Ecological Entomology (2004) 29, 6065

Linking the bacterial community in pea aphids with

host-plant use and natural enemy resistance
JULIA FERRARI1, ALISTAIR C. DARBY2,4, TIM J. DANIELL3,
H . C H A R L E S J . G O D F R A Y 1 and A N G E L A E . D O U G L A S 2 1NERC Centre
for Population Biology and Department of Biological Sciences, Imperial College London, Ascot, 2Department of Biology,
University of York and 3PlantSoil Interactions, Scottish Crop Research Institute, Invergowrie, Dundee, U.K.

Abstract. 1. Pea aphids, Acyrthosiphon pisum, harbour a range of facultative

accessory bacteria (secondary symbionts), including those informally known as
PASS (R-type), PAR, PABS (T-type), and PAUS (U-type).
2. To explore the relationship between possession of these bacteria and ecologic-
ally important traits of A. pisum, correlations between the accessory bacteria
found in 47 parthenogenetic clones of A. pisum and the host plant on which each
clone was collected and its susceptibility to natural enemies were surveyed.
3. The bacterial complement varied with plant of collection. PAUS (U) was
present in all of 12 clones affiliated to Trifolium but was otherwise rare, while
PABS (T) and PASS (R) occurred at significantly higher frequency in clones from
Lotus and Vicia, respectively, than clones from other plants.
4. Possession of PABS (T) was associated strongly with resistance to the parasitoid
Aphidius eadyi and weakly with resistance to Aphidius ervi. Aphids carrying
PAUS (U) were more resistant to the fungal pathogen Pandora (Erynia) neoaphidis,
although this correlation was complicated by a strong association with host-plant use.
Key words. Accessory bacteria, Acyrthosiphon pisum, Aphidius eadyi, Aphidius
ervi, host-plant use, Pandora neoaphidis, pea aphid, resistance, secondary sym-
bionts, specialisation.

Introduction
It has long been known that certain insect groups (e.g.
aphids and other homopterans, cockroaches, anoplurans,
various curculionid beetles) are absolutely dependent on
specific microorganisms in their tissues (Buchner, 1965;
Douglas, 1989). For example, Buchnera, the obligate bac-
terial associate of aphids, provides nutrients that are in
short supply in the insects diet (Buchner, 1965; Douglas,
1998; Moran & Baumann, 2000). Many insects additionally
bear one to several additional microbial taxa, informally
known as accessory microorganisms or secondary sym-
bionts, which are only facultatively associated with their
hosts (Buchner, 1965; Douglas, 1989). The significance of
Correspondence: Julia Ferrari, NERC Centre for Population
Biology and Department of Biological Sciences, Imperial College
London, Silwood Park Campus, Ascot, Berkshire, SL5 7PY, U.K.
E-mail: julia.ferrari@imperial.ac.uk.
4
Current address: Centre for Tropical Veterinary Medicine,
University of Edinburgh, Scotland, U.K.
these accessory microorganisms to the insect has until
recently been obscure; however, recent research on aphids
suggests that one possible explanation for the occurrence of
facultative forms is that they may influence ecologically
important traits, including plant utilisation characteristics
and susceptibility to parasitoids (Douglas et al., 2002).
The accessory bacteria of aphids have been most closely
studied in the pea aphid, Acyrthosiphon pisum, where at
least five different taxa have been recorded in natural popu-
lations worldwide (Chen et al., 1996; Chen & Purcell, 1997;
Fukatsu et al., 2000; Darby et al., 2001; Sandstrom et al.,
2001; Tsuchida et al., 2002; Russell et al., 2003). The bac-
teria have not been described formally and four widely
investigated forms are known by two alternative systems
of informal names: PASS (R-type), PAR, PABS (T-type),
and PAUS (U-type) (Sandstrom et al., 2001; Tsuchida et al.,
2002), which have been abbreviated here to PASS (R), etc.
PASS (R), PABS (T), and PAUS (U) belong to the
-proteo-
bacteria, while PAR is a Rickettsia sp. The fifth bacterium is
a Spiroplasma known at low densities (Fukatsu et al., 2001).

60 # 2004 The Royal Entomological Society


PASS (R), PABS (T), PAUS (U), and PAR are vertically
transmitted from mother to offspring, and there is also
horizontal transmission (Sandstrom et al., 2001; Darby &
Douglas, 2003), though the mechanism is not known.
The last few years have seen important new evidence
about the possible effects of accessory bacteria on aphid
fitness. Chen et al. (2000) found that artificially infecting
pea aphid clones with PASS (R) or PAR had neutral or
negative effects on components of host fitness. A single
clone of the related species Acyrthosiphon kondoi experi-
enced much more severely reduced fitness after the injection
of these bacteria; however, aphids that had been artificially
infected with PASS (R) were in most cases better able to
recover from a heat-stress episode than individuals from the
same clone without accessory bacteria (Chen et al., 2000;
Montllor et al., 2002). A likely but not proven mechanism
for this is that PASS (R) rescues the obligate symbionts
from heat damage (Montllor et al., 2002). This beneficial
effect of PASS (R) was negated by the presence of PAR,
which at least under the conditions studied appeared to
have a negative effect on host fitness (Montllor et al.,
2002). Oliver et al. (2003) explored the impact of
PASS (R), PABS (T), and PAUS (U) on the response of a
single A. pisum clone to parasitoid attack, and revealed that
aphids with PABS (T) were approximately twice as likely to
survive parasitism by the hymenopteran parasitoid Aphidius
ervi than controls. Aphids with PASS (R) showed lesser but
still significantly increased survival over aphids lacking
accessory bacteria, while PAUS (U) had no effect.
Experimental studies such as those summarised above
provide the strongest evidence of the effects of accessory
bacteria on host biology, but are restricted to relatively few
aphid genotypes. Correlative studies of infection status and
aphid ecology or phenotype provide the opportunity to
assess the generality of patterns and can indicate interesting
effects of aphid and/or bacterial genotype. For example,
Tsuchida et al. (2002) assessed the accessory bacteria in
119 clones collected from two host-plant genera (Trifolium
and Vicia) throughout the main Japanese islands (except
Hokkaido). They found PAUS (U) infections to be more
frequent on Trifolium than Vicia, and more common in
cool, dry localities. No pattern was found for PASS (R),
while PAR and the Spiroplasma were rare, and PABS (T)
absent. Similarly, Simon et al. (2003) assessed the infection
status of 335 individual aphids from 21 populations
collected on Medicago, Trifolium, and Pisum in France.
PASS (R) and PAR were disproportionately common on
Pisum, while PABS (T) and PAUS (U) were significantly
more common on Medicago and Trifolium, respectively.
Leonardo and Muirus (2003) study on pea aphids on
Medicago and Trifolium in California confirmed this pat-
tern; however, Darby et al. (2003) identified no correlation
between host-plant affiliation and possession of PABS (T)
in 329 clones from U.K. populations of A. pisum on five
host-plant species (including Medicago) across 2 years.
Here the infection status of 47 clones of pea aphid
collected from four genera of host plant (Vicia, Trifolium,
Lotus, Pisum) in the south of England is reported. The
# 2004 The Royal Entomological Society, Ecological Entomology, 29, 6065
Pea aphid accessory bacteria 61
collections were made as part of a study of genetic variation
in aphid resistance to natural enemies two species of
parasitoid wasp and an entomopathogenic fungus and
possible trade-offs between resistance and other fitness
components (Ferrari et al., 2001; Ferrari & Godfray,
2003). Thus natural host plant, and a variety of measures
of resistance and other life-history parameters are known
from some or all of the clones. The goals of the study were
two-fold: first to detect novel correlations between posses-
sion of accessory bacteria and aphid phenotype or ecology.
Second, to test the generality of the relationships identified
in other recent studies across multiple clones in different
geographical regions of the world. Of particular interest are
(i) whether hosts collected on Trifolium are more likely to
harbour PAUS (U), as Tsuchida et al. (2002), Simon et al.
(2003) and Leonardo and Muiru (2003) found and (ii)
whether resistance to parasitoids is correlated with posses-
sion of PASS (R) or PABS (T), as Oliver et al.s (2003) work
suggests.
Materials and methods
The aphids
Forty-seven clonal isolates of Acyrthosiphon pisum
(Harris) derived from single parthenogenetic females col-
lected in the summers of 1998 and 1999 in Berkshire (U.K.)
were maintained as vigorous cultures on pre-flowering Vicia
faba cv. The Sutton at 15
C, 60% RH and LD 16:8 h. Each
clonal culture was started with an individual that was
collected at least 50 m away from any other aphid used to
avoid sampling the same genotype twice. All of these
clones have been used previously in studies of natural
enemy resistance in pea aphids (Ferrari et al., 2001; Ferrari
& Godfray, 2003).
Microbial communities
The presence of accessory bacteria in each of the 47 clones
was assessed using terminal restriction fragment length
polymorphism (T-RFLP) (e.g. Moeseneder et al., 1999;
Leser et al., 2000; Osborn et al., 2000). Differences in the
size of a terminal restriction-enzyme fragment of a PCR-
amplified portion of the 16S rRNA gene allow the different
taxa to be distinguished. Full details of the application of
this technique to the accessory bacteria of pea aphid is given
in Darby et al. (2001) and Haynes et al. (2003) and hence are
not repeated here. Briefly, a part of the 16S rDNA is ampli-
fied using universal primers (Y2MOD [ACT-YCT-ACG-
GRA-GGC-AGC-AGT-RGG] modified from Young et al.
(1991) and B16S-30 ) and the cycling parameters of Darby
et al. (2001). The length of restriction enzyme digests was
analysed on an automatic sequencer. Validation studies
using denaturing gradient gel electrophoresis and diagnostic
PCR have shown that Buchnera, PASS (R), PAR, PABS (T),
and PAUS (U) can all be distinguished using this technique
62 Julia Ferrari et al.

(Haynes et al., 2003). Spiroplasma is rare in U.K. pea aphid
clones (A. E. Douglas, unpubl. data) and is not discussed
further here.
Aphid traits
The procedures to quantify aphid performance on differ-
ent plant species and resistance to natural enemies have
been described in detail in Ferrari et al. (2001) and Ferrari
and Godfray (2003). Here, all aphid clones from those
studies are used; reasons why any particular clone was
chosen for an experiment are outlined there. The methods
used to quantify the six aphid traits studied are outlined
briefly here. In each experiment, aphid clones were stratified
across temporal blocks. The experiments were performed at
20
C, 60% RH and LD 16:8 h.
(i) The collection plant. The aphid clones were collected
from four plant genera; the details are given in Table 1.
(ii) Nymphal survival on different host plants. This
experiment used 12 clones collected from Lotus pedunculatus
(uliginosus) Cav. and a further 12 from Trifolium pratense L.
For each replicate, one wingless female was allowed to pro-
duce nymphs over a 24-h period on the test plant species. Five
of these offspring were kept and the proportion that survived
to adulthood was scored after 10 days. This was repeated four
times for each clone and test plant combination.
(iii) Lifetime fecundity on Vicia faba. For reasons that
are not understood, all pea aphid clones perform well on
Vicia faba. Lifetime fecundity of six replicates of 14 clones
on pre-flowering V. faba, on which aphid populations have
a high rate of increase, and of eight replicates of nine clones
on flowering V. faba, which support relatively poor perfor-
mance, were measured.
(iv) Resistance to the parasitoid wasp Aphidius ervi
Haliday. This species attacks A. pisum and related aphids.
The wasp lays an egg into an aphid nymph and its offspring
develops inside the aphid, eventually causing the aphid to
mummify and become attached to the substrate. Pupation
occurs within the aphid mummy. Some aphid clones can
resist parasitoid attack by an unknown physiological
mechanism that does not involve cellular encapsulation
(Henter & Via, 1995; Ferrari et al., 2001; Li et al., 2002; Oliver
et al., 2003). Five or six replicate groups of 30 nymphs
(34 days old) from each of 27 clones were exposed to single
parasitoids for 9 h and the fraction of mummies produced
after 14 days was used as a measure of susceptibility.
(v) Resistance to the parasitoid wasp Aphidius eadyi
Stary, Gonzalez & Hall. The biology of this species is very
similar to A. ervi except that it is a more specific parasitoid
of pea aphid and the nature of aphid resistance to this
species is more complicated in the aphid populations
studied here, as some resistant clones can survive parasitism
with no changes in their fecundity, while other clones can
survive with severely reduced fecundity (Ferrari et al., 2001;
J. Ferrari, unpubl. data). The 41 clones tested for resistance
to A. eadyi comprise two data-sets, one of 22 clones studied
previously (Ferrari et al., 2001) and one of 19 clones. The
bioassay for resistance to A. eadyi is identical to that used
for A. ervi. The mean and variance in susceptibility of the
aphids in the two data-sets did not differ significantly and
they were therefore pooled for analysis.
(vi) Susceptibility to the fungal pathogen Pandora
(Erynia) neoaphidis Humber. After germinating on the
cuticle, the fungus invades the aphid, eventually causing it
to mummify, whereupon sporulation occurs. Susceptibility
was measured as the proportion of 20 young adults that
exhibited fungal sporulation within 6 days of exposure to a
fungal spore shower from two sporulating aphid cadavers
for 90 min in each of four replicates of 44 clones. The clonal
means presented here were corrected for the spore dose
received by the aphids (details in Ferrari et al., 2001; Ferrari
& Godfray, 2003).
Statistical analysis
A generalised linear modelling framework was used to
analyse the data (McCullagh & Nelder, 1989). Pea aphids
collected from different plant species differ not only in their
performance on different plant species (e.g. Muller, 1980;
Via, 1991), but also in other ecologically important traits
(Hufbauer & Via, 1999; Ferrari & Godfray, 2003). To con-
trol for this effect, the collection plant was included in all
analyses before adding the presence of the bacterial taxa to
the model. These factors were treated as fixed effects.
Whenever possible, an interaction between the presence of
two bacterial taxa was also tested for. In the results, the
statistical model including host plant and single bacterial
taxa is presented, but further information is given when the
addition of other explanatory variables changed the out-
come of the analyses. Data were transformed prior to the
analyses to ensure a normal distribution of errors: propor-
tion data were arcsine-square root and count data when

Table 1. Frequencies of accessory bacteria in pea aphid clones collected from different host plants. In no case did all three bacterial taxa
occur in the same aphid clone.

Collection plant No. of clones None PASS PABS PAUS PASS PABS PASS PAUS PABS PAUS
Lotus pedunculatus 14 3 1 6 0 0 1 3
Pisum sativum 5 1 1 2 0 0 0 1
Trifolium pratense 12 0 0 0 9 0 0 3
Vicia spp. 16 5 7 1 1 0 1 1
Total 47 9 9 9 10 0 2 8

# 2004 The Royal Entomological Society, Ecological Entomology, 29, 6065


necessary square root transformed. All analyses were per-
formed using R (version 1.5.1; http://www.r-project.org/).
Results
The T-RFLP analysis of the bacterial community in the
aphids revealed that all 47 pea aphid clones bore Buchnera
and that 38 clones (81%) had one or two of the previously
described accessory bacteria PASS (R), PABS (T), and
PAUS (U) (Table 1). None of the clones generated a peak
that could be attributed to PAR, or to the accessory bac-
teria recently found in other species of aphids by Russell
et al. (2003) and placed in clades called So-So and Ars, or to
any other bacteria. Randomisation tests to investigate
whether single infections were more common than expected
or whether specific bacteria types tended to co-occur
revealed no statistically significant relationships. The correl-
ation between the bacterial community and the six traits of
aphids was as follows.
(i) Collection plant. The bacterial community varied
among pea aphid clones collected from different host-
plant genera (Table 1). Most strikingly, PAUS (U) was
universal in pea aphids from T. pratense, but rare in aphid
clones collected from the other plant species (2 25.96,
P < 0.001). In clones collected from Vicia spp., PASS (R)
was significantly more common than in clones collected
from other plants (2 9.57, 0.001 < P < 0.01) and
PABS (T) was significantly more common in clones
from L. pedunculatus than other plants (2 6.83,
0.001 < P < 0.01).
(ii) Nymphal survival on collection plants. Aphid clones
collected from L. pedunculatus varied in their performance on
both this plant species and T. pratense, but no clone per-
formed significantly better on the latter (Ferrari & Godfray,
2003). PABS (T), but none of the other accessory bacteria,
was significantly associated with increased survival on
L. pedunculatus (Fig. 1; F1,10 6.58, P 0.028), but not on
T. pratense (F1,10 1.81, P 0.21). The aphid clones col-
lected from T. pratense performed uniformly well on
this plant species and poorly on L. pedunculatus, with no
detectable differences between clones bearing or lacking
PABS (T). The association between other accessory bacteria
Fig. 1. Survival of clones collected from Lotus pedunculatus on (a)
L. pedunculatus and (b) Trifolium pratense. Bars represent average
clonal means SE.
# 2004 The Royal Entomological Society, Ecological Entomology, 29, 6065
Pea aphid accessory bacteria 63
on performance of clones from T. pratense could not be
quantified because all clones bore PAUS (U) and none had
PASS (R).
(iii) Lifetime fecundity on Vicia faba. This trait was not
significantly correlated with the presence of any bacterial
taxon.
(iv) Resistance to the parasitoid wasp Aphidius
ervi. PABS (T) was significantly associated with increased
resistance to A. ervi (Fig. 2a; F1,23 7.73, P 0.011). There
was also a weak, non-significant association between
increased resistance to A. ervi and possession of PAUS (U)
(F1,23 3.67; P 0.068) or PASS (R) (F1,23 3.41,
P 0.078), and therefore, a further analysis was performed
controlling for the presence of PAUS (U) and PASS (R).
The addition of the presence of PABS (T) did not lead to a
statistically significant increase in this models explanatory
power (F1,21 2.64, P 0.12), which suggests that accessory
bacteria in general are associated with increased resistance
to A. ervi, but that it was not possible to identify which of
the taxa was most important.
(v) Resistance to the parasitoid wasp Aphidius eadyi. This
trait was significantly associated with the presence of PABS (T)
(Fig. 2b; F1,36 21.80, P < 0.001) and PAUS (U) (F1,36 6.77,
P 0.014), collection plant (F3,37 3.66, P 0.021), and sur-
vival on L. pedunculatus (r 0.49, d.f. 24, P 0.011). Con-
trolling for plant affiliation, the addition of PABS (T) to a
statistical model containing PAUS (U) was highly significant
(F1,35 17.23, P < 0.001), while the reverse did not improve
the fit of the model (F1,35 3.65, P 0.064). This analysis
suggests that PABS (T) is the main bacterium associated with
resistance to A. eadyi.
(vi) Resistance to the fungal pathogen Pandora
neoaphidis. Susceptibility to the pathogen P. neoaphidis
was tested in two experiments (clones collected in 1999 or
1998, respectively), which had to be analysed separately
since the response to the pathogen is very sensitive to envir-
onmental conditions. All clones collected from T. pratense
in 1999 were both fully resistant to the pathogen P. neo-
aphidis and contained PAUS (U). It was therefore not pos-
sible to disentangle the separate associations of host plant
and bacterial composition on fungal resistance. There was
overall little variation in susceptibility to P. neoaphidis
among clones collected from L. pedunculatus (in 1999) and
Fig. 2. Association between the susceptibility to parasitoids and
the presence of the accessory bacterium PABS (T). (a) Suscept-
ibility to Aphidius ervi and (b) susceptibility to Aphidius eadyi; the
bars represent average clonal means SE.
64 Julia Ferrari et al.
there was no association with any accessory bacteria.
The aphids collected in 1998 (which did not include any
clones from T. pratense) also displayed a significant asso-
ciation between PAUS (U) and resistance to P. neoaphidis
(F1,20 4.83, P 0.040) in an analysis that controlled for
the collection plant.
Discussion
This study shows that two ecologically important traits in
pea aphids, plant affiliation and susceptibility to natural
enemies, are correlated with the possession of particular
accessory bacteria.
Tsuchida et al. (2002) in Japan, Simon et al. (2003) in
France, and Leonardo and Muiru (2003) in California
found that aphid clones collected from Trifolium tended to
carry PAUS (U). In the work reported here an even stronger
relationship was found with all 12 clones collected on
Trifolium having this bacterium, which was present in only
three of the other 35 clones. This thus seems a consistent
and geographically widespread association. The second
hypothesis to do with host-plant affiliation that was
explored involved the link between PASS (R), PAR, and
Pisum (Simon et al., 2003). With only five clones from
Pisum there was limited statistical power to explore this
link, but PAR was entirely absent and no strong association
with PASS (R) was discovered. Furthermore, the incidence
of PABS (T) in Pisum-derived A. pisum in U.K. is high, at
3/5 (60%) in this study and 5090% in Darby et al. (2003),
in contrast to the low incidence observed by Simon et al.
(2003) in France.
In Japan, Tsuchida et al. (2002) found no bacterium to be
particularly common in clones collected on Vicia, while here
PASS (R) was found to be preferentially associated with this
plant genus. Aphids from Lotus, a host-plant genus from
which pea aphid bacteria had not previously been examined
were also studied. Many of these aphids bore PABS (T) and,
interestingly, clones from this plant species with this bacter-
ium tended to have higher fitness on Lotus.
Oliver et al. (2003) found that injecting PABS (T) into an
aphid clone without bacteria markedly increased its resist-
ance to the parasitoid A. ervi, while injecting PASS (R) had
a similar but weaker effect. A correlation between resistance
to this parasitoid and possession of PABS (T) was found,
although statistically the possible effects of the other bac-
teria could not be excluded. With the closely related para-
sitoid A. eadyi, more unambiguous evidence that PABS (T)
is associated with resistance was obtained. The results thus
support Oliver et al.s (2003) findings and suggest (i) that
their results are not due to a particularity of the single clone
they worked with and (ii) that the beneficial effects they
identified extend to resistance to more than one species of
parasitoid. The presence of PASS (R) did not correlate with
resistance to either parasitoid in the present study, contrary
to what Oliver et al.s (2003) experimental results suggest.
Ferrari and Godfray (2003) found that all the clones
collected on Trifolium were resistant to the common
# 2004 The Royal Entomological Society, Ecological Entomology, 29, 6065
entomopathogenic fungus Pandora neoaphidis. Here it is
shown that these aphids all carry PAUS (U). Clearly
PAUS (U) might be involved in resistance to Pandora in
the same way PABS (T) is to parasitoids, but experiments
are required to exclude possible other factors associated
with pea aphid genotypes on Trifolium (Ferrari & Godfray,
2003, excluded direct effects of food plants). In the 1998
collection of clones, which did not include aphids from
Trifolium, there was an association between resistance to
the fungus and possession of PAUS (U), which supports the
further investigation of the role of this bacterium.
To conclude, the survey reported here, along with those
of Tsuchida et al. (2002), Darby et al. (2003), Simon et al.
(2003), and Leonardo and Muiru (2003), point to a variety
of possible effects of accessory bacteria on host plant use
and natural enemy resistance. Purely correlative studies
such as these, however, cannot demonstrate the role of
accessory bacteria in the aphids interaction with its
resources or enemies it identifies patterns and hypotheses
that can subsequently be tested experimentally. To date the
only one that has been tested is the effect of accessory
bacteria on pea aphid resistance to parasitoids (Oliver
et al., 2003) and more such studies are needed. Analysis of
the relationship between accessory bacteria and host-plant
affiliation in A. pisum needs to be set within the context of
the genetic complexity of the aphid, long known to consist
of a set of host-plant adapted populations of uncertain
relatedness (e.g. Muller, 1980; Sandstrom, 1996). Recent
studies by Via (1999), Hawthorne and Via (2001), and
Simon et al. (2003) are beginning to resolve this, and will
pave the way for a better understanding of bacteriaaphid
genetic interactions. As pea aphid fast becomes a model
system for the study of a variety of problems in ecology,
genetics, and evolution the authors believe it will prove
particularly valuable for exploring general issues in obligate
and facultative symbiosis.
Acknowledgements
We are grateful to K. Oliver, M. Hunter, J. Russell, and
C. Gibson for constructive comments on an earlier version
of this manuscript. This research was conducted with
financial support from NERC grant NER/B/S/2000/00054.
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