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Reproductive failure and the free amino acid pools in pea aphids (Acyrthosiphon pisum) lacking symbiotic bacteria
Vol. 42, No. 3, pp. 241-255, 1996 Copyright 0 1996 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0022-1910196 $15.00 + 0.00
J. Insect Physiol.
Pergamon
0022-1910(95)00105-0
Reproductive Failure and the Free Amino Acid Pools in Pea Aphids (Acyrthosiphon pisum) Lacking Symbiotic Bacteria A. E. DOUGLAS* Received
16 June 1995; revised
1 August
1995
Pea aphids (Acyrthosiphon pisum) treated at birth with the antibiotic, rifampicin, to disrupt the symbiotic bacteria (i.e. aposymbiotic aphids) grow very slowly. The growth of embryos in the aposymbiotic aphids was particularly depressed, such that, by adulthood, the embryo fraction represented 65% of the total protein in symbiotic aphids, but just 12% of the protein in aposymbionts. Profile analysis of the free amino acid pools of the aphids and their embryos indicated that the concentrations of the aromatic amino acids phenylalanine and tryptophan were very low in the embryos of aposymbionts, and it is proposed that these amino acids may limit embryo growth in aposymbiotic aphids. There are also indications that lysine may be limiting to the growth of the maternal tissues of 4-day-old larval aposymbionts, and methionine may be in short supply in older aposymbiotic aphids. Aphid
Acyrthosiphon
pisum
Buchneru
Embryo
Symbiosis
INTRODUCTION The symbiotic bacteria Buchneru in aphids are maternally inherited. In parthenogenetic morphs of aphids, the bacteria are transferred from cells, called mycetocytes, in the insect haemocoel to blastoderm embryos in the ovaries (Hinde, 197 l), and they are subsequently incorporated into the embryo’s mycetocytes, as these cells differentiate (Buchner, 1965). The spatial organization of the symbiosis in the parthenogenetic morphs is, however, complex because embryogenesis is initiated in larvae and, in some species, in embryos. An aphid therefore comprises at least two spatially-distinct symbioses: the ‘maternal symbiosis’, represented by the mycetocytes in the aphid haemocoel, and the ‘embryo symbiosis’ comprising all the bacteria-bearing embryos in the aphid’s ovaries. To date, virtually all research on the physiology of the aphid symbiosis has ignored the spatial organization of the symbiosis. In particular, none of the various studies that have demonstrated bacterial provision of essential amino acids to the aphid tissues have differentiated between the maternal symbiosis and embryo symbiosis (e.g. Douglas, 1988; Douglas and Prosser, 1992; Sasaki
*Department of Biology, YOl 5YW, U.K.
University
of York,
P.O.
Box 373, York,
247
Amino
acids
et al., 1993; Sasaki and Ishikawa, 1995; Febvay et al., 1995). As a consequence, the possibility that both the profile of amino acids translocated from the bacteria to the aphid and the rate of amino acid release may differ between the maternal and embryo symbioses has not been addressed experimentally. The purpose of the study reported here was to identify whether the maternal and embryo symbioses in pea aphids are physiologically distinct. The strategy was to compare the response of the maternal tissues and embryos of pea aphids to the elimination of the bacteria. It was reasoned that any difference would be indicative of a difference in the association between the two symbioses. This approach requires separate analysis of the maternal and embryo fractions of the aphids. Preliminary experiments, however, revealed: first, that only the embryo fraction could be obtained reliably from the aphids, while some of the maternal contents, especially haemolymph and fat body cells, were lost during dissections; and second, that the biomass of the embryos could reliably be quantified by protein content, but not fresh weight. Therefore, the study concentrated on the protein content of the embryos and total aphid tissues and, where appropriate, information on the maternal fraction was obtained by subtraction. Two parameters were measured: the growth of the aphids, and their free amino acid content. These were
248
A. E. DOUGLAS
chosen on the basis of the substantial body of information already available on the characteristics of pea aphids lacking their symbiotic bacteria (also known as ‘aposymbiotic’ aphids). These aphids are very small. They grow slowly as larvae, and the adults are essentially reproductively-sterile (Sasaki et al., 1991; Douglas, 1992). Aposymbiosis also substantially changes the profile of free amino acids in aphids (Prosser and Douglas, 1991; Liadouze et al., 1995). Aposymbionts have low levels of essential amino acids, presumably as a consequence of the loss of the bacterial supply of these nutrients. In particular, Prosser and Douglas (199 1) noted the very low concentration of the aromatic amino acid phenylalanine in the aposymbiotic pea aphids, and suggested that this amino acid may be “the crucial limiting amino acid”. Aposymbiotic aphids also have elevated concentrations of the nonessential amino acid glutamine, and this has been linked to the possible role of the bacteria in recycling nitrogen (Prosser and Douglas, 199 1; Sasaki and Ishikawa, 1993). Specifically, there is now substantial evidence that the bacteria utilize aphid waste ammonia as a nitrogen source (Whitehead et al., 1992), and the resultant increase in ammonia in aposymbionts is reduced by its assimilation via glutamine synthetase to glutamine (Wilkinson and Douglas, 1995). In this study, the protein growth of the aphids and their embryos were quantified over larval development. The free amino acid content of the total and embryo fraction was also examined in 4-day-old and S-day-old larval aphids.
MATERIALS
AND METHODS
The aphids The standard culture of pea aphids (Acyrthosiphon pisum) clone 0x2 was maintained at 20°C with 18 h L:6 h D on 3-4-week-old Vicia faba grown in cocofibre compost. The experimental aphids were collected within 24 h of deposition from apterous mothers; they are described as O-day-old aphids, and the time of collection as day-0 of the experiment. Aposymbiotic pea aphids were generated by treatment with the antibiotic rifampicin, administered via plants using a method modified from Douglas (1992). The roots of 3-week-old Vicia faba plants were cleaned of excess compost by careful washing in tapwater, and the plants were transferred to distilled water containing 200 pg rifampicin ml-‘, with control plants in antibiotic-free distilled water. One day later, the O-day-old aphids were applied to the plants: those on the rifampicin-treated plants are described as aposymbiotic aphids, and those on control plants as symbiotic aphids. After 2 days treatment (i.e. on day-2 of the experiment), all aphids were returned to plants in compost under the standard culture conditions. This treatment was fully effective in disrupting the symbiotic bacteria in both the maternal tissues and embryos, as indicated by light microscopical
examination of the antibiotic-treated insects (unpublished results). The experiments were conducted on 2-l l-day-old symbiotic and aposymbiotic aphids. Alatiform aphids (which accounted for 5-10% of the 8- and 1l-day-old aphids) were excluded, and larvae and adults, respectively, were selected on day-8 and day-l 1 (2-6% of the symbiotic aphids and none of the aposymbionts were adult on day-8, and all symbiotic aphids and >95% of the aposymbionts were adult on day-l 1). Older aposymbiotic aphids were unsuitable for these studies because their embryos could not be distinguished or separated from other tissues in the dissections. Number and size of embryos On days 2, 4, 6, 8 and 11 of the experiment, 10 replicate symbiotic and aposymbiotic aphids were fixed in alcoholic Bouin’s solution (Ponsen, 1976) for 34 weeks. Each aphid was then dissected in a drop of water with fine pins, using a dissecting microscope. Two parameters were scored: (a) the number of embryos greater than 0.1 mm long (in some of the dissections, embryos CO.1 mm could not be identified unambiguously); and (b) the length of the longest embryo, as determined with the aid of an eyepiece micrometer at x32 magnification. Protein and amino acid assays A full factorial design was adopted for the study of protein and amino acid content of aphids. The factors were: antibiotic (symbiotic or aposymbiotic aphids), aphid fraction (total or embryo), and aphid age (days 2, 4, 6, 8 and 11 for protein assays, and days 4 and 8 for amino acid assays). Every treatment group comprised 10 replicate aphids, each of which was weighed to the nearest microgram on a Mettler MT5 balance prior to analysis. The protein content of aphids was quantified with the protein assay kit of the Biorad Chemical Co., following manufacturer’s instructions for the microassay, with bovine serum albumin as standard. Each ‘total’ sample was a single aphid homogenized in 0.2 ml ice-cold 50 mM Tris-HCl pH 7.5. For the samples of embryo fraction, the complement of embryos was dissected from each aphid in ice-cold Tris buffer, carefully separated from contaminating fat body and other maternal tissues, and homogenized in 0.2 ml Tris buffer. The total protein content and protein content of the embryo fraction were calculated per aphid and per unit aphid fresh weight. The amino acids were quantified by reverse-phase HPLC after o-phthaldialdehyde derivatization (Jones et al., 1981) using a Beckman System Gold solvent delivery system with C,,-ultrasphere column and Shimadzu RF-551 fluorescence detector. The reference amino acid mixture was AA-S-18 (Sigma), supplemented with asparagine, glutamine and tryptophan. For the total aphid samples, each aphid was homogenized in 0.2 ml ice-cold 80% methanol. For the embryo fraction samples, the embryos were dissected in Tris buffer (as for protein
REPRODUCTIVE
FAILURE
assays), centrifuged at 1,000 g for 5 min, and the supernatant was replaced by 0.2 ml ice-cold 80% methanol, and the embryos were homogenized. All methanol extracts were centrifuged at 13,000 g for 10 min, and the supernatants were stored at -20°C for up to 6 weeks prior to analysis. The amino acids in the total aphid samples were expressed as nmol/pg total protein, and those in the embryo fraction as nmol/unit embryo protein, using the data obtained from the protein assays for total and embryo protein content/unit fresh weight. All proteinamino acids in the aphids were assayed except cysteine and proline, which cannot be detected by this method. The combined concentrations of alanine + tyrosine and of glycine + threonine are shown because these amino acids could not be resolved with confidence in all samples. Statistical
analysis
Parametric statistical tests were conducted, to explore the effects of antibiotic treatment and aphid age on the number and size of embryos, and on the protein content of the embryo and total aphid tissues. The technique of profile analysis, as described in detail by Morrison (1976) and Seber (1984) was used to analyse the impact of antibiotic, aphid age (4- and 8-day-old aphids) and tissue fraction (embryo and total) on the amino acid content of the aphids. The data were log,-transformed prior to the analysis so that the tests for homogeneity of the distributions would correspond to tests for parallelism in the profile analysis. Preliminary tests on each variable for each treatment combination revealed no significant outliers, but other univariate tests (skewness, kurtosis and homogeneity of variances across treatments) revealed departures from the standard assumptions and standard multivariate tests for homogeneity of covariance matrices were unavailable due to sample size restrictions. The patterns revealed by this analysis (see results) should, there1.2
1
IN PEA APHIDS
fore, be interpreted with due statistical caution. The patterns obtained (see results) proved to be complex and, therefore, the three independent variables (antibiotic, aphid age and tissue) were expressed as a single factor in the detailed comparisons between profiles. The comparisons were achieved by calculating confidence intervals for every simple contrast involving a pair of amino acids and a pair of treatments. Overall 95% protection was achieved using Bonferroni intervals.
RESULTS
The number
and size of embryos
in the aphids
Every 2-l 1-day-old aphid examined contained embryos. All the embryos in the 2-day-old aphids were CO.1 mm long (they were not studied further, see Methods), but by day-4, the largest embryos had grown, on average, to 0.3 mm in length, in both the symbiotic and aposymbiotic aphids [Fig. l(a)]. Between days 4 and 11, the largest embryos in symbiotic aphids increased four-fold in length to 1.16 5 0.016 mm, but the length of these embryos in the aposymbionts did not vary significantly with time. The aposymbiotic aphids contained significantly fewer embryos than the symbiotic aphids [Fig. l(b)], and the discrepancy between the two groups of aphids increased with aphid age. In the symbiotic aphids, the number of embryos increased by 75% between days 4 and 11. In the aposymbionts, no increase in embryo number was obtained, and the number in the 1l-day-old aphids was significantly lower than in the 48-day-old aphids (0.001 < P < 0.01). The growth
of aphids and their embryos
The protein content of the aphids and their embryos was used to explore the contribution of embryos to aphid
4ol
(4
249
(b)
/
0
2
4
6
8
Aphid age (days)
10
12
4
6
8
10
12
Aphid age (days)
FIGURE 1. The embryos in symbiotic (0) and aposymbiotic (0) pea aphids. (a) The length of the longest embryo per aphid. antibiotic: F,,,, = 351.1, P < 0.0001; aphid age: F3,72 = 185.9, P < 0.0001; interaction: F372 = 102.0, P < 0.0001. (b) The number of embryos greater than 0.1 mm in length. antibiotic: F,,,, = 432.6, P < 0.0001; aphid age: Fx,72= 17.1, P < 0.0001; interaction: F3,72 = 75.4, P < 0.0001.
250
A. E. DOUGLAS
growth. On day-2, the embryo fraction of the symbiotic and aposymbiotic aphids contained 0.5 f 0.032 and 0.48 f 0.034 pg protein, respectively, representing 24 25% of the total protein of the aphids (Fig. 2). In the symbiotic aphids, the embryo protein increased nearly 400-fold over the course of the experiment, accounting for 45% of the total protein in the 8-day-old larvae and 65% of total protein in the 1l-day-old adults. In the aposymbiotic aphids, the embryo protein increased 5-fold and contributed 12% of the total protein in the 1l-day-old aphids. The amino acid content of aphids and their embvos The total amino acid content of the aphids is shown in Table l(a). It is significantly elevated in aposymbiotic aphids [see also Prosser and Douglas (1991) and Wilkinson and Douglas (1995)]; and, for both symbiotic and aposymbiotic aphids, it is significantly lower in the embryo fraction than in the total aphid samples. The statistically significant interaction term “antibiotic x aphid age” arises from the 80% reduction in amino acid content of both the total and embryo fractions of symbiotic aphids between days 4 and 8, and this result is consistent with the inverse relationship between body size and amino acid concentration in pea aphids identified by Liadouze et al. (1995). The composition of the amino acids in the aphids is displayed in Fig. 3. The data for total aphid tissues are in broad agreement with previous analyses (e.g. Prosser and Douglas, 1991; Liadouze et al., 1995). In particular, glutamine was the dominant amino acid in all samples analysed, and its concentration was higher in aposymbiotic aphids than in symbiotic aphids (20-23% of the amino acids in symbiotic aphids and 30-34% in aposymbionts). In the embryo fraction, however, glutamine was not the amino acid at the highest concentration. On both day-4 and day-8, glutamate and glutamine are at closely similar concentrations in embryos of symbiotic 350
1 (a)
TABLE
acid content
Day-8
Day-4 total tissues Symbiotic aphids Aposymbiotic aphids
embryos
total tissues
embryos
2.87kO.184
2.72f0.113
0.64~kO.019
0.60+0.024
4.91 kO.240
4.10 f 0.165
4.61 f 0.187
4.11 f 0.235
(b) F-values Source of variation Antibiotic Aphid tissue Aphid age Antibiotic x aphid tissue Antibiotic x aphid age Aphid tissue x age Antibiotic x aphid tissue
of ANOVA
F (1,72 dj) P 13322 P < 0.0001 12 0.0001 < P < 0.001 419 P < 0.0001 3.57 P > 0.05 360 P < 0.0001 P > 0.05 0.32 P > 0.05 0.03
x
age *The weight (mean k SE 10 reps) of the symbiotic and aposymbiotic aphids, respectively, were: 0.60 k 0.024 and 0.42 f 0.073 mg on day-4, and 2.97 + 0.088 and 0.82 f 0.023 mg on day-g. (The data in Table l(a) were log-transformed prior to statistical analysis).
aphids, and asparagine was the dominant amino acid in embryos of aposymbiotic aphids. To explore further the variation in amino acid composition of the aphids, a profile analysis of the data set was conducted (see Table 2). The significance of all the main effects and the interaction terms indicate that the concentrations of the various amino acids vary in a complex fashion with aphid age and tissue and between symbiotic
1(b)
250 $ 0 2 Q) z k UJ 3.
700 / 150-
/ ,..’ .,r2....
p
100
50
1
Aphid age (days)
of
(a) Amino acid content of aphids nmol amino acids pg-’ protein* (mean + SE 10 replicates)
25
300 -
1. The effect of aposymbiosis on the amino pea aphids and their embryos
2
3
4
5
6
7
8
9
10
II
Aphid age (days)
FIGURE 2. Protein growth of pea aphids and their embryos. For (a) symbiotic aphids and (b) aposymbiotic aphids, the total and the protein fraction of the maternal aphid protein (0) and protein content of the embryo fraction (0) were determined, tissues (0) was obtained by subtraction. Mean values are displayed (the coefficient of variation (SD/mean) did not exceed 0.09, and was uniform between symbiotic and aposymbiotic aphids). Note difference of scale between (a) and (b).
REPRODUCTIVE
FAILURE
IN PEA APHIDS
251
2 Symbiotic aphids
1.8
1.8 -
1.6
Aposymbiotic total tissues
aphids
1.6 1.4 1.2 1
0.8
0.8
0.6
0.6 0.4 0.2 0
2 1.8
1.6
,
I Symbiotic aphids embryos day 4
1.4
1.4
1.2
1.2
1
1
0.8
0.8
0.6
0.6
0.4
0.4
0.2
0.2
0
0
FIGURE
3. The amino
acid content
of pea aphids
and aposymbiotic aphids. All the statistically significant pairwise comparisons of amino acid concentrations between treatments are shown in Fig. 4. The differences in amino acid profiles between symbiotic and aposymbiotic aphids are dominated by a reduction in certain essential amino acids in the aposymbionts. The most consistent effects concern the two aromatic essential amino acids, tryptophan (W) and phenylalanine (F). These amino acids are depressed in aposymbionts, relative to an array of other amino acids, in both the total and embryo fractions. However, the reduction of these amino acids is more pronounced in the embryo fraction, as is indicated by: first, the reduction in aposymbiotic embryos, relative to total tissues of aposymbionts, of phenylalanine on day-4 and both phenyl-
and their embryos
(continued
on p. 252).
alanine and tryptophan on day-8; and second, the reduction in both amino acids in aposymbiotic embryos, but not total tissues between day-4 and day-8. Further essential amino acids that are depressed (relative to other amino acids) in aposymbiotic aphids include: methionine (M) in both tissue types on day-& isoleucine (I) in total aphid samples on day-4 and day-8 and in the embryos on day-4; and leucine (L) in the embryo tissues on day-4 and total tissues on day-8. The pattern of variation in lysine (K) is complex. On day-4, it is depressed in the total tissues, but not embryo fraction, of aposymbionts; and, on day-8, it is increased (relative to other amino acids) in the embryo, but not total, tissues of symbiotic aphids and decreased in the total, but not embryo, tissues of aposymbiotic aphids.
252
A. E. DOUGLAS
(b) 2 1.8
Symbiotic aphids
1.6
1 0.8
1.8 1.6-
Symbiotic aphids embryos day8
1.4 1.2 l0.8 0.6 0.4 0.2 -
FIGURE
3. Continued.
Of all the essential amino acids, valine (V) is notable in that it is not significantly depressed in aposymbiotic aphids of either age or tissue type. Histidine (H) is also little-affected by aposymbiosis; it is significantly reduced in S-day-old aposymbionts, only relative to asparagine (N) in the total tissues, and asparagine and glutamine (Q) in embryos. DISCUSSION
One of the principal findings of this study is that elimination of the bacteria from young larval aphids results in a far greater reduction in the growth of the embryo fraction than of the maternal tissues. As Fig. 2 illustrates, most of the protein growth of symbiotic aphids is in the
TABLE
2. Multivariate
Main effects Aphid tissue Antibiotic Aphid age Antibiotic x aphid age Antibiotic x tissue Aphid age x tissue Aphid tissue x antibiotic x aphid age
profile analysis of the amino pea aphids
Wilks h Roys 4
*F,, 56
acid content
P
0.105 0.045 0.375
8.524 20.740 1.660
32.89 81.68 6.44
<0.0001 ~0.0001 <0.0001
0.423 0.492 0.593
1.364 1.033 0.683
5.28 4.00 2.64
<0.0001 <0.0001 0.001 > P > 0.0001
0.375
1.667
6.43
10.0001
*s = 1, m = 61, n = 28.
of
REPRODUCTIVE
FAILURE
IN PEA APHIDS
253
WI (AY,R,N,D.Q,E,GT,s,H.K) F t(AY,R.N,D,Cj,GT,S,H,K) MI (R,N,D,K) 1.L I (AY,E,S,H) c
KI (AY,R,N.D.Q,E,GT,s.I.L,M,W,V,F) MI (AY,R,N,D,Q.E,GT,S,H,K,W)
Mt (R,ND,Q.E,GT,KS,V)
F~(AY,R,N,D,Q,E,GT,s,H,K)
\
KI(AY,R.N.D,Q.E.GT.s) F t (N,D,Q,GT) Wt 0W.Q)
/
E,IIW)
7 Aposymbiotic total day 4
c
AY,Rt(N,D,H,E,V) L t(N,D,E,H) GT,F t (N,D,H)
AY t (N,D,E,H,I) S I(N.D,E,H)
K,W,FW,D,Q,E WJLV) RI U’LDJLLLW
QI (ND,W
AY I (D,H,I,L)
GT t(H)
. Ft(AY,R.N,D,Q,E,GT,S,H.K,W.V) I +(N,D,Q.GT,S,H,K,W,V) L,WI (ND,Q,V
R&I ND,Q) AY I (N)
Ff(R1.L)
/
\
W~(AY,RN,Q,GT, W-LLLK) I.Lt U’J,D,Q,E,GT,M)
Wt (Q.V)
Kt (AY,R.N,D,Q.s,E,I,V,F)
\
/
M.W,FI(AY,R,N,D,Q,E,GT,S,H,I.L.K.V) E,H,KI(N,Q) FIGURE 4. Representation of statistically significant differences in the profiles of amino acids between symbiotic biotic aphids and with aphid age and tissue type. The nomenclature for amino acids is the standard single-letter with the following arrangement: A
d!3)
and aposymdesignations,
B
TT(Q
In the comparison
of profile B, compared to profile A, the amino acid ‘01’ is depressed, acid ‘y’ is elevated, relative to amino acid ‘6’.
embryo fraction, but in the aposymbiotic aphids, it is in the maternal fraction of aposymbiotic aphids. The implication is that embryos are more dependent than the maternal tissues on the symbiotic bacteria. This dependence cannot be attributed to any specific developmental defect (see Douglas, 1992), but it is expressed as a dramatic curtailment of growth of individual embryos. In this study, the largest embryos did not increase significantly in length, and recruitment of embryos to the size class ‘>O.l mm’ was not observed. Ultrastructural stud-
relative
to amino acid ‘p’, and amino
ies have revealed generalised breakdown of the embryos, possibly associated with embryo resorption, in lo-15 day-old pea aphids (Douglas, unpublished), and this may be the basis of both the recorded decrease in the number of embryos in 11 -day-old aposymbionts (Fig. 1) and the difficulties in the identification and dissection of the embryo fraction in older aposymbionts (see Methods). There is now substantial biochemical evidence that the symbiotic bacteria provide essential amino acids to the aphid tissues (see references in Introduction); and the sig-
254
A. E. DOUGLAS
nificance of this interaction is further emphasized by the finding that genes coding certain enzymes in essential amino acid synthesis are amplified on bacterial plasmids (Lai et al., 1994; Bracho et al., 1995). It is both parsimonious and plausible to suggest that the depressed embryo growth in aposymbionts may be limited by certain essential amino acids (which are provided by the bacteria in symbiotic aphids, see introduction); and that the limiting nutrients are specifically reduced in the free amino acid pools of aposymbionts. From this reasoning and the analyses of the free amino acids (Figs 3 and 4), it is proposed that embryo growth in aposymbiotic aphids is limited by the aromatic amino acids, tryptophan and phenylalanine. The analysis of amino acid pools also suggest that lysine may be limiting to the growth of maternal tissues in aposymbiotic aphids, at least on day-4; that bacterial provision of histidine and valine may not be critical to aphid growth; and that ammonia detoxification (as indicated by high glutamine levels) may be more important in the maternal tissues than in the embryos of aposymbiotic aphids. All these interpretations require confirmation by direct metabolic study. More generally, this study illustrates the usefulness of analyses of the amino acid pools in the study of the aphid-bacterial symbiosis; but it also highlights the limitations and potential pitfalls of the approach. The principal difficulty relates to the amino acid profiles of total aphid tissues. The various differences, especially the day8 data set, between the symbiotic and aposymbiotic aphids can be attributed to both the effects of aposymbiosis on the free amino acid profiles of the maternal and embryo fractions and to the different contribution of the maternal and embryo fractions to the total biomass (the embryos account for 45 and 15% of the protein in 8-dayold symbiotic and aposymbiotic aphids, respectively). The compounding of these two factors probably contributes to the complexity of the variation in amino acids, both in this and other studies (e.g. Liadouze et al., 1995). There are biological, as well as methodological, implications of the difference in amino acid pools between the maternal and embryo fractions; namely, that the maternal and embryo pools are not in equilibrium and that the amino acids probably do not pass freely between the two pools. At present, very little is known about the metabolic and nutritional relationship between aphid embryos and the surrounding maternal tissues, beyond the truism that the embryos, of necessity, derive all their requirements from the maternal fraction. It is possible that essential amino acids are not rapidly translocated into embryos and that embryos in symbiotic aphids may derive their requirement for many essential amino acids from their own complement of bacteria. There are two broad implications. First, essential amino acids provided to aphids via the diet may not be available to embryos (this could account for the failure of dietary supplements of essential amino acids to ‘rescue’ embryo production in aposymbiotic aphids, e.g. Prosser and Douglas, 1992). Second, the bacterial provision of nutrients to aphid tis-
sues may be under separate metabolic controls in the maternal tissues and embryos, with the opportunity for the extent and profile of amino acids released from the bacteria to be regulated in relation to the growth rate and developmental age of each symbiosis. Such fine-scale regulation of the nutritional relationship between aphids and their symbiotic bacteria would be totally obscured by the current methods to analyse the nutritional physiology of the aphid symbiosis. In conclusion, this study has provided evidence that the symbiosis in the maternal tissues and embryos of pea aphids are not physiologically equivalent, and specifically that embryo growth in aphids may be critically dependent on bacterial-derived phenylalanine and tryptophan. This proposal is open to testing by direct study of the metabolism of embryos in symbiotic and aposymbiotic aphids. REFERENCES Bracho A. M., Martinez-Torres D., Moya A. and Latorre A. (1995) Discovery and molecular characterisation of a plasmid localised in Buchneru sp. bacterial endosymbiont of the aphid Rhopalosiphum padi. J. molec. Evol. 41, 67773. Buchner P. (1965) Endosymbiosis of Animals with Plant Microorganisms. John Wiley and Sons, New York. Douglas A. E. (1988) Sulphate utilisation in an aphid symbiosis. Insect Biochem. 18, 599-605. Douglas A. E. (1992) Requirement of pea aphids (Acyrthosiphon pisum) for their symbiotic bacteria. Ent. Exp. Appl. 65, 1955198. Douglas A. E. and Prosser W. A. (1992) Synthesis of the essential amino acid tryptophan in the pea aphid (Acyrthosiphon pisum) symbiosis. J. Insect Physiol. 38, 5655568. Febvay G., Liadouze I., Guillaud J. and Bonnot G. (1995)Analysis of energetic amino acid metabolism in Acyrthosiphon pisum: a multidimensional approach to amino acid metabolism in aphids. Arch. Insect Biochem. Physiol. 29, 45-69. Hinde R (1971) The control of the mycetome symbiotes of the aphids Brevicotyne brassicae, Myzus persicae and Macrosiphum rosae. J. Insect Physiol. 17, 1791-1800. Jones B. N., Paabo S. and Stein S. (1981) Amino acid analysis and enzymatic sequence determination of peptides by an improved ophthaldialdehyde precolumn labelling procedure. J. Liq. Chromatogr. 4, 565-586. Lai C.-Y., Baumann L. and Baumann P. (1994) Amplification of trpEG: adaptation of Buchneru uphidicolu to an endosymbiotic association with aphids. Proc. Nat1 Acad. Sci. U.S.A. 91, 38193823. Liadouze I., Febvay G., Guillaud J. and Bonnot G. (1995) Effect of diet on the free amino acid pools of symbiotic and aposymbiotic pea aphids. J. Insect Physiol. 41, 3340. Morrison D. F. (1976) Multivariate Statistical Methods. McGraw-Hill, New York. Ponsen M. B. (1976) Anatomy of an aphid vector: Myzus persicae. In Aphids as Virus Vectors (Eds. Harris K. F. and Maramorosch K.), pp. 63-82. Academic Press, New York. Prosser W. A. and Douglas A. E. (1991) The aposymbiotic aphid: an analysis of chlortetracycline-treated pea aphid, Acyrthosiphon pisum. J. Insect Physiol. 37, 7 13-7 19. Prosser W. A. and Douglas A. E. (1992) A test of the hypotheses that nitrogen is upgraded and recycled in an aphid (Acyrthosiphon pisum) symbiosis. J. Insect Physiol. 3, 93-99. Sasaki T. and Ishikawa H. (1993) Nitrogen recycling in the endosymbiotic system of the pea aphid, Acyrthosiphon pisum. 2001. Sci. 10, 7799785. Sasaki T. and Ishikawa H. (1995) Production of essential amino acids
REPRODUCTIVE FAILURE IN PEA APHIDS from glutamate by mycetocyte symbionts of the pea aphid, Acyrthosiphon pisum. J. Insect Physiol. 41, 4146. Sasaki T., Fukuchi N. and Ishikawa H. (1993) Amino acid flow through aphid and its symbiont: studies with 15N-labelled glutamin. Zool. Sci. 10, 787-791. Sasaki T., Hayashi H. and Ishikawa H. (1991) Growth and reproduction of the symbiotic and aposymbiotic pea aphids, Acyrthosiphon pisum maintained on artificial diets. J. Insect Physiol. 37, 749-756. Seber G. A. F. (1984) Multivariate Observations. Wiley, New York. Whitehead L. F., Wilkinson T. L. and Douglas A. E. (1992) Nitrogen
255
recycling in the pea aphid (Acyrthosiphon pisum) symbiosis. Proc. R. Sot. Lond. B 250, 115-l 17. Wilkinson T. L. and Douglas A. E. (1995) Why pea aphids (Acyrthosiphon pisum) lacking symbiotic bacteria have elevated levels of the amino acid glutamine. J. Insect Physiol. 41, 92 I-927.
Acknowledgements-I versity of York for his Dr T. L. Wilkinson for Society of London for
thank John Byrne, Computing Services, Uniadvice and support with the statistical analysis; his comments on the manuscript; and the Royal financial support,
J. Insect Physiol.
Pergamon
0022-1910(95)00105-0
Reproductive Failure and the Free Amino Acid Pools in Pea Aphids (Acyrthosiphon pisum) Lacking Symbiotic Bacteria A. E. DOUGLAS* Received
16 June 1995; revised
1 August
1995
Pea aphids (Acyrthosiphon pisum) treated at birth with the antibiotic, rifampicin, to disrupt the symbiotic bacteria (i.e. aposymbiotic aphids) grow very slowly. The growth of embryos in the aposymbiotic aphids was particularly depressed, such that, by adulthood, the embryo fraction represented 65% of the total protein in symbiotic aphids, but just 12% of the protein in aposymbionts. Profile analysis of the free amino acid pools of the aphids and their embryos indicated that the concentrations of the aromatic amino acids phenylalanine and tryptophan were very low in the embryos of aposymbionts, and it is proposed that these amino acids may limit embryo growth in aposymbiotic aphids. There are also indications that lysine may be limiting to the growth of the maternal tissues of 4-day-old larval aposymbionts, and methionine may be in short supply in older aposymbiotic aphids. Aphid
Acyrthosiphon
pisum
Buchneru
Embryo
Symbiosis
INTRODUCTION The symbiotic bacteria Buchneru in aphids are maternally inherited. In parthenogenetic morphs of aphids, the bacteria are transferred from cells, called mycetocytes, in the insect haemocoel to blastoderm embryos in the ovaries (Hinde, 197 l), and they are subsequently incorporated into the embryo’s mycetocytes, as these cells differentiate (Buchner, 1965). The spatial organization of the symbiosis in the parthenogenetic morphs is, however, complex because embryogenesis is initiated in larvae and, in some species, in embryos. An aphid therefore comprises at least two spatially-distinct symbioses: the ‘maternal symbiosis’, represented by the mycetocytes in the aphid haemocoel, and the ‘embryo symbiosis’ comprising all the bacteria-bearing embryos in the aphid’s ovaries. To date, virtually all research on the physiology of the aphid symbiosis has ignored the spatial organization of the symbiosis. In particular, none of the various studies that have demonstrated bacterial provision of essential amino acids to the aphid tissues have differentiated between the maternal symbiosis and embryo symbiosis (e.g. Douglas, 1988; Douglas and Prosser, 1992; Sasaki
*Department of Biology, YOl 5YW, U.K.
University
of York,
P.O.
Box 373, York,
247
Amino
acids
et al., 1993; Sasaki and Ishikawa, 1995; Febvay et al., 1995). As a consequence, the possibility that both the profile of amino acids translocated from the bacteria to the aphid and the rate of amino acid release may differ between the maternal and embryo symbioses has not been addressed experimentally. The purpose of the study reported here was to identify whether the maternal and embryo symbioses in pea aphids are physiologically distinct. The strategy was to compare the response of the maternal tissues and embryos of pea aphids to the elimination of the bacteria. It was reasoned that any difference would be indicative of a difference in the association between the two symbioses. This approach requires separate analysis of the maternal and embryo fractions of the aphids. Preliminary experiments, however, revealed: first, that only the embryo fraction could be obtained reliably from the aphids, while some of the maternal contents, especially haemolymph and fat body cells, were lost during dissections; and second, that the biomass of the embryos could reliably be quantified by protein content, but not fresh weight. Therefore, the study concentrated on the protein content of the embryos and total aphid tissues and, where appropriate, information on the maternal fraction was obtained by subtraction. Two parameters were measured: the growth of the aphids, and their free amino acid content. These were
248
A. E. DOUGLAS
chosen on the basis of the substantial body of information already available on the characteristics of pea aphids lacking their symbiotic bacteria (also known as ‘aposymbiotic’ aphids). These aphids are very small. They grow slowly as larvae, and the adults are essentially reproductively-sterile (Sasaki et al., 1991; Douglas, 1992). Aposymbiosis also substantially changes the profile of free amino acids in aphids (Prosser and Douglas, 1991; Liadouze et al., 1995). Aposymbionts have low levels of essential amino acids, presumably as a consequence of the loss of the bacterial supply of these nutrients. In particular, Prosser and Douglas (199 1) noted the very low concentration of the aromatic amino acid phenylalanine in the aposymbiotic pea aphids, and suggested that this amino acid may be “the crucial limiting amino acid”. Aposymbiotic aphids also have elevated concentrations of the nonessential amino acid glutamine, and this has been linked to the possible role of the bacteria in recycling nitrogen (Prosser and Douglas, 199 1; Sasaki and Ishikawa, 1993). Specifically, there is now substantial evidence that the bacteria utilize aphid waste ammonia as a nitrogen source (Whitehead et al., 1992), and the resultant increase in ammonia in aposymbionts is reduced by its assimilation via glutamine synthetase to glutamine (Wilkinson and Douglas, 1995). In this study, the protein growth of the aphids and their embryos were quantified over larval development. The free amino acid content of the total and embryo fraction was also examined in 4-day-old and S-day-old larval aphids.
MATERIALS
AND METHODS
The aphids The standard culture of pea aphids (Acyrthosiphon pisum) clone 0x2 was maintained at 20°C with 18 h L:6 h D on 3-4-week-old Vicia faba grown in cocofibre compost. The experimental aphids were collected within 24 h of deposition from apterous mothers; they are described as O-day-old aphids, and the time of collection as day-0 of the experiment. Aposymbiotic pea aphids were generated by treatment with the antibiotic rifampicin, administered via plants using a method modified from Douglas (1992). The roots of 3-week-old Vicia faba plants were cleaned of excess compost by careful washing in tapwater, and the plants were transferred to distilled water containing 200 pg rifampicin ml-‘, with control plants in antibiotic-free distilled water. One day later, the O-day-old aphids were applied to the plants: those on the rifampicin-treated plants are described as aposymbiotic aphids, and those on control plants as symbiotic aphids. After 2 days treatment (i.e. on day-2 of the experiment), all aphids were returned to plants in compost under the standard culture conditions. This treatment was fully effective in disrupting the symbiotic bacteria in both the maternal tissues and embryos, as indicated by light microscopical
examination of the antibiotic-treated insects (unpublished results). The experiments were conducted on 2-l l-day-old symbiotic and aposymbiotic aphids. Alatiform aphids (which accounted for 5-10% of the 8- and 1l-day-old aphids) were excluded, and larvae and adults, respectively, were selected on day-8 and day-l 1 (2-6% of the symbiotic aphids and none of the aposymbionts were adult on day-8, and all symbiotic aphids and >95% of the aposymbionts were adult on day-l 1). Older aposymbiotic aphids were unsuitable for these studies because their embryos could not be distinguished or separated from other tissues in the dissections. Number and size of embryos On days 2, 4, 6, 8 and 11 of the experiment, 10 replicate symbiotic and aposymbiotic aphids were fixed in alcoholic Bouin’s solution (Ponsen, 1976) for 34 weeks. Each aphid was then dissected in a drop of water with fine pins, using a dissecting microscope. Two parameters were scored: (a) the number of embryos greater than 0.1 mm long (in some of the dissections, embryos CO.1 mm could not be identified unambiguously); and (b) the length of the longest embryo, as determined with the aid of an eyepiece micrometer at x32 magnification. Protein and amino acid assays A full factorial design was adopted for the study of protein and amino acid content of aphids. The factors were: antibiotic (symbiotic or aposymbiotic aphids), aphid fraction (total or embryo), and aphid age (days 2, 4, 6, 8 and 11 for protein assays, and days 4 and 8 for amino acid assays). Every treatment group comprised 10 replicate aphids, each of which was weighed to the nearest microgram on a Mettler MT5 balance prior to analysis. The protein content of aphids was quantified with the protein assay kit of the Biorad Chemical Co., following manufacturer’s instructions for the microassay, with bovine serum albumin as standard. Each ‘total’ sample was a single aphid homogenized in 0.2 ml ice-cold 50 mM Tris-HCl pH 7.5. For the samples of embryo fraction, the complement of embryos was dissected from each aphid in ice-cold Tris buffer, carefully separated from contaminating fat body and other maternal tissues, and homogenized in 0.2 ml Tris buffer. The total protein content and protein content of the embryo fraction were calculated per aphid and per unit aphid fresh weight. The amino acids were quantified by reverse-phase HPLC after o-phthaldialdehyde derivatization (Jones et al., 1981) using a Beckman System Gold solvent delivery system with C,,-ultrasphere column and Shimadzu RF-551 fluorescence detector. The reference amino acid mixture was AA-S-18 (Sigma), supplemented with asparagine, glutamine and tryptophan. For the total aphid samples, each aphid was homogenized in 0.2 ml ice-cold 80% methanol. For the embryo fraction samples, the embryos were dissected in Tris buffer (as for protein
REPRODUCTIVE
FAILURE
assays), centrifuged at 1,000 g for 5 min, and the supernatant was replaced by 0.2 ml ice-cold 80% methanol, and the embryos were homogenized. All methanol extracts were centrifuged at 13,000 g for 10 min, and the supernatants were stored at -20°C for up to 6 weeks prior to analysis. The amino acids in the total aphid samples were expressed as nmol/pg total protein, and those in the embryo fraction as nmol/unit embryo protein, using the data obtained from the protein assays for total and embryo protein content/unit fresh weight. All proteinamino acids in the aphids were assayed except cysteine and proline, which cannot be detected by this method. The combined concentrations of alanine + tyrosine and of glycine + threonine are shown because these amino acids could not be resolved with confidence in all samples. Statistical
analysis
Parametric statistical tests were conducted, to explore the effects of antibiotic treatment and aphid age on the number and size of embryos, and on the protein content of the embryo and total aphid tissues. The technique of profile analysis, as described in detail by Morrison (1976) and Seber (1984) was used to analyse the impact of antibiotic, aphid age (4- and 8-day-old aphids) and tissue fraction (embryo and total) on the amino acid content of the aphids. The data were log,-transformed prior to the analysis so that the tests for homogeneity of the distributions would correspond to tests for parallelism in the profile analysis. Preliminary tests on each variable for each treatment combination revealed no significant outliers, but other univariate tests (skewness, kurtosis and homogeneity of variances across treatments) revealed departures from the standard assumptions and standard multivariate tests for homogeneity of covariance matrices were unavailable due to sample size restrictions. The patterns revealed by this analysis (see results) should, there1.2
1
IN PEA APHIDS
fore, be interpreted with due statistical caution. The patterns obtained (see results) proved to be complex and, therefore, the three independent variables (antibiotic, aphid age and tissue) were expressed as a single factor in the detailed comparisons between profiles. The comparisons were achieved by calculating confidence intervals for every simple contrast involving a pair of amino acids and a pair of treatments. Overall 95% protection was achieved using Bonferroni intervals.
RESULTS
The number
and size of embryos
in the aphids
Every 2-l 1-day-old aphid examined contained embryos. All the embryos in the 2-day-old aphids were CO.1 mm long (they were not studied further, see Methods), but by day-4, the largest embryos had grown, on average, to 0.3 mm in length, in both the symbiotic and aposymbiotic aphids [Fig. l(a)]. Between days 4 and 11, the largest embryos in symbiotic aphids increased four-fold in length to 1.16 5 0.016 mm, but the length of these embryos in the aposymbionts did not vary significantly with time. The aposymbiotic aphids contained significantly fewer embryos than the symbiotic aphids [Fig. l(b)], and the discrepancy between the two groups of aphids increased with aphid age. In the symbiotic aphids, the number of embryos increased by 75% between days 4 and 11. In the aposymbionts, no increase in embryo number was obtained, and the number in the 1l-day-old aphids was significantly lower than in the 48-day-old aphids (0.001 < P < 0.01). The growth
of aphids and their embryos
The protein content of the aphids and their embryos was used to explore the contribution of embryos to aphid
4ol
(4
249
(b)
/
0
2
4
6
8
Aphid age (days)
10
12
4
6
8
10
12
Aphid age (days)
FIGURE 1. The embryos in symbiotic (0) and aposymbiotic (0) pea aphids. (a) The length of the longest embryo per aphid. antibiotic: F,,,, = 351.1, P < 0.0001; aphid age: F3,72 = 185.9, P < 0.0001; interaction: F372 = 102.0, P < 0.0001. (b) The number of embryos greater than 0.1 mm in length. antibiotic: F,,,, = 432.6, P < 0.0001; aphid age: Fx,72= 17.1, P < 0.0001; interaction: F3,72 = 75.4, P < 0.0001.
250
A. E. DOUGLAS
growth. On day-2, the embryo fraction of the symbiotic and aposymbiotic aphids contained 0.5 f 0.032 and 0.48 f 0.034 pg protein, respectively, representing 24 25% of the total protein of the aphids (Fig. 2). In the symbiotic aphids, the embryo protein increased nearly 400-fold over the course of the experiment, accounting for 45% of the total protein in the 8-day-old larvae and 65% of total protein in the 1l-day-old adults. In the aposymbiotic aphids, the embryo protein increased 5-fold and contributed 12% of the total protein in the 1l-day-old aphids. The amino acid content of aphids and their embvos The total amino acid content of the aphids is shown in Table l(a). It is significantly elevated in aposymbiotic aphids [see also Prosser and Douglas (1991) and Wilkinson and Douglas (1995)]; and, for both symbiotic and aposymbiotic aphids, it is significantly lower in the embryo fraction than in the total aphid samples. The statistically significant interaction term “antibiotic x aphid age” arises from the 80% reduction in amino acid content of both the total and embryo fractions of symbiotic aphids between days 4 and 8, and this result is consistent with the inverse relationship between body size and amino acid concentration in pea aphids identified by Liadouze et al. (1995). The composition of the amino acids in the aphids is displayed in Fig. 3. The data for total aphid tissues are in broad agreement with previous analyses (e.g. Prosser and Douglas, 1991; Liadouze et al., 1995). In particular, glutamine was the dominant amino acid in all samples analysed, and its concentration was higher in aposymbiotic aphids than in symbiotic aphids (20-23% of the amino acids in symbiotic aphids and 30-34% in aposymbionts). In the embryo fraction, however, glutamine was not the amino acid at the highest concentration. On both day-4 and day-8, glutamate and glutamine are at closely similar concentrations in embryos of symbiotic 350
1 (a)
TABLE
acid content
Day-8
Day-4 total tissues Symbiotic aphids Aposymbiotic aphids
embryos
total tissues
embryos
2.87kO.184
2.72f0.113
0.64~kO.019
0.60+0.024
4.91 kO.240
4.10 f 0.165
4.61 f 0.187
4.11 f 0.235
(b) F-values Source of variation Antibiotic Aphid tissue Aphid age Antibiotic x aphid tissue Antibiotic x aphid age Aphid tissue x age Antibiotic x aphid tissue
of ANOVA
F (1,72 dj) P 13322 P < 0.0001 12 0.0001 < P < 0.001 419 P < 0.0001 3.57 P > 0.05 360 P < 0.0001 P > 0.05 0.32 P > 0.05 0.03
x
age *The weight (mean k SE 10 reps) of the symbiotic and aposymbiotic aphids, respectively, were: 0.60 k 0.024 and 0.42 f 0.073 mg on day-4, and 2.97 + 0.088 and 0.82 f 0.023 mg on day-g. (The data in Table l(a) were log-transformed prior to statistical analysis).
aphids, and asparagine was the dominant amino acid in embryos of aposymbiotic aphids. To explore further the variation in amino acid composition of the aphids, a profile analysis of the data set was conducted (see Table 2). The significance of all the main effects and the interaction terms indicate that the concentrations of the various amino acids vary in a complex fashion with aphid age and tissue and between symbiotic
1(b)
250 $ 0 2 Q) z k UJ 3.
700 / 150-
/ ,..’ .,r2....
p
100
50
1
Aphid age (days)
of
(a) Amino acid content of aphids nmol amino acids pg-’ protein* (mean + SE 10 replicates)
25
300 -
1. The effect of aposymbiosis on the amino pea aphids and their embryos
2
3
4
5
6
7
8
9
10
II
Aphid age (days)
FIGURE 2. Protein growth of pea aphids and their embryos. For (a) symbiotic aphids and (b) aposymbiotic aphids, the total and the protein fraction of the maternal aphid protein (0) and protein content of the embryo fraction (0) were determined, tissues (0) was obtained by subtraction. Mean values are displayed (the coefficient of variation (SD/mean) did not exceed 0.09, and was uniform between symbiotic and aposymbiotic aphids). Note difference of scale between (a) and (b).
REPRODUCTIVE
FAILURE
IN PEA APHIDS
251
2 Symbiotic aphids
1.8
1.8 -
1.6
Aposymbiotic total tissues
aphids
1.6 1.4 1.2 1
0.8
0.8
0.6
0.6 0.4 0.2 0
2 1.8
1.6
,
I Symbiotic aphids embryos day 4
1.4
1.4
1.2
1.2
1
1
0.8
0.8
0.6
0.6
0.4
0.4
0.2
0.2
0
0
FIGURE
3. The amino
acid content
of pea aphids
and aposymbiotic aphids. All the statistically significant pairwise comparisons of amino acid concentrations between treatments are shown in Fig. 4. The differences in amino acid profiles between symbiotic and aposymbiotic aphids are dominated by a reduction in certain essential amino acids in the aposymbionts. The most consistent effects concern the two aromatic essential amino acids, tryptophan (W) and phenylalanine (F). These amino acids are depressed in aposymbionts, relative to an array of other amino acids, in both the total and embryo fractions. However, the reduction of these amino acids is more pronounced in the embryo fraction, as is indicated by: first, the reduction in aposymbiotic embryos, relative to total tissues of aposymbionts, of phenylalanine on day-4 and both phenyl-
and their embryos
(continued
on p. 252).
alanine and tryptophan on day-8; and second, the reduction in both amino acids in aposymbiotic embryos, but not total tissues between day-4 and day-8. Further essential amino acids that are depressed (relative to other amino acids) in aposymbiotic aphids include: methionine (M) in both tissue types on day-& isoleucine (I) in total aphid samples on day-4 and day-8 and in the embryos on day-4; and leucine (L) in the embryo tissues on day-4 and total tissues on day-8. The pattern of variation in lysine (K) is complex. On day-4, it is depressed in the total tissues, but not embryo fraction, of aposymbionts; and, on day-8, it is increased (relative to other amino acids) in the embryo, but not total, tissues of symbiotic aphids and decreased in the total, but not embryo, tissues of aposymbiotic aphids.
252
A. E. DOUGLAS
(b) 2 1.8
Symbiotic aphids
1.6
1 0.8
1.8 1.6-
Symbiotic aphids embryos day8
1.4 1.2 l0.8 0.6 0.4 0.2 -
FIGURE
3. Continued.
Of all the essential amino acids, valine (V) is notable in that it is not significantly depressed in aposymbiotic aphids of either age or tissue type. Histidine (H) is also little-affected by aposymbiosis; it is significantly reduced in S-day-old aposymbionts, only relative to asparagine (N) in the total tissues, and asparagine and glutamine (Q) in embryos. DISCUSSION
One of the principal findings of this study is that elimination of the bacteria from young larval aphids results in a far greater reduction in the growth of the embryo fraction than of the maternal tissues. As Fig. 2 illustrates, most of the protein growth of symbiotic aphids is in the
TABLE
2. Multivariate
Main effects Aphid tissue Antibiotic Aphid age Antibiotic x aphid age Antibiotic x tissue Aphid age x tissue Aphid tissue x antibiotic x aphid age
profile analysis of the amino pea aphids
Wilks h Roys 4
*F,, 56
acid content
P
0.105 0.045 0.375
8.524 20.740 1.660
32.89 81.68 6.44
<0.0001 ~0.0001 <0.0001
0.423 0.492 0.593
1.364 1.033 0.683
5.28 4.00 2.64
<0.0001 <0.0001 0.001 > P > 0.0001
0.375
1.667
6.43
10.0001
*s = 1, m = 61, n = 28.
of
REPRODUCTIVE
FAILURE
IN PEA APHIDS
253
WI (AY,R,N,D.Q,E,GT,s,H.K) F t(AY,R.N,D,Cj,GT,S,H,K) MI (R,N,D,K) 1.L I (AY,E,S,H) c
KI (AY,R,N.D.Q,E,GT,s.I.L,M,W,V,F) MI (AY,R,N,D,Q.E,GT,S,H,K,W)
Mt (R,ND,Q.E,GT,KS,V)
F~(AY,R,N,D,Q,E,GT,s,H,K)
\
KI(AY,R.N.D,Q.E.GT.s) F t (N,D,Q,GT) Wt 0W.Q)
/
E,IIW)
7 Aposymbiotic total day 4
c
AY,Rt(N,D,H,E,V) L t(N,D,E,H) GT,F t (N,D,H)
AY t (N,D,E,H,I) S I(N.D,E,H)
K,W,FW,D,Q,E WJLV) RI U’LDJLLLW
QI (ND,W
AY I (D,H,I,L)
GT t(H)
. Ft(AY,R.N,D,Q,E,GT,S,H.K,W.V) I +(N,D,Q.GT,S,H,K,W,V) L,WI (ND,Q,V
R&I ND,Q) AY I (N)
Ff(R1.L)
/
\
W~(AY,RN,Q,GT, W-LLLK) I.Lt U’J,D,Q,E,GT,M)
Wt (Q.V)
Kt (AY,R.N,D,Q.s,E,I,V,F)
\
/
M.W,FI(AY,R,N,D,Q,E,GT,S,H,I.L.K.V) E,H,KI(N,Q) FIGURE 4. Representation of statistically significant differences in the profiles of amino acids between symbiotic biotic aphids and with aphid age and tissue type. The nomenclature for amino acids is the standard single-letter with the following arrangement: A
d!3)
and aposymdesignations,
B
TT(Q
In the comparison
of profile B, compared to profile A, the amino acid ‘01’ is depressed, acid ‘y’ is elevated, relative to amino acid ‘6’.
embryo fraction, but in the aposymbiotic aphids, it is in the maternal fraction of aposymbiotic aphids. The implication is that embryos are more dependent than the maternal tissues on the symbiotic bacteria. This dependence cannot be attributed to any specific developmental defect (see Douglas, 1992), but it is expressed as a dramatic curtailment of growth of individual embryos. In this study, the largest embryos did not increase significantly in length, and recruitment of embryos to the size class ‘>O.l mm’ was not observed. Ultrastructural stud-
relative
to amino acid ‘p’, and amino
ies have revealed generalised breakdown of the embryos, possibly associated with embryo resorption, in lo-15 day-old pea aphids (Douglas, unpublished), and this may be the basis of both the recorded decrease in the number of embryos in 11 -day-old aposymbionts (Fig. 1) and the difficulties in the identification and dissection of the embryo fraction in older aposymbionts (see Methods). There is now substantial biochemical evidence that the symbiotic bacteria provide essential amino acids to the aphid tissues (see references in Introduction); and the sig-
254
A. E. DOUGLAS
nificance of this interaction is further emphasized by the finding that genes coding certain enzymes in essential amino acid synthesis are amplified on bacterial plasmids (Lai et al., 1994; Bracho et al., 1995). It is both parsimonious and plausible to suggest that the depressed embryo growth in aposymbionts may be limited by certain essential amino acids (which are provided by the bacteria in symbiotic aphids, see introduction); and that the limiting nutrients are specifically reduced in the free amino acid pools of aposymbionts. From this reasoning and the analyses of the free amino acids (Figs 3 and 4), it is proposed that embryo growth in aposymbiotic aphids is limited by the aromatic amino acids, tryptophan and phenylalanine. The analysis of amino acid pools also suggest that lysine may be limiting to the growth of maternal tissues in aposymbiotic aphids, at least on day-4; that bacterial provision of histidine and valine may not be critical to aphid growth; and that ammonia detoxification (as indicated by high glutamine levels) may be more important in the maternal tissues than in the embryos of aposymbiotic aphids. All these interpretations require confirmation by direct metabolic study. More generally, this study illustrates the usefulness of analyses of the amino acid pools in the study of the aphid-bacterial symbiosis; but it also highlights the limitations and potential pitfalls of the approach. The principal difficulty relates to the amino acid profiles of total aphid tissues. The various differences, especially the day8 data set, between the symbiotic and aposymbiotic aphids can be attributed to both the effects of aposymbiosis on the free amino acid profiles of the maternal and embryo fractions and to the different contribution of the maternal and embryo fractions to the total biomass (the embryos account for 45 and 15% of the protein in 8-dayold symbiotic and aposymbiotic aphids, respectively). The compounding of these two factors probably contributes to the complexity of the variation in amino acids, both in this and other studies (e.g. Liadouze et al., 1995). There are biological, as well as methodological, implications of the difference in amino acid pools between the maternal and embryo fractions; namely, that the maternal and embryo pools are not in equilibrium and that the amino acids probably do not pass freely between the two pools. At present, very little is known about the metabolic and nutritional relationship between aphid embryos and the surrounding maternal tissues, beyond the truism that the embryos, of necessity, derive all their requirements from the maternal fraction. It is possible that essential amino acids are not rapidly translocated into embryos and that embryos in symbiotic aphids may derive their requirement for many essential amino acids from their own complement of bacteria. There are two broad implications. First, essential amino acids provided to aphids via the diet may not be available to embryos (this could account for the failure of dietary supplements of essential amino acids to ‘rescue’ embryo production in aposymbiotic aphids, e.g. Prosser and Douglas, 1992). Second, the bacterial provision of nutrients to aphid tis-
sues may be under separate metabolic controls in the maternal tissues and embryos, with the opportunity for the extent and profile of amino acids released from the bacteria to be regulated in relation to the growth rate and developmental age of each symbiosis. Such fine-scale regulation of the nutritional relationship between aphids and their symbiotic bacteria would be totally obscured by the current methods to analyse the nutritional physiology of the aphid symbiosis. In conclusion, this study has provided evidence that the symbiosis in the maternal tissues and embryos of pea aphids are not physiologically equivalent, and specifically that embryo growth in aphids may be critically dependent on bacterial-derived phenylalanine and tryptophan. This proposal is open to testing by direct study of the metabolism of embryos in symbiotic and aposymbiotic aphids. REFERENCES Bracho A. M., Martinez-Torres D., Moya A. and Latorre A. (1995) Discovery and molecular characterisation of a plasmid localised in Buchneru sp. bacterial endosymbiont of the aphid Rhopalosiphum padi. J. molec. Evol. 41, 67773. Buchner P. (1965) Endosymbiosis of Animals with Plant Microorganisms. John Wiley and Sons, New York. Douglas A. E. (1988) Sulphate utilisation in an aphid symbiosis. Insect Biochem. 18, 599-605. Douglas A. E. (1992) Requirement of pea aphids (Acyrthosiphon pisum) for their symbiotic bacteria. Ent. Exp. Appl. 65, 1955198. Douglas A. E. and Prosser W. A. (1992) Synthesis of the essential amino acid tryptophan in the pea aphid (Acyrthosiphon pisum) symbiosis. J. Insect Physiol. 38, 5655568. Febvay G., Liadouze I., Guillaud J. and Bonnot G. (1995)Analysis of energetic amino acid metabolism in Acyrthosiphon pisum: a multidimensional approach to amino acid metabolism in aphids. Arch. Insect Biochem. Physiol. 29, 45-69. Hinde R (1971) The control of the mycetome symbiotes of the aphids Brevicotyne brassicae, Myzus persicae and Macrosiphum rosae. J. Insect Physiol. 17, 1791-1800. Jones B. N., Paabo S. and Stein S. (1981) Amino acid analysis and enzymatic sequence determination of peptides by an improved ophthaldialdehyde precolumn labelling procedure. J. Liq. Chromatogr. 4, 565-586. Lai C.-Y., Baumann L. and Baumann P. (1994) Amplification of trpEG: adaptation of Buchneru uphidicolu to an endosymbiotic association with aphids. Proc. Nat1 Acad. Sci. U.S.A. 91, 38193823. Liadouze I., Febvay G., Guillaud J. and Bonnot G. (1995) Effect of diet on the free amino acid pools of symbiotic and aposymbiotic pea aphids. J. Insect Physiol. 41, 3340. Morrison D. F. (1976) Multivariate Statistical Methods. McGraw-Hill, New York. Ponsen M. B. (1976) Anatomy of an aphid vector: Myzus persicae. In Aphids as Virus Vectors (Eds. Harris K. F. and Maramorosch K.), pp. 63-82. Academic Press, New York. Prosser W. A. and Douglas A. E. (1991) The aposymbiotic aphid: an analysis of chlortetracycline-treated pea aphid, Acyrthosiphon pisum. J. Insect Physiol. 37, 7 13-7 19. Prosser W. A. and Douglas A. E. (1992) A test of the hypotheses that nitrogen is upgraded and recycled in an aphid (Acyrthosiphon pisum) symbiosis. J. Insect Physiol. 3, 93-99. Sasaki T. and Ishikawa H. (1993) Nitrogen recycling in the endosymbiotic system of the pea aphid, Acyrthosiphon pisum. 2001. Sci. 10, 7799785. Sasaki T. and Ishikawa H. (1995) Production of essential amino acids
REPRODUCTIVE FAILURE IN PEA APHIDS from glutamate by mycetocyte symbionts of the pea aphid, Acyrthosiphon pisum. J. Insect Physiol. 41, 4146. Sasaki T., Fukuchi N. and Ishikawa H. (1993) Amino acid flow through aphid and its symbiont: studies with 15N-labelled glutamin. Zool. Sci. 10, 787-791. Sasaki T., Hayashi H. and Ishikawa H. (1991) Growth and reproduction of the symbiotic and aposymbiotic pea aphids, Acyrthosiphon pisum maintained on artificial diets. J. Insect Physiol. 37, 749-756. Seber G. A. F. (1984) Multivariate Observations. Wiley, New York. Whitehead L. F., Wilkinson T. L. and Douglas A. E. (1992) Nitrogen
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recycling in the pea aphid (Acyrthosiphon pisum) symbiosis. Proc. R. Sot. Lond. B 250, 115-l 17. Wilkinson T. L. and Douglas A. E. (1995) Why pea aphids (Acyrthosiphon pisum) lacking symbiotic bacteria have elevated levels of the amino acid glutamine. J. Insect Physiol. 41, 92 I-927.
Acknowledgements-I versity of York for his Dr T. L. Wilkinson for Society of London for
thank John Byrne, Computing Services, Uniadvice and support with the statistical analysis; his comments on the manuscript; and the Royal financial support,