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How an aphid (Acyrthosiphon pisum) symbiosis responds to variation in dietary nitrogen
J. /meet Physiol. Vol. 38, No. 4, pp. 301-307, Printed in Great Britain. All rights reserved
1992 Copyright
0
0022-1910/92 $5.00 + 0.00 1992 Pergamon Press Ltd
HOW AN APHID (ACYRTHOSIPHON PISUM) SYMBIOSIS RESPONDS TO VARIATION IN DIETARY NITROGEN W. A. PROSSER,S. J. SIMPSONand A. E. DOUGLAS* Department of Zoology, University of Oxford, South Parks Road, Oxford OX1 3PS, U.K. (Received 16 October 1991)
Abstract-This study concerns the responses of the pea aphid Acyrthosiphon pisum to chemically-defined diets containing different concentrations of nitrogen (45-270 mM amino acids) and how these responses are influenced by the antibiotic chlortetracycline, which selectively disrupts the aphids’ symbiotic bacteria. The chlortetracycline-treated aphids ingested less diet than untreated controls, and the difference between the two groups of insects could be ascribed largely to the greater size of the untreated aphids. Diet uptake increased with decreasing dietary amino acid concentration, in the range 202-89 mM for untreated aphids and 202-135 mM for chlortetracycline-treated aphids. This compensatory feeding response contributed to the close similarity of aphid growth rates on these diets. The honeydew of untreated and chlortetracycline-treated aphids contained 6-10 and 10-30 mol% of the dietary amino acid concentration, respectively. and the values (in percentage terms) did not vary consistently with dietary nitrogen. The concentration of non-essential amino acids, but not essential amino acids, was significantly higher in the honeydew of chlortetracycline-treated aphids than in untreated aphids’ honeydew. The selective utilization of non-essential amino acids by untreated aphids is consistent with the hypothesis that the symbiotic bacteria utilize non-essential amino acids as a nitrogen source for essential amino acid synthesis, a process known as nitrogen upgrading. Key Word Index: Aphid; symbiosis; mycetocyte; Acyrthosiphon pisum; bacteria
INTRODUCHON
Ecologists have, for many years, considered nitrogen as a major determinant of the abundance and population increase of plant sap-sucking insects of the order Homoptera (McNeil1 and Southwood, 1978). For example, the faster growth rates of aphids on forbs than trees and the evolution of complex aphid life cycles involving migration from trees to forbs in the summer have been ascribed to a higher nitrogen content in the phloem sap of forbs than trees (Dixon, 1985). However, the ecologists’ appreciation of the significance of nitrogen has not been matched by an understanding among insect physiologists of how these insects respond to variations in dietary nitrogen. Many phytophagous insects of the orders Lepidoptera and Orthoptera are known to respond to a reduction in dietary nitrogen by increased consumption of food and/or by enhanced retention of ingested *To whom all correspondence
should be addressed.
nitrogen (Slansky and Scriber, 1985; Simpson and Simpson, 1990). As yet, the evidence for comparable processes occurring in Homoptera is largely anecdotal, e.g. tree-feeding aphids produce more honeydew than forb-feeders. An additional parameter to consider in the Homoptera is their symbiotic bacteria, located in specialized insect cells, mycetocytes, in the haemocoel (Douglas, 1989). These bacteria have been implicated in the nitrogen nutrition of aphids, particularly by the provision of essential amino acids (Douglas, 1990), and one would therefore anticipate some interplay between the physiological responses of the insect and metabolic responses of the bacteria to variations in dietary nitrogen. For example, do aphids lacking functional bacteria attempt to compensate for the loss of their bacteria-derived amino acids by ingesting larger volumes of diet? The study reported here is a first detailed consideration of these issues in phloem sap-sucking insects. The principal nitrogenous compounds in phloem are free amino acids. The experiments were conducted on 301
302
W. A. TOSSER et al.
larvae of the pea aphid Acyrthosiphon pisum, introduced to chemically-defined diets of uniform amino acid composition but variable concentration, so that the effects of nitrogen quantity could be assessed independently of nitrogen quality. This is undoubtedly a simplified experimental system, but it enables the basic physiological principles underlying the aphids’ responses to be investigated. MATERIALS AND METHODS
The experiments were conducted on fourth-instar larvae of the pea aphid A. pisum clone 0X-2, derived from a long-standing clonal culture of virginoparae (Prosser and Douglas, 1992), according to the following protocol. Within 24 h of deposition from apterous adults, groups of 10 larvae were transferred to chemically-defined diet containing 135 mM amino acids and 550 mM sucrose [diet composition and procedures are described in Prosser and Douglas (1991)]. The bacterial symbionts in half of the aphids were disrupted by treatment with the antibiotic chlortetracycline, at 50 pg ml-’ diet for the first 5 days of larval development (Douglas, 1988). The larvae were maintained at 20°C with 18 h light-6 h dark regime and 75% r.h. They developed to fourth larval instar on the sixth day and, on the seventh day, they were transferred to chlortetracyclinefree diets containing 270, 202, 13.5, 89 or 45 mM amino acids. At this age, the chlortetracyclinetreated aphids were approximately half the weight of untreated aphids. All experiments were of 24 h duration. Diet uptake was assayed by a modification of the method of Wright et al. (1985) using [‘4C]methylated inulin (Sigma) at specific activity 10 PCi per 0.1 ml
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200
300
acid concentration ImM) of diet
Fig. 1. Diet uptake by untreated (solid symbols) and (open symbols) fourth-instar A. pisum maintained on diets containing 45-270mM amino acids for 24 h. The aphids were weighed at the start and end of the 24 h experiment, and the average of these two values was used as covariate in the analysis of covariance of diet uptake data: aphid weight (covariate) F,,@= 69.64, P < 0.001; diet composition F4.@= 2.45, P > 0.05; chlortetracycline treatment F,,@ = 1.81, P > 0.05; interaction between diet composition and chlortetracycline treatment chlortetracycline-treated
F4,@= 2.72, 0.01 < P < 0.05:
diet (see Douglas, 1988). The honeydew was collected on GF/C glass fibre filters (Whatman), which were then dried and shaken with Optiscint Hisafe-l scintillant (LKB Ltd). The radioactivity was counted in a LKB 1219 Rackbeta scintillation counter with preset 14C window. The honeydew produced by each cage of aphids was collected on a sheet of tinfoil. The samples were analysed by reversed-phase high performance liquid chromatography after derivatization with o-phthalaldehyde (Jones et al., 1981) as described in detail by Prosser and Douglas (1991). The aphids were weighed individually on an ElmerParker AD4 electrobalance to the nearest pg.
RESULTS
Diet uptake
Each fourth-instar larva of A. pisum ingested between 0.1 and 1.O~1 of diet over 24 h after introduction to the test diets containing amino acids at the total concentration of 45-270mM (Fig. 1). The aphids, whose symbiotic bacteria had been disrupted by chlortetracycline treatment during early larval development, fed more slowly than the untreated control aphids. This was largely a consequence of the small size of the treated aphids (see also Prosser and Douglas, 1991) as is indicated by the non-significance of the main effect, chlortetracycline treatment, once aphid weight is included in the analysis as a covariate (see legend to Fig. 1). The feeding rate of untreated aphids increased by 6&80% with decreasing dietary amino acid concentration from 202-89 mM, but no further increase was evident on the diet containing 45 mM amino acids. Diet consumption by chlortetracycline-treated aphids also increased with declining dietary nitrogen in the range 270-135 mM, but declined progressively with further reduction in dietary nitrogen. This difference between untreated and chlortetracycline-treated aphids is statistically significant (see interaction term in analysis, legend to Fig. 1). Amino acid content of aphid honeydew
The total concentration of protein amino acids in the honeydew produced by the aphids lay in the range 3-30mM. On all test diets, the honeydew of untreated aphids contained less than lOmol% of the amino acids in the diet. The honeydew of chlortetracycline-treated aphids had higher amino acid contents, representing l&20 and 30mol% on the dietary amino acid contents on diets containing 270-89 mM and 45 mM amino acids, respectively [Fig. 2(a)],
Responses of the pea aphid A. pisum This difference between the amino acid content of honeydew from untreated and chlortetracyclinetreated aphids is mediated by the differences in their non-essential amino acid content. The total essential amino acid content of their honeydew was closely similar [see Fig. 2(b)], but the non-essential amino acids represented 52-57 moi% of the amino acids in the honeydew of chlortetracycline-treated aphids but only 7-22 mol% of the amino acids of untreated aphid honeydew. Since the diet contains non-essential and essential amino acids in equal proportions, these data indicate that untreated aphids selectively utilize non-essential amino acids in the diet. The concentration of the individual amino acids in the honeydew is shown in Fig. 3. Every non-essential amino acid conformed to the pattern of, first, increasing concentration with increasing dietary concentration and, second, higher concentrations in the honeydew of chlortetracycline-treated than untreated aphids. The glutamine content of honeydew from
,
I
0
100 Total om~no ac~d concentration
200 (mM)
300 of diet
(b) 20 d 8
_A
Total om~m and ccncentrotlon
(mM)
of diet
Fig. 2. The amino acid concentration in honeydew produced by untreated (solid symbols) and chlortetracycline-treated (open symbols) fourth-instar A. pisum maintained on diets containing 4%270mM amino acids for 24 h. (a) Total
amino acid concentration, expressed as percentage of amino acid concentration in the diet. (b) Concentration of essential (squares) and non-essential (circles) amino acids. The F values for analysis of variance conducted on essential amino acid concentration are: diet composition F, m = 9.05. P < O.OOl;chlortetracycline treatment;,, = 1.91,‘j > 0.05; interaction F,.._, m = 2.71. 0.01 < P < 0.05. Euuivalent values for a separate analysis of non-essential amino acids are: chlortetracycline treatment F,,, = 6.71, P c 0.001; diet composition F,., = 155.63, P < 0.001; interaction F4,* = 8.90, P < 0.001.
303
chlortetracycline-treated aphids was exceptionally high, representing 22-27mol% of the total amino acid content; glutamine accounted for less than 2 mol% of untreated aphid honeydew. The effect of chlortetracycline treatment on the concentration of individual essential amino acids in the honeydew was more complex. By visual inspection of the data, the amino acids could be divided into three groups:
(a) The sole amino acid at closely-similar concentrations in the honeydew of untreated and chlortetracycline-treated aphids was arginine, which varied from approx. 5 mM in aphids on diets containing 270 mM amino acids to 2 mM on diets with 45 mM amino acids. However, because the total amino acid concentration was higher in the honeydew of chlortetracycline-treated arginine aphids, represented 30-50 and 14-18 mol% of the total honeydew amino acids from untreated and chlortetracycline-treated aphids, respectively. (b) The amino acids at higher concentration in the honeydew of untreated than chlortetracycline-treated aphids were tryptophan. histidine and phenylalanine. Tryptophan represented 20-23 and 2-5 mol% of the amino acids in the honeydew of untreated and chlortetracycline-treated aphids, respectively, and equivalent values for histidine are 7-19 and 3-5 mol%, respectively. Tryptophan is a minor amino acid in the diet (accounting for 2mol% of the amino acids). The honeydew of untreated aphids on diet containing 45 mM amino acids is 0.88mM, just greater than the dietary concentration (0.8 mM). The final amino acid in this group, phenylalanine, is the amino acid at lowest concentration in the honeydew of chlortetracycline-treated aphids (where it is invariably x0.1 mM); and, together with valine and isoleucine, it is also a minor component of untreated aphid honeydew. On every test diet, phenylalanine was 25-500% higher in the honeydew of untreated than chlortetracycline-treated aphids. (c) The remaining essential amino acids, namely isoleucine, leucine, lysine, methionine and threonine, were at higher concentrations in the honeydew of chlortetracycline-treated than untreated aphids.
W. A. PROSSERet al.
304
Growth and eficiency
of food utilization
The aphids on all test diets increased in weight over the 24 h experiment. Consistent with all previous studies linking the disruption of symbionts
with reduced aphid growth (Douglas, 1989), the relative growth rate of untreated aphids was significantly greater than that of chlortetracyclinetreated aphids (Table 1). The concentration of dietary nitrogen also influenced growth, especially of
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Fig. 3. The concentration (mean f SE) of each amino acid in the honeydew of utreated (a) and chlortetracycline-treated (b) fourth-instar A. pisum maintained on diets containing 270mM (m), 202mM (a),
135 mM (0),_89 mM (m) and 45 mM (0) amino acids for 24 h.
GABA
Responses of the pea aphid A. pisum
305
Table 1. Performance of fourth-instar A. pisum larvae maintained on diets of different amino acid concentrations for 24 h RGR
Dietary amino acid concentration (mW
Untreated aphids
270 202 135 89 45
0.45 + 0.22 f 0.3 1 + 0.26 k 0.14 *
0.045 0.068 0.036 0.036 0.037
EC1
Chlortetracycline treated aphids 0.37 f 0.099 0.13 &-0.075 0.16 + 0.039 0.12 & 0.038 0.05 + 0.022
Untreated aphids 0.78 + 0.53 f 0.59 * 0.38 + 0.22 +
0.066 0.072 0.053 0.044 0.025
Chlortetracycline treated aphids 0.59 + 0.088 0.38 _+0.109 0.38 + 0.063 0.47 + 0.172 0.13 +_0.056
Values of mean k SE are shown. RGR: relative growth rate (mg mg-’ aphid fresh weight) over the 24 h period on test diets. ECI: efficiency of conversion of ingesta (mg dry weight increase of aphids per mg dry weight of diet ingested). The results of analysis of variance for RGR are: diet composition F4,57= 9.75, P < 0.001; chlortetracycline treatment F, 57= 12.03, P i 0.001; interaction Fd r, = 0.71, P > 0.05. Values for the analysis for EC1 are: diet composition Fd ,; = 11.10, P < 0.001; chlortetracychne treatment F,.57= 4.94, 0.01 > P > 0.001; mteraction F4.5,= 1.43, P > 0.05.
chlortetracycline-treated three-fold
difference
aphids, as is indicated between
by the
the relative growth rate
aphids on diets containing 45 and 720 mM amino acids, and IO-fold difference in relative growth rate of chlortetracycline-treated aphids on these diets. However, no consistent variation in this growth rate with amino acid concentration in the range 89-202 mM was evident, suggesting that both untreated and chlortetracycline-treated aphids compensated for dietary nitrogen in this concentration range. The efficiency with which ingested food was converted into aphid biomass was derived from the weight gain of aphids and their diet uptake over 24 h. Most of the values of efficiency of conversion of ingested food were in the range 0.38-0.6pg pg-‘, with a single high value of 0.78 pg pg-’ for untreated aphids on diet with 270mM amino acids and low values of 0.22 and 0.13 pg pg-’ for untreated and chlortetracycline-treated aphids, respectively, on the 45mM diet (Table 1). As with relative growth rate, the efficiency of conversion of ingested food of aphids was significantly depressed by chlortetracycline-treatment. of untreated
DISCUSSION
Response of untreated aphid
(containing symbiotic
bacteria) to dietary nitrogen
The present study includes the first unambiguous demonstration that a plant sap-sucking insect responds to variations in dietary nitrogen by increased consumption. This compensatory feeding response substantially mitigated the deleterious effects of low nitrogen diets (in the range 89-202 mM amino acids) on the growth of larval A. pisum (Table 1). However,
at the lowest concentration tested (45 mM), the feeding rate of untreated aphids did not increase and the aphid growth rate was reduced substantially. Perhaps on this diet, the absolute concentration of phagostimulatory amino acids in the diet is too low to stimulate the aphids’ gustatory receptors adequately. These data, obtained for diet-reared aphids, give credence to the accepted interpretation of several published studies on feeding rates of plant sapsucking insects on plants. For example, the reduction in honeydew production by the pear psyllid Psylla sp. when nitrogen fertilizer is applied to the host pear trees (Pfeiffer and Burts, 1984) may be a consequence of increased nitrogen concentration in the phloem sap; and the copious amounts of dilute honeydew produced by the aphid Amphoropha agathonica on the resistant raspberry variety “Candy” (Kennedy and Schaefers, 1975) may be indicative of a low nitrogen content in ‘Candy” phloem sap. However, the responses of insects to dietary nitrogen in plants is undoubtedly influenced by the composition of amino acids in the phloem sap and other chemical and physical features of the plant. For example, honeydew production by the aphid Rhopalosiphum padi was greater on the cultivated spring oats Avena sativa with phloem sap containing 1.2-1.7 nmol amino acids mg-’ than on the wild relative A. macrostachya containing 0.5 nmol amino acids mg-’ (Weibull, 1988a, b). Potentially, there are two detrimental consequences for an insect which responds to low nitrogen diets by feeding faster. First, the food may pass more rapidly through the gut and therefore the efficiency with which the amino acids are absorbed may be reduced (digestive efficiency is not relevant to aphids because the dietary nitrogen comprises amino acids).
306
w. A.
hOSSER
This is not significant for diet-reared A. pisum, which retained more than 90 mol% of ingested amino acids on all diets [Fig. 2(a)], but it may be important for plant-reared aphids, including A. pisum which feed S-10 times faster on plants than on artificial diets (Slansky and Scriber, 1985; Douglas, unpublished). The second consequence is metabolic, including enhanced metabolic transformations associated with reduced nitrogen : carbon ratio and absolute shortfall of certain limiting amino acids on the low nitrogen diets. The metabolic consequences of feeding on low nitrogen diets are appreciable for diet-reared A. pisum, as is indicated by the markedly depressed efficiency of conversion of ingested food of aphids on diets containing 45 mM amino acids (Table 1). Slansky and Wheeler (1991) have also reported a substantial metabolic “cost” but no detectable digestive/absorptive “cost” of increased feeding rates by the velvetbean caterpillar Anticarsia gemmatalis on nutrient-poor diets. The response of chlortetracycline-treated
aphidr to
variation in dietary nitrogen
The data on diet utpake by chlortetracyclinetreated aphids provide insight into the role of the symbiotic bacteria in aphids. The conclusion that aphid weight is a major determinant of the difference in feeding rate between untreated and chlortetracycline-treated aphids is consistent with the view that the antibiotic does not have a direct deleterious effect on the aphids. This confirms the substantial structural and metabolic evidence that the primary target of the antibiotic is the bacteria (e.g. Griffiths and Beck, 1974; Douglas, 1988). The possibility that the aphids attempt to compensate for loss of the bacterial-derived nutrients by increased consumption (see Introduction) can also be discounted, at least for diet-reared A. pisum. A related issue is the effect of chlortetracycline treatment on the compensatory feeding response of aphids. The untreated and chlortetracycline-treated aphids responded similarly to dietary dilution in the range 270-135 mM amino acids, but the feeding rate of chlortetracycline-treated aphids (and not untreated aphids) declined progressively with further reduction in dietary nitrogen. As yet, the mechanisms underlying regulation of food intake by aphids are obscure, but one physiological cue that triggers feeding in other insects is declining free amino acid content of the body fluids (Abisgold and Simpson, 1988). Chlortetracycline-treated A. pisum have considerably higher free amino acid content than untreated aphids (Prosser and Douglas, 1991a) and, as a consequence, they may be less able than untreated aphids to detect variations in dietary nitrogen. Simpson and Simpson
et al.
(1990) described this effect as “jamming” of the feeding control system. Despite the reduced feeding rates of chlortetracycline-treated aphids on the diet with 89 mM amino acids, these aphids increased in weight over the experimental period at a closely similar rate to those on the diet with 202 mM amino acids (See Figs 1 and Table 1). Comparison of the efficiency of conversion of ingested food for the aphids on diets with 89 mM and 202 mM amino acids (0.45 and 0.38, respectively) suggests that the former aphids may have displayed a compensatory increase in metabolic efficiency. These data illustrates how insect growth can be influenced by a potentially complex interplay between feeding and postingestive responses (see also Simpson and Simpson, 1990). The second respect in which chlortetracycline affects A. pisum concerns the amino acid composition of the aphid honeydew. The higher concentrations of all non-essential amino acids in the honeydew of chlortetracycline-treated than untreated aphids suggests that the bacteria may enhance aphid utilization of non-essential amino acids. Furthermore, the bacteria increase the availability of essential amino acids to the aphid, to the extent that untreated aphids can use the essential amino acids arginine, histidine and tryptophan, as vehicles of nitrogen excretion; the non-essential amino acid glutamine adopts this role in chlortetracycline-treated aphids [this issue is discussed in detail by Prosser and Douglas (1991), see also Cloutier (1986)]. Together, these data are consistent with the view that the bacteria utilize nonessential amino acids and synthesize essential amino acids, which are released to the aphid tissues. Other lines of evidence for the importance of this process, known as nitrogen upgrading, in the aphid symbiosis are considered in Prosser and Douglas (1992). Implicit in the proposed role of amino acids in nitrogen excretion of aphids is that a proportion of the amino acids in the honeydew is of metabolic origin. Further evidence is that the honeydew, but not diet, contains GABA [see Fig. 3 and Prosser and Douglas (1991)] and the concentration of tryptophan is lower in the diet with 45 mM amino acids than in the honeydew of untreated aphids feeding on this diet. (The possibility that excess tryptophan in these aphids is synthesized by the bacteria is considered by Douglas and Prosser, 1992.) Thus, the amino acids in the aphid honeydew are not only those molecules which escape absorption in the gut; and accurate estimates of nitrogen assimilation efficiency of aphids cannot be obtained from comparisons of the nitrogen content of diet and honeydew. This study has more general implications for the use of chlortetracycline-treated aphids in the study of
307
Responses of the pea aphid A. pisum the aphid symbiosis. At one level, the results vindicate their use, in that the antibiotic does not cause general malaise, resulting in impaired feeding or severely reduced absorption of nutrients. However, they also point to the very widespread and diverse effects of symbiont loss on the overall biology of the insect. As the diet uptake data illustrate, chlortetracyclinetreated aphids are not simply aphids whose bacteria have been deleted. Acknowledgements--We
thank NERC, AFRC, The Royal
Society of London, British Federation of University Women and Jesus College Fund for financial support. REFERENCES
Abisgold J. D. and Simpson S. J. (1988) The effect of dietary protein levels and haemolymph composition on the sensitivity of the maxilliary palp chemoreceptors of locusts. J. exp. Biol. 135, 215-228. Cloutier C. (1986) Amino acid utilisation in the aphid Acyrthosiphon pisum infected by the parasitoid Aphidium smithi. J. Insect Physiol.
32, 263-261.
Dixon A. F. G. (1985) Aphid Ecology. Blackie & Son, Glasgow. Douglas A. E. (1988) Sulphate utilisation in an aphid symbiosis. Insect Biochem. 18, 599605. Douglas A. E. (1989) Mycetocyte symbiosis in insects. Biol. Rev. 64, 4099434. Douglas A. E. (1990) Nutritional Interactions between Myzus persicae and its symbionts. In Aphid-Plant Genotype Interactions (Eds Campbell R. K. and Eikenbary R. D.), pp. 319-328. Elsevier, Amsterdam. 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. In press. Griffiths G. W. and Beck S. D. (1974) Effects of antibiotics on intracellular symbiotes in the pea aphid Acyrthosiphon pisum.
Cell Tissue Res. 48, 287-300.
Jones B. N., Paabo S. and Stein S. (1981) Amino acid analysis and enzymatic sequence determination of peptides by an improved o-phthalaldehyde precolumn labeling procedure.. J. Liq. Chromatogr. -4, 565-586. Kennedy G. G. and Schaefers G. A. (1975) Role of nutrition in the immunity of red raspberry to Amphoropha agathon ica Hottes. Envir. Ent. 4, 115-119. McNeil1 S. and Southwood T. R. E. (1978) The role of . nitrogen in the development of insect/plant relationships. In Biochemical Aspects of Plant and Animal Coevolution (Ed. Harbome J. B.), pp. 77-98. Academic Press, New York. Pfeiffer D. G. and Burts E. C. (1984) Effect of tree fertihsation on protein and free amino acid content and feeding rate of Dear osvlla (Homootera: Pvslhdae). Envir. Ent. 13. 1487-1490. - . Presser W. A. and Douglas A. E. (1991) The aposymbiotic aphid: an analysis of chlortetracycline-treated pea aphid. I
Acyrthosiphon
pisum. J. Insect Physiol. 31, 7 13-7 19.
Prosser W. A. and Douglas A. E. (1992) A test of the hypotheses that nitrogen is recycled and upgraded in an aphid symbiosis. J. Insect Physiol. 38, 93-99. Simpson S. J. and Simpson C. L. (1990) The mechanisms of nutritional compensation by phytophagous insects. In Focus on Insect/Plant Interactions (Ed. Bernays E. A.), Vol. 2, pp. 11 I-160. CRC Press, Orlando, FL. Slansky F. and Scriber J. M. (1985) Food consumption and utilisation. In Comprehensive Insect Physiology, Biochemistry and Pharmacology (Eds Kerkut G. A. and Gilbert L. I.), Vol. 4, pp. 87Ii63. Pergamon Press, Oxford. Slanskv F. and Wheeler G. S. (1991) Food comoensation and utilisation responses to dietary dilution with cellulose and water by velvetbean caterpillars Anticarsia gemmatalis.
Physiol. Ent. 16, 99-116.
Weibull J. H. W. (1988a) Free amino acids in the phloem sap from oats and barley resistant to Rhopalosiphum padi. Phytochemistry
21, 2069-2072.
Weibull J. H. W. (1988b) Resistance in the wild crop relatives Avena macrostachya and Hordeum bogdani to the aphid pest Rhopalosiphum padi. Entomologia exp. appl. 48, 2255232.
Wright J. P., Fisher D. B. and Mittler T. E. (1985) Measurement of aphid feeding rates of artificial diets using ‘H-inulin. Entomologia exp. appl. 31, 9-11.
1992 Copyright
0
0022-1910/92 $5.00 + 0.00 1992 Pergamon Press Ltd
HOW AN APHID (ACYRTHOSIPHON PISUM) SYMBIOSIS RESPONDS TO VARIATION IN DIETARY NITROGEN W. A. PROSSER,S. J. SIMPSONand A. E. DOUGLAS* Department of Zoology, University of Oxford, South Parks Road, Oxford OX1 3PS, U.K. (Received 16 October 1991)
Abstract-This study concerns the responses of the pea aphid Acyrthosiphon pisum to chemically-defined diets containing different concentrations of nitrogen (45-270 mM amino acids) and how these responses are influenced by the antibiotic chlortetracycline, which selectively disrupts the aphids’ symbiotic bacteria. The chlortetracycline-treated aphids ingested less diet than untreated controls, and the difference between the two groups of insects could be ascribed largely to the greater size of the untreated aphids. Diet uptake increased with decreasing dietary amino acid concentration, in the range 202-89 mM for untreated aphids and 202-135 mM for chlortetracycline-treated aphids. This compensatory feeding response contributed to the close similarity of aphid growth rates on these diets. The honeydew of untreated and chlortetracycline-treated aphids contained 6-10 and 10-30 mol% of the dietary amino acid concentration, respectively. and the values (in percentage terms) did not vary consistently with dietary nitrogen. The concentration of non-essential amino acids, but not essential amino acids, was significantly higher in the honeydew of chlortetracycline-treated aphids than in untreated aphids’ honeydew. The selective utilization of non-essential amino acids by untreated aphids is consistent with the hypothesis that the symbiotic bacteria utilize non-essential amino acids as a nitrogen source for essential amino acid synthesis, a process known as nitrogen upgrading. Key Word Index: Aphid; symbiosis; mycetocyte; Acyrthosiphon pisum; bacteria
INTRODUCHON
Ecologists have, for many years, considered nitrogen as a major determinant of the abundance and population increase of plant sap-sucking insects of the order Homoptera (McNeil1 and Southwood, 1978). For example, the faster growth rates of aphids on forbs than trees and the evolution of complex aphid life cycles involving migration from trees to forbs in the summer have been ascribed to a higher nitrogen content in the phloem sap of forbs than trees (Dixon, 1985). However, the ecologists’ appreciation of the significance of nitrogen has not been matched by an understanding among insect physiologists of how these insects respond to variations in dietary nitrogen. Many phytophagous insects of the orders Lepidoptera and Orthoptera are known to respond to a reduction in dietary nitrogen by increased consumption of food and/or by enhanced retention of ingested *To whom all correspondence
should be addressed.
nitrogen (Slansky and Scriber, 1985; Simpson and Simpson, 1990). As yet, the evidence for comparable processes occurring in Homoptera is largely anecdotal, e.g. tree-feeding aphids produce more honeydew than forb-feeders. An additional parameter to consider in the Homoptera is their symbiotic bacteria, located in specialized insect cells, mycetocytes, in the haemocoel (Douglas, 1989). These bacteria have been implicated in the nitrogen nutrition of aphids, particularly by the provision of essential amino acids (Douglas, 1990), and one would therefore anticipate some interplay between the physiological responses of the insect and metabolic responses of the bacteria to variations in dietary nitrogen. For example, do aphids lacking functional bacteria attempt to compensate for the loss of their bacteria-derived amino acids by ingesting larger volumes of diet? The study reported here is a first detailed consideration of these issues in phloem sap-sucking insects. The principal nitrogenous compounds in phloem are free amino acids. The experiments were conducted on 301
302
W. A. TOSSER et al.
larvae of the pea aphid Acyrthosiphon pisum, introduced to chemically-defined diets of uniform amino acid composition but variable concentration, so that the effects of nitrogen quantity could be assessed independently of nitrogen quality. This is undoubtedly a simplified experimental system, but it enables the basic physiological principles underlying the aphids’ responses to be investigated. MATERIALS AND METHODS
The experiments were conducted on fourth-instar larvae of the pea aphid A. pisum clone 0X-2, derived from a long-standing clonal culture of virginoparae (Prosser and Douglas, 1992), according to the following protocol. Within 24 h of deposition from apterous adults, groups of 10 larvae were transferred to chemically-defined diet containing 135 mM amino acids and 550 mM sucrose [diet composition and procedures are described in Prosser and Douglas (1991)]. The bacterial symbionts in half of the aphids were disrupted by treatment with the antibiotic chlortetracycline, at 50 pg ml-’ diet for the first 5 days of larval development (Douglas, 1988). The larvae were maintained at 20°C with 18 h light-6 h dark regime and 75% r.h. They developed to fourth larval instar on the sixth day and, on the seventh day, they were transferred to chlortetracyclinefree diets containing 270, 202, 13.5, 89 or 45 mM amino acids. At this age, the chlortetracyclinetreated aphids were approximately half the weight of untreated aphids. All experiments were of 24 h duration. Diet uptake was assayed by a modification of the method of Wright et al. (1985) using [‘4C]methylated inulin (Sigma) at specific activity 10 PCi per 0.1 ml
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200
300
acid concentration ImM) of diet
Fig. 1. Diet uptake by untreated (solid symbols) and (open symbols) fourth-instar A. pisum maintained on diets containing 45-270mM amino acids for 24 h. The aphids were weighed at the start and end of the 24 h experiment, and the average of these two values was used as covariate in the analysis of covariance of diet uptake data: aphid weight (covariate) F,,@= 69.64, P < 0.001; diet composition F4.@= 2.45, P > 0.05; chlortetracycline treatment F,,@ = 1.81, P > 0.05; interaction between diet composition and chlortetracycline treatment chlortetracycline-treated
F4,@= 2.72, 0.01 < P < 0.05:
diet (see Douglas, 1988). The honeydew was collected on GF/C glass fibre filters (Whatman), which were then dried and shaken with Optiscint Hisafe-l scintillant (LKB Ltd). The radioactivity was counted in a LKB 1219 Rackbeta scintillation counter with preset 14C window. The honeydew produced by each cage of aphids was collected on a sheet of tinfoil. The samples were analysed by reversed-phase high performance liquid chromatography after derivatization with o-phthalaldehyde (Jones et al., 1981) as described in detail by Prosser and Douglas (1991). The aphids were weighed individually on an ElmerParker AD4 electrobalance to the nearest pg.
RESULTS
Diet uptake
Each fourth-instar larva of A. pisum ingested between 0.1 and 1.O~1 of diet over 24 h after introduction to the test diets containing amino acids at the total concentration of 45-270mM (Fig. 1). The aphids, whose symbiotic bacteria had been disrupted by chlortetracycline treatment during early larval development, fed more slowly than the untreated control aphids. This was largely a consequence of the small size of the treated aphids (see also Prosser and Douglas, 1991) as is indicated by the non-significance of the main effect, chlortetracycline treatment, once aphid weight is included in the analysis as a covariate (see legend to Fig. 1). The feeding rate of untreated aphids increased by 6&80% with decreasing dietary amino acid concentration from 202-89 mM, but no further increase was evident on the diet containing 45 mM amino acids. Diet consumption by chlortetracycline-treated aphids also increased with declining dietary nitrogen in the range 270-135 mM, but declined progressively with further reduction in dietary nitrogen. This difference between untreated and chlortetracycline-treated aphids is statistically significant (see interaction term in analysis, legend to Fig. 1). Amino acid content of aphid honeydew
The total concentration of protein amino acids in the honeydew produced by the aphids lay in the range 3-30mM. On all test diets, the honeydew of untreated aphids contained less than lOmol% of the amino acids in the diet. The honeydew of chlortetracycline-treated aphids had higher amino acid contents, representing l&20 and 30mol% on the dietary amino acid contents on diets containing 270-89 mM and 45 mM amino acids, respectively [Fig. 2(a)],
Responses of the pea aphid A. pisum This difference between the amino acid content of honeydew from untreated and chlortetracyclinetreated aphids is mediated by the differences in their non-essential amino acid content. The total essential amino acid content of their honeydew was closely similar [see Fig. 2(b)], but the non-essential amino acids represented 52-57 moi% of the amino acids in the honeydew of chlortetracycline-treated aphids but only 7-22 mol% of the amino acids of untreated aphid honeydew. Since the diet contains non-essential and essential amino acids in equal proportions, these data indicate that untreated aphids selectively utilize non-essential amino acids in the diet. The concentration of the individual amino acids in the honeydew is shown in Fig. 3. Every non-essential amino acid conformed to the pattern of, first, increasing concentration with increasing dietary concentration and, second, higher concentrations in the honeydew of chlortetracycline-treated than untreated aphids. The glutamine content of honeydew from
,
I
0
100 Total om~no ac~d concentration
200 (mM)
300 of diet
(b) 20 d 8
_A
Total om~m and ccncentrotlon
(mM)
of diet
Fig. 2. The amino acid concentration in honeydew produced by untreated (solid symbols) and chlortetracycline-treated (open symbols) fourth-instar A. pisum maintained on diets containing 4%270mM amino acids for 24 h. (a) Total
amino acid concentration, expressed as percentage of amino acid concentration in the diet. (b) Concentration of essential (squares) and non-essential (circles) amino acids. The F values for analysis of variance conducted on essential amino acid concentration are: diet composition F, m = 9.05. P < O.OOl;chlortetracycline treatment;,, = 1.91,‘j > 0.05; interaction F,.._, m = 2.71. 0.01 < P < 0.05. Euuivalent values for a separate analysis of non-essential amino acids are: chlortetracycline treatment F,,, = 6.71, P c 0.001; diet composition F,., = 155.63, P < 0.001; interaction F4,* = 8.90, P < 0.001.
303
chlortetracycline-treated aphids was exceptionally high, representing 22-27mol% of the total amino acid content; glutamine accounted for less than 2 mol% of untreated aphid honeydew. The effect of chlortetracycline treatment on the concentration of individual essential amino acids in the honeydew was more complex. By visual inspection of the data, the amino acids could be divided into three groups:
(a) The sole amino acid at closely-similar concentrations in the honeydew of untreated and chlortetracycline-treated aphids was arginine, which varied from approx. 5 mM in aphids on diets containing 270 mM amino acids to 2 mM on diets with 45 mM amino acids. However, because the total amino acid concentration was higher in the honeydew of chlortetracycline-treated arginine aphids, represented 30-50 and 14-18 mol% of the total honeydew amino acids from untreated and chlortetracycline-treated aphids, respectively. (b) The amino acids at higher concentration in the honeydew of untreated than chlortetracycline-treated aphids were tryptophan. histidine and phenylalanine. Tryptophan represented 20-23 and 2-5 mol% of the amino acids in the honeydew of untreated and chlortetracycline-treated aphids, respectively, and equivalent values for histidine are 7-19 and 3-5 mol%, respectively. Tryptophan is a minor amino acid in the diet (accounting for 2mol% of the amino acids). The honeydew of untreated aphids on diet containing 45 mM amino acids is 0.88mM, just greater than the dietary concentration (0.8 mM). The final amino acid in this group, phenylalanine, is the amino acid at lowest concentration in the honeydew of chlortetracycline-treated aphids (where it is invariably x0.1 mM); and, together with valine and isoleucine, it is also a minor component of untreated aphid honeydew. On every test diet, phenylalanine was 25-500% higher in the honeydew of untreated than chlortetracycline-treated aphids. (c) The remaining essential amino acids, namely isoleucine, leucine, lysine, methionine and threonine, were at higher concentrations in the honeydew of chlortetracycline-treated than untreated aphids.
W. A. PROSSERet al.
304
Growth and eficiency
of food utilization
The aphids on all test diets increased in weight over the 24 h experiment. Consistent with all previous studies linking the disruption of symbionts
with reduced aphid growth (Douglas, 1989), the relative growth rate of untreated aphids was significantly greater than that of chlortetracyclinetreated aphids (Table 1). The concentration of dietary nitrogen also influenced growth, especially of
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Fig. 3. The concentration (mean f SE) of each amino acid in the honeydew of utreated (a) and chlortetracycline-treated (b) fourth-instar A. pisum maintained on diets containing 270mM (m), 202mM (a),
135 mM (0),_89 mM (m) and 45 mM (0) amino acids for 24 h.
GABA
Responses of the pea aphid A. pisum
305
Table 1. Performance of fourth-instar A. pisum larvae maintained on diets of different amino acid concentrations for 24 h RGR
Dietary amino acid concentration (mW
Untreated aphids
270 202 135 89 45
0.45 + 0.22 f 0.3 1 + 0.26 k 0.14 *
0.045 0.068 0.036 0.036 0.037
EC1
Chlortetracycline treated aphids 0.37 f 0.099 0.13 &-0.075 0.16 + 0.039 0.12 & 0.038 0.05 + 0.022
Untreated aphids 0.78 + 0.53 f 0.59 * 0.38 + 0.22 +
0.066 0.072 0.053 0.044 0.025
Chlortetracycline treated aphids 0.59 + 0.088 0.38 _+0.109 0.38 + 0.063 0.47 + 0.172 0.13 +_0.056
Values of mean k SE are shown. RGR: relative growth rate (mg mg-’ aphid fresh weight) over the 24 h period on test diets. ECI: efficiency of conversion of ingesta (mg dry weight increase of aphids per mg dry weight of diet ingested). The results of analysis of variance for RGR are: diet composition F4,57= 9.75, P < 0.001; chlortetracycline treatment F, 57= 12.03, P i 0.001; interaction Fd r, = 0.71, P > 0.05. Values for the analysis for EC1 are: diet composition Fd ,; = 11.10, P < 0.001; chlortetracychne treatment F,.57= 4.94, 0.01 > P > 0.001; mteraction F4.5,= 1.43, P > 0.05.
chlortetracycline-treated three-fold
difference
aphids, as is indicated between
by the
the relative growth rate
aphids on diets containing 45 and 720 mM amino acids, and IO-fold difference in relative growth rate of chlortetracycline-treated aphids on these diets. However, no consistent variation in this growth rate with amino acid concentration in the range 89-202 mM was evident, suggesting that both untreated and chlortetracycline-treated aphids compensated for dietary nitrogen in this concentration range. The efficiency with which ingested food was converted into aphid biomass was derived from the weight gain of aphids and their diet uptake over 24 h. Most of the values of efficiency of conversion of ingested food were in the range 0.38-0.6pg pg-‘, with a single high value of 0.78 pg pg-’ for untreated aphids on diet with 270mM amino acids and low values of 0.22 and 0.13 pg pg-’ for untreated and chlortetracycline-treated aphids, respectively, on the 45mM diet (Table 1). As with relative growth rate, the efficiency of conversion of ingested food of aphids was significantly depressed by chlortetracycline-treatment. of untreated
DISCUSSION
Response of untreated aphid
(containing symbiotic
bacteria) to dietary nitrogen
The present study includes the first unambiguous demonstration that a plant sap-sucking insect responds to variations in dietary nitrogen by increased consumption. This compensatory feeding response substantially mitigated the deleterious effects of low nitrogen diets (in the range 89-202 mM amino acids) on the growth of larval A. pisum (Table 1). However,
at the lowest concentration tested (45 mM), the feeding rate of untreated aphids did not increase and the aphid growth rate was reduced substantially. Perhaps on this diet, the absolute concentration of phagostimulatory amino acids in the diet is too low to stimulate the aphids’ gustatory receptors adequately. These data, obtained for diet-reared aphids, give credence to the accepted interpretation of several published studies on feeding rates of plant sapsucking insects on plants. For example, the reduction in honeydew production by the pear psyllid Psylla sp. when nitrogen fertilizer is applied to the host pear trees (Pfeiffer and Burts, 1984) may be a consequence of increased nitrogen concentration in the phloem sap; and the copious amounts of dilute honeydew produced by the aphid Amphoropha agathonica on the resistant raspberry variety “Candy” (Kennedy and Schaefers, 1975) may be indicative of a low nitrogen content in ‘Candy” phloem sap. However, the responses of insects to dietary nitrogen in plants is undoubtedly influenced by the composition of amino acids in the phloem sap and other chemical and physical features of the plant. For example, honeydew production by the aphid Rhopalosiphum padi was greater on the cultivated spring oats Avena sativa with phloem sap containing 1.2-1.7 nmol amino acids mg-’ than on the wild relative A. macrostachya containing 0.5 nmol amino acids mg-’ (Weibull, 1988a, b). Potentially, there are two detrimental consequences for an insect which responds to low nitrogen diets by feeding faster. First, the food may pass more rapidly through the gut and therefore the efficiency with which the amino acids are absorbed may be reduced (digestive efficiency is not relevant to aphids because the dietary nitrogen comprises amino acids).
306
w. A.
hOSSER
This is not significant for diet-reared A. pisum, which retained more than 90 mol% of ingested amino acids on all diets [Fig. 2(a)], but it may be important for plant-reared aphids, including A. pisum which feed S-10 times faster on plants than on artificial diets (Slansky and Scriber, 1985; Douglas, unpublished). The second consequence is metabolic, including enhanced metabolic transformations associated with reduced nitrogen : carbon ratio and absolute shortfall of certain limiting amino acids on the low nitrogen diets. The metabolic consequences of feeding on low nitrogen diets are appreciable for diet-reared A. pisum, as is indicated by the markedly depressed efficiency of conversion of ingested food of aphids on diets containing 45 mM amino acids (Table 1). Slansky and Wheeler (1991) have also reported a substantial metabolic “cost” but no detectable digestive/absorptive “cost” of increased feeding rates by the velvetbean caterpillar Anticarsia gemmatalis on nutrient-poor diets. The response of chlortetracycline-treated
aphidr to
variation in dietary nitrogen
The data on diet utpake by chlortetracyclinetreated aphids provide insight into the role of the symbiotic bacteria in aphids. The conclusion that aphid weight is a major determinant of the difference in feeding rate between untreated and chlortetracycline-treated aphids is consistent with the view that the antibiotic does not have a direct deleterious effect on the aphids. This confirms the substantial structural and metabolic evidence that the primary target of the antibiotic is the bacteria (e.g. Griffiths and Beck, 1974; Douglas, 1988). The possibility that the aphids attempt to compensate for loss of the bacterial-derived nutrients by increased consumption (see Introduction) can also be discounted, at least for diet-reared A. pisum. A related issue is the effect of chlortetracycline treatment on the compensatory feeding response of aphids. The untreated and chlortetracycline-treated aphids responded similarly to dietary dilution in the range 270-135 mM amino acids, but the feeding rate of chlortetracycline-treated aphids (and not untreated aphids) declined progressively with further reduction in dietary nitrogen. As yet, the mechanisms underlying regulation of food intake by aphids are obscure, but one physiological cue that triggers feeding in other insects is declining free amino acid content of the body fluids (Abisgold and Simpson, 1988). Chlortetracycline-treated A. pisum have considerably higher free amino acid content than untreated aphids (Prosser and Douglas, 1991a) and, as a consequence, they may be less able than untreated aphids to detect variations in dietary nitrogen. Simpson and Simpson
et al.
(1990) described this effect as “jamming” of the feeding control system. Despite the reduced feeding rates of chlortetracycline-treated aphids on the diet with 89 mM amino acids, these aphids increased in weight over the experimental period at a closely similar rate to those on the diet with 202 mM amino acids (See Figs 1 and Table 1). Comparison of the efficiency of conversion of ingested food for the aphids on diets with 89 mM and 202 mM amino acids (0.45 and 0.38, respectively) suggests that the former aphids may have displayed a compensatory increase in metabolic efficiency. These data illustrates how insect growth can be influenced by a potentially complex interplay between feeding and postingestive responses (see also Simpson and Simpson, 1990). The second respect in which chlortetracycline affects A. pisum concerns the amino acid composition of the aphid honeydew. The higher concentrations of all non-essential amino acids in the honeydew of chlortetracycline-treated than untreated aphids suggests that the bacteria may enhance aphid utilization of non-essential amino acids. Furthermore, the bacteria increase the availability of essential amino acids to the aphid, to the extent that untreated aphids can use the essential amino acids arginine, histidine and tryptophan, as vehicles of nitrogen excretion; the non-essential amino acid glutamine adopts this role in chlortetracycline-treated aphids [this issue is discussed in detail by Prosser and Douglas (1991), see also Cloutier (1986)]. Together, these data are consistent with the view that the bacteria utilize nonessential amino acids and synthesize essential amino acids, which are released to the aphid tissues. Other lines of evidence for the importance of this process, known as nitrogen upgrading, in the aphid symbiosis are considered in Prosser and Douglas (1992). Implicit in the proposed role of amino acids in nitrogen excretion of aphids is that a proportion of the amino acids in the honeydew is of metabolic origin. Further evidence is that the honeydew, but not diet, contains GABA [see Fig. 3 and Prosser and Douglas (1991)] and the concentration of tryptophan is lower in the diet with 45 mM amino acids than in the honeydew of untreated aphids feeding on this diet. (The possibility that excess tryptophan in these aphids is synthesized by the bacteria is considered by Douglas and Prosser, 1992.) Thus, the amino acids in the aphid honeydew are not only those molecules which escape absorption in the gut; and accurate estimates of nitrogen assimilation efficiency of aphids cannot be obtained from comparisons of the nitrogen content of diet and honeydew. This study has more general implications for the use of chlortetracycline-treated aphids in the study of
307
Responses of the pea aphid A. pisum the aphid symbiosis. At one level, the results vindicate their use, in that the antibiotic does not cause general malaise, resulting in impaired feeding or severely reduced absorption of nutrients. However, they also point to the very widespread and diverse effects of symbiont loss on the overall biology of the insect. As the diet uptake data illustrate, chlortetracyclinetreated aphids are not simply aphids whose bacteria have been deleted. Acknowledgements--We
thank NERC, AFRC, The Royal
Society of London, British Federation of University Women and Jesus College Fund for financial support. REFERENCES
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Dixon A. F. G. (1985) Aphid Ecology. Blackie & Son, Glasgow. Douglas A. E. (1988) Sulphate utilisation in an aphid symbiosis. Insect Biochem. 18, 599605. Douglas A. E. (1989) Mycetocyte symbiosis in insects. Biol. Rev. 64, 4099434. Douglas A. E. (1990) Nutritional Interactions between Myzus persicae and its symbionts. In Aphid-Plant Genotype Interactions (Eds Campbell R. K. and Eikenbary R. D.), pp. 319-328. Elsevier, Amsterdam. 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. In press. Griffiths G. W. and Beck S. D. (1974) Effects of antibiotics on intracellular symbiotes in the pea aphid Acyrthosiphon pisum.
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Wright J. P., Fisher D. B. and Mittler T. E. (1985) Measurement of aphid feeding rates of artificial diets using ‘H-inulin. Entomologia exp. appl. 31, 9-11.