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Amino acid composition of phloem sap and the relation to intraspecific variation in pea aphid (Acyrthosiphon pisum) performance
Pergamon
J. Insect Physiol. Vol. 40, No. I I, pp. 947-955. 1994 Copyright 0 1994 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0022-1910194 $7.00 + 0.00
0022-1910(94)00059-X
Amino Acid Composition of Phloem Sap and the Relation to Intraspecific Variation in Pea Aphid (Acyrthosiphon pisum) Performance J. SANDSTRijM,*
J. PETTERSSON*
Received 9 March 1994; revised 4 May 1994
The performance of five clones of the pea aphid, Acyrthosiphon pisum, was evaluated on red clover, Trifolium pratense, alfalfa, Medicago sativa, broad bean, Vicia faba and five genotypes of pea, Pisum satiuum. Phloem sap samples were collected from excised aphid stylets on plants of each species and analysed for amino acids. The pea aphid clones displayed large differences in performance patterns on the plant species, but more uniform patterns on the pea genotypes. Performance patterns of the aphid clones were related to the host plant species from which they had been collected originally. The total concentration of amino acids varied considerably between phloem samples, but there were no significant differences in the means between plant species or pea genotypes. Plant species differed distinctly in their amino acid composition, whereas pea genotypes did not. Several of the amino acids were present in similar compositions in all plant species and pea genotypes. The total concentration of amino acids in the phloem sap could not explain the differences in the performance of pea aphid clones on pea genotypes. However, differences in the performance of two out of five of the pea aphid clones could be explained by a part of the amino acid composition.
Pea aphid
Acyrthosiphon pisum
Amino
acid
Phloem
INTRODUCTION
Insects that feed on plant sap utilize a food source rich in carbohydrates but with a relatively low nitrogen concentration. Thus the nitrogen concentration is often an important nutritional factor, as reflected in the strong correlations found between this variable and individual growth rates, fecundity, survival and population growth rates for aphids on plants (Auclair et al., 1957; Van Emden and Bashford, 1969; Dixon, 1970; Jansson et al., 1987; Weibull, 1987) and on artificial diets (Prosser et al., 1992). Most aphids feed exclusively on sap from the vascular tissues of plants, and most of the nitrogen is obtained from the phloem. For correct evaluation of the relationship between aphid performance and the nitrogen concentration of their food source it is preferable to use pure phloem sap from severed aphid stylets. However, such material is difficult to obtain, and for practical reasons most studies concerning aphids are based on whole-leaf extracts. In addition to the nitrogen concentration, another factor that could affect aphid performance is the form in which the nitrogen is available. Nitrogen is present primarily as free amino acids in phloem, so the avail*Swedish University of Agricultural Sciences, Department Entomology P.O. Box 7044. S-750 07 Uppsala, Sweden.
of
Genetic
variation
ability depends mainly on the balance between the various amino acids. Experiments with artificial diets have shown that the amino acid balance affects the growth and survival of the pea aphid Acyrthosiphon pisum (Prosser and Douglas, 1992), and there are also indications that individual amino acids can stimulate or deter its feeding (Srivastava and Auclair, 1983). The pea aphid shows high intraspecific variability with regards to host plant utilization even within local areas (Via, 1991; Sandstriim, 1994). The underlying causes of this variation might include adaptation to special amino acid compositions present in the phloem sap of their host plants. The aim of this study was to describe the free amino acid composition of pure phloem sap from different food plants of the pea aphid and relate it to intraspecific variation in host utilization by the pea aphid. Such knowledge should help us to determine if aphids are adapted physiologically and/or behaviourally to the amino acid composition of their diets. MATERIALS AND METHODS Aphids
Five clones of A. pisum were established from single parthenogenetic females collected from four fields near 947
948
J. SANDSTROM
and J. PETTERSSON
Uppsala, Sweden (Table 1). The aphid clones were chosen from a larger sample with the purpose of obtaining clones with disparate host plant affiliations. Aphid stock cultures were kept in cages in a greenhouse at a temperature of 18-25°C and with a minimum photoperiod of 17 h of daylight supplemented, when needed, by artificial light (Philips HPI-T 400 W). All clones were reared on broad beans (Viciufuba, cv. Major). Plants The following food plants were used; broad bean V. fuba cv. Major (Weibull AB, Landskrona, Sweden); red clover Trifolium pratense cv. Hermes II; alfalfa, Medicago sativa cv. Sverre; and five pea genotypes of Pisum sativum, i.e. cv. Rigel, Patriot and Vreta (Svaliif AB, Uppsala, Sweden) and Gorkovskij 186, Prevoshodnyj 240 (Vavilov Institute, St Petersburg, Russia). Vreta was used as a reference. Three of the pea genotypes had short internodes and a compact growth form (Patriot, Prevoshodnyj 240, Rigel); two of them were leafless (Patriot, Rigel) and also genetically halfsibs. The pea genotypes were selected based on earlier screening tests evaluating the performance of different pea aphid clones (Sandstrbm, 1994). All plants were cultivated under the same environmental conditions as the aphid cultures were exposed to. All test plants were grown in 12 cm dia plastic pots in soil, P-jord (Hasselfors garden, Hasselfors, Sweden), supplemented with fertilizer (Osmocote plus, 200 g/100 dm3 soil). The plants were watered once a day with tap water. Aphid performance The experiments were performed in a greenhouse using the conditions and procedures described by SandStrom (1994, second experiment). At the start of the experiment all plants were of the same age as the plants used for phloem sap collection. To circumvent the loss of some values owing to high mortality, performance was scored from 1 to 10, where 1 corresponds to 100% mortality and 2 to 10 is based on intrinsic rate of increase, r,, values. The performance tests on plant species and pea genotypes were done in separate experiments. Phloem sap collection The broad bean and pea plants used for phloem collection were 23 &-2 days old, while the red clover alfalfa were 44 + 2 days of age. Five to ten adult aphids were placed on each plant l-6 h before collection. Pea aphid clone No. 3 was used on pea TABLE
1. Pea aphid, A. pisum, clones used in this study
Clone number 1 2 3 4 5
Collection
crop
Alfalfa Alfalfa Pea Pea Red clover
Colour
form
Red Green Green Green Red
sap and pea sap and
broad bean, clone No. 1 on alfalfa and clone No. 5 on red clover. Phloem sap was collected between 10 a.m. and 4p.m., which means that the plants had been exposed to 5-l 1 h of light. The aphid stylets were cut off by microcautery according to the method described by Unwin (1978). All stylets were cut in a growth chamber under artificial light at 15-16°C and 97-99% RH. The high relative humidity minimized evaporative losses from the samples. Samples were collected from new growth from veins on the underside of leaves. In addition, in peas samples were also collected from veins on the underside of petioles. The duration of the flow of phloem sap from the cut aphid stylets ranged from a few minutes up to several hours, depending on plant species, but samples for amino acid analysis were always completed within 1 h. Samples smaller than 20 nl were discarded. The phloem sap was collected in 0.5 ~1 micropipettes (Drummond Scientific Inc., U.S.A.) which were rinsed into vials several times with 10 ~1 of a mixture 0.1 M HCl-95% ethanol (1: 1, v/v). The vials were stored at -20°C until analysis. The amount of sap was determined by measuring the length of liquid column in the micropipette. Amino acid analysis Free amino acids in the phloem sap were analysed by high performance liquid chromatography (HPLC) after precolumn derivatization with o-phtalaldehyde according to Weibull et al. (1986). The amino acids were identified by comparing their retention times with a reference amino acid mixture. Concentrations were measured with an external standard because standard curves were partly non-linear. The standard consisted of 26 different amino acids. The detection limit for a single amino acid in an original phloem sample of 20 nl was about 0.05 mM. All amino acids were of the L-form and were obtained from Merck, except for homoserine (Sigma) and o-phosphoserine (FLUKA). For details about chemicals used in the derivatization and mobile phases, see Weibull et al. (1986). Statistical analysis Data on total free amino acid content and aphid performance were analysed by analysis of variance (ANOVA) using the SAS software (SAS Institute, Cary, NC, U.S.A.) procedure GLM. If the F test was significant (P < 0.05), the means were compared using Tukey’s test. The amino acid pattern and its relation to aphid performance was evaluated by means of multivariate data analysis with the Sirius software program (ver. 3.0, Umeb, Sweden) using principal component analysis (PCA) and partial least squares regression (PLS). The data were centred and standardized before analysis (Wold et al., 1984). Concentrations of individual amino acids were used as independent variables in the PLS runs. The pea aphid performance score, based on life history variables, was used as the dependent variable. In
TABLE
2. Means
AMINO
ACIDS
IN PHLOEM-PEA
of life history
variables
for one of the five pea aphid pea genotypes
Nymphal survival
Plant species
Genotype
Pea Broad bean Alfalfa Red clover
Vreta Major Sverre Hermes
Pea
Vreta Rigel Prevoshodnyj 240 Gorkovskij 186 Patriot
100 a 95 a 46 b 0 c 100 100 98 94 83
949
PERFORMANCE clones,
No. 4, on selected
plant
species and
Intrinsic rate of natural increase
Teneral weight
Fecundity
(days)
(mg)
(nymphs/day)
8.27 b 7.67 b 10.92 a
3.03 a 3.18 a 1.34 b
9.43 a 9.00 a 0.43 b
0.390 a 0.401 a 0.109 b
3.22 a 2.94 ab 3.42 a 2.88 b 2.60 b
8.11 a 8.09 a 7.52 ab 6.70 b 5.18 c
0.380 ab 0.402 a 0.359 bc 0.355 bc 0.324 c
Prereproductive period
W)
II
APHID
a a a ab b
8.20 bc 7.63 c 8.51 ab 8.59 ab 8.96 a
Results from two separate experiments, with pea cv. Vreta used as a reference in both. The intrinsic rate of increase values for all five pea aphid clones are shown in Fig. 1. Values within each experiment followed by different letters are significantly different according to Tukey’s test (P i 0.05) (n = 15).
our comparison between aphid performance and phloem amino acid composition only means of these variables could be compared. For practical reasons, it was impossible to use the same aphid individual for measuring aphid performance and collecting phloem sap in the same plant. This resulted in a loss of variation and reduced the strength of the PLS analysis. Level of statistical significance in the models were determined by cross-validation.
RESULTS
Pea aphid performance Each pea aphid clone showed large differences in performance between plant species, whereas their performance on the various pea genotypes was more uniform (Table 2). In aphid clone/plant combinations where performance was good, the differences were most pro-
nounced in the length of the prereproductive period, teneral weight and fecundity. For combinations in which performance was low, increased mortality was also evident. Although the life history variables shown for clone 4 in Table 2 were also recorded for the other four clones on the same plants, only the intrinsic rate of increase are shown for these clones (Fig. 1). In some aphid clone/plant combinations all pea aphids died, so mortality was the only recorded variable (asterisks in Fig. 1). The pea aphid clones displayed very large differences in performance patterns on the plant species. Differences in patterns on the pea genotypes were smaller, even though the general performance level on the pea genotypes varied between aphid clones (Fig. 1). Clones collected from the same crop showed similar performance patterns. Based on these pattern similarities the clones can be divided into three groups: clones 1 and 2, which were collected in alfalfa; clones 3 and 4 in pea, and
Clone 3
Clone 1
Clone 5
*,II
0.4 0.3 0.2 0.1
Atfalfa B. baan
R. clover
GcWPatPmRiiVra Paa
Gor Pat Pm Bit Vra
B. bean
Clone 2
R. CICQW
0. bean
Alfalfa Gor Pat Pm Ri Vre R. clover PSi
Clone 4
0.4
0.4
0.3
0.3
0.2
0.2
0.1
0.1 Alfalfa
B. bean
Peel
R. clover
B. bean
Gor Pat Pre Rig Vre R. clmer
Paa
FIGURE 1. Performance, measured as intrinsic rate of increase, of five different clones of pea aphid on broad bean, alfalfa, red clover and five genotypes of pea, Gor, Gorkovskij 186; Pat. Patriot; Pre, Prevoshodnyj 240; Rig, Rigel; Vre, Vreta. Clones I and 2 were collected on alfalfa, clones 3 and 4 on pea and clone 5 on red clover. Asterisks (*) denote aphid clone-plant combination with 100% mortality.
950
J. SANDSTROM TABLE Plant
3. Mean
concentration
species:
Genotype:
Amino
(mM) of individual Broad
bean
and J. PETTERSSON
amino
acids in phloem
Red clover
Alfalfa
Major (n = 10)
Hermes II (n = 10)
Sverre (n = 10)
1.6 10.7 107.6 4.4 0.2 7.4 32.8 0.6 2.2 ND 4.4 5.1 5.1 1.6 0.5 2.8 25.5 6.9 0.9 2.9 7.1 230.5 29.9
4.3 6.0 114.2 9.3 0.2 13.8 16.2 0.5 1.4 ND 4.2 3.9 3.7 1.0 0.4 2.7 11.2 5.8 0.8 1.6 6.9 210.0 25.9
9.1 4.4 140.3 6.8 0.1 12.7 11.2 1.0 I.0 ND 3.8 3.4 3.0 1.6 0.6 1.7 18.1 8.4 0.4 0.9 6.6 235.6 27.1
sap from selected
food plants
of the pea aphid
Pea Gorkovskij (n = 11)
Prevoshodnyj (n = 9)
Rigel (n = 11)
Patriot (n = 9)
Vreta (II = 9)
1.8 11.0 71.7 9.2 0.2 15.4 29.7 1.4 1.9 72.3 3.3 3.5 4.3 1.5 0.3 2.1 13.0 5.0 0.5 0.9 5.3 254.4 24.0
2.4 13.1 67.9 8.9 0.2 13.7 20.2 1.2 1.7 40.8 7.1 8.7 6.8 2.3 0.4 5.3 20.1 3.8 1.6 3.1 13.2 242.7 31.2
3.3 15.2 76.1 6.4 0.5 12.9 31.3 2.0 1.6 69.5 4.1 4.6 4.4 2.0 0.4 3.1 18.6 6.5 0.9 1.2 9.0 274.1 21.3
acid
Alanine (ALA) Arginine (ARG) Asparagine (ASN) Aspartic acid (ASP) y-Aminobutyric acid (GABA) Glutamic acid (GLU) Glutamine (GLN) Glycine (GLY) Histidine (HIS) Homoserine (HOM) Isoleucine (ILE) Leucine (LEU) Lysine (LYS) Methionine (MET) o-Phosphoserine (PSER) Phenylalanine (PHE) Serine (SER) Threonine (THR) Tryptophan (TRP) Tyrosine (TYR) Valine (VAL) Total concentration Standard error
3.4 14.3 64.5 8.4 0.4 13.8 17.4 1.7 5.6 52.3 4.2 4.1 4.9 1.4 0.5 3.8 13.5 6.1 1.1 1.8 7.9 230.2 14.7
1.6 10.9 82.7 8.3 0.3 8.3 21.5 1.3 1.1 69.0 4.4 5.8 4.6 1.5 0.2 3.8 17.4 4.6 I.0 1.6 8.3 256.5 28.1
ND = not detected.
clone 5 in red clover (Fig. 1). All pea aphid clones performed well on broad bean and on the plant species from which they had originally been collected. Phloem sap analysis
The total concentration of amino acids varied considerably (from 89 to 476 mM) between individual phloem samples. There was no significant difference in the mean concentration of total amino acids between plant species (F = 0.830, P = 0.484, df = 35) or pea genotypes (F = 0.500, P = 0.738, df = 44). Twenty-one different amino acids were identified in the phloem sap samples (Table 3). The content of secondary amino acids such as proline could not be determined since these amino acids do not form a detectable fluorescent complex with OPA (Benson and Hare, 1975). Because of low detector response to cysteine this compound could not be quantified (Jones et al., 1981). A few unidentified peaks also appeared, but they were always very small, and their combined peak area never exceeded 0.2% of the total peak area. Twenty of the amino acids were found in all the plant species, only homoserine was unique for peas. Concentrations of several individual amino acids were closely correlated with each other in all plant species. The concentrations of aspartic acid and glutamic acid are well correlated (linear regression; P < 0.001, r2 = 0.56, n = 78). Intercorrelations between concentrations of leucine, isoleucine, lysine and valine were even higher (P < 0.001, r2 > 0.80, n = 78), they also showed high intercorrelations to tyrosine, tryptophan
and phenylalanine (P < 0.001, r2 > 0.60, n = 78). All correlations were found to be positive. Accordingly, the covarying amino acids can be arranged into two groups, the first one comprising aspartic and glutamic acids and the second comprising isoleucine, leucine, lysine, valine, tryptophan, tyrosine and phenylalanine. The amino acid concentration data for plant species and pea genotypes were subjected to separate PCA (Fig. 2). To obtain a clearer picture of the differences, the influence of the second group of covarying amino acids was reduced by replacing them with a single variable in the PCA shown. The variables o-phosphoserine and y-aminobutyric acid were omitted because of a high signal-to-noise ratio. As can be seen in Fig. 2(A) the various plant species can be separated based on their amino acid composition, although the differences between alfalfa and red clover were not very distinct. On the other hand, no clear differences between the pea genotypes can be seen in Fig. 2(B), although 65% (41 + 21%) of the amino acid variation is displayed in the figure. The amino acid composition of the pea phloem sap, irrespective of genotype, was characterized by the presence of homoserine and by relatively high levels of arginine and glycine. Alfalfa and red clover both had relatively high levels of alanine and asparagine. However, alfalfa generally had higher levels of asparagine and alanine and lower levels of glutamine compared with red clover. The composition of broad bean phloem sap, with its relatively high levels of serine, was intermediate between that of pea and red clover-alfalfa. Broad bean phloem sap resembles pea in
AMINO
ACIDS
IN PHLOEM-PEA
APHID
TABLE 4. Mean total amino acid concentration phloem sap samples from all pea genotypes collected different locations on the plants
its relatively high levels of arginine and glutamine, but was also similar to red clover and alfalfa since all three species were found to have relatively high levels of asparagine [Fig. 2(A), Table 31. The variation in the second group of covarying amino acids together with methionine, aspartic and glutamic acid were not characteristic for any plant species. The amino acid composition of phloem was similar to that reported by Sasaki et al. (1990) for broad bean, by Girousse et al. (1991) for alfalfa and by Urquhart and Joy (1981) for pea. The composition of phloem from red clover does not appear to have been analysed previously. The phloem samples from pea were collected from different locations on the new growth depending on the
A
951
PERFORMANCE in in
Amino acid concentration Location Underside Underside
of leaf of petiole
Primary vein Secondary vein Tertiary vein
Sample
(mM)
21 22
227.2 a 219.9 b
39 I 3
245.8 a 263.0 a 290.1 a
All samples from new growth. Values within each group with different letters are significantly different according to Tukey’s test (P < 0.05).
feeding site chosen by the aphids. When these locations were compared, significant differences in mean total amino acid concentrations were found between leaf and petiole (F = 7.23, P = 0.011, df = 47). No significant differences were found between primary, secondary or tertiary veins on leaves and petioles (F = 0.81, P = 0.449, df = 46), but more peripheral veins tended to have higher concentrations (Table 4). The above-mentioned feeding sites could not be separated on the basis of amino acid composition in the PCA-analysis.
Plant species Horn
Gln
Relationship between amino acids and aphid performance -.
‘\
--._
‘.
&.!!.__--_-~&n
,
1 (36%)
Component
B
Pea genotypes
0 0 l
X
X
0 a
l.
0
X0
.
XX
+
ix
n
X +
l
,
+n++
+ n
+ n
x+
0
BXA
ii
n
x
A q
A+
Component
q
No significant correlation between the total concentration of free amino acids in the phloem and aphid performance was found for any of the five pea aphid clones. To test whether the individual amino acids in the phloem sap had any influence on pea aphid performance, the amino acid data were subjected to PLS regression using the amino acid concentrations from both plant species and pea genotypes as independent variables. Pea aphid performance score was used as the dependent variable for one aphid clone at the time. PLS models for two of the five aphid clones were significant and explained about 60% of the variation in pea aphid performance on the test plants (Table 5). These two aphid clones were both collected in peas and showed rather similar performance patterns (clones 3 and 4,
0
0 0
1 (41%)
FIGURE 2. Amino acid composition of phloem sap samples from different plant species (A) and pea genotypes (B). Plot of the two first principal components from principal component analysis (PCA) based on most of the amino acid variables. The percentage values on the axis denotes the share of the total variation that the component accounts for. The individual phloem samples are in A plotted together with the weights of the most influential amino acid separating the plant species, because of no separation in B no amino acid variables are plotted. The amino acid variables positions and distance relative to the origin is proportional to their contribution to the variation shown in A. Amino acid abbreviations see Table 3. A, broad bean; 0, red clover; n , alfalfa; 0, pea cv. Vreta; 0, pea cv. Patriot; x , pea cv. Gorkovskij 186; A, pea cv. Prevoshodnyj 240; f, pea cv. Rigel.
TABLE between
5. Summary of results from PLS-analysis of relationships amino acid composition in plant phloem sap and individual pea aphid clones performance on the same plants
Dependent variable (pea aphid
clone)
Number of significant components
I
0
2 3 4 5
0 1 1 0
% Explained variance of pea aphid performance
% Of amino acid variance accounted for
65 62
28 19
Independent variables in all tests are mean concentrations of all individual amino acids from all plants tested. Dependent variable is the performance score for the individual pea aphid clones. For a more detailed description of the model for clone 4, see Fig. 3.
952
J. SANDSTROM
and J. PETTERSSON
‘r
GASA ARG
ASP
GLN
GLU
HIS GLY
ILE HOM
LYS LEU
PSER SER TFIP VAL MET PHE THR TYFI
enormance score
FIGURE 3. Degree to which variation in amino acid composition explains differences in the performance of pea aphid clone No. 4 among pea genotypes and plant species. PLS-analysis with phloem concentrations of individual amino acids as independent variables (left) and the performance score of pea aphid clone No. 4 as the dependent variable (right). The first and only significant component is shown. The length of each bar in the left part of the figure corresponds to the degree to which variation in the concentration of each amino acid explains differences in aphid performance. The length of the bar in the right part corresponds to the extent to which aphid performance is explained by the variation in amino acid concentrations. Amino acid abbreviations see Table 3.
Fig. 1). In both models about one-fourth of the variation in amino acid concentration explained the variation in aphid clones performance. The model for clone 4 is shown in more detail in Fig. 3, which is also representative of the situation for clone 3. In both models arginine, alanine and asparagine were the most influential of the amino acids. Arginine concentration was positively correlated with performance, whereas alanine and asparagine were negatively correlated. Arginine explained much of the variation in performance patterns among the plant species but not among the pea genotypes. The cross-validation revealed only one significant principal component in both models.
DISCUSSION Total concentration of amino acids in phloem No significant differences in total amino acid concentration in phloem sap were found between pea genotypes or leguminous plant species. The total concentration of amino acids was about 240 mM, or 3.5% w/v, in all plants. This is somewhat lower than values reported in other studies with regard to amino acid concentrations in phloem sap collected from severed aphid stylets on leguminous plants pea (ca 340 mM) (Barlow and Randolph, 1978) and alfalfa (ca 350 mM) (Girousse et al., 1991). The discrepancy could be due to differences in plant genotypes, plant age or treatment, e.g. fertilizer, temperature. Another probable explanation is that the studies differed in the extent to which water was lost through evaporation during sap collection. Other investigations made on phloem sap from oats (Avena sativa) (Kuo-Sell, 1989) and rice (Oryza sativa) (Fukumorita and Chino, 1982) have reported concentrations in the same range. The total concentration of free amino acids varied considerably between samples within all plant genotypes
and species studied. Variation of this magnitude has also been reported by several workers who collected phloem sap from severed aphid stylets (Weibull, 1987; Kuo-Sell, 1989; Girousse et al., 1991). This variation could be a result of a diurnal rhythm or changes in the physiological condition of the plant (Sharkey and Pate, 1976). The variation could also be due to concentration differences between sieve tubes, which may reflect the photosynthetic activity of the area where the sieve tube is loaded (Weibull and Melin, 1990). If sieve tubes with different concentrations are available aphids would have an opportunity to choose among them. Histological examinations of stylet paths made by feeding Aphis fabae on V. faba show that the aphid probes several sieve tubes prior to sustained feeding (Tjallingi and Hogen Esch, 1993) indicating that aphids discriminate between sieve tubes. If the observed variation in amino acid concentrations reflect choices made by the pea aphids, this would indicate that they accept a very broad range of amino acid concentrations. The variability in amino acid concentration found in this study can also be ascribed partly to differences between plant parts; i.e. concentrations in sap obtained from excised stylets in the pea genotypes were significantly higher on petioles than on leaves, and they tended to be higher in peripheral veins than in more central ones. Adult pea aphid feeds equally well on petioles and leaves but prefer primary veins over more peripheral ones (unpublished results). Thus, the adult pea aphids’ preference for certain feeding sites does not seem to be influenced by concentrations of amino acids. No correlation was found between performance of the pea aphid and total phloem amino acid concentrations in the pea genotypes or plant species. Furthermore, in similar studies on aphids no such correlation was found in phloem samples from lucerne (Girousse et al., 1991), oats or barley (Weibull, 1988) or Brassica (Weibull and Melin, 1990). Low nitrogen concentrations
AMINO
ACIDS
IN PHLOEM-PEA
have repeatedly been proposed as an important source of resistance against aphids in peas (Auclair et al., 1957; Auclair, 1976) and other plants (Quiros et al., 1977; Jansson et al., 1987). However, these studies were based on wholeleaf extracts; thus the relevance of such measurements is questionable since the relationship between the nitrogen content of such extracts and concentrations of nitrogen in the phloem is unknown. In light of our results we do not question the importance of nitrogen level as a factor influencing pea aphid performance but we doubt that the intraspecific variation in phloem nitrogen levels is large enough to contribute to aphid resistance in pea genotypes. As seen in this study, variation is larger within genotypes than between them. Thus, for an individual pea aphid, concentration differences that it would encounter when moving between sieve tubes or as a result of a change in plant condition would probably be greater than those encountered when moving between different pea genotypes or even host plant species. Amino
acid composition
of phloem
The composition of all plants studied was dominated by non-essential amino acids, especially asparagine. This is in accordance with the pattern found in other plants within the family Fabaceae e.g. Lupinus sp. (Rhabe et al., 1991) and in rice (Fukumorita and Chino, 1982). However, leguminous phloem sap differs from that of cereals and Brassica. in which the sap is dominated by the non-essential amino acids glutamine and glutamic acid (Weibull, 1988; Kuo-Sell, 1989; Weibull and Melin, 1990). Even if there are differences in composition among plants tested in this study and among plants in other studies a general pattern in amino acid composition of phloem exist, a few for animals non-essential amino acids dominate with 50 mol% or more and essential amino acids seldom constitute more than 25 mol%. Analysis of the amino acid composition of the phloem sap revealed strong covariance between several amino acids, both within and between plant species. A similar pattern of covariance was observed in barley and oat phloem sap (Weibull, unpublished data). The covariance could be a result of linkage in the biosynthesis or in phloem loading of the amino acids in the plants. It is noteworthy that six out of the ten amino acids essential for animals belong to this covarying group. Non-protein amino acids are widely distributed among leguminous plants, being found in both wholeplant extracts and seeds (Hunt, 1985). It has been suggested that these amino acids function as defensive “allelochemicals”, and they are generally toxic to insects (Rosenthal and Bell, 1979). Non-protein amino acids from P. sativum and Lathrys sp. have shown activity as both phagostimulants and feeding deterrents when tested on A. pisum (Srivastava et al., 1988). Furthermore, unspecified non-protein amino acids have been suggested to contribute to resistance against A. pisum and A. ,fabae in Vicia sp. (Birch and Holt, 1981). The
APHID
PERFORMANCE
953
only non-protein amino acids detected in the phloem of the plants used in this study were those acting as intermediate compounds in the biosynthesis of the protein amino acids homoserine, y -aminobutyric acid and o-phosphoserine, which are not known to function as defensive “allelochemicals”. However, the presence of other non-protein amino acids in phloem sap cannot be excluded since some may not be detectable with the present methods or they could have been present at levels below the detection threshold. Aphids might also encounter such amino acids outside the phloem while penetrating the plant tissues with their stylets. In two of the pea aphid clones with similar host plant affiliations, relationships were found between aphid performance and amino acid composition. The variation in performance was explained mainly by differences in concentrations of arginine, alanine and asparagine among the plant species. Arginine was positively correlated to better aphid performance. This may be because arginine is an essential amino acid for animals, in general, and was one of the dominant essential amino acids in our test plants (17-28% of essential amino acids). It should be noted that this relationship with amino acids was only found with pea aphid clones originally collected from peas. This could mean that the other clones’ performance is not affected by the amino acid composition. However, it is also possible that relationships existed but that our tests were unable to discern them. After studying the relations between the composition of phloem exudates from alfalfa genotypes and pea aphid performance, Febvay et al. (1988) suggested that sugar/amino acid ratios affect aphid performance. However, their results were not confirmed by those obtained in a later study with phloem samples from aphid stylets (Girousse et al., 1991). In a study with another aphid species, Rhopalosiphum padi, Weibull (1988) found a negative relationship between performance and the concentration of glutamic acid in phloem sap exuding from severed stylets cut on oats and barley. In addition to the knowledge gained from the PLSanalysis, a couple of general conclusions can be drawn by relating phloem composition to aphid performance. In broad bean, the only species that all aphid clones performed well on, the composition of amino acids was intermediate between those of the other species. One might hypothesize that broad bean, with its intermediate composition, is a suitable host for several pea aphid clones differing in the amino acid compositions to which they are optimally adapted. On the other hand. there is little evidence that aphids on alfalfa and red clover have become adapted to the respective amino acid compositions of these species. To the contrary, although these two plant species have similar amino acid compositions, pea aphid clones adapted to alfalfa perform poorly on red clover, and vice versa (Via, 1991, this study). This study revealed some between-species differences in the amino acid composition of phloem sap. However, the similarities are more pronounced: a few non-essential amino acids dominate, and most of the amino acids
954
J. SANDSTROM
and J. PETTERSSON
essential to animals are present in relatively low proportions. Thus although phloem sap does not provide a balanced diet for animals, aphids have succeeded in exploiting it as a food source, probably with help from their intracellular bacterial symbionts (Douglas, 1993). Thus, variation in phloem amino acid composition among plant species is perhaps not an insurmountable physiological problem for aphids. The adaptations underlying the performance patterns discerned in this study need not be of physiological origin. Rather, they could also be behavioural adaptations that, because of our experimental design, could not be distinguished from physiological adaptations. The amino acid composition of the phloem sap may provide a behavioural cue for the pea aphid, helping it to evaluate a feeding site once it has reached a sieve tube in the phloem. Amino acids have been proposed to provide such a cue for pea aphids based on experiments with artificial diets (Srivastava and Auclair, 1983). However, the variability we found among phloem samples chosen by continuously feeding aphids suggests that pea aphids are not very choosy when it comes to amino acid composition. Our study has provided indications that certain pea aphid genotypes with particular host plant affiliations are physiologically and/or behaviourally adapted to the amino acids compositions present in their host plants phloem sap. The results also indicate that the plants, inside or outside phloem, contain other chemical components, to which host-specific pea aphid genotypes are differently adapted. REFERENCES Auclair J. L. (1976) Feeding and nutrition of the pea aphid, Acyrthosiphon pisum (Harris), with special reference to amino acids. Symp. Biol. Hung. 16, 29-34. Auclair J. L., Maltaise J. B. and Cartier J. J. (1957) Factors in resistance of peas to the pea aphid, Acyrthosiphon pisum (Harr.) (Homoptera: Aphididae). II. Amino acids. Can. Ent. 89, 457464. Barlow C. A. and Randolph P. A. (1978) Quality and quantity of plant sap available to the pea aphid. Ann. em. Sot. Am. 71, 4648. Benson J. R. and Hare P. E. (1975) o-Phtalaldehyde: Fluorogenic detection of primary amines in the picomole range. Comparison with fluoroescamine and ninhydrin. Proc. natn. Acad. Sci. U.S.A. 72, 619-622. Birch N. and Holt J. (1981) Aphid resistance in Viciu in relation to non-protein amino acids. ZOBC/WPRS Bull. IV/l, l-7. Dixon A. F. G. (1970) Quality and availability of food for a sycamore aphid population. In Animal Populations in Relation to Their Resources (Ed. Watson A.), pp. 271-287. Blackwell Scientific Publication, Oxford. Douglas A. E. (1993) The nutritional quality of phloem sap utilized by natural aphid populations. Ecol. Ent. 18, 31-38. Febvay G., Bonnin J., Rahbe Y., Bournoville R., Delrot S. and Bonnemain J. L. (1988) Resistance of different lucerne cultivars to the pea aphid Acyrthosiphonpisum: influence of phloem composition on aphid fecundity. Entomologia exp. appl. 48, 127-134. Fukumorita T. and Chino M. (1982) Sugar, amino acid and inorganic contents in rice phloem sap. Plant Cell Physiol. 23, 273-283. Girousse C., Febvay G., Rahbe Y. and Bournoville R. (1991) Reproductive rate of pea aphid related to phloem sap composition of alfalfa. In Proceedings from the Congress Aphid-Plant Interactions: Population to Molecules. Stillwater, OK, U.S.A. August 12-17, 1990.
Hunt S. (1985) The non-protein amino acids. In Chemistry and Biochemistry of the Amino Acids (Ed. Barret G. C.), pp. 55-138. Chapman and Hall, London. Jansson R. K., Elliott G. C., Smilowitz Z. and Cole R. H. (1987) Influence of cultivar maturity time and foliar nitrogen on population growth of Myzus persicae on potato. Entomologia exp. appl. 42, 297-300. Jones B. N., Plabo S. and Stein S. (1981) Amino acid analysis and enzymatic sequence determination of peptides by an improved o-phtaldialdehyde precolumn labeling procedure. J. Liq. Chromat. 4, X-586. Kuo-Sell H.-L. (1989) Aminosiiuren und Zucker im Phloemsaft verschiedener Pllanzenteile von Hafer (Avena sativa) in Beziehung zur Saugortpriiferenz von Getreideblattlausen (Horn., Aphididae). J. appl. Ent. 108, 54463. 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. 38, 93-99. Prosser W. A., Simpson S. J. and Douglas A. E. (1992) How an aphid (Acyrthosiphon pisum) symbiosis responds to variation in dietary nitrogen. J. Insect Physiol. 38, 301-307. Quiros C. F., Stevens M. A., Rick C. M. and Kok-Yokomi M. L. (1977) Resistance in tomato to the pink form of the potato aphid (Mucrosiphum euphorbiae Thomas): The role of anatomy, epidermal hairs, and foliage composition. J. Am. Sot. hort. Sci. 102, 166171. Rahbi Y., Delobel B., Calatayud P.-A. and Febvay G. (1991) Phloem sap composition of lupine analyzed by aphid stylectomy: Methodology, variations in major constituents and detection of minor solutes. In Proceedings from the Congress Aphid-Plant Interactions: Populations to Molecules. Stillwater, OK, U.S.A. August 12-17, 1990. Rosenthal G. A. and Bell E. A. (1979) Naturally occurring, toxic nonprotein amino acids. In Herbivores: Their Interaction with Secondary Plant Metabolites (Eds Rosenthal G. A. and Janzen D. H.), pp. 353-385. Academic Press, New York. Sandstrom J. (1994) High variation in host adaptation among clones of the pea aphid, Acyrthosiphon pisum, on peas, Pisum sativum. Entomologia exp. appl. 71, 245-256. Sasaki T., Aoki T., Hayashi H. and Ishikawa H. (1990) Amino acid composition of the honeydew of symbiotic and aposymbiotic pea aphids Acyrthosiphon pisum. J. Insect Physiol. 36, 3540. Sharkey P. J. and Pate J. S. (1976) Translocation from leaves to fruits of a legume, studied by phloem bleeding technique: Diurnal changes and effects on continuous darkness. Planta (Berl.) 128, 63-72. Srivastava P. N., Auclair J. L. and Srivastava V. (1983) Effect of nonessential amino acids on phagostimulation and maintenance of the pea aphid, Acyrthosiphon pisum. Can. J. Zool. 61, 22242229. Srivastava P. N., Lambein F. and Auclair J. L. (1988) Nonprotein amino acid-aphid interaction: Phagostimulatory effects and survival of the pea aphid, Acyrthosiphon pisum. Entomologia exp. appl. 48, 109-l 15. Tjallingi W. F. and Hogen Esch T. (1993) Fine structure of aphid stylet routes in plant tissues in correlation with EPG signals. Physiol. Enr. 18, 317-328. Unwin D. M. (1978) A versatile high frequency radio microcautery. Physiol. Ent. 3, 71-73. Urquhart A. A. and Joy K. W. (1981) Use of phloem exudate technique in the study of amino acid transport in pea plants. Plant Physiol. 68, 756754. Van Emden H. F. and Bashford M. A. (1969) A comparison of the reproduction of Brevicoryne brassicae and Myzus persicae in relation to soluble nitrogen concentration and leaf age (leaf position) in the Brussels sprout plant. Entomologiu exp. uppl. 12, 351-364. Via S. (1991) The genetic structure of host plant adaptation in a spatial patchwork: Demographic variability among reciprocally transplanted pea aphid clones. Evolution 45, 8277852. Weibull J., Brishammar S. and Pettersson J. (1986) Amino acid analysis of phloem sap from oats and barley: A combination of aphid stylet excision and high performance liquid chromatography. Entomologia exp. appl. 42, 27730.
AMINO
ACIDS
IN PHLOEM-PEA
Weibull J. (1987) Seasonal changes in the free amino acids of oat and barley phloem sap in relation to plant growth stage and growth of Rhopalosiphum padi. Ann. appl. Biol. 111, 729-737. Weibull J. (1988) Free amino acids in the phloem sap from oats and barley resistant to Rhopalosiphum padi. Phytochemistry 21, 2069-2072. Weibull J. and Melin G. (1990) Free amino acid content of phloem sap from Brassica plants in relation to performance of Lipaphis erysimi (Hemiptera: Aphididae). Ann. uppl. Biol. 116, 417423. Weld S., Albano C., Dunn W., Edlund U., Esbensen K., Geladi P., Hellberg S., Johansson E., Lindberg W. and Sjiistrom M. (1984)
APHID
PERFORMANCE
9.55
Multivariate data analysis in chemistry. In Chemomefrics-Mathematics and Stntistics in Chemistry (Ed. Kowalski B. R.), pp. 17-95. D. Reidel Publ. Comp., Dordrecht, Holland.
Acknowledgements-We would like to thank Barbara Ekbom, Christer Solbreck, David Tilles, Jens Weibull and Inger Ahman for helpful comments on the manuscript. We also thank Jens Weibull for introducing us to the techniques used for cutting aphid stylet. This study was supported by a grant from the Swedish Council for Forestry and Agricultural Research.
J. Insect Physiol. Vol. 40, No. I I, pp. 947-955. 1994 Copyright 0 1994 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0022-1910194 $7.00 + 0.00
0022-1910(94)00059-X
Amino Acid Composition of Phloem Sap and the Relation to Intraspecific Variation in Pea Aphid (Acyrthosiphon pisum) Performance J. SANDSTRijM,*
J. PETTERSSON*
Received 9 March 1994; revised 4 May 1994
The performance of five clones of the pea aphid, Acyrthosiphon pisum, was evaluated on red clover, Trifolium pratense, alfalfa, Medicago sativa, broad bean, Vicia faba and five genotypes of pea, Pisum satiuum. Phloem sap samples were collected from excised aphid stylets on plants of each species and analysed for amino acids. The pea aphid clones displayed large differences in performance patterns on the plant species, but more uniform patterns on the pea genotypes. Performance patterns of the aphid clones were related to the host plant species from which they had been collected originally. The total concentration of amino acids varied considerably between phloem samples, but there were no significant differences in the means between plant species or pea genotypes. Plant species differed distinctly in their amino acid composition, whereas pea genotypes did not. Several of the amino acids were present in similar compositions in all plant species and pea genotypes. The total concentration of amino acids in the phloem sap could not explain the differences in the performance of pea aphid clones on pea genotypes. However, differences in the performance of two out of five of the pea aphid clones could be explained by a part of the amino acid composition.
Pea aphid
Acyrthosiphon pisum
Amino
acid
Phloem
INTRODUCTION
Insects that feed on plant sap utilize a food source rich in carbohydrates but with a relatively low nitrogen concentration. Thus the nitrogen concentration is often an important nutritional factor, as reflected in the strong correlations found between this variable and individual growth rates, fecundity, survival and population growth rates for aphids on plants (Auclair et al., 1957; Van Emden and Bashford, 1969; Dixon, 1970; Jansson et al., 1987; Weibull, 1987) and on artificial diets (Prosser et al., 1992). Most aphids feed exclusively on sap from the vascular tissues of plants, and most of the nitrogen is obtained from the phloem. For correct evaluation of the relationship between aphid performance and the nitrogen concentration of their food source it is preferable to use pure phloem sap from severed aphid stylets. However, such material is difficult to obtain, and for practical reasons most studies concerning aphids are based on whole-leaf extracts. In addition to the nitrogen concentration, another factor that could affect aphid performance is the form in which the nitrogen is available. Nitrogen is present primarily as free amino acids in phloem, so the avail*Swedish University of Agricultural Sciences, Department Entomology P.O. Box 7044. S-750 07 Uppsala, Sweden.
of
Genetic
variation
ability depends mainly on the balance between the various amino acids. Experiments with artificial diets have shown that the amino acid balance affects the growth and survival of the pea aphid Acyrthosiphon pisum (Prosser and Douglas, 1992), and there are also indications that individual amino acids can stimulate or deter its feeding (Srivastava and Auclair, 1983). The pea aphid shows high intraspecific variability with regards to host plant utilization even within local areas (Via, 1991; Sandstriim, 1994). The underlying causes of this variation might include adaptation to special amino acid compositions present in the phloem sap of their host plants. The aim of this study was to describe the free amino acid composition of pure phloem sap from different food plants of the pea aphid and relate it to intraspecific variation in host utilization by the pea aphid. Such knowledge should help us to determine if aphids are adapted physiologically and/or behaviourally to the amino acid composition of their diets. MATERIALS AND METHODS Aphids
Five clones of A. pisum were established from single parthenogenetic females collected from four fields near 947
948
J. SANDSTROM
and J. PETTERSSON
Uppsala, Sweden (Table 1). The aphid clones were chosen from a larger sample with the purpose of obtaining clones with disparate host plant affiliations. Aphid stock cultures were kept in cages in a greenhouse at a temperature of 18-25°C and with a minimum photoperiod of 17 h of daylight supplemented, when needed, by artificial light (Philips HPI-T 400 W). All clones were reared on broad beans (Viciufuba, cv. Major). Plants The following food plants were used; broad bean V. fuba cv. Major (Weibull AB, Landskrona, Sweden); red clover Trifolium pratense cv. Hermes II; alfalfa, Medicago sativa cv. Sverre; and five pea genotypes of Pisum sativum, i.e. cv. Rigel, Patriot and Vreta (Svaliif AB, Uppsala, Sweden) and Gorkovskij 186, Prevoshodnyj 240 (Vavilov Institute, St Petersburg, Russia). Vreta was used as a reference. Three of the pea genotypes had short internodes and a compact growth form (Patriot, Prevoshodnyj 240, Rigel); two of them were leafless (Patriot, Rigel) and also genetically halfsibs. The pea genotypes were selected based on earlier screening tests evaluating the performance of different pea aphid clones (Sandstrbm, 1994). All plants were cultivated under the same environmental conditions as the aphid cultures were exposed to. All test plants were grown in 12 cm dia plastic pots in soil, P-jord (Hasselfors garden, Hasselfors, Sweden), supplemented with fertilizer (Osmocote plus, 200 g/100 dm3 soil). The plants were watered once a day with tap water. Aphid performance The experiments were performed in a greenhouse using the conditions and procedures described by SandStrom (1994, second experiment). At the start of the experiment all plants were of the same age as the plants used for phloem sap collection. To circumvent the loss of some values owing to high mortality, performance was scored from 1 to 10, where 1 corresponds to 100% mortality and 2 to 10 is based on intrinsic rate of increase, r,, values. The performance tests on plant species and pea genotypes were done in separate experiments. Phloem sap collection The broad bean and pea plants used for phloem collection were 23 &-2 days old, while the red clover alfalfa were 44 + 2 days of age. Five to ten adult aphids were placed on each plant l-6 h before collection. Pea aphid clone No. 3 was used on pea TABLE
1. Pea aphid, A. pisum, clones used in this study
Clone number 1 2 3 4 5
Collection
crop
Alfalfa Alfalfa Pea Pea Red clover
Colour
form
Red Green Green Green Red
sap and pea sap and
broad bean, clone No. 1 on alfalfa and clone No. 5 on red clover. Phloem sap was collected between 10 a.m. and 4p.m., which means that the plants had been exposed to 5-l 1 h of light. The aphid stylets were cut off by microcautery according to the method described by Unwin (1978). All stylets were cut in a growth chamber under artificial light at 15-16°C and 97-99% RH. The high relative humidity minimized evaporative losses from the samples. Samples were collected from new growth from veins on the underside of leaves. In addition, in peas samples were also collected from veins on the underside of petioles. The duration of the flow of phloem sap from the cut aphid stylets ranged from a few minutes up to several hours, depending on plant species, but samples for amino acid analysis were always completed within 1 h. Samples smaller than 20 nl were discarded. The phloem sap was collected in 0.5 ~1 micropipettes (Drummond Scientific Inc., U.S.A.) which were rinsed into vials several times with 10 ~1 of a mixture 0.1 M HCl-95% ethanol (1: 1, v/v). The vials were stored at -20°C until analysis. The amount of sap was determined by measuring the length of liquid column in the micropipette. Amino acid analysis Free amino acids in the phloem sap were analysed by high performance liquid chromatography (HPLC) after precolumn derivatization with o-phtalaldehyde according to Weibull et al. (1986). The amino acids were identified by comparing their retention times with a reference amino acid mixture. Concentrations were measured with an external standard because standard curves were partly non-linear. The standard consisted of 26 different amino acids. The detection limit for a single amino acid in an original phloem sample of 20 nl was about 0.05 mM. All amino acids were of the L-form and were obtained from Merck, except for homoserine (Sigma) and o-phosphoserine (FLUKA). For details about chemicals used in the derivatization and mobile phases, see Weibull et al. (1986). Statistical analysis Data on total free amino acid content and aphid performance were analysed by analysis of variance (ANOVA) using the SAS software (SAS Institute, Cary, NC, U.S.A.) procedure GLM. If the F test was significant (P < 0.05), the means were compared using Tukey’s test. The amino acid pattern and its relation to aphid performance was evaluated by means of multivariate data analysis with the Sirius software program (ver. 3.0, Umeb, Sweden) using principal component analysis (PCA) and partial least squares regression (PLS). The data were centred and standardized before analysis (Wold et al., 1984). Concentrations of individual amino acids were used as independent variables in the PLS runs. The pea aphid performance score, based on life history variables, was used as the dependent variable. In
TABLE
2. Means
AMINO
ACIDS
IN PHLOEM-PEA
of life history
variables
for one of the five pea aphid pea genotypes
Nymphal survival
Plant species
Genotype
Pea Broad bean Alfalfa Red clover
Vreta Major Sverre Hermes
Pea
Vreta Rigel Prevoshodnyj 240 Gorkovskij 186 Patriot
100 a 95 a 46 b 0 c 100 100 98 94 83
949
PERFORMANCE clones,
No. 4, on selected
plant
species and
Intrinsic rate of natural increase
Teneral weight
Fecundity
(days)
(mg)
(nymphs/day)
8.27 b 7.67 b 10.92 a
3.03 a 3.18 a 1.34 b
9.43 a 9.00 a 0.43 b
0.390 a 0.401 a 0.109 b
3.22 a 2.94 ab 3.42 a 2.88 b 2.60 b
8.11 a 8.09 a 7.52 ab 6.70 b 5.18 c
0.380 ab 0.402 a 0.359 bc 0.355 bc 0.324 c
Prereproductive period
W)
II
APHID
a a a ab b
8.20 bc 7.63 c 8.51 ab 8.59 ab 8.96 a
Results from two separate experiments, with pea cv. Vreta used as a reference in both. The intrinsic rate of increase values for all five pea aphid clones are shown in Fig. 1. Values within each experiment followed by different letters are significantly different according to Tukey’s test (P i 0.05) (n = 15).
our comparison between aphid performance and phloem amino acid composition only means of these variables could be compared. For practical reasons, it was impossible to use the same aphid individual for measuring aphid performance and collecting phloem sap in the same plant. This resulted in a loss of variation and reduced the strength of the PLS analysis. Level of statistical significance in the models were determined by cross-validation.
RESULTS
Pea aphid performance Each pea aphid clone showed large differences in performance between plant species, whereas their performance on the various pea genotypes was more uniform (Table 2). In aphid clone/plant combinations where performance was good, the differences were most pro-
nounced in the length of the prereproductive period, teneral weight and fecundity. For combinations in which performance was low, increased mortality was also evident. Although the life history variables shown for clone 4 in Table 2 were also recorded for the other four clones on the same plants, only the intrinsic rate of increase are shown for these clones (Fig. 1). In some aphid clone/plant combinations all pea aphids died, so mortality was the only recorded variable (asterisks in Fig. 1). The pea aphid clones displayed very large differences in performance patterns on the plant species. Differences in patterns on the pea genotypes were smaller, even though the general performance level on the pea genotypes varied between aphid clones (Fig. 1). Clones collected from the same crop showed similar performance patterns. Based on these pattern similarities the clones can be divided into three groups: clones 1 and 2, which were collected in alfalfa; clones 3 and 4 in pea, and
Clone 3
Clone 1
Clone 5
*,II
0.4 0.3 0.2 0.1
Atfalfa B. baan
R. clover
GcWPatPmRiiVra Paa
Gor Pat Pm Bit Vra
B. bean
Clone 2
R. CICQW
0. bean
Alfalfa Gor Pat Pm Ri Vre R. clover PSi
Clone 4
0.4
0.4
0.3
0.3
0.2
0.2
0.1
0.1 Alfalfa
B. bean
Peel
R. clover
B. bean
Gor Pat Pre Rig Vre R. clmer
Paa
FIGURE 1. Performance, measured as intrinsic rate of increase, of five different clones of pea aphid on broad bean, alfalfa, red clover and five genotypes of pea, Gor, Gorkovskij 186; Pat. Patriot; Pre, Prevoshodnyj 240; Rig, Rigel; Vre, Vreta. Clones I and 2 were collected on alfalfa, clones 3 and 4 on pea and clone 5 on red clover. Asterisks (*) denote aphid clone-plant combination with 100% mortality.
950
J. SANDSTROM TABLE Plant
3. Mean
concentration
species:
Genotype:
Amino
(mM) of individual Broad
bean
and J. PETTERSSON
amino
acids in phloem
Red clover
Alfalfa
Major (n = 10)
Hermes II (n = 10)
Sverre (n = 10)
1.6 10.7 107.6 4.4 0.2 7.4 32.8 0.6 2.2 ND 4.4 5.1 5.1 1.6 0.5 2.8 25.5 6.9 0.9 2.9 7.1 230.5 29.9
4.3 6.0 114.2 9.3 0.2 13.8 16.2 0.5 1.4 ND 4.2 3.9 3.7 1.0 0.4 2.7 11.2 5.8 0.8 1.6 6.9 210.0 25.9
9.1 4.4 140.3 6.8 0.1 12.7 11.2 1.0 I.0 ND 3.8 3.4 3.0 1.6 0.6 1.7 18.1 8.4 0.4 0.9 6.6 235.6 27.1
sap from selected
food plants
of the pea aphid
Pea Gorkovskij (n = 11)
Prevoshodnyj (n = 9)
Rigel (n = 11)
Patriot (n = 9)
Vreta (II = 9)
1.8 11.0 71.7 9.2 0.2 15.4 29.7 1.4 1.9 72.3 3.3 3.5 4.3 1.5 0.3 2.1 13.0 5.0 0.5 0.9 5.3 254.4 24.0
2.4 13.1 67.9 8.9 0.2 13.7 20.2 1.2 1.7 40.8 7.1 8.7 6.8 2.3 0.4 5.3 20.1 3.8 1.6 3.1 13.2 242.7 31.2
3.3 15.2 76.1 6.4 0.5 12.9 31.3 2.0 1.6 69.5 4.1 4.6 4.4 2.0 0.4 3.1 18.6 6.5 0.9 1.2 9.0 274.1 21.3
acid
Alanine (ALA) Arginine (ARG) Asparagine (ASN) Aspartic acid (ASP) y-Aminobutyric acid (GABA) Glutamic acid (GLU) Glutamine (GLN) Glycine (GLY) Histidine (HIS) Homoserine (HOM) Isoleucine (ILE) Leucine (LEU) Lysine (LYS) Methionine (MET) o-Phosphoserine (PSER) Phenylalanine (PHE) Serine (SER) Threonine (THR) Tryptophan (TRP) Tyrosine (TYR) Valine (VAL) Total concentration Standard error
3.4 14.3 64.5 8.4 0.4 13.8 17.4 1.7 5.6 52.3 4.2 4.1 4.9 1.4 0.5 3.8 13.5 6.1 1.1 1.8 7.9 230.2 14.7
1.6 10.9 82.7 8.3 0.3 8.3 21.5 1.3 1.1 69.0 4.4 5.8 4.6 1.5 0.2 3.8 17.4 4.6 I.0 1.6 8.3 256.5 28.1
ND = not detected.
clone 5 in red clover (Fig. 1). All pea aphid clones performed well on broad bean and on the plant species from which they had originally been collected. Phloem sap analysis
The total concentration of amino acids varied considerably (from 89 to 476 mM) between individual phloem samples. There was no significant difference in the mean concentration of total amino acids between plant species (F = 0.830, P = 0.484, df = 35) or pea genotypes (F = 0.500, P = 0.738, df = 44). Twenty-one different amino acids were identified in the phloem sap samples (Table 3). The content of secondary amino acids such as proline could not be determined since these amino acids do not form a detectable fluorescent complex with OPA (Benson and Hare, 1975). Because of low detector response to cysteine this compound could not be quantified (Jones et al., 1981). A few unidentified peaks also appeared, but they were always very small, and their combined peak area never exceeded 0.2% of the total peak area. Twenty of the amino acids were found in all the plant species, only homoserine was unique for peas. Concentrations of several individual amino acids were closely correlated with each other in all plant species. The concentrations of aspartic acid and glutamic acid are well correlated (linear regression; P < 0.001, r2 = 0.56, n = 78). Intercorrelations between concentrations of leucine, isoleucine, lysine and valine were even higher (P < 0.001, r2 > 0.80, n = 78), they also showed high intercorrelations to tyrosine, tryptophan
and phenylalanine (P < 0.001, r2 > 0.60, n = 78). All correlations were found to be positive. Accordingly, the covarying amino acids can be arranged into two groups, the first one comprising aspartic and glutamic acids and the second comprising isoleucine, leucine, lysine, valine, tryptophan, tyrosine and phenylalanine. The amino acid concentration data for plant species and pea genotypes were subjected to separate PCA (Fig. 2). To obtain a clearer picture of the differences, the influence of the second group of covarying amino acids was reduced by replacing them with a single variable in the PCA shown. The variables o-phosphoserine and y-aminobutyric acid were omitted because of a high signal-to-noise ratio. As can be seen in Fig. 2(A) the various plant species can be separated based on their amino acid composition, although the differences between alfalfa and red clover were not very distinct. On the other hand, no clear differences between the pea genotypes can be seen in Fig. 2(B), although 65% (41 + 21%) of the amino acid variation is displayed in the figure. The amino acid composition of the pea phloem sap, irrespective of genotype, was characterized by the presence of homoserine and by relatively high levels of arginine and glycine. Alfalfa and red clover both had relatively high levels of alanine and asparagine. However, alfalfa generally had higher levels of asparagine and alanine and lower levels of glutamine compared with red clover. The composition of broad bean phloem sap, with its relatively high levels of serine, was intermediate between that of pea and red clover-alfalfa. Broad bean phloem sap resembles pea in
AMINO
ACIDS
IN PHLOEM-PEA
APHID
TABLE 4. Mean total amino acid concentration phloem sap samples from all pea genotypes collected different locations on the plants
its relatively high levels of arginine and glutamine, but was also similar to red clover and alfalfa since all three species were found to have relatively high levels of asparagine [Fig. 2(A), Table 31. The variation in the second group of covarying amino acids together with methionine, aspartic and glutamic acid were not characteristic for any plant species. The amino acid composition of phloem was similar to that reported by Sasaki et al. (1990) for broad bean, by Girousse et al. (1991) for alfalfa and by Urquhart and Joy (1981) for pea. The composition of phloem from red clover does not appear to have been analysed previously. The phloem samples from pea were collected from different locations on the new growth depending on the
A
951
PERFORMANCE in in
Amino acid concentration Location Underside Underside
of leaf of petiole
Primary vein Secondary vein Tertiary vein
Sample
(mM)
21 22
227.2 a 219.9 b
39 I 3
245.8 a 263.0 a 290.1 a
All samples from new growth. Values within each group with different letters are significantly different according to Tukey’s test (P < 0.05).
feeding site chosen by the aphids. When these locations were compared, significant differences in mean total amino acid concentrations were found between leaf and petiole (F = 7.23, P = 0.011, df = 47). No significant differences were found between primary, secondary or tertiary veins on leaves and petioles (F = 0.81, P = 0.449, df = 46), but more peripheral veins tended to have higher concentrations (Table 4). The above-mentioned feeding sites could not be separated on the basis of amino acid composition in the PCA-analysis.
Plant species Horn
Gln
Relationship between amino acids and aphid performance -.
‘\
--._
‘.
&.!!.__--_-~&n
,
1 (36%)
Component
B
Pea genotypes
0 0 l
X
X
0 a
l.
0
X0
.
XX
+
ix
n
X +
l
,
+n++
+ n
+ n
x+
0
BXA
ii
n
x
A q
A+
Component
q
No significant correlation between the total concentration of free amino acids in the phloem and aphid performance was found for any of the five pea aphid clones. To test whether the individual amino acids in the phloem sap had any influence on pea aphid performance, the amino acid data were subjected to PLS regression using the amino acid concentrations from both plant species and pea genotypes as independent variables. Pea aphid performance score was used as the dependent variable for one aphid clone at the time. PLS models for two of the five aphid clones were significant and explained about 60% of the variation in pea aphid performance on the test plants (Table 5). These two aphid clones were both collected in peas and showed rather similar performance patterns (clones 3 and 4,
0
0 0
1 (41%)
FIGURE 2. Amino acid composition of phloem sap samples from different plant species (A) and pea genotypes (B). Plot of the two first principal components from principal component analysis (PCA) based on most of the amino acid variables. The percentage values on the axis denotes the share of the total variation that the component accounts for. The individual phloem samples are in A plotted together with the weights of the most influential amino acid separating the plant species, because of no separation in B no amino acid variables are plotted. The amino acid variables positions and distance relative to the origin is proportional to their contribution to the variation shown in A. Amino acid abbreviations see Table 3. A, broad bean; 0, red clover; n , alfalfa; 0, pea cv. Vreta; 0, pea cv. Patriot; x , pea cv. Gorkovskij 186; A, pea cv. Prevoshodnyj 240; f, pea cv. Rigel.
TABLE between
5. Summary of results from PLS-analysis of relationships amino acid composition in plant phloem sap and individual pea aphid clones performance on the same plants
Dependent variable (pea aphid
clone)
Number of significant components
I
0
2 3 4 5
0 1 1 0
% Explained variance of pea aphid performance
% Of amino acid variance accounted for
65 62
28 19
Independent variables in all tests are mean concentrations of all individual amino acids from all plants tested. Dependent variable is the performance score for the individual pea aphid clones. For a more detailed description of the model for clone 4, see Fig. 3.
952
J. SANDSTROM
and J. PETTERSSON
‘r
GASA ARG
ASP
GLN
GLU
HIS GLY
ILE HOM
LYS LEU
PSER SER TFIP VAL MET PHE THR TYFI
enormance score
FIGURE 3. Degree to which variation in amino acid composition explains differences in the performance of pea aphid clone No. 4 among pea genotypes and plant species. PLS-analysis with phloem concentrations of individual amino acids as independent variables (left) and the performance score of pea aphid clone No. 4 as the dependent variable (right). The first and only significant component is shown. The length of each bar in the left part of the figure corresponds to the degree to which variation in the concentration of each amino acid explains differences in aphid performance. The length of the bar in the right part corresponds to the extent to which aphid performance is explained by the variation in amino acid concentrations. Amino acid abbreviations see Table 3.
Fig. 1). In both models about one-fourth of the variation in amino acid concentration explained the variation in aphid clones performance. The model for clone 4 is shown in more detail in Fig. 3, which is also representative of the situation for clone 3. In both models arginine, alanine and asparagine were the most influential of the amino acids. Arginine concentration was positively correlated with performance, whereas alanine and asparagine were negatively correlated. Arginine explained much of the variation in performance patterns among the plant species but not among the pea genotypes. The cross-validation revealed only one significant principal component in both models.
DISCUSSION Total concentration of amino acids in phloem No significant differences in total amino acid concentration in phloem sap were found between pea genotypes or leguminous plant species. The total concentration of amino acids was about 240 mM, or 3.5% w/v, in all plants. This is somewhat lower than values reported in other studies with regard to amino acid concentrations in phloem sap collected from severed aphid stylets on leguminous plants pea (ca 340 mM) (Barlow and Randolph, 1978) and alfalfa (ca 350 mM) (Girousse et al., 1991). The discrepancy could be due to differences in plant genotypes, plant age or treatment, e.g. fertilizer, temperature. Another probable explanation is that the studies differed in the extent to which water was lost through evaporation during sap collection. Other investigations made on phloem sap from oats (Avena sativa) (Kuo-Sell, 1989) and rice (Oryza sativa) (Fukumorita and Chino, 1982) have reported concentrations in the same range. The total concentration of free amino acids varied considerably between samples within all plant genotypes
and species studied. Variation of this magnitude has also been reported by several workers who collected phloem sap from severed aphid stylets (Weibull, 1987; Kuo-Sell, 1989; Girousse et al., 1991). This variation could be a result of a diurnal rhythm or changes in the physiological condition of the plant (Sharkey and Pate, 1976). The variation could also be due to concentration differences between sieve tubes, which may reflect the photosynthetic activity of the area where the sieve tube is loaded (Weibull and Melin, 1990). If sieve tubes with different concentrations are available aphids would have an opportunity to choose among them. Histological examinations of stylet paths made by feeding Aphis fabae on V. faba show that the aphid probes several sieve tubes prior to sustained feeding (Tjallingi and Hogen Esch, 1993) indicating that aphids discriminate between sieve tubes. If the observed variation in amino acid concentrations reflect choices made by the pea aphids, this would indicate that they accept a very broad range of amino acid concentrations. The variability in amino acid concentration found in this study can also be ascribed partly to differences between plant parts; i.e. concentrations in sap obtained from excised stylets in the pea genotypes were significantly higher on petioles than on leaves, and they tended to be higher in peripheral veins than in more central ones. Adult pea aphid feeds equally well on petioles and leaves but prefer primary veins over more peripheral ones (unpublished results). Thus, the adult pea aphids’ preference for certain feeding sites does not seem to be influenced by concentrations of amino acids. No correlation was found between performance of the pea aphid and total phloem amino acid concentrations in the pea genotypes or plant species. Furthermore, in similar studies on aphids no such correlation was found in phloem samples from lucerne (Girousse et al., 1991), oats or barley (Weibull, 1988) or Brassica (Weibull and Melin, 1990). Low nitrogen concentrations
AMINO
ACIDS
IN PHLOEM-PEA
have repeatedly been proposed as an important source of resistance against aphids in peas (Auclair et al., 1957; Auclair, 1976) and other plants (Quiros et al., 1977; Jansson et al., 1987). However, these studies were based on wholeleaf extracts; thus the relevance of such measurements is questionable since the relationship between the nitrogen content of such extracts and concentrations of nitrogen in the phloem is unknown. In light of our results we do not question the importance of nitrogen level as a factor influencing pea aphid performance but we doubt that the intraspecific variation in phloem nitrogen levels is large enough to contribute to aphid resistance in pea genotypes. As seen in this study, variation is larger within genotypes than between them. Thus, for an individual pea aphid, concentration differences that it would encounter when moving between sieve tubes or as a result of a change in plant condition would probably be greater than those encountered when moving between different pea genotypes or even host plant species. Amino
acid composition
of phloem
The composition of all plants studied was dominated by non-essential amino acids, especially asparagine. This is in accordance with the pattern found in other plants within the family Fabaceae e.g. Lupinus sp. (Rhabe et al., 1991) and in rice (Fukumorita and Chino, 1982). However, leguminous phloem sap differs from that of cereals and Brassica. in which the sap is dominated by the non-essential amino acids glutamine and glutamic acid (Weibull, 1988; Kuo-Sell, 1989; Weibull and Melin, 1990). Even if there are differences in composition among plants tested in this study and among plants in other studies a general pattern in amino acid composition of phloem exist, a few for animals non-essential amino acids dominate with 50 mol% or more and essential amino acids seldom constitute more than 25 mol%. Analysis of the amino acid composition of the phloem sap revealed strong covariance between several amino acids, both within and between plant species. A similar pattern of covariance was observed in barley and oat phloem sap (Weibull, unpublished data). The covariance could be a result of linkage in the biosynthesis or in phloem loading of the amino acids in the plants. It is noteworthy that six out of the ten amino acids essential for animals belong to this covarying group. Non-protein amino acids are widely distributed among leguminous plants, being found in both wholeplant extracts and seeds (Hunt, 1985). It has been suggested that these amino acids function as defensive “allelochemicals”, and they are generally toxic to insects (Rosenthal and Bell, 1979). Non-protein amino acids from P. sativum and Lathrys sp. have shown activity as both phagostimulants and feeding deterrents when tested on A. pisum (Srivastava et al., 1988). Furthermore, unspecified non-protein amino acids have been suggested to contribute to resistance against A. pisum and A. ,fabae in Vicia sp. (Birch and Holt, 1981). The
APHID
PERFORMANCE
953
only non-protein amino acids detected in the phloem of the plants used in this study were those acting as intermediate compounds in the biosynthesis of the protein amino acids homoserine, y -aminobutyric acid and o-phosphoserine, which are not known to function as defensive “allelochemicals”. However, the presence of other non-protein amino acids in phloem sap cannot be excluded since some may not be detectable with the present methods or they could have been present at levels below the detection threshold. Aphids might also encounter such amino acids outside the phloem while penetrating the plant tissues with their stylets. In two of the pea aphid clones with similar host plant affiliations, relationships were found between aphid performance and amino acid composition. The variation in performance was explained mainly by differences in concentrations of arginine, alanine and asparagine among the plant species. Arginine was positively correlated to better aphid performance. This may be because arginine is an essential amino acid for animals, in general, and was one of the dominant essential amino acids in our test plants (17-28% of essential amino acids). It should be noted that this relationship with amino acids was only found with pea aphid clones originally collected from peas. This could mean that the other clones’ performance is not affected by the amino acid composition. However, it is also possible that relationships existed but that our tests were unable to discern them. After studying the relations between the composition of phloem exudates from alfalfa genotypes and pea aphid performance, Febvay et al. (1988) suggested that sugar/amino acid ratios affect aphid performance. However, their results were not confirmed by those obtained in a later study with phloem samples from aphid stylets (Girousse et al., 1991). In a study with another aphid species, Rhopalosiphum padi, Weibull (1988) found a negative relationship between performance and the concentration of glutamic acid in phloem sap exuding from severed stylets cut on oats and barley. In addition to the knowledge gained from the PLSanalysis, a couple of general conclusions can be drawn by relating phloem composition to aphid performance. In broad bean, the only species that all aphid clones performed well on, the composition of amino acids was intermediate between those of the other species. One might hypothesize that broad bean, with its intermediate composition, is a suitable host for several pea aphid clones differing in the amino acid compositions to which they are optimally adapted. On the other hand. there is little evidence that aphids on alfalfa and red clover have become adapted to the respective amino acid compositions of these species. To the contrary, although these two plant species have similar amino acid compositions, pea aphid clones adapted to alfalfa perform poorly on red clover, and vice versa (Via, 1991, this study). This study revealed some between-species differences in the amino acid composition of phloem sap. However, the similarities are more pronounced: a few non-essential amino acids dominate, and most of the amino acids
954
J. SANDSTROM
and J. PETTERSSON
essential to animals are present in relatively low proportions. Thus although phloem sap does not provide a balanced diet for animals, aphids have succeeded in exploiting it as a food source, probably with help from their intracellular bacterial symbionts (Douglas, 1993). Thus, variation in phloem amino acid composition among plant species is perhaps not an insurmountable physiological problem for aphids. The adaptations underlying the performance patterns discerned in this study need not be of physiological origin. Rather, they could also be behavioural adaptations that, because of our experimental design, could not be distinguished from physiological adaptations. The amino acid composition of the phloem sap may provide a behavioural cue for the pea aphid, helping it to evaluate a feeding site once it has reached a sieve tube in the phloem. Amino acids have been proposed to provide such a cue for pea aphids based on experiments with artificial diets (Srivastava and Auclair, 1983). However, the variability we found among phloem samples chosen by continuously feeding aphids suggests that pea aphids are not very choosy when it comes to amino acid composition. Our study has provided indications that certain pea aphid genotypes with particular host plant affiliations are physiologically and/or behaviourally adapted to the amino acids compositions present in their host plants phloem sap. The results also indicate that the plants, inside or outside phloem, contain other chemical components, to which host-specific pea aphid genotypes are differently adapted. REFERENCES Auclair J. L. (1976) Feeding and nutrition of the pea aphid, Acyrthosiphon pisum (Harris), with special reference to amino acids. Symp. Biol. Hung. 16, 29-34. Auclair J. L., Maltaise J. B. and Cartier J. J. (1957) Factors in resistance of peas to the pea aphid, Acyrthosiphon pisum (Harr.) (Homoptera: Aphididae). II. Amino acids. Can. Ent. 89, 457464. Barlow C. A. and Randolph P. A. (1978) Quality and quantity of plant sap available to the pea aphid. Ann. em. Sot. Am. 71, 4648. Benson J. R. and Hare P. E. (1975) o-Phtalaldehyde: Fluorogenic detection of primary amines in the picomole range. Comparison with fluoroescamine and ninhydrin. Proc. natn. Acad. Sci. U.S.A. 72, 619-622. Birch N. and Holt J. (1981) Aphid resistance in Viciu in relation to non-protein amino acids. ZOBC/WPRS Bull. IV/l, l-7. Dixon A. F. G. (1970) Quality and availability of food for a sycamore aphid population. In Animal Populations in Relation to Their Resources (Ed. Watson A.), pp. 271-287. Blackwell Scientific Publication, Oxford. Douglas A. E. (1993) The nutritional quality of phloem sap utilized by natural aphid populations. Ecol. Ent. 18, 31-38. Febvay G., Bonnin J., Rahbe Y., Bournoville R., Delrot S. and Bonnemain J. L. (1988) Resistance of different lucerne cultivars to the pea aphid Acyrthosiphonpisum: influence of phloem composition on aphid fecundity. Entomologia exp. appl. 48, 127-134. Fukumorita T. and Chino M. (1982) Sugar, amino acid and inorganic contents in rice phloem sap. Plant Cell Physiol. 23, 273-283. Girousse C., Febvay G., Rahbe Y. and Bournoville R. (1991) Reproductive rate of pea aphid related to phloem sap composition of alfalfa. In Proceedings from the Congress Aphid-Plant Interactions: Population to Molecules. Stillwater, OK, U.S.A. August 12-17, 1990.
Hunt S. (1985) The non-protein amino acids. In Chemistry and Biochemistry of the Amino Acids (Ed. Barret G. C.), pp. 55-138. Chapman and Hall, London. Jansson R. K., Elliott G. C., Smilowitz Z. and Cole R. H. (1987) Influence of cultivar maturity time and foliar nitrogen on population growth of Myzus persicae on potato. Entomologia exp. appl. 42, 297-300. Jones B. N., Plabo S. and Stein S. (1981) Amino acid analysis and enzymatic sequence determination of peptides by an improved o-phtaldialdehyde precolumn labeling procedure. J. Liq. Chromat. 4, X-586. Kuo-Sell H.-L. (1989) Aminosiiuren und Zucker im Phloemsaft verschiedener Pllanzenteile von Hafer (Avena sativa) in Beziehung zur Saugortpriiferenz von Getreideblattlausen (Horn., Aphididae). J. appl. Ent. 108, 54463. 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. 38, 93-99. Prosser W. A., Simpson S. J. and Douglas A. E. (1992) How an aphid (Acyrthosiphon pisum) symbiosis responds to variation in dietary nitrogen. J. Insect Physiol. 38, 301-307. Quiros C. F., Stevens M. A., Rick C. M. and Kok-Yokomi M. L. (1977) Resistance in tomato to the pink form of the potato aphid (Mucrosiphum euphorbiae Thomas): The role of anatomy, epidermal hairs, and foliage composition. J. Am. Sot. hort. Sci. 102, 166171. Rahbi Y., Delobel B., Calatayud P.-A. and Febvay G. (1991) Phloem sap composition of lupine analyzed by aphid stylectomy: Methodology, variations in major constituents and detection of minor solutes. In Proceedings from the Congress Aphid-Plant Interactions: Populations to Molecules. Stillwater, OK, U.S.A. August 12-17, 1990. Rosenthal G. A. and Bell E. A. (1979) Naturally occurring, toxic nonprotein amino acids. In Herbivores: Their Interaction with Secondary Plant Metabolites (Eds Rosenthal G. A. and Janzen D. H.), pp. 353-385. Academic Press, New York. Sandstrom J. (1994) High variation in host adaptation among clones of the pea aphid, Acyrthosiphon pisum, on peas, Pisum sativum. Entomologia exp. appl. 71, 245-256. Sasaki T., Aoki T., Hayashi H. and Ishikawa H. (1990) Amino acid composition of the honeydew of symbiotic and aposymbiotic pea aphids Acyrthosiphon pisum. J. Insect Physiol. 36, 3540. Sharkey P. J. and Pate J. S. (1976) Translocation from leaves to fruits of a legume, studied by phloem bleeding technique: Diurnal changes and effects on continuous darkness. Planta (Berl.) 128, 63-72. Srivastava P. N., Auclair J. L. and Srivastava V. (1983) Effect of nonessential amino acids on phagostimulation and maintenance of the pea aphid, Acyrthosiphon pisum. Can. J. Zool. 61, 22242229. Srivastava P. N., Lambein F. and Auclair J. L. (1988) Nonprotein amino acid-aphid interaction: Phagostimulatory effects and survival of the pea aphid, Acyrthosiphon pisum. Entomologia exp. appl. 48, 109-l 15. Tjallingi W. F. and Hogen Esch T. (1993) Fine structure of aphid stylet routes in plant tissues in correlation with EPG signals. Physiol. Enr. 18, 317-328. Unwin D. M. (1978) A versatile high frequency radio microcautery. Physiol. Ent. 3, 71-73. Urquhart A. A. and Joy K. W. (1981) Use of phloem exudate technique in the study of amino acid transport in pea plants. Plant Physiol. 68, 756754. Van Emden H. F. and Bashford M. A. (1969) A comparison of the reproduction of Brevicoryne brassicae and Myzus persicae in relation to soluble nitrogen concentration and leaf age (leaf position) in the Brussels sprout plant. Entomologiu exp. uppl. 12, 351-364. Via S. (1991) The genetic structure of host plant adaptation in a spatial patchwork: Demographic variability among reciprocally transplanted pea aphid clones. Evolution 45, 8277852. Weibull J., Brishammar S. and Pettersson J. (1986) Amino acid analysis of phloem sap from oats and barley: A combination of aphid stylet excision and high performance liquid chromatography. Entomologia exp. appl. 42, 27730.
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Weibull J. (1987) Seasonal changes in the free amino acids of oat and barley phloem sap in relation to plant growth stage and growth of Rhopalosiphum padi. Ann. appl. Biol. 111, 729-737. Weibull J. (1988) Free amino acids in the phloem sap from oats and barley resistant to Rhopalosiphum padi. Phytochemistry 21, 2069-2072. Weibull J. and Melin G. (1990) Free amino acid content of phloem sap from Brassica plants in relation to performance of Lipaphis erysimi (Hemiptera: Aphididae). Ann. uppl. Biol. 116, 417423. Weld S., Albano C., Dunn W., Edlund U., Esbensen K., Geladi P., Hellberg S., Johansson E., Lindberg W. and Sjiistrom M. (1984)
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Acknowledgements-We would like to thank Barbara Ekbom, Christer Solbreck, David Tilles, Jens Weibull and Inger Ahman for helpful comments on the manuscript. We also thank Jens Weibull for introducing us to the techniques used for cutting aphid stylet. This study was supported by a grant from the Swedish Council for Forestry and Agricultural Research.