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J. Insect Physid. Vol. 41, No. 1, pp. 71-75, 1995 Copyright 0 1995 Elsevier Science Ltd

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Response of the Pea Aphid (Acyrthosiphon pisum) to Variation in Dietary Levels of Sugar and Amino Acids: the Significance of Amino Acid Quality S. J. SIMPSON,*$ J. D. ABISGOLD,* A. E. DOUGLAS?

Received 28 March 1994; revised 15 June 1994

Growth, survivorship and food intake were measured for newborn pea aphids maintained on one of 25 chemically-defined diets varying in sucrose and amino acid content (200-1000 and 50-250 mM, respectively), with amino acids comprising 20 : 80 essential/non-essentials (e : n). Survivorship varied positively with dietary amino acid concentration, while dry weight growth was strongly affected by concentration of amino acids and sucrose, and also by the ratio of sucrose to amino acids (s: aa). Peak performance was achieved on the diet containing 800 mM sucrose and 200 mM amino acids. This was a lower s:aa ratio than that which supported maximal growth in previous experiments for aphids reared on diets containing 50:50 e:n. Comparison of plots for final dry weight against s:aa ratio indicated that the 20 : 80 e: n amino acid mixture supported growth ca. 60% of that on the 50 : 50 e: n mix. When the scaling of the s: aa ratio was adjusted accordingly, the plots for 20:80 and 50:50 e: n converged in all but elevation; a difference which represented the extra growth costs of feeding on 20:80 e: n diets. Analysis of diet consumption showed that these costs were associated with, and perhaps attributable to, inhibition of compensatory feeding.

Aphid Acyrthosiphon pisum Nutrition Feeding Symbiosis

INTRODUCTION

Recent studies on insects have emphasized the mutli- dimensionality of nutritional homeostasis and have pro- vided a framework for integrating functional and mechanistic aspects of feeding behaviour (Raubenheimer and Simpson, 1993; Simpson and Raubenheimer, 1993a, b). In an earlier study we have used this approach to investigate sucrose and amino acid regulation in first-instar pea aphids, Acyrthosiphon pisum (Harris) (Abisgold et al., 1994). Aphids were tested on 31 chemi- cally-defined diets varying in sucrose and amino acid concentration. Performance criteria (size, survivorship and body composition) were used to establish the pos- ition in nutrient space of functional “targets” for intake and growth. The behavioural and physiological mechan- isms used by aphids to approach these targets were investigated by reference to the geometry of arrays of sucrose versus amino acid intake and growth.

*Department of Zoology and University Museum, University of Oxford, South Parks Road, Oxford OX1 3PS and TDepartment of

Biology, University of York, York, England. $To whom correspondence should be addressed.

In such geometric arrays, amino acid consumption was represented as a single, compound axis (as has also been the case for protein in studies on chewing phyto- phages). It would be expected, however, that the position of the point of intake (in nutrient space) leading to maximal performance will vary according to the amino acid composition of the diet. This is because amino acids differ in nutritional value, and the utility of each amino acid depends on its concentration relative to that of others in the diet (see Simpson and Raubenheimer, 1993a). The geometric framework provides a powerful means of investigating these effects of nitrogen quality.

In the previous study (Abisgold et al., 1994) we maintained first-instar pea aphids on diets containing an amino acid mixture comprised of equal proportions of essential and non-essential amino acids, and found that performance was maximal on a diet containing 600 mM sucrose and 75 mM amino acids. A ratio which is more typical of host-plant phloem sap is 20: 80 essential to non-essential amino acids (e: n) (Sasaki et al., 1990; Douglas, 1993). We predicted that providing aphids with such a phloem-based mixture would result in the per- formance peak moving towards a lower ratio of sucrose to amino acids (Abisgold et al., 1994). The aim of the

IP 41,1--D 71

12 S. J. SIMPSON et al.

present paper was to investigate this prediction quanti- tatively, and thereby to begin to explore the fundamental issues of scaling in nutritional responses to dietary amino acids (Simpson and Raubenheimer, 1993a).

MATERIALS AND METHODS

Experimental insects came from a long-term culture of Acyrthosiphon pisum, clone 0X-2, reared on Vicia faba var. The Sutton, as described in Prosser and Douglas (1991). Newborn larvae were transferred to chemically- defined diets and kept under LD 16 : 8 at 19-21 “C and 75% RH. Foods were based on diet B in Prosser and Douglas (1992), formulated to contain an amino acid profile similar to that of phloem sap in V. faba (Sasaki et al., 1990). A total of 25 diets was tested, representing all combinations of 200, 400, 600, 800 and 1000 mM sucrose with 50, 100, 150, 200 and 250 mM amino acids. Accordingly, sucrose : amino acid (s : aa, mol/mol) ratios ranged from 0.8 : 1 to 20: 1. Diets were presented as described in Prosser and Douglas (1991).

The experiment followed the protocol established in Abisgold et al. (1994), which used aphids from the same clone, reared in the same manner. Duplicates of 10 aphids were maintained on each diet for 48 h, after which the number of surviving insects was recorded. The dry weight of individual survivors was established by weighing them to the nearest pg on an Elmer-Parker AD4 electrobalance, following oven drying at 50°C for 24 h.

The volume of diet ingested over 48 h was measured in a separate experiment by collecting and counting the radioactivity in honeydew produced by aphids fed diets labelled with i4C-methylated inulin (as described in Abisgold et al., 1994). Three replicates of 10 aphids were run for each diet formulation, with the amount ingested per aphid being estimated as the total volume consumed per group divided by the number of surviving aphids. Amounts of amino acids and sucrose consumed were calculated from the volume of diet ingested.

RESULTS

The performance of aphids varied with dietary nutri- ents. Survivorship was significantly influenced by the amino acid concentration of the diet (ANOVA, F4,25 = 14.01, P -C O.OOOl), but not its sucrose concen- tration (F4,?, = 1.35, P = 0.28) or the ratio of sucrose to amino acids (as evidenced by the lack of significant interaction between sucrose and amino acids, F - 1.29, P = 0.28) [Fig. l(a)]. Final dry weight was 16,25 -

strongly affected by dietary amino acid content (F4,3,,9 = 17.6, P < O.OOOl), sucrose concentration

- 108.8,

E,.,09 -

P < 0.00001) and s:aa ratio

,63W = 5.73, P < 0.0001) [Fig. l(b)]. Final dry weight rose sharply as the s: aa ratio in-

creased from 0.8 : 1 to 4: 1, then declined as the ratio

increased further (Fig. 2). The graph in Fig. 2 conflates the main effects of the two nutrients with that due to their ratio, as is evident for the additional effect of concentration of nutrients within a given ratio. For the ratio 4: 1 s: aa, maximal final dry weight (70.7 pg) was achieved by aphids fed the diet containing 800 : 200 s: aa and declined as concentrations increased or decreased relative to this (24.4, 40.1, 65.6 and 55.8 pg for diets 50 : 200, 100 : 400, 150 : 600 and 250 : 1000, respectively) (Fig. 2).

For the purposes of comparison with the data from Abisgold et al. (1994), curve fitting procedures were used to summarize the relationship between final dry weight and dietary s : aa ratio. Both the present and the earlier data sets were subjected to log-transformation of the ratio s: aa in the diet. This resulted in the relationship plotted in Fig. 2 (and its more extensive equivalent in Abisgold et al., 1994) becoming bell shaped rather

@ .- .z 2 C? u) P

P s

FIGURE 1. Three dimensional plots for two measures of performance

lpercentage of aphids surviving (a), and final dry weight (b)], against the sucrose and amino acid concentration of the diet.

SUCROSE AND AMINO ACID REGULATION IN PEA APHIDS 13

80 ,

0 5 10 15 20 25

Ratio sucrose:total amino acids in diet (mM:mM)

FIGURE 2. Variation in final dry weight (mean 5 SE) of aphids

against the ratio of sucrose to total amino acids in the diet. The dotted

line shows the mean initial dry weight of a sample of newborn aphids.

than skewed. An appropriate model, of the form y = exp( - ((x - rn)/~)~/2)/((2h)~~~s), was then fitted to each set of data using iterative curve-fitting procedures in SigmaPlot. The parameters m, h and s represent the point of maximum final weight, the elevation of the curve and the shape of the curve, respectively. Calculated parameter values, their asymptotic standard errors and coefficients of variation are listed in Table 1. The two curves were similar in shape but differed in m and h [Fig. 3(a)]. Aphids fed the 20: 80 e: n mixture reached a maximum weight at a s : aa ratio which was about 60% of that for insects fed the 50: 50 e: n mix. Additionally, aphids were consistently smaller after 48 h on diets containing the 20: 80 rather than the 50 : 50 e : n mixture, despite having begun the experiment at similar birth weights (26.4 f 1.80 ,ug dry wt for 20:80 and 30 f 2.08 pg for 50:50 e:n).

A sub-set of 12 diets which best supported growth and survival was chosen for investigating diet intake (as in Abisgold et al., 1994). These were the combinations of 150,200 and 250 mM amino acids with 400,600,800 and 1000 mM sucrose. Analysis of volume of diet ingested showed no response to amino acid concentration

80

I (a) 50x50

80

70

20

10

l-

1 lb) I’

-0.2 0.0 0.2 0.4 0.8 0.8 1.0 1.2 1.4 1.8 1.8

Log ratio sucrose:total amino acids in diet (mM:mM)

r

0.0

-

-

:

- I I I I I I I I 1 -I

0.2 0.4 0.8 0.8 1.0 1.2 1.4 1.8 1.8 2.0

Log ratio sucrose:“usable” amino acids in diet (mM:mM)

FIGURE 3. Fitted curves for mean dry weight of aphids against the log-transformed ratio of sucrose to amino acids in the food. Curves are

fitted to data from the present experiment (20 : 80 e: n amino acids) and

from Abisgold et 01. (1994) (SO: 50). (a) Shows curves when the x-axis

is log (s: total aa), while in (b) the data are plotted against

log(s:“usable” aa) (see text and also Table 1).

TABLE 1. Parameters from non-linear regressions for final dry weight of aphids against

log-transformed s:aa ratio in the diet

Amino acid

composition

of the diet m

Coefficient:

Value & SE (CV%)

h s

20:80 0.61 f 0.036 (5.8%) 5.6 k 0.09 x 1O-4 (16.3%) 0.50 + 0.049 (10.0%)

50:50 0.85 k 0.036 (4.2%) 2.6 f 0.04 x 1O-4 (16.0%) 0.60 + 0.058 (9.6%)

Equation:

y = exp( - ((x-m)/s)2/2)/((2h)0,5s)

where y, dry weight growth; x, log s:aa ratio in food; m, point of maximum weight gain; h, elevation of the curve; s, shape of the curve.

SE, asymptotic standard error, CV%, coefficient of variation (SE x loo/parameter value).

74 S. J. SIMPSON et al.

0.35

0.30

I

; 0.25 ‘j; % .E

f 0.20

3

0.15

0.10

T T

I I I I

400 600 800 1000

Sucrose concentration of diet (mM)

FIGURE 4. Mean & SE volume ingested by aphids on diets varying

in sucrose and amino acid content. The curved line is the best-fitting

quadratic model for intake versus sucrose concentration (r2 = 0.47).

Data are presented for the 12 diets which best supported survival and

growth. Numbers next to the means represent amino acid concen-

tration.

(ANOVA, F2,24 = 0.52, P = 0.60), a significant response to level of dietary sucrose (F3,24 = 3.8, P = 0.02), and no significant interaction between the nutrients (F6,24 = 1.5, P = 0.22). The effect of sucrose was due to decreased consumption of diets containing 1000 mM sucrose rela- tive to lower concentrations (Fig. 4). Intake of sucrose versus amino acids is plotted on a 2-dimensional nutrient plane in Fig. 5.

90

80 T

0 2 4 8 8 10 12

Amino acid intake (pg)

FIGURE 5. Plot of the intake array (means f bivariate SEs for the 12 best diets) on the sucrose-amino acid nutrient plane. Numbers 1-12

refer to the following dietary s: aa ratios: 1000: 150, 800: 150,

1000:200, 600: 150, 1000:250, 800:200 (performance peak achieved for this point of intake), 800 : 250,600 : 200,400 : 150,600 : 250,400 : 200,

and 400 : 250.

DISCUSSION

The prediction of Abisgold et al. (1994) that pea aphids fed diets containing a phloem-based amino acid mixture would achieve peak performance on a lower sucrose to amino acid ratio than insects reared on diets rich in essential amino acids was clearly met by the present results. The assumption made is that dry weight growth is commensurate with fitness. Although not necessarily true for other insects (e.g. locusts, see Raubenheimer and Simpson, 1993) this is likely to be the case for larval aphids, especially given their nutri- tional investment in embryos (Dixon et al., 1982).

Data summarized in Fig. 3a indicate that the 20: 80 e : n amino acid mixture had an overall utility for growth only 60% of that for the 50: 50 e:n mix. When consid- ering growth, the utility of an amino acid mixture is defined as the proportion of ingested amino acids which is converted to growth, for levels of intake less than or equal to the combined growth target for amino acids (see Raubenheimer and Simpson, 1994 for detailed discus- sion). Utility will vary not only with the composition of the mixture, but also with changes in the biochemical capabilities of the insect, as is apparent in the pea aphid after experimental removal of its symbiotic micro- organisms (Douglas and Prosser, 1992; Prosser and Douglas, 1992; Whitehead et al., 1992).

Utility values can be derived from utilization plots based on measurements of nitrogen consumption and egestion (Raubenheimer and Simpson, 1994). Prosser et al. (1992) found that less than 10% of ingested protein amino acids appeared in the honeydew of fourth-instar pea aphids reared on diets with 50: 50 e:n. When levels in the honeydew of GABA (the other major source of excreted nitrogen) were also considered, the utility of the 50: 50 e: n mixture was about 90% for untreated aphids and around 70% for aphids whose bacterial symbionts had been removed by treatment with antibiotics. A utility of 90% is also consistent with data for first-instar aphids (assuming a growth target over the first stadium of ca 2.5 pg amino acids, see Figs 5 and 6 in Abisgold et al., 1994). It would therefore be anticipated that the utility of the 20: 80 e: n mixture is ca 50%.

Resealing the x-axis of Fig. 3(a) in accordance with these estimates unites the two data sets in all but their elevation, with the curve for 20 : 80 e : n being consistently lower by about 15 pg than that for 50: 50 e:n [Fig. 3(b)]. This quantifies the extra growth consequences associated with feeding on the 20: 80 e: n mix.

Prosser and Douglas (1992) discussed various possible explanations for the relatively poor performance of fourth-instar pea aphids on a phloem-based artifical diet. Some of these relate to the control of feeding behaviour and others to post-ingestive physiology. The foods are not under positive pressure and lack many of the physical and chemical cues associated with the host plant. Also, bacterial symbionts may not thrive in aphids reared on chemically-defined diets. Such disadvantages could require that artificial foods contain a better

SUCROSE AND AMINO ACID REGULATION IN PEA APHIDS 75

balanced mixture of amino acids for aphids to perform well.

If bacterial symbionts in diet-reared aphids do not function as effectively as in aphids feeding on plants, then to this list of disadvantages might be added the metabolic costs of removing unused non-essential amino acids from the body, and the “jamming” by such amino acids of feeding control systems, leading to inhibition of compensatory feeding responses in aphids fed diets with 20 : 80 e : n (Simpson and Simpson, 1990; Simpson and Raubenheimer, 1993b).

It is certainly the case that compensatory responses were considerably less apparent in the present exper- iment than in the earlier work with the 50: 50 e: n mixture. Aphids on the subset of diets which best supported growth and survivorship increased consump- tion with dilution of dietary sucrose. However, while insects provided with the 50: 50 e: n mix responded across the full range of concentrations by increasing intake linearly from 22 ,~I/48 h at 1000 mM sucrose to 37 ~1 at 400 mM, aphids on the phloem-based mixture only increased ingestion with dilution from 1000 to 800 mM sucrose (18 to 25 ,~1/48 h), after which intake reached a plateau. It seems as if compensatory feeding for sucrose (and also amino acids) was inhibited when the diet contained a phloem-based mixture of amino acids.

Because of the limited compensatory response shown to variation in dietary nutrients, the array of sucrose versus amino acid intakes was not consistent with the behavioural rule “closest distance optimization” (i.e. move to the geometrically closest point in nutrient space to the intake target), whatever the scaling of the axes. This differs markedly from the pattern found in the experiments with 50 : 50 e : n (Abisgold et al., 1994), and also probably does not reflect the response of aphids on plants (see discussion in Prosser and Douglas, 1992).

While it is true that aphids perform better on plants than on chemically-defined diets (whatever their amino acid quality), and plant sap contains 20 : 80 e : n, this does not necessarily mean that aphids would not perform even better were plant sap to contain 50 : 50 e : n. In other words, there need not be any interaction between amino acid quality (20 : 80 or 50 : 50) and food source (plant or artificial diet), simply main effects of both. Discussion to date has implied that an interaction exists.

The principal value of the present study is that it illustrates how the problem of the scaling of axes in

geometric analysis of nutrition is amenable to exper- imental investigation. The results meet the general pre- dictions made in an earlier theoretical treatment (Simpson and Raubenheimer, 1993a) and also the specific predictions arising from our previous study on the pea aphid (Abisgold et al., 1994).

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Acknowledgements-Many thanks to David Raubenheimer for valu-

able discussion and comments on the manuscript, and to AFRC for

financial support.