization with o-phthaldialdehyde (Jones ef al.. 1981). The method is described in Prosser and Douglas (1991). RESULTS Performance
of A. pisum on a tryptophan-free diet
A. pisum clone Ox-2 persisted for two generations on the chemically-defined diet containing the full complement of protein amino acids (the complete diet) and on the diet from which tryptophan was omitted. In the first generation, there was no significant difference in the preadult mortality or larval relative growth rate between the two groups of aphids. Ninety per cent (27/30) of aphids on the complete diet and 97% (29/30) of aphids on the tryptophan-free diets survived to adulthood; and the relative growth rate of apterae over larval development was 0.20 f 0.004 (26) and 0.19 + 0.005 (14) mg mg-’ d-’ [mean + SE (No. aphids)] on the complete and tryptophan-free diets, respectively (t,, = 1.25, P > 0.05). However, 14 of the 29 adults (52%) on tryptophan-free diet were alate, whereas all but one of the 27 aphids on the complete diet were apterous. Aphids of the second generation on diets developed more slowly than the first generation, such that the mean larval relative growth rate was 0.17 mg mg-r d-’ for apterae on both the complete and tryptophan-free diets. As with the first generation, about 50 and 5% of aphids on the tryptophanfree diet and complete diets, respectively, were alate. Detailed data were not obtained for the third generation of aphids, but these insects grew and developed very slowly. When the experiment was terminated
Amino acid tryptophan in A. pisum Table 1. Tryptophan synthetase activity in extracts of A. pisum and their symbiotic bacteria Units enzyme activity* (mean & SE, 4 reps)
Untreated aphids Chlortetracycline-treated aphids Isolated bacteria
567
complete diet contained 1.69 f 0.12 mM tryptophan, and those on tryptophan-free diet produced honeydew with 0.32 + 0.08 mM tryptophan.
12 k 0.8 <0.3 86k 1.5
*One unit of enzyme utilizes I nmol indole mg-’ protein min-‘. The protein content of extracts was determined by the method of Bradford (1976). with bovine serum albumin as standard.
(7 weeks after the first generation aphids were transferred to diets), a few aphids on the complete diet and none on the tryptophan-free diet had reached adulthood. The aphids treated with chlortetracycline to disrupt their symbiotic bacteria grew and developed very slowly on the diets. On the complete diet, 67% (20/30) of the aphids survived to adulthood, and all but one of these were apterous. Their relative growth rate was 0.09 + 0.005 (19) mgmg-’ d-‘. On the tryptophan-free diet, only one of 30 chlortetracyclinetreated aphids survived to adulthood; most of these aphids died in the fourth larval stadium. Tryptophan synthetase activity of A. pisum
Extracts from aphids containing symbiotic bacteria had tryptophan synthetase activity (Table 1). The enzyme activity was abolished by heating the extract to 90°C for 15 min and it was reduced by 90% when the substrate, pyridoxal phosphate, was omitted from the reaction medium. The preparations of symbiotic bacteria also contained tryptophan synthetase, with activity (per unit protein) seven times that of the crude aphid extracts. Tryptophan synthetase was not detectable in aphids that had been treated with chlortetracycline to disrupt their symbionts. The detection limit of the assay was 2.5% of the activity recorded in extracts of untreated aphids. Tryptophan content of A. pisum honeydew
The honeydew produced by aphids reared for 7 days on tryptophan-free diet contained a significantly higher concentration of amino acids (17.80 + 0.44 mM) than in the honeydew of untreated aphids (13.66 f 0.42 mM) (mean f SE, five replicates; ts = 6.83, P < 0.001). In terms of amino acid composition, the honeydew of the two groups of aphids was closely similar, both to each other and to a previously published data set for diet-reared A. pisum clone Ox-2 (Prosser and Douglas, 1991). The sole important difference concerned tryptophan. The honeydew of aphids on the
DISCUSSION
The “individual deletion” method, i.e. comparison of insect performance on chemically-defined diets containing and lacking the nutrient of interest, is a widely-accepted approach to investigate the nutritional requirements of insects. However, maternallyderived nutrients may mask an insect’s requirement for a nutrient over several days. To avoid this problem, Dadd and Krieger (1968) recommend that, for aphids, the experiment be conducted over two generations. By this criterion, tryptc . an is not a required nutrient for A. pisum clone ( A !. The impt’, ...tion. that this essential amino acid is synthesized oy the symbiotic bacteria in clone 0x-2, is supported by the death of almost all chlortetracychne-treated aphids on tryptophan-free diets as first generation larvae. However, this conclusion cannot be generalized to all clones of A. pisum. For example, the growth of the A. pisum clone studied by Retnakaran and Beck (1968) was deleteriously affected by the individual deletion of any essential amino acid, including tryptophan; and Srivastava et al. (1963) found that one biotype (‘C”) was independent of dietary tryptophan, but that weight gain by a second biotype (“J”) was substantially reduced on tryptophan-free diets, even in the first generation. Many factors may contribute to the diversity in responses of A. pisum to tryptophan-free diets, including diet composition, aphid genotype and possibly characteristics of the symbiotic bacteria. The IO-fold increase in the proportion of alates, when clone Ox-2 was maintained on tryptophan-free diets, has not been reported for other A. pisum clones. However, omission of dietary tryptophan does promote alate development in the green peach aphid, Myzus persicae (e.g. Mittler and Kleinjan, 1970; Harrewijn, 1972) prompting Harrewijn (1976) to speculate that biogenic amines derived from tryptophan contribute to the regulation of wing dimorphism in M. persicae. In the present study, it was considered important to underpin the dietary studies with a direct investigation of tryptophan synthesis in A. pisum. The supposition, on which the study of tryptophan synthetase activity was based, was that the enzyme is exclusively of bacterial origin (i.e. aphids, like other animals, lack tryptophan synthetase). Two lines of evidence are consistent with this view. First, in this study, the activity of tryptophan synthetase was abolished by chlortetracycline treatment, and its
568
A. E.
DOUGLAS
specific activity was considerably higher in extracts of the bacterial preparations than in crude aphid extracts (Table 1). Second, a portion of the gene for the fi-subunit of tryptophan synthetase (?I@) has recently been detected in the primary symbionts of Schizaphis graminum (Lei et al., unpublished; cited by Munson et al., 1992). The most parsimonious explanation for the tryptophan in the honeydew of aphids on tryptophan-free diets is that it is of bacterial origin, and “excess” to the insect’s nutritional requirements. On this basis, it is possible to estimate the rate of tryptophan synthesis in diet-reared A. pisum clone 0x-2. Each fourth-instar larva on the complete diet consumes approx. 0.6~1 diet per day (Prosser et al., 1992). Assuming that the aphids on tryptophan-free diet feed at similar rates, and given that they produce honeydew containing 0.32 mM tryptophan (this study), each aphid produces 192 pmol tryptophan d-’ in its honeydew. Each apterous fourth-instar A. pisum contains approx. 5 x lo6 cells of Buchnera (Douglas, unpublished); consequently, 0.04 fmol tryptophan synthesized by each bacterium is diverted to honeydew per day. As yet there is no information on the mechanisms underlying tryptophan transfer from the symbiotic bacteria to the aphid. By analogy with other symbioses, it is probably released from intact bacterial cells, perhaps as the free amino acid, and not by lysis of bacteria (Smith and Douglas, 1987). Acknowledgement-We
thank NERC, AFRC Royal Society of London for financial support.
and the
REFERENCES Akey D. H. and Beck S. D. (1972) Nutrition of the pea aphid, Acyrthosiphon pisum: requirements for trace metals, sulphur and cholesterol. J. Insect Physiol. 18, 1901-1914. Bradford M. M. (1976) A rapid sensitive method for the quantitation of microgram quantities of protein using the principle of protein dye binding. Analyt. Biochem. 72, 248-254.
Breznak J. A. (1982) Intestinal microbiota of termites and other xylophagous insects. A. Rev. Microbial. 36,323-343. Dadd R. H. (1985) Nutrition: organisms. In Comprehensive Insect Physiology, Biochemistry and Pharmacology (Eds Kerkut G. A. and Gilbert L. I.), Vol. 4, pp. 313-391.
Pergamon Press, Oxford. Dadd R. H. and Krieger D. L. (1968) Dietary amino acid requirements of the aphid, h4yzu.s persicae. J. Insect Physiol. 14, 741-764.
Douglas A. E. (1988a) Sulphate utilisation in an aphid symbiosis. Insect Biochem. 18, 599-605.
anId W. A.
PROSSER
Douglas A. E. (1988b) On the source of sterols for the green peach aphid Myzus persicae, on holidic diets. J. Znsec? Physiol. 34, 403408.
Douglas A. E. (1988~) Experimental studies on the symbiosis in the leafhopper Euscelis incisus. J. Insect Physiol. 34, 1043-1053.
Douglas A. E. (1989) Mycetocyte symbiosis in insects. Biol. Rev. 64, 409434.
Douglas A. E. (1990) Nutritional interaction between Myzus persicae and its symbionts. In Ahid-Plant Genotype Znleractions (Eds Campbell R. K. and Eikenbary R. D.), pp. 319-328. Elsevier, Amsterdam. Douglas A. E. (1992) Symbiosis in evolution. Oxford Surveys in Evolutionary Biology (Eds Futuyama D. and Antonovics J.), Vol. 8. OUP, Oxford. Gange A. C. and Brown V. K. (1991) Culturing root aphids using hydroponics. Ent. Gazette 42, 165-169. Hara E. & Ishikawa H. (1990) Purification and partial characterisation of symbionin, an aphid endosymbiontspecific protein. Insect Biochem. 20, 421-427. Harrewijn P. (1972) Wing production by the aphid Myzus persicae related to nutritional factors in potato plants and artificial diets. In Insect and Mite Nutrition (Ed. Rodriguez G.), pp. 372-379. Elsevier, Amsterdam. Harrewijn P. (1976) Role of monoamine metabolism in wing dimorphism of the aphid Myzuspersicae. Camp. Biochem. Physiol. SK,
147-153.
Harrison C. P., Douglas A. E. and Dixon A. F. G. (1989) A rapid method to isolate symbiotic bacteria from aphids. J. Invert. Path. 53, 427428.
Jones B. N., Paabo S. and Stein S. (198 1) Amino acid analysis and enzymatic sequence determination of peptides by an improved o-phthaldialdehyde precolumn labelling procedure. J. Liq. Chromatogr. 4, 565-586. Mittler T. E. and Kleinjan J. E. (1970) Effects of artificial diet composition on wing production by the aphid Myzus persicae. J. Insect Physiol. 16, 833-850.
Munson M. A., Baumann P. and Kinsey M. G. (1991) Buchnera gen. nov. and Buchnera aphidicola sp. nov., a taxon consisting of the mycetocyte-associated, primary endosymbionts of aphids. Znt. J. Syst. Bat. 41, 566-568. Munson M. A., Baumann L. and Baumann P. (1992) Buchnera aphidicola, the endosymbiont of aphids, contains genes for four ribosomal RNA proteins, initiation factor3, and the alpha-subunit of RNA polymerase. Curr. Microbial. 24, 23-28.
Prosser W. A. and Douglas A. E. (1991) The aposymbiotic aphid: an analysis of chlortetracycline-treated pea aphid, Acyrthosiphon pisum. J. Insect Physiol. 37,713-719. Presser W. A., Simpson S. J. and Douglas A. E. (1992) How an aphid responds to variation in dietary nitrogen. J. Insect Physiol. 38, 301-307.
Retnakaran A. and Beck S. D. (1968) Amino acid requirements and sulphur amino acid metabolism in the pea aphid, Acyrihosiphon pisum (Harris). Comp. Biochem. Physiol. 24, 611-619.
Smith D. C. and Douglas A. E. (1987) The Biology of Symbiosis. Edward Arnold, London. Smith 0. H. and Yanofsky C. (1962) Enzymes involved in the biosynthesis of tryptophan. Metho& Enzym. 5, 794-806.
Srivastava P. N., Gao Y., Levesque J. and Auclair J. L. (1984) Differences in amino acid requirements between two biotypes of the pea aphid, Acyrthosiphon pisum. Can. J. Zool. 63, 603-606.
Vol. 38, No. 8, pp. 565-568, Printed in Great Britain. All rights reserved
1992
0022-1910/92 $5.00 + 0.00
Pergamon Press Ltd
SYNTHESIS OF THE ESSENTIAL AMINO ACID TRYPTOPHAN IN THE PEA APHID (ACYRTHOSIPHON PISUA4) SYMBIOSIS A. E. DOUGLAS and W. A. PROSSER Department of Zoology, University of Oxford, South Parks Road, Oxford OX1 3PS, U.K. (Received 17 December 1991; revised 4 February 1992)
aphids (Acyrthosiphonpisum) containing their symbiotic bacteria do not require a dietary supply of the essential amino acid tryptophan over two generations on chemicallydefined diets; but aphids, whose bacteria are disrupted by chlortetracycline, die as firstgeneration larvae on tryptophan-free diet. Consistent with the interpretation that the symbiotic bacteria in the aphids synthesize tryptophan, tryptophan synthetase activity was detected in both symbiont-bearing aphids and isolated bacteria, but not in chlortetracyclinetreated aphids. Aphids reared on tryptophan-free diet produced honeydew containing 0.32 mM tryptophan, from which it can be calculated that each bacterial cell in the aphid releases at least 0.04 fmol tryptophan d-‘. Abstract-Pea
Key Word Index: Symbiosis; aphids; essential amino acids; tryptophan
INTRODUCTION
Symbiotic microorganisms can be regarded as “vehicles” by which animals gain access to complex metabolic capabilities (Douglas, 1992). For example, some xylophagous insects (e.g. lower termites) utilize the cellulolytic capabilities of the protists in their guts (reviewed by Breznak, 1982); and many insects use the ability of intracellular microorganisms to synthesize “high value” nutrients, such as essential amino acids, sterols and vitamins (Douglas, 1989). The latter associations are commonly described as mycetocyte symbioses because the microorganisms are located in the cytoplasm of specialized insect cells called mycetocytes. A relatively well-characterized mycetocyte symbiosis is in aphids. The microorganisms are bacteria, which have recently been assigned to a new genus, Buchneru, in the yproteobacteria (Munson et al., 1991). Evidence is gradually accumulating that these bacteria provide essential amino acids to the aphid tissues, and that this contributes to the aphids’ ability to utilize phloem sap, a diet of low essential amino acid content. In particular, Douglas (1988a, 1990) has demonstrated that the bacteria in one clone of the green peach aphid Myzus persicae can synthesize the essential amino acid methionine, which is released to the insect; this clone can persist for at least five generations on methionine-free diets. More generally,
aphids, unlike most insects, appear not to require a dietary supply of the full complement of essential amino acids [see review of Dadd (1985)], suggesting that the bacteria may contribute to the aphids’ requirements of most, perhaps all, essential amino acids. In a recent study of honeydew production by the pea aphid Acyrthosiphon pisum, Prosser et al. (1992) found that aphids reared on diets of low nitrogen content produce honeydew with a higher concentration of tryptophan than in the diet. They hypothesized that the bacterial symbionts may have synthesized the “excess” tryptophan. (A. pisum bears two morphologically-distinct bacteria, the primary symbionts, Buchnera, and small numbers of secondary symbionts, either or both of which could contribute to tryptophan synthesis.) This paper describes dietary and metabolic investigations which confirm the interpretation of Prosser et al. (1992), specifically that the bacteria are capable of tryptophan synthesis and make this amino acid available to the aphid tissues in A. pisum.
MATERIALS
AND METHODS
The aphids were a clonal culture of A. pisum, known as “clone OX-~“, and maintained on Viciu faba var. The Sutton at 20°C and 18 h light-6 h dark 565
566
A. E. DOUGLAS and W. A. PROSSER
regime. For some experiments, the aphids were transferred to chemically-defined diets within 24 h of birth. The diets were prepared as in Douglas (1988b) and diet composition was that of Akey and Beck (1972), as modified by Prosser and Douglas (1991). The symbiotic bacteria were routinely disrupted by allowing the aphids to feed for 5 days on the chemically-defined diet supplemented with the antibiotic chlortetracycline at 50 pg ml-’ (Douglas, 1988b). For aphids used in enzyme assays (see below), chlortetracycline was administered via the plant. V. f&a were raised to the three-leaf stage in a substratum of expanded clay (“Hydroleca”: Sinclair Horticulture and Leisure Ltd, Lincoln, U.K.) (Gange and Brown, 1991). The roots of these plants were separated from the Hydroleca particles, and immersed in nutrient solution comprising: 0.05 mM (NH,), S04, 2 mM 0.1 mM CaCl,. 2H,O, 50pM MgSO,. 7H,O, Fe,Na.EDTA, 2mM K,HP04/KH2P0, buffer pH 7, and 250 pg chlortetracycline ml-‘. Preliminary experiments showed that the concentration of chlortetracycline in the leaf tissues [assayed as in Douglas (1988c)] rose to 50-7Opg g-’ leaf tissue within 24 h, and remained stable at this level for a further 4 days. The following protocol was therefore adopted; day 1, first group of plants transferred to nutrient solution containing chlortetracycline; day 2, aphids introduced to chlortetracycline-treated plants; day 4, second group of plants transferred to nutrient solution with chlortetracycline; day 5, aphids transferred from first to second group of chlortetracyclinetreated plants; day 7, chlortetracycline-treated aphids transferred to antibiotic-free plants. The aphids were assayed on day 9. For studies of the dietary requirement of A. pisum for tryptophan, the aphids were raised on chemicallydefined diets either containing or lacking the standard concentration of tryptophan (2.5 mM), such that the total concentration of amino acids in the diet were 135 mM and 132.5 mM, respectively. Within 24 h of reaching adulthood, each aphid was weighed on an electrobalance (Elmer-Parker-AT4) to the nearest pg, and the relative growth rate of apterae over larval development was determined from the equation: [log(teneral weight/birth weight)J/[development
time].
The offspring produced over 5 days by apterae were retained; and the relative growth rate of these second generation aphids was assessed on diets of the same formulation as used for their mothers. The tryptophan synthetase activity of aphids and their symbiotic bacteria was determined by the spectrophotometric assay of Smith and Yanofsky (1962). Each sample of 0.5-1.0 g aphids was ground in a pestle and mortar with ice-cold 50 mM Tris-HCl
pH 7.8, and passed through two layers of muslin. For total aphid enzyme activity, the filtrate was sonicated at 4°C in six 10 s bursts at full power over 3 min, with a W-375 sonicator (Heat-Systems, Ultrasonics Inc.); the sonicate was centrifuged at 13,OOOgfor 15 min; and the supernatant was assayed. For assays of enzyme activity in the bacteria, the bacteria were purified by a method modified after Harrison et al. (1989), as follows: the filtrate was combined with an equal volume of ice-cold 0.01% (v/v) Nonidet P-40 in 50 mM Tris-HCl, pH 7.8, and washed five times, by centrifugation at 6000g for 5 min and resuspension of the pellet in fresh ice-cold buffer. The final pellet, which, by phase contrast microscopy, comprised predominantly intact bacterial cells, was sonicated and centrifuged as for the aphid material. Honeydew produced over 24 h by aphids raised to the fourth larval instar on the complete and tryptophan-free diets was allowed to collect on tinfoil strips placed below each diet cage. The amino acids in the honeydew were quantified by reversed phase high performance liquid chromatography, after derivatization with o-phthaldialdehyde (Jones ef al.. 1981). The method is described in Prosser and Douglas (1991). RESULTS Performance
of A. pisum on a tryptophan-free diet
A. pisum clone Ox-2 persisted for two generations on the chemically-defined diet containing the full complement of protein amino acids (the complete diet) and on the diet from which tryptophan was omitted. In the first generation, there was no significant difference in the preadult mortality or larval relative growth rate between the two groups of aphids. Ninety per cent (27/30) of aphids on the complete diet and 97% (29/30) of aphids on the tryptophan-free diets survived to adulthood; and the relative growth rate of apterae over larval development was 0.20 f 0.004 (26) and 0.19 + 0.005 (14) mg mg-’ d-’ [mean + SE (No. aphids)] on the complete and tryptophan-free diets, respectively (t,, = 1.25, P > 0.05). However, 14 of the 29 adults (52%) on tryptophan-free diet were alate, whereas all but one of the 27 aphids on the complete diet were apterous. Aphids of the second generation on diets developed more slowly than the first generation, such that the mean larval relative growth rate was 0.17 mg mg-r d-’ for apterae on both the complete and tryptophan-free diets. As with the first generation, about 50 and 5% of aphids on the tryptophanfree diet and complete diets, respectively, were alate. Detailed data were not obtained for the third generation of aphids, but these insects grew and developed very slowly. When the experiment was terminated
Amino acid tryptophan in A. pisum Table 1. Tryptophan synthetase activity in extracts of A. pisum and their symbiotic bacteria Units enzyme activity* (mean & SE, 4 reps)
Untreated aphids Chlortetracycline-treated aphids Isolated bacteria
567
complete diet contained 1.69 f 0.12 mM tryptophan, and those on tryptophan-free diet produced honeydew with 0.32 + 0.08 mM tryptophan.
12 k 0.8 <0.3 86k 1.5
*One unit of enzyme utilizes I nmol indole mg-’ protein min-‘. The protein content of extracts was determined by the method of Bradford (1976). with bovine serum albumin as standard.
(7 weeks after the first generation aphids were transferred to diets), a few aphids on the complete diet and none on the tryptophan-free diet had reached adulthood. The aphids treated with chlortetracycline to disrupt their symbiotic bacteria grew and developed very slowly on the diets. On the complete diet, 67% (20/30) of the aphids survived to adulthood, and all but one of these were apterous. Their relative growth rate was 0.09 + 0.005 (19) mgmg-’ d-‘. On the tryptophan-free diet, only one of 30 chlortetracyclinetreated aphids survived to adulthood; most of these aphids died in the fourth larval stadium. Tryptophan synthetase activity of A. pisum
Extracts from aphids containing symbiotic bacteria had tryptophan synthetase activity (Table 1). The enzyme activity was abolished by heating the extract to 90°C for 15 min and it was reduced by 90% when the substrate, pyridoxal phosphate, was omitted from the reaction medium. The preparations of symbiotic bacteria also contained tryptophan synthetase, with activity (per unit protein) seven times that of the crude aphid extracts. Tryptophan synthetase was not detectable in aphids that had been treated with chlortetracycline to disrupt their symbionts. The detection limit of the assay was 2.5% of the activity recorded in extracts of untreated aphids. Tryptophan content of A. pisum honeydew
The honeydew produced by aphids reared for 7 days on tryptophan-free diet contained a significantly higher concentration of amino acids (17.80 + 0.44 mM) than in the honeydew of untreated aphids (13.66 f 0.42 mM) (mean f SE, five replicates; ts = 6.83, P < 0.001). In terms of amino acid composition, the honeydew of the two groups of aphids was closely similar, both to each other and to a previously published data set for diet-reared A. pisum clone Ox-2 (Prosser and Douglas, 1991). The sole important difference concerned tryptophan. The honeydew of aphids on the
DISCUSSION
The “individual deletion” method, i.e. comparison of insect performance on chemically-defined diets containing and lacking the nutrient of interest, is a widely-accepted approach to investigate the nutritional requirements of insects. However, maternallyderived nutrients may mask an insect’s requirement for a nutrient over several days. To avoid this problem, Dadd and Krieger (1968) recommend that, for aphids, the experiment be conducted over two generations. By this criterion, tryptc . an is not a required nutrient for A. pisum clone ( A !. The impt’, ...tion. that this essential amino acid is synthesized oy the symbiotic bacteria in clone 0x-2, is supported by the death of almost all chlortetracychne-treated aphids on tryptophan-free diets as first generation larvae. However, this conclusion cannot be generalized to all clones of A. pisum. For example, the growth of the A. pisum clone studied by Retnakaran and Beck (1968) was deleteriously affected by the individual deletion of any essential amino acid, including tryptophan; and Srivastava et al. (1963) found that one biotype (‘C”) was independent of dietary tryptophan, but that weight gain by a second biotype (“J”) was substantially reduced on tryptophan-free diets, even in the first generation. Many factors may contribute to the diversity in responses of A. pisum to tryptophan-free diets, including diet composition, aphid genotype and possibly characteristics of the symbiotic bacteria. The IO-fold increase in the proportion of alates, when clone Ox-2 was maintained on tryptophan-free diets, has not been reported for other A. pisum clones. However, omission of dietary tryptophan does promote alate development in the green peach aphid, Myzus persicae (e.g. Mittler and Kleinjan, 1970; Harrewijn, 1972) prompting Harrewijn (1976) to speculate that biogenic amines derived from tryptophan contribute to the regulation of wing dimorphism in M. persicae. In the present study, it was considered important to underpin the dietary studies with a direct investigation of tryptophan synthesis in A. pisum. The supposition, on which the study of tryptophan synthetase activity was based, was that the enzyme is exclusively of bacterial origin (i.e. aphids, like other animals, lack tryptophan synthetase). Two lines of evidence are consistent with this view. First, in this study, the activity of tryptophan synthetase was abolished by chlortetracycline treatment, and its
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DOUGLAS
specific activity was considerably higher in extracts of the bacterial preparations than in crude aphid extracts (Table 1). Second, a portion of the gene for the fi-subunit of tryptophan synthetase (?I@) has recently been detected in the primary symbionts of Schizaphis graminum (Lei et al., unpublished; cited by Munson et al., 1992). The most parsimonious explanation for the tryptophan in the honeydew of aphids on tryptophan-free diets is that it is of bacterial origin, and “excess” to the insect’s nutritional requirements. On this basis, it is possible to estimate the rate of tryptophan synthesis in diet-reared A. pisum clone 0x-2. Each fourth-instar larva on the complete diet consumes approx. 0.6~1 diet per day (Prosser et al., 1992). Assuming that the aphids on tryptophan-free diet feed at similar rates, and given that they produce honeydew containing 0.32 mM tryptophan (this study), each aphid produces 192 pmol tryptophan d-’ in its honeydew. Each apterous fourth-instar A. pisum contains approx. 5 x lo6 cells of Buchnera (Douglas, unpublished); consequently, 0.04 fmol tryptophan synthesized by each bacterium is diverted to honeydew per day. As yet there is no information on the mechanisms underlying tryptophan transfer from the symbiotic bacteria to the aphid. By analogy with other symbioses, it is probably released from intact bacterial cells, perhaps as the free amino acid, and not by lysis of bacteria (Smith and Douglas, 1987). Acknowledgement-We
thank NERC, AFRC Royal Society of London for financial support.
and the
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