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Keywords:

  • Acyrthosiphon pisum;
  • aphid;
  • Buchnera;
  • symbiosis;
  • trpEG;
  • tryptophan

Abstract.

  1. Top of page
  2. Abstract.
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The symbiotic bacteria Buchnera provide their aphid hosts with tryptophan and other essential amino acids. Tryptophan production by Buchnera varied among 12 parthenogenetic clones of the pea aphid Acyrthosiphon pisum (Harris), as determined from both the incorporation of radioactivity from 14C-anthranilate into tryptophan and the protein-tryptophan growth rate of larval aphids on tryptophan-free diet. The values of tryptophan production obtained for the two methods were correlated significantly with each other but not with the level of amplification of the Buchnera genes trpEG, which code for anthranilate synthase, a key enzyme in tryptophan biosynthetic pathway. This study provides the first direct demonstration of interclonal variation in production of any nutrient in an aphid–Buchnera symbiosis and indicates that a key aspect of Buchnera phenotype (tryptophan production) does not vary in a simple fashion with Buchnera genotype.


Introduction

  1. Top of page
  2. Abstract.
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Aphids derive essential amino acids from their symbiotic microorganisms, thereby enabling them to feed through the life cycle on phloem sap, a diet deficient in these nutrients (Baumann et al., 1995; Douglas, 1998). The dominant microbial partner in virtually all aphids is a γ-proteobacterium known as Buchnera aphidicola (Munson et al., 1991). The association is apparently very ancient, 180–250 million years old (Moran et al., 1993) and is maintained by obligate vertical transmission involving the transfer of Buchnera cells to the early embryo or unfertilized eggs in the ovaries of parthenogenetic and oviparous females, respectively (Buchner, 1965; Hinde, 1971). The relationship is obligate for both partners; aphids deprived of their bacteria grow slowly and are reproductively sterile (Douglas, 1992) and Buchnera, which have very small genomes (Charles & Ishikawa, 1999; Wernegreen et al., 2000; Gil et al., 2002), are unculturable and unknown apart from within aphids.

This paper concerns a largely unstudied issue, the variation among aphids in essential amino acid provisioning by Buchnera. Several published studies are relevant. First, Srivastava et al. (1985) found that two parthenogenetic clones of the pea aphid, Acyrthosiphon pisum, differed in their essential amino acid requirements. Both clones required histidine, methionine and threonine, and the additional requirements of clone J were phenylalanine and valine and of clone C were leucine, lysine and tryptophan. These data suggest that Buchnera may provide the amino acids required in the diet at inadequate rates to support growth and reproduction, with the implication that the provisioning by Buchnera varies between the two clones. Second, Sandström & Moran (2001) showed that the tryptophan content of honeydew varied among aphid species feeding from wheat: the honeydew of Diuraphis noxia contained undetectable levels of tryptophan, but Schizaphis graminum and Rhopalosiphum padi produced honeydew containing 0.5–1.0 nmol tryptophan/mg/day, equivalent to 5 and 22%, respectively, of ingested tryptophan. Finally, Wernegreen & Moran (2000) found that the tryptophan concentration in the free amino acid pool of Diuraphis noxia is very low. Both Sandström & Moran (2001) and Wernegreen & Moran (2000) suggested that Buchnera in D. noxia synthesize tryptophan at low rates, and they related their interpretation to an aspect of genomic organization in this species. The relevant genes are trpEG, which code for the enzyme anthranilate synthase in the tryptophan biosynthetic pathway and are amplified as multiple tandem repeats on a plasmid (Lai et al., 1994). The level of amplification of trpEG, relative to the chromosomal gene trpB (trpE/trpB), is 14 : 1 for S. graminum and 1.8 : 1 for D. noxia (Thao et al., 1998). These data are consistent with the proposal of Lai et al. (1994) that trpEG amplification promotes tryptophan production by Buchnera. Anthranilate synthase is traditionally considered as the ‘pacemaker enzyme’ in the tryptophan biosynthetic pathway because its activity is subject to allosteric inhibition by tryptophan, i.e. anthranilate synthase activity determines the rate of tryptophan synthesis (Crawford, 1989). However, an important limitation to the studies of Srivastava et al. (1985), Wernegreen & Moran (2000) and Sandström & Moran (2001) is that the rates of amino acid production were not quantified directly.

In the pea aphid, A. pisum, the level of amplification of Buchnera trpEG (as determined by the trpE/trpB ratio) varies between two-fold and 16-fold among parthenogenetic clones (Birkle et al., unpublished data). The variation may arise from both variation in the number of trpEG repeats per plasmid and number of plasmids per Buchnera chromosome; the chromosomal copy number of Buchnera is high, and can exceed 100 copies per cell (Komaki & Ishikawa, 1999). This raises two linked questions which are addressed in this study. First, does the rate of tryptophan production vary among A. pisum clones? Second, is the variation in tryptophan production rates correlated positively with the level of trpEG amplification of Buchnera? This study explores the relationship between specific aspects of Buchnera genotype, the level of trpEG amplification, and phenotype, the rate of tryptophan production.

Materials and methods

  1. Top of page
  2. Abstract.
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Twelve clonal cultures of Acyrthosiphon pisum, each derived from a single alate female colonizing Vicia faba plants at the Central Science Laboratory, North Yorkshire, U.K., during the summer of 1999, were selected for study. All of the clones were green. They could be maintained readily on chemically defined diets and encompassed the full known range of trpE/trpB. In particular, molecular analysis confirmed that all clones bear the plasmid ptrp, with no within- or between-clone variation detectable by extensive restriction analysis (Minto & Douglas, unpublished results). The aphids were maintained at 20 °C in a LD 18 : 6 h photocycle on excised leaves of V. faba cv. The Sutton.

TrpE/trpB of Buchnera was determined by quantitative DNA hybridization. The DNA of four replicate aphids was extracted with the DNeasy Tissue kit (Qiagen, Crawley, U.K.) and applied to a Zeta-probe GT blotting membrane (Bio-Rad, Hemel Hempstead, U.K.) in a 90-well dot blot apparatus. Each sample was hybridized sequentially against trpB (0.72 kb) and trpE (0.52 kb) probes that had been generated by PCR (Munson & Baumann, 1993; Lai et al., 1994) and labelled with chemiluminescence by the ECL kit (Amersham, Hemel Hempstead, U.K.). Chemiluminescence was quantified directly with the AlphaImager Documentation & Analysis System (Alpha Innotech Corp., Lichfield, U.K.), and the trpE/trpB ratio calculated by interpolation from calibration standards comprising 1–100 ng PCR-generated trpE and trpB probes.

Tryptophan production was quantified by two methods, ‘radiotracer’ and ‘growth rate’, using aphids reared from birth (day 0) on a chemically defined diet modified from formulation A (0.5 m sucrose, 0.15 m amino acids) of Prosser & Douglas (1992) by the omission of tryptophan. (The concentration of tryptophan in the diet was <1 nm, as quantified by HPLC.) All experiments included aphids deprived experimentally of their bacteria by supplementing the diet with rifampicin at 50 µg/mL over days 0–2 of the experiment (Rahbe et al., 1994; Wilkinson et al., 2001). The aphids that were not treated with rifampicin are termed ‘untreated aphids’.

For the radiotracer method, three replicate groups of five 2-day-old rifampicin-treated and untreated aphids were transferred to diet supplemented with 10 µCi (0.8 µmol) [U-14C]-anthranilate/mL. The radiochemical (Sigma, 12.5 mCi/mmol anthranilate) was of >99% chemical purity as confirmed by thin layer chromatography with autoradiography. At day 8, each group of aphids was weighed, homogenized in 0.4 mL ice-cold 80% methanol in a hand-held glass homogeniser and centrifuged at 20 000 g for 15 min. The supernatant was dried in a vacuum centrifuge, resuspended in 20 µL of 80% methanol and frozen at −20 °C prior to analysis. The amino acid and 14C content of the tryptophan peak of the methanol extracts were quantified by HPLC and scintillation counting following the methods of Douglas et al. (2001) precisely, from which the incorporation of anthranilate carbon atoms per mol tryptophan in the free amino acid fraction of the aphids was determined. The radioactivity content of the eluate sample corresponding to the HPLC tryptophan peak for the rifampicin-treated aphids was low and adopted as the background level, i.e. the mean value of mol anthranilate incorporated/mol tryptophan for rifampicin-treated aphids was subtracted from the equivalent value for untreated aphids of each clone. The background-subtracted value for the untreated aphids was then divided by the proportion ‘free tryptophan content/total tryptophan’ for each sample, to obtain the incorporation of anthranilate into the total tryptophan content of the untreated aphids. This calculation assumes that aphid metabolism was in equilibrium (see also Febvay et al., 1999, and Douglas et al., 2001), such that the 14C content of free and protein tryptophan was equal on a per mole basis. The free tryptophan content of the aphids, which varies among aphids and with conditions, was determined by the method of Douglas et al. (2001), and the value 0.8% (g/g), equivalent to 39 nmols tryptophan/mg aphid protein, as used previously (Douglas et al., 2001), was adopted for the tryptophan content of aphid protein, which is relatively invariant among aphids (e.g. Sandström & Moran, 2001).

For the growth rate method, 20 replicate rifampicin-treated and untreated 2-day-old aphids of each clone were weighed individually, reared singly to day 8 on tryptophan-free diet, and then re-weighed. All the 8-day-old aphids were final-stadium apterous larvae. The weights were transformed to protein content, using values of protein content per unit fresh weight obtained from 10 replicate samples of each of 2-day-old aphids and 8-day-old untreated and rifampicin-treated aphids of the clones reared under the same conditions as the aphids used for the growth experiment. (The experimental design precluded protein quantification of the aphids used to determine growth rates.) The protein growth rate of each aphid was calculated as [ln(final protein content/initial protein content)]/6. The rate of increase in protein-tryptophan was determined from the product of protein growth rate and tryptophan content of aphid protein (see above), and this value was transformed to moles of protein-tryptophan synthesized per day. The contribution of Buchnera-derived tryptophan to the net increase in protein-tryptophan was obtained as the difference between the mean rate of increase in protein-tryptophan of untreated and rifampicin-treated aphids.

Data sets were analysed by parametric tests after logarithmic transformation, which were required to obtain normal distributions (Anderson–Darling test) and homogeneity of variances (Bartlett test).

Results

  1. Top of page
  2. Abstract.
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The trpE/trpB ratio of Buchnera in the 12 clones of A. pisum varied from 2.4 to 16.2 (Fig. 1). This variation was statistically significant (F11,36 = 6.18, P < 0.001, after logarithmic transformation of the data) and the clones could be divided into three significantly different groups by Tukey's post-hoc analysis: clones A and B with ‘high’trpE/trpB, clones C–I with ‘intermediate’trpE/trpB, and clones J–L with ‘low’trpE/trpB.

image

Figure 1. The gene ratio of trpE/trpB of the symbiotic bacteria Buchnera in 12 clones of A.pisum.

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The untreated and rifampificin-treated 2-day-old aphids of all clones settled readily onto the tryptophan-free diets and all the aphids displayed net growth over the 6-day experiments. The protein growth rates of the aphids are shown in Table 1. Consistent with previous studies demonstrating that elimination of the symbiotic bacteria by antibiotic treatment depresses aphid growth rates (reviewed in Douglas, 1998), the protein growth rates of the aphids were significantly depressed by treatment with the antibiotic rifampicin (F1,383 = 623.29, P < 0.001), although the extent of this effect varied significantly among the clones (F11,383 = 5.41, P < 0.001). The mean contribution of Buchnera to the tryptophan-protein growth rate of untreated aphids varied three-fold among the clones, from 3.1 (clone B) to 10.5 (clone C) pmol/µg aphid protein/day (Table 1).

Table 1.  Net synthesis of protein-tryptophan by Buchnera in clones of A. pisum, as determined by the growth rate method.
 Rate of protein synthesis (mean±SE) (µgµg−1d−1)Mean rate of protein-tryptophan
Aphid cloneUntreated aphidsRifampicin-treated aphidssynthesis by Buchnera (pmolµg−1proteind−1)*
  1. *Obtained by subtraction of protein synthesis rates of rifampicin-treated aphids from those of untreated aphids, and corrected for tryptophan content of protein (39nmol/mg) (see Materials and methods for details).

A0.46±0.0130.27±0.0127.4
B0.33±0.0080.25±0.0103.1
C0.34±0.0180.21±0.00710.5
D0.46±0.0120.22±0.0079.3
E0.49±0.0130.22±0.0065.1
F0.35±0.0220.23±0.0064.7
G0.41±0.0200.26±0.0065.9
H0.30±0.0160.20±0.0063.9
I0.40±0.0190.24±0.0096.2
J0.39±0.0190.22±0.0126.6
K0.38±0.0220.24±0.0055.4
L0.39±0.0190.26±0.0105.1

The incorporation of 14C-anthranilate into tryptophan varied 24-fold between aphid clones, from 0.47 ± 0.047 (clone L) to 11.31 ± 0.656 (clone C) moles anthranilate carbon/mole aphid tryptophan (mean ± SE, n = 3). Across the 12 aphid clones, the mean values of tryptophan production obtained by the radiotracer method and growth rate method were correlated significantly (r = 0.709, d.f. = 10, P < 0.05; Fig. 2).

image

Figure 2. Variation in tryptophan production in aphids with trpE/trpB of Buchnera as determined by (a) the growth rate method and (b) the radiotracer method.

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The relationship between tryptophan production and trpE/trpB of Buchnera was analysed in two complementary ways. First, tryptophan production was regressed on trpE/trpB; the slopes did not differ from zero for both the growth rate method (F1,10 = 0.01, P = 0.927; r2 = 0.1%) and the radiotracer method (F1,10 = 1.50, P = 0.248; r2 = 13.1%). Second, the variation in tryptophan production rates between the aphids bearing Buchnera of the three classes of trpE/trpB identified in Fig. 1 were analysed by anova, and found not to vary significantly for either the growth rate method (F2,9 = 0.36, P = 0.707) or the radiotracer method (F2,9 = 2.00, P = 0.191).

Discussion

  1. Top of page
  2. Abstract.
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

This study has revealed considerable variation in the rate of tryptophan production among clones of the pea aphid, A. pisum, but this variation is not correlated with the level of Buchnera trpEG amplification, as quantified by trpE/trpB. The correlated values of tryptophan production obtained by the two independent methods gives added confidence to the conclusion that this aspect of Buchnera genotype and phenotype is not related in a simple fashion. A likely explanation for the greater interclonal variation in tryptophan production obtained by the radiotracer method than the growth rate method is that the growth of aphids with high tryptophan production rates is limited by factors other than tryptophan availability.

A poor correlation either between trpEG and the level of the product, anthranilate synthase, or between the level of anthranilate synthase and tryptophan synthesis rates, could account for the observed independence of tryptophan production and trpEG copy number. Unfortunately, direct analysis of this issue is precluded for the present because anthranilate synthase in Buchnera is intractable to direct biochemical analysis (Lai et al., 1994) for unknown reasons. However, data accumulating for a range of systems are contrary to the rationale underlying the proposed link between trpEG amplification and tryptophan production rates, namely that the rate of tryptophan biosynthesis is controlled principally by anthranilate synthase activity (see Introduction). There is increasing evidence for a range of metabolic pathways, including tryptophan synthesis, that control over flux is distributed across multiple enzymes, such that a change in the activity of one enzyme is unlikely to alter flux substantially (Fell, 1997). Directly relevant to this study, Niederberger et al. (1992) were able to engineer elevated tryptophan production into yeast by amplifying four or five of the 5 TRP genes, but not by amplifying any one gene, including TRP2 coding for anthranilate synthase. The present data suggest that the inflexibility in metabolic networks is not restricted to novel gene constructions generated artificially, but is also evident in the naturally occurring genome of Buchnera, which has been exposed to selection over millions of years.

These results raise the issue of the determinants of interclonal variation in tryptophan production. The density of Buchnera does not vary significantly between the clones (unpublished data), but the variation in nutrient release by Buchnera with aphid genotype remains to be explored. It would also be of interest to establish the quantitative relationship between the production of tryptophan and other amino acids by Buchnera. Perhaps there is considerable interclonal variation in the overall ‘productivity’ of Buchnera (i.e. rates of essential amino acid provisioning) or, alternatively, Buchnera which produce tryptophan at high rates may display low rates of production of other amino acids (e.g. phenylalanine, which like tryptophan is an aromatic essential amino acid). More generally, this study raises the possibility that variation in Buchnera function may be an important factor contributing to differences in the nutritional physiology and performance among aphids.

Acknowledgements

  1. Top of page
  2. Abstract.
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

We thank Kim Simpson for excellent technical support, and the Natural Environment Research Council, U.K. for financial support (Grant GST/02/1842).

References

  1. Top of page
  2. Abstract.
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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Accepted 2 October 2002