See

discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/227788482

Phloem amino acids and the host plant range of

the polyphagous aphid, Aphis fabae
Article in Entomologia Experimentalis et Applicata February 2003
DOI: 10.1046/j.1570-7458.2003.00014.x

CITATIONS

READS

71

44

2 authors, including:
Thomas L Wilkinson
University College Dublin
40 PUBLICATIONS 1,486 CITATIONS
SEE PROFILE

All content following this page was uploaded by Thomas L Wilkinson on 20 November 2015.
The user has requested enhancement of the downloaded file. All in-text references underlined in blue are added to the original document
and are linked to publications on ResearchGate, letting you access and read them immediately.

Blackwell Science, Ltd

Phloem amino acids and the host plant range

of the polyphagous aphid, Aphis fabae
T. L. Wilkinson* & A. E. Douglas
Department of Biology, University of York, PO Box 373, York, YO10 5DD, UK
Accepted: 12 November 2002

Key words : amino acids, Aphis fabae, Hemiptera, Aphididae, phytophagous insect, insect nutrition,
phloem sap, plant range

Abstract

This study investigated the relationship between the essential amino acid requirement of the aphid
Aphis fabae Scop. and the phloem sap amino acid composition of its host plants. The dietary amino
acid requirement of A. fabae varied between clones. One or more of the eight clones of A. fabae tested
displayed depressed larval survival, larval growth rate, or rm on diets lacking histidine, methionine,
threonine, and valine, but none of the other five essential amino acids. The required amino acids
corresponded closely to the essential amino acids that varied in relative concentrations among 16
plant species tested: histidine, threonine, tryptophan, and valine. It is suggested that the interclonal
variation in the dietary requirements of an aphid species may contribute to the intraspecific variation
in plant utilisation patterns. The phloem sap amino acid composition and sucrose : amino acid ratio
did not differ consistently between host plant species of A. fabae and non-host species, indicating that
phloem amino acid composition is not an important factor in determining the host plant range of
this aphid species.

Introduction
Of the many factors defining the suitability of a plant
for phytophagous insects (e.g., Jaenike, 1990; Jermy, 1993;
Bernays & Wcislo, 1994), the most fundamental is that the
plant is of adequate nutritional quality. It has long been
recognised that the plant nitrogen and carbon : nitrogen
(C : N) ratio may limit the abundance and population
growth of many phytophagous insects, and that the dietary
requirement of insects for nitrogen comprises both the total
amount of nitrogen (nitrogen quantity) and the composition
of nitrogenous compounds (nitrogen quality) of the food
ingested (e.g., Mattson, 1980; Brodbeck & Strong, 1987).
With respect to nitrogen quality, the key nitrogenous
compounds are the nine essential amino acids which animals
generally cannot synthesize de novo: histidine, isoleucine,
leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine (Morris, 1991). Although the total nitrogen
content of plant tissue is commonly used as the sole index
of the nutritional value of plants for insects (e.g., Schoonhoven
et al., 1998), nitrogen quality can be of crucial nutritional
*Correspondence: Department of Zoology, University College
Dublin, Belfield, Dublin 4, Ireland. Tel.: 353 017 162264,
Fax: 353 017 061152. E-mail: tom.wilkinson@ucd.ie

importance. If the concentration of just one essential

amino acid is inadequate, an insect will not grow, whatever
its total dietary nitrogen intake.
Phloem sap-feeding aphids are particularly suitable for
studying the impact of dietary nitrogen quality on plant
utilisation by phytophagous insects because the principal
phloem-mobile nitrogenous compounds are free amino
acids which can be readily quantified. Although the amino
acid composition of phloem sap is variable (e.g., Winter et al.,
1992; Blackmer & Byrne, 1999; Brugire et al., 1999), it is
generally of very low nitrogen quality, i.e., it has very low
concentrations of essential amino acids (Douglas, 1993;
Sandstrm & Moran, 1999). The aphid utilisation of plant
phloem sap has been linked to their possession of symbiotic
bacteria of the genus Buchnera, from which they obtain
essential amino acids (Douglas, 1998). However, this bacterial
symbiosis does not invariably meet the total aphid requirement for essential amino acids. For example, the clone of
Myzus persicae studied by Mittler (1971) had a dietary
requirement for methionine, and the Acyrthosiphon pisum
clones used by Srivastava et al. (1985) required several
essential amino acids.
The purpose of this study was to investigate the relationship between the dietary requirement of an aphid for
particular essential amino acids and the pattern of plant

2003 The Netherlands Entomological Society Entomologia Experimentalis et Applicata 106: 103 113, 2003

103

104

Wilkinson & Douglas

utilisation by the aphid. The experimental material comprised

eight clones of the polyphagous aphid Aphis fabae Scop.
(Hemiptera: Aphididae) and 16 plant species, 10 of which
were host plants of this aphid species. Aphis fabae comprises
four known subspecies [A. f. cirsiacanthoidis Scop. (= A. acanthi
Schrank), A. f. fabae Scop., A. f. mordwilkoi Bmer and
Janish, and A. f. solanella Theob.] with distinct but overlapping plant ranges, such that some plant species are
common hosts to all subspecies and other plant species, the
subspecies-specific hosts, are utilized by just one subspecies
(Stroyan, 1984; von Thieme, 1987). It was therefore
possible to explore the link between the essential amino
acid requirements of aphids and the phloem amino acid
composition of host plants for both A. fabae sensu lato and
its constituent subspecies.

Materials and methods

The aphids and plants

The aphids studied were Aphis fabae fabae and A. f.

mordwilkoi, the two A. fabae subspecies that can be reared
in the laboratory on both plants and diets (Douglas, 1997;
Douglas et al., 2001; unpubl. results). Each clonal culture
was derived from a single parthenogenetic aphid collected
from the field (Table 1) and was maintained on excised
leaves of Rumex obtusifolius at 20 C with a L18:D6 cycle.
The clones were identified to subspecies by their production
of vigorous long-term cultures on preflowering plants
of either Vicia faba (A. f. fabae) or Tropaeolum majus
(A. f. mordwilkoi) (von Thieme, 1988). Their morphological
characteristics (Stroyan, 1984) were consistent with this
designation (A. E. Douglas, unpubl.). The clone reference
numbers (see Table 1), which are consistent with previous
publications from this laboratory, have been replaced by
single letter codes to avoid confusion in the text.

Aphid clone
Reference codea

The 16 plant species listed in Table 2 were grown from

seed in John Innes F2 compost (medium nutrient) under
glass at 20 5 C. All experiments were conducted on
preflowering plants of uniform age for each species (3
6 weeks post sowing, varying between species).
Aphid performance on chemically defined diets

The chemically defined diet formulation A of Prosser &

Douglas, 1992) with 0.5 sucrose and 0.15 amino acids
was used as the complete diet (containing all protein amino
acids: 16.5 m glutamine, 14.3 m asparagine, aspartic
acid, and arginine, 8.7 m histidine, isoleucine, leucine,
lysine, threonine, and valine, 8.4 m glutamic acid, 5.7 m
alanine, proline, and serine, 2.9 m methionine, phenylalanine, and tryptophan, 2.7 m cysteine, 1.2 m glycine,
and 0.6 m tyrosine), following confirmation of its
suitability for one aphid clone (A. f. fabae A) used in
this study (Douglas et al., 2001). Each amino acid was
individually deleted without replacement, such that the
amino acid concentration of the diets varied from 133.5 m
(glutamine-free diet) to 149.4 m (tyrosine-free diet).
Parafilm sachets of the filter-sterilized diet were prepared
aseptically and applied to Perspex cages of internal diameter
3 cm. Adult apterae from the cultures on excised Rumex
obtusifolius leaves were allowed to larviposit on the complete
diet over 24 h and their offspring were applied to the test
diets when 2 days old.
Two experimental designs were used to explore aphid
performance over one generation. In the first, groups of 10
2-day-old larvae were transferred to the complete diet and
to each of the 20 diets with an amino acid deletion, and
screened to identify dietclone combinations with a high
mortality or low fecundity. In the second experiment, 10
replicate 2-day-old aphids of each clone were individually
weighed and transferred singly to the complete diet and

Table 1 The aphid clones

Collection details
Plant species

Location

(a) Aphis fabae fabae

HR91/3
A
AED94/81
B
AED95/31
C
AED95/33
D

Vicia faba
Vicia faba
Euonymus europaeus
Euonymus europaeus

Abingdon, Oxon, UK
Bletchingdon, Oxon, UK
Rennes, France
Rennes, France

(b) Aphis fabae mordwilkoi

AED94/82
E
AED94/84
F
DY94/109
G
AED95/19
H

Tropaeolum majus
Tropaeolum majus
Tropaeolum majus
Viburnum opulus

Brockenhurst, Hants, UK
York, N. Yorks, UK
Claxton, N. Yorks, UK
Rennes, France

To avoid confusion, the clones are referred to in the text by the single letter abbreviations
AH.

Phloem amino acids and A. fabae 105

Table 2 The plant species

Plant species

Order

Family

Host plant statusa

Arabidopsis thaliana
Arctium minus
Chenopodium album
Chenopodium hybridum
Cirsium arvense
Lamium purpureum
Papaver dubium
Plantago lanceolata
Rumex acetosa
Rumex acetosella
Rumex obtusifolius
Solanum nigrum
Trifolium repens
Tropaeolum majus
Vicia faba
Viola arvensis

Brassicales
Asterales
Caryophyllales
Caryophyllales
Asterales
Lamiales
Ranunculales
Lamilaes
Polygonales
Polygonales
Polygonales
Solanales
Fabales
Brassicales
Fabales
Malpighiales

Brassicaceae
Asteraceae
Chenopodiaceae
Chenopodiaceae
Asteraceae
Lamiaceae
Papaveraceae
Plantaginaceae
Polygonaceae
Polygonaceae
Polygonaceae
Solanaceae
Fabaceae
Tropaeoleaceae
Fabaceae
Violaceae

non-host
A. f. mordwilkoi
A. f. fabae
non-host
A. f. cirsiacanthoides
A. f. fabae
A. f. fabae
non-host
A. fabae (s.l.)
non-host
A. fabae (s.l.)
A. f. solanella
non-host
A. f. mordwilkoi
A. f. fabae
non-host

Host plants of A. fabae (sensu lato) or individual subspecies refer to pre-flowering species
only [Blackman (1974), Stroyan (1984), von Thieme (1987), and unpubl. observations].

diets lacking one amino acid. Each aphid was monitored

daily until it either died or reached adulthood, when it was
re-weighed and its morph was scored. Each apterous adult
was checked daily until it larviposited, from which the prereproductive period (the time from birth to the initiation
of reproduction) was determined and the number of offspring produced over a period equal to the pre-reproductive
period was scored. Two indices of the performance of
apterae were adopted: relative growth rate from day 2 to
adulthood, RGR = ln (adult weight/day-2 weight)/(days
as larva on test diet); and the intrinsic rate of population
increase, rm = 0.738(lnMd)/T, where Md is the number of
offspring produced in the time equivalent to the prereproductive period (T) (Wyatt & White, 1977). Apterae
that died before reaching age 2T were excluded from the
quantification of rm.
Plant phloem sap analysis

The methodology adopted in the definitive analyses was

informed by preliminary experiments comparing the two
widely used methods of obtaining plant phloem sap: from
severed stylets of feeding aphids (Fisher & Frame, 1984),
or from excised plant tissue into EDTA solution, which
chelates calcium ions and so prevents sieve element sealing
(King & Zeevaart, 1974). The EDTA exudates from excised
leaves of all species listed in Table 2 contained sugars and
amino acids, indicative of phloem sap release. Stylectomy
was not feasible for the non-host species (as A. fabae did
not feed from most of them) and failed for some host
species, notably Chenopodium album, Rumex obtusifolius,
and Papaver dubium, for which the stylets consistently failed

to exude. Quantitative comparisons between the amino

acid composition of the stylet- and EDTA-exudates from
Vicia faba and Lamium purpureum, however, confirmed
the conclusions of previous studies on various plant species
(e.g., Weibull et al., 1990; Girousse et al., 1991; Sandstrm
et al., 2000) that EDTA exudates offer a reliable measure of
phloem amino acid composition.
The EDTA exudation method followed the procedure of
King & Zeevart (1974) as modified by Douglas (1993) to
avoid generalised damage to plant tissues and non-specific
amino acid release, caused by high EDTA concentrations
or long incubation periods. The youngest fully expanded
leaf was excised from each of 10 plants of each species using
clean sharp scissors and the cut surface was immediately
immersed in 200 l 5 m Na2EDTA. The sample was
incubated in a light-proof box at 25 C for 90 min, with a
saturated solution of KH2PO4 to maintain high humidity.
The leaf was discarded and the phloem exudates in the EDTA
solution were stored at 20 C.
The amino acids in individual exudate samples were
analysed by reverse-phase high performance liquid
chromatography with pre-column derivatisation using ophthaldialdehyde (Jones et al., 1981). The amino acids were
separated on a Xorbax Eclipse XDB-C8 column at
20 C, using a Hewlett-Packard HP 1100 delivery system
and fluorescence detector. Amino acids were quantified by
comparison of sample peak areas to a three-level calibration
plot of the reference amino acid mixture AA-S-18 (Sigma
Chemical Co.) supplemented with glutamine, asparagine,
and tryptophan, using linear regression forced through the
origin. All protein amino acids except proline and cysteine

106

Wilkinson & Douglas

could be detected by this method, with a detection limit of

approximately 0.5 pmol.
The sucrose concentration in the phloem exudates was
quantified by a glucose assay from Sigma Chemical Co.
(procedure no. 510) following enzymatic hydrolysis. In brief,
10 l volumes of phloem exudate and invertase (10 U ml1)
were mixed and incubated for 30 min at 37 C, to which
was added 300 l reaction mixture containing glucose
oxidase, peroxidase, and o-dianisidine (see the manufacturers procedures for full details). Following incubation
at 37 C for 30 min, the absorbance of the sample was
measured at 450 nm and compared to glucose standards.
Glucose equivalents were converted to sucrose equivalents
for calculation of the sucrose : amino acid ratios in the
exudates. It was thought justified to use sucrose : amino
acid ratio as a measure of the C : N content of phloem sap
because, first, sucrose is the dominant phloem transport
sugar in plants of the same family as all species listed in
Table 2, except the Lamiaceae, which are reported to have
the raffinose family of oligosaccharides as the main phloem
sugars (Zimmermann & Ziegler, 1975), and, second, the
phloem exudates of Lamium purpureum plants used in
our study had sucrose as the sole disaccharide, with no
detectable higher-order oligosaccharides (T.L. Wilkinson
& A.E. Douglas, unpubl. results obtained using thin-layer
chromatography).
Statistical analysis

The variation in amino acid composition between EDTA

exudates of plant species was analysed by multivariate analysis
of variance (MANOVA) followed by principal components
analysis (PCA) to explore the underlying trends in the
dataset. The MANOVA was a balanced, single factor design
with 10 replicates in each factor group (plant species), and
the data (nmol amino acid exuded per sample) were natural
log-transformed prior to analysis to achieve normality and
homogenous variance (the exponent for transformation
following Taylors power law was 0.07). PCA was performed
using the relative amounts of each amino acid (expressed
as mol %) in each of 10 replicate samples from 16 plant
species, using a correlation matrix to standardise variables
(Randerson, 1996).
Other datasets were examined by univariate methods.
ANOVA was applied where the data conformed to the
assumptions of normality and homogenous variances, as
indicated by KolmogorovSmirnoff one-sample test and
Bartletts test, respectively. Differences between groups in
one-way ANOVA were examined post hoc using Tukeys
difference test, with a Bonferroni correction for multiple
comparisons. Where the assumptions of normality and
homogenous variance could not be met, non-parametric
tests were employed.

Results
Requirement of Aphis fabae clones for dietary amino acids

The analysis was initiated by monitoring the response of

the eight clones of A. f. fabae and A. f. mordwilkoi to the
complete diet (containing all amino acids) and diets in
which each of the 20 amino acids were individually
omitted. The 2-day-old larvae of every clone settled onto
all diets and fed, as indicated by honeydew production.
On the complete diet and most diets with an individual
amino acid deletion, the aphids of all clones developed to
adulthood within 2 weeks and produced offspring. However,
on diets lacking histidine, methionine, threonine, tryptophan,
or valine, the survival or reproductive output of one or
more clones appeared to be depressed. In some diet-clone
combinations (especially on the tryptophan-free diet), low
fecundity could be linked to the production of alates, many
of which did not settle after reaching adulthood and died
within a few days, apparently without feeding.
The performance of the aphids on diets lacking histidine,
methionine, threonine, tryptophan, or valine was analysed
in detail, with the complete diet as the control treatment.
All clones performed sufficiently well on the complete diet
for the relative growth rate (RGR; Figure 1) and intrinsic
rate of increase (rm; data not shown) to be quantified. At
least nine of the 10 individuals of each clone survived to
adulthood, all but one of the 78 aphids were apterous, and
the mean RGR of the apterae varied from 0.161 mg mg1
day1 (A. f. mordwilkoi G) to 0.252 mg mg1 day1 (A. f. fabae C).
The interclonal variation in RGR was statistically significant
(F7,69 = 18.69, P < 0.001), and the pair-wise comparison
revealed that the RGR of two clones of A. f. fabae (C and A)
were significantly higher than that of A. f. fabae B and three
clones of A. f. mordwilkoi (H, F and G). Every aphid that
survived to twice the pre-reproductive period produced
offspring. The rm ranging from 0.11 0.015 (A. f. mordwilkoi
G) to 0.17 0.013 (A. f. fabae C) aphids aphid1 day1 (mean
SE), varied significantly between clones (F7,66 = 4.38, P
< 0.001), with a pattern of individual pair-wise differences
among the clones similar to that for RGR, such that the
RGR and rm were significantly correlated whether expressed
on the basis of per-aphid (rs = 0.505, P < 0.01, n = 77) or
per-clone (rs = 0.839, P < 0.01, n = 8). It was concluded
from this analysis that the complete diet could support all
the clones through one generation and, consequently, the
poor performance of any clone on a diet with an amino
acid deletion could be attributed to a dietary requirement
for that amino acid, and not to a non-specific malaise
linked to a general nutritional inadequacy of the chemically
defined diets.
Certain dietary deletions promoted alate production,
but not consistently across aphid clones. Alate production

Phloem amino acids and A. fabae 107

diet, larval mortality was very high for five clones; of the
remaining three clones, A. f. fabae D and A. f. mordwilkoi
H displayed RGR and rm on the methionine-free diet which
were at least comparable to values for the complete diet,
and A. f. fabae C generated predominantly alates (whose
performance was not analysed further). The histidine-free
diet supported the development to adulthood of seven or
more aphids of all clones except A. f. fabae B, which died as
larvae, but the aphids of most clones on this diet were
small and produced few offspring, resulting in significantly
depressed RGR and rm values. Exceptionally, aphids of
A. f. mordwilkoi F and H performed well without dietary
histidine.
Generally, the aphids performed well on the diets lacking
threonine or tryptophan (Figure 1), although it was not
possible to establish the dietary requirements of A. f. fabae
C definitively because of its high alate production on diets
lacking these amino acids. Larviposition by A. f. fabae B
was depressed on the threonine-free diet, resulting in a
significant reduction in rm relative to the complete diet.
In summary, depressed larval survival, larval growth rate,
or adult fecundity were displayed by certain clones of A. fabae
reared on diets lacking histidine, methionine, threonine,
valine, and possibly tryptophan, but no effect was consistent
across all clones.
Phloem sap amino acids

Figure 1 Relative growth rate of apterous Aphis fabae to

adulthood on chemically defined diets with individual amino
acid deletions. Clone designations (AH) are as shown in Table 1
(all data mean SE, sample size at base of bar).

varied from A. f. mordwilkoi clones F and G, each of which

produced just one alatiform adult, to A. f. fabae C, of which
half or more aphids were alate on the diets lacking histidine,
methionine, threonine, or tryptophan but not valine.
Dietary omission of valine, methionine, or histidine had
a striking effect on the performance of some clones
(Figure 1 and Table 3). All 10 aphids of A. f. fabae D died
as larvae on the valine-free diet, but the performance of all
other clones on this diet was equivalent to (and for clones
B and H significantly better than) their performance on
the complete diet (Table 3). On the methionine-free

The full complement of protein amino acids was detected

in every phloem exudate sample from all 16 plant species,
and the amino acids were consistently separated with
no co-eluting peaks. The non-protein amino acid aminobutyric acid was recovered from all samples, but at
varying concentrations, and the phloem exudates from
Tropaeolum majus contained an unidentified peak that eluted
after lysine (retention time 32.7 min) and represented
1730% of the total peak area. Previous experiments have
suggested that -aminobutyric acid is an artefact of the
exudation technique (see Girousse et al., 1991), and there
was no detector response at a retention time equivalent
to the unidentified peak in an analysis of the honeydew
of aphids feeding on Tropaeolum majus (T.L. Wilkinson
& A.E. Douglas, unpubl.). It was concluded that these
compounds were unlikely to be phloem-mobile, and further
analysis was restricted to the 18-protein amino acids.
Table 4 shows the amino acid composition of the exudates from 16 plant species (the sucrose : amino acid ratio,
and the nonessential amino acid composition is shown in
Table 4a, and the percentage essential amino acid content, the essential amino acid composition in Table 4b).
The composition of the amino acids varied significantly between plant species, as revealed by MANOVA
(Wilks (s=15, m=1, n=62.5) < 0.01, F270,1509 = 24.3, P < 0.001). The

108

Wilkinson & Douglas

Table 3 Statistical analysis for relative

growth rate

ANOVA
Aphid clone

Degrees
of freedom

A. f. fabae
A
B
Cc
D

4,26
3,24
3,20
4,33

A. f. mordwilkoi
E
F
G
H

4,32
4,38
4,33
5,42

Pa

Tukeys test
Pair-wise differencesb

5.44
9.79
4.79
6.23

0.003*
< 0.001*
0.011
0.001*

trpo > hiso

valo > complete

thro > hiso

13.38
2.61
5.29
14.10

< 0.001*
0.051
0.002*
< 0.001

complete, thro, valo > hiso

no individual differences
meto, thro, valo > complete, hiso

* Significant after Bonferroni correction for eight tests (critical P = 0.00625).

trpo refers to tryptophan-free diet, hiso to histidine-free diet, etc. (standard abbreviations
for amino acids are provided in legend to Figure 2).
c
Data for some diets were excluded from statistical analysis because sample sizes were < 3.
b

phloem exudates were dominated by nonessential amino

acids, particularly glutamine or asparagine. The mean contribution of essential amino acids to the total amino acid
content in the exudates did not exceed 20 mol % in any of
the plant species examined. The dominance of nonessential
amino acids in the phloem exudates was confirmed by
principal component analysis, in which over 90% of the
variance in the dataset could be accounted for by the first
two principal components (Figure 2a,b). The first principal
component axis, explaining 64.5% of the variance in the data,
was strongly correlated with the amount of glutamine in the
phloem exudate. The second principal component axis,
accounting for 26.1% of the variance, divided the data into
two groups on the basis of the relative amount of asparagine in the phloem exudates. Thus, the 16 plant species
were separated on the basis of the dominance of either
glutamine (in 5/6 non-hosts and 6/10 hosts) or asparagine
[in one non-host (Trifolium repens) and certain subspecies
specific hosts of A. f. fabae (Vicia faba), A. f. cirsiacanthoidis
(Cirsium arvense), and A. f. mordwilkoi (Arctium minus and
Tropaeolum majus)]. These results suggest that host and
non-host plants of A. fabae do not differ consistently in their
dominant amino acids.
Analysis of the nine essential amino acids in phloem
exudates revealed significant variation between the plant
species (Wilks (s=9, m=2.5, n=67) < 0.01, F135,1073 = 21.1,
P < 0.001), with 80.8% of the variance in the data accounted
for by the first two axes of the principal component analysis
(Figure 2c,d). The first principal component (accounting
for 64.4% of the variance) separated the data on the
basis of the relative amounts of threonine and histidine
in the phloem exudates. For example, the essential amino
acids from the phloem exudates of 11/16 plant species

had relatively high concentrations of threonine, as

represented by the cluster of data points at the negative end
of the first principal component axis, whereas the essential amino acids in the phloem exudates from Vicia faba
were dominated by histidine, resulting in the isolation of
9/10 exudate samples from Vicia faba with respect to the
first principal component. Threonine was also relatively
abundant, but not the dominant essential amino acid, in the
phloem exudates from Chenopodium hybridum (non-host),
Chenopodium album (specific host of A. f. fabae), and Rumex
acetosa (common host), and the position of these species
in the analysis was strongly influenced by the second
principal component axis (16.4% of the total variance)
which, from the plot of the loading scores, was correlated
with tryptophan. The separation of 9/10 exudate samples
of Arctium minus from the main cluster of points could be
attributed to the relatively high concentration of valine in
its phloem exudates.
The phloem exudates of host and non-host species of
A. fabae did not differ significantly in either the median
essential amino acid content (MannWhitney U-test: W
= 83.5, P > 0.05) or in the median sucrose : amino acid
ratio (W = 81.0, P > 0.05).
In summary, among the plants we studied, the phloem
sap composition of the host plants of A. fabae did not differ
consistently from that of non-host plant species, by the
criteria of amino acid composition or sucrose : amino acid
ratio of phloem sap. However, the data do reveal striking
differences between amino acids in their variability among
plant species. Of particular relevance to the phloemfeeding insects, the levels of four essential amino acids
(threonine, histidine, tryptophan, and valine) varied between
plant species, while those of the other five essential amino

Phloem amino acids and A. fabae 109

Figure 2 Principal component analysis of phloem exudates from

16 plant species. (a, b) All protein amino acids, (c, d) essential
amino acids only. (a) and (c) are plots of principal components 1
and 2, and (b) and (d) are the loading scores of the amino acids.
Plant species are grouped as non-host plants (+), common host
plants (h), and subspecies specific host plants (s). The values on
the axes in (b) and (d) refer to the proportion of the total variance
accounted for by each principal component. In (b), ess refers to
the nine essential amino acids (see Introduction). In (c), the

subspecies-specific host plants Vicia faba and Arctium minus

are highlighted by filled and stippled circles, respectively.
Amino acid concentrations were converted to mol % prior
to analysis. Standard abbreviations are used as follows: ala,
alanine; arg, arginine; asn, asparagine; asp, aspartate;
glu, glutamate; gln, glutamine; gly, glycine; his, histidine;
ile, isoleucine; leu, leucine; lys, lysine; met, methionine;
phe, phenylalanine; ser, serine; thr, threonine; trp, tryptophan;
tyr, tyrosine; val, valine.

110

Wilkinson & Douglas

Table 4 The amino acid composition of phloem exudates from 16 plant species. All data are mean mol % of 10 replicates
(a) Non-essential amino acids

Host plant status

Plant species

Sugar :
amino
acid ratio
(mol /mol)

Non-host plants

Arabidopsis thaliana
Chenopodium hybridum
Plantago lanceolata
Rumex acetosella
Trifolium repens
Viola arvensis

2.2
1.6
6.7
1.0
5.2
3.3

Common host plants

Rumex acetosa
Rumex obtusifolius

Sub species-specific host plants

Arctium minus
Chenopodium album
Cirsium arvense
Lamium purpureum
Papaver dubium
Solanum nigrum
Tropaeolum majus
Vicia faba

(b) Essential amino acids

Relative amount of nonessential amino

acid in exudates (mol%)a
ala

arg

asn

asp

glu

gln

gly

ser

tyr

9.7
6.4
9.4
5.2
7.5
5.4

1.6
0.9
8.2
0.8
0.7
1.2

10.1
1.9
2.6
4.3
45.5
3.5

9.5
7.9
7.7
9.5
8.3
11.1

11.4
17.5
20.8
18.5
11.3
11.7

33.2
42.4
27.0
30.9
2.3
46.4

0.7
2.0
1.2
2.9
2.0
1.2

7.7
4.0
7.6
14.0
10.2
9.3

0.6
1.6
1.1
1.8
1.1
1.0

7.9
3.9

5.0
4.3

0.8
1.5

1.5
1.6

6.4
10.8

21.8
21.5

44.6
36.1

1.1
0.9

6.3
7.8

2.4
1.2

1.3
3.6
1.2
0.1
0.4
3.0
8.7
1.1

9.7
5.1
10.1
6.3
6.1
9.3
7.5
3.0

1.3
1.2
1.0
0.7
0.4
1.4
1.0
0.6

39.3
2.8
32.2
2.8
3.9
5.4
41.7
68.7

5.0
7.7
3.9
2.1
23.1
6.6
9.1
3.4

15.0
21.5
11.8
8.1
11.2
12.4
11.2
3.7

9.1
32.2
18.8
61.5
33.4
32.8
3.9
6.7

1.3
2.0
2.4
5.4
0.8
2.7
1.2
0.8

7.1
5.7
8.2
6.1
7.7
9.8
7.95
4.4

1.4
1.5
0.9
0.5
1.2
1.5
1.4
0.5

Relative amount of essential

amino acid in exudates (mol%)a

Host plant status

Plant species

% essential
amino
acid content

Non-host plants

Arabidopsis thaliana
Chenopodium hybridum
Plantago lanceolata
Rumex acetosella
Trifolium repens
Viola arvensis

15.3
15.4
14.4
12.0
9.5
9.1

0.4
0.9
0.3
0.6
0.4
0.3

1.2
1.6
1.3
1.1
0.8
0.9

1.2
1.7
1.6
1.5
1.1
1.1

1.8
2.0
1.9
1.3
1.3
1.3

0.7
1.1
0.8
0.7
0.4
0.4

1.1
1.2
1.0
0.8
0.7
0.7

5.6
1.1
3.4
2.6
2.2
1.7

0.9
3.1
0.8
1.2
0.9
0.6

2.4
2.7
3.3
2.2
1.8
2.0

Common host plants

Rumex acetosa
Rumex obtusifolius

11.7
14.4

0.3
0.5

0.6
1.0

0.8
1.5

0.8
1.3

0.4
1.0

0.8
1.3

2.5
4.2

3.5
1.3

1.4
2.3

Sub species-specific host plants

Arctium minus
Chenopodium album
Cirsium arvense
Lamium purpureum
Papaver dubium
Solanum nigrum
Tropaeolum majus
Vicia faba

10.9
19.8
10.7
6.5
12.3
18.1
15.1
8.2

0.7
0.5
0.6
0.3
0.2
0.7
0.7
3.3

0.9
2.4
1.0
0.5
1.0
2.2
1.3
0.5

1.3
3.0
1.2
0.7
0.9
2.3
2.3
0.6

1.4
2.5
1.1
0.6
0.6
1.9
2.0
1.0

0.6
1.2
0.3
0.4
0.3
1.0
0.5
0.2

1.9
0.9
1.3
0.3
0.3
1.6
1.4
0.4

1.8
3.3
2.2
2.2
4.9
3.6
3.2
1.0

0.4
2.8
0.7
0.6
1.2
2.0
1.4
0.2

2.0
3.1
2.2
0.9
2.8
2.7
1.9
1.0

his

ile

leu

lys

met

phe

thr

trp

val

Standard abbreviations for amino acids are provided in legend to Figure 2.

acids (isoleucine, leucine, lysine, methionine and phenylalanine) were relatively invariant.

Discussion
The variable essential amino acids in aphidplant interactions

This study has revealed significant variation in both the

essential amino acid requirements among eight clones

of A. fabae, and the essential amino acid composition of

phloem sap exudates among 16 plant species.
The single amino acid deletion technique, as used here
to identify the essential amino acid requirements of A. fabae,
provides accurate information on the dietary requirements
of insects, and has been widely employed in aphids (Dadd,
1977, 1985). Classical dietary studies were conducted on
single clones of each aphid species (e.g., Dadd & Krieger,

Phloem amino acids and A. fabae 111

1967, 1968; Retnakaran & Beck, 1968; Leckstein & Llewellyn,

1973) with the, often implicit, assumption that the nutritional requirements of all members of any one aphid species are uniform. As Figure 1 illustrates, this assumption is
not justified. However, the present study on A. fabae is not
the first report of intraspecific variation in the essential
amino acid requirements of aphids. Srivastava et al. (1985)
found that two clones of the pea aphid Acyrthosiphon
pisum had different essential amino acid requirements, and
attributed the difference to a variation in the pattern of amino
acids produced by their symbiotic bacteria Buchnera (see
below). The interclonal variation in aphid requirement for
methionine is of particular interest because the complete
genome sequences of Buchnera (from Acyrthosiphon pisum
and Schizaphis graminum) suggest that these bacteria lack the
capacity to synthesize methionine (Shigenobu et al., 2000;
Tamas et al., 2002). If the Buchnera in Aphis fabae also
lack the capacity to synthesize methionine, then the interclonal variation in the requirement for dietary methionine
could reflect interclonal differences in the abundance of
other bacteria (secondary symbionts) with this biosynthetic
capability (Douglas et al., in press).
To our knowledge, the data in Table 2 represent the first
analysis of phloem sap amino acid composition for most
of the plant species used in this study. The dataset is in
excellent accord with published data showing that phloem
amino acids are generally dominated by glutamate/glutamine
and aspartate/asparagine, and have low concentrations
of essential amino acids (Ziegler, 1975). The analysis of
Sandstrm & Moran (1999) focused on the phloem profile
of essential amino acids. From published data on nine plant
species, they identified certain essential amino acids whose
relative concentrations tended to be uniform (isoleucine,
leucine, lysine, and phenylalanine) and other amino acids,
notably histidine, methionine, and threonine, which were
more variable. In close, but not complete, agreement, the
essential amino acids that varied most among the 16
species analysed here were histidine, threonine, tryptophan,
and valine. Brodbeck & Strong (1987) also pinpointed
histidine, methionine, and tryptophan as the plant amino
acids most likely to be limiting for phytophagous insects,
including phloem-feeders. Taken together, these datasets
indicate that polyphagous aphids, including Aphis fabae,
are likely to encounter variation in the abundance of five
essential amino acids, histidine, methionine, threonine,
tryptophan, and valine, among the plants on which they
feed.
The essential amino acids required by one or more clones
of A. fabae were histidine, methionine, threonine, and valine.
A striking aspect to these results is their close correspondence with the amino acids that are generally variable in
plant phloem sap.

Essential amino acids and the plant range of aphids

A key result of this study is that the host plant species of

A. fabae and non-host plants did not differ consistently
in their essential amino acid content or sucrose : amino
acid ratio (i.e., indices of the quality and quantity of dietary
nitrogen, respectively). For example, the histidine content
varied 19-fold among the species tested, with the highest
and lowest mean concentrations in Vicia faba and Papaver
dubium, both of which are specific hosts of A. f. fabae (Stroyan,
1984; von Thieme, 1987). These data provide strong evidence
against a role for phloem essential amino acid composition as a determinant of the host plant range of A. fabae.
Consistent with this conclusion, other analyses have
attributed the compatibility between A. fabae and particular
plant species to non-nutritional factors. In particular, the
utilization of Lamium purpureum by A. fabae, despite poor
aphid performance (Adams & Douglas, 1997; Wilkinson
et al., 2001) can be linked to low natural enemy abundance
on this plant species (Raymond et al., 2000), and the
high mortality of A. f. fabae on Tropaeolum majus, a host
plant of A. f. mordwilkoi, arises from a barrier at the sieve
element phase of feeding, causing premature withdrawal of
the stylets of A. f. fabae clones (Tosh et al., 2001).
The variation in essential amino acid requirements
among the A. fabae clones strongly indicates that, if these
dietary requirements influence plant utilization patterns,
then they operate at a finer phylogenetic scale than species
and subspecies in A. fabae. The interclonal differences
may be linked to the symbiotic bacteria Buchnera, which
provide essential amino acids for the insect (Baumann
et al., 1995; Douglas, 1998), and could arise from either
genetic differences or stable differences in gene expression
between the Buchnera in different aphid clones. Whatever
its molecular basis, the dietary requirement for an amino
acid is probably not an evolutionarily conserved trait in
A. fabae, because the pattern of variation in amino acid
requirements is not well-correlated with either aphid
subspecies (see Figure 1) or interclonal variation in mtDNA,
which, like Buchnera, is maternally inherited (Raymond
et al., 2001).
What are the selection pressures involved in maintaining
the intraspecific variation in amino acid requirements
in natural populations of A. fabae? An important clue may
be that the amino acids required by certain aphid clones
are generally the amino acids which vary among plant
species, i.e., the phloem-variable amino acids (see above).
An aphid clone with a dietary requirement for a particular
amino acid is predicted to perform well on plants that are
relatively rich in that amino acid, and poorly on plants
with low levels of the amino acid. However, such a clone
may perform better than clones without the dietary
requirement on plants with high levels of the relevant amino

112

Wilkinson & Douglas

acid. For example, the bacterial symbiosis in aphids without the dietary requirement may continue to produce the
amino acid at high rates, diverting potentially limiting
dietary nitrogen from other essential functions.
The broad implication of these considerations is that
different members of an aphid species may have distinct
nutritional requirements, possibly underlain by differences in the nutritional capabilities of their vertically transmitted symbiotic bacteria. This variation may account for
some of the variation in plant utilization within individual
subspecies of A. fabae (e.g., Stroyan, 1984) and within other
aphid species (e.g., Pilson, 1992; Dixon, 1998; Lushai et al.,
2002).

Acknowledgements
We thank Dr J.-C. Simon for providing aphids from
Rennes and Dr D. Yurman for clone G, Ms. N. Burdon and
Mrs L. Minto for technical assistance, and Dr A. J. Karley
and Dr C. Cloutier for helpful comments on the manuscript.
Financial support was provided by The Natural Environment
Research Council.

References
Adams D & Douglas AE (1997) How symbiotic bacteria influence
plant utilisation by the polyphagous aphid, Aphis fabae.
Oecologia 110: 528 532.
Baumann P, Baumann L, Lai C-Y, Rouhbakhsh D, Moran NA
& Clark MA (1995) Genetics, physiology, and evolutionary
relationships of the genus Buchnera: intracellular symbionts of
aphids. Annual Review of Microbiology 49: 55 94.
Bernays EA & Wcislo WT (1994) Sensory capabilities, information
processing and resource specialisation. Quarterly Review of
Biology 69: 187204.
Blackman RL (1974) Invertebrate Types Aphids. Ginn & Company, London, pp 175.
Blackmer JL & Byrne DN (1999) Changes in amino acids in Cucumis
melo in relation to life-history traits and flight propensity of
Bemisia tabaci. Entomologia Experimentalis et Applicata 93:
29 40.
Brodbeck B & Strong (1987) Amino acid nutrition of herbivorous
insects and stress to host plants. Insect Outbreaks (ed. by P Barbosa
& J C Schultz), pp. 347 364. Academic Press, New York.
Brugire N, Dubois F, Limami AM, Lelandais M, Roux Y,
Sangwan RS & Hirel B (1999) Glutamine synthetase in the
phloem plays a major role in controlling proline production.
Plant Cell 11: 1995 2001.
Dadd RH (1977) Qualitative requirements and utilization of
nutrients: insects. Handbook Series in Nutrition and Food,
Section D, Vol. 1. Nutritional Requirements. (ed. by M Rechcigl),
pp. 305 346. CRC Press, Cleveland, OH.
Dadd RH (1985) Nutrition: organisms. Comprehensive Insect
Physiology, Biochemistry and Pharmacology (ed. by G A Kerkut

& L I Gilbert), Vol. 4, pp. 313 391. Pergamon Press, Oxford,

UK.
Dadd RH & Krieger DL (1967) Continuous rearing of aphids of
the Aphis fabae complex on sterile synthetic diet. Journal of
Economic Entomology 60: 1512 1517.
Dadd RH & Krieger DL (1968) Dietary amino acid requirements
of the aphid, Myzus persicae. Journal of Insect Physiology 14:
741 755.
Dixon AFG (1998) Aphid Ecology. Chapman & Hall, London,
UK.
Douglas AE (1993) The nutritional quality of phloem sap utilized
by natural aphid populations. Ecological Entomology 18:
3138.
Douglas AE (1997) Provenance, experience and host plant
affiliation in the polyphagous aphid, Aphis fabae. Entomologia
Experimentalis et Applicata 83: 161170.
Douglas AE (1998) Nutritional interactions in insect-microbial
symbioses: aphids and their symbiotic bacteria Buchnera. Annual
Review of Entomology 43: 17 37.
Douglas AE, Darby AC, Birkle LM & Walters KFA (2002) The
ecological significance of symbiotic micro-organisms in animals
perspectives from the microbiota of aphids. Genes in the
Environment (ed. by R M Hails, J Beringer & H C J Godfray),
pp 306325 Blackwell Scientific Publishers, Oxford, UK.
Douglas AE, Minto LB & Wilkinson TL (2001) Quantifying
nutrient production by the microbial symbionts in an aphid.
Journal of Experimental Biology 204: 349 358.
Fisher DB & Frame JM (1984) A guide to the use of the exudingstylet technique in phloem physiology. Planta 161: 385
393.
Girousse C, Bonnemain J-L, Delrot S & Bournoville R (1991)
Sugar and amino acid composition of phloem sap of Medicago
sativa: a comparative study of two collecting methods. Plant
Physiology and Biochemistry 29: 41 48.
Jaenike J (1990) Host specialisation in phytophagous insects.
Annual Review of Ecology and Systematics 21: 243 273.
Jermy T (1993) Evolution of insect-plant relationships a devils
advocate approach. Entomologia Experimentalis et Applicata
66: 3 12.
Jones BN, Paabo S & Stein S (1981) Amino acid analysis and
enzymatic sequence determination of peptides by an improved
o-phthaldialdehyde precolumn labelling procedure. Journal of
Liquid Chromatography 4: 565 586.
King RW & Zeevaart JAD (1974) Enhancement of phloem exudation from cut petioles by chelating agents. Plant Physiology
53: 96 103.
Leckstein PM & Llewellyn M (1973) Effect of dietary amino acids
on the size and alary polymorphism of Aphis fabae. Journal of
Insect Physiology 19: 973 980.
Lushai G, Markovitch O & Loxdale HD (2002) Host-based
genotype variation in insects revisited. Bulletin of Entomological Research 92: 159 164.
Mattson WJ (1980) Herbivory in relation to plant nitrogen content.
Annual Reviews of Ecology and Systematics 11: 119 161.
Mittler TE (1971) Dietary amino acid requirement of the aphid
Myzus persicae affected by antibiotic uptake. Journal of Nutrition
101: 1023 1028.

Phloem amino acids and A. fabae 113

Morris JG (1991) Nutrition. Environmental and Metabolic

Animal Physiology (ed. by C L Prosser), pp. 231 276. John Wiley
& Sons, New York.
Pilson D (1992) Insect distribution patterns and evolution of
host use. Plant Resistance to Herbivores and Pathogens (ed. by
R S Fritz & E L Simms), pp. 120 139. University of Chicago
Press, Chicago, IL.
Prosser WA & Douglas AE (1992) A test of the hypotheses that
nitrogen is upgraded and recycled in an aphid (Acyrthosiphon
pisum) symbiosis. Journal of Insect Physiology 38: 93 99.
Randerson PF (1996) Ordination. Biological Data Analysis, a
Practical Approach (ed. by J C Fry). IRL Press, Oxford, UK.
Raymond B, Darby AC & Douglas AE (2000) Intraguild predation and the spatial distribution of a parasitoid. Oecologia 124:
367 372.
Raymond B, Searle JB & Douglas AE (2001) On the processes
shaping reproductive isolation in aphids of the Aphis fabae
(Scop.) complex (Aphididae: Homoptera). Biological Journal
of the Linnean Society 74: 205 215.
Retnakaran A & Beck SD (1968) Amino acid requirements and
sulfur amino acid metabolism in the pea aphid Acyrthosiphon
pisum (Harris). Comparative Biochemistry and Physiology 24:
611619.
Sandstrm J & Moran NA (1999) How nutritionally imbalanced
is phloem sap for aphids? Entomologia Experimentalis et
Applicata 91: 203 210.
Sandstrm J, Telang A & Moran NA (2000) Nutritional
enhancement of host plants by aphids a comparison of three
aphid species on grasses. Journal of Insect Physiology 46:
33 40.
Schoonhoven LM, Jermy T & van Loon JJA (1998) InsectPlant
Biology. Chapman & Hall, London.
Shigenobu S, Watanabe H, Hattori M, Sakaki Y & Ishikawa H
(2000) Genome sequence of the endocellular bacterial symbiont of aphids Buchnera sp. APS. Nature 407: 81 86.
Srivastava PN, Gao Y, Levesque J & Auclair JL (1985) Differences
in amino acid requirements between two biotypes of the pea
aphid, Acyrthosiphon pisum. Canadian Journal of Zoology 3:
603 606.

Stroyan HLG (1984) Aphids Pterocommatinae and Aphidinae

(Aphidini). Handbooks for the Identification of British Insects,
Vol. 2. Part 6. Royal Entomological Society of London, UK.
Tamas I, Klasson L, Canback B, Naslund AK, Eriksson AS,
Wernegreen JJ, Sandstrom JP, Moran NA & Andersson SGE
(2002) 50 million years of genomic stasis in endosymbiotic
bacteria. Science 296: 2376 2379.
von Thieme T (1987) Members of the complex of Aphis fabae
Scop. & their host plants. Population Structure, Genetics and
Taxonomy of Aphids (ed. by J Holman, J Pelekan, A F G Dixon
& C Wensmen), pp. 314 323. SPB. Academic Publishers, The
Hague.
von Thieme, T (1988) Zur Biologie von Aphis fabae mordwilkoi
Bmer und Janisch 1922 (Homoptera: Aphididae). Journal of
Applied Entomology 105: 510 515.
Tosh CR, Walters KFA & Douglas AE (2001) On the mechanistic
basis of plant affiliation in the black bean aphid (Aphis fabae)
species complex. Entomologia Experimentalis et Applicata 99:
121125.
Weibull J, Ronquist F & Brishammer S (1990) Free amino acid
composition of leaf exudates and phloem sap. A comparative
study in oats and barley. Plant Physiology 92: 222 226.
Wilkinson TL, Adams D, Minto LB & Douglas AE (2001) The
impact of host plant on the abundance and function of symbiotic bacteria in an aphid. Journal of Experimental Biology 204:
30273038.
Winter H, Lohaus G & Heldt HW (1992) Phloem transport of
amino acids in relation to their cytosolic levels in barley leaves.
Plant Physiology 99: 996 1004.
Wyatt IJ & White PF (1977) Simple estimation of intrinsic
increase rates for aphids and tetranychid mites. Journal of
Applied Ecology 14: 757 766.
Ziegler H (1975) Nature of transported substances. Encyclopedia
of Plant Physiology Newseries (ed. by M H Zimmerman &
J A Milburn), Vol. 1, pp. 59 100. Springer Verlag, Berlin.
Zimmermann MH & Ziegler (1975) List of sugars and sugar alcohols
in sieve-tube exudates. Encyclopedia of Plant Physiology New
Series (ed. by M H Zimmerman & J A Milburn), pp. 480 496,
Vol. 1. Springer Verlag, Berlin.