Bulletin of Entomological Research (2002) 92, 159–164 DOI: 10.

1079/BER2001138

Host-based genotype variation in

insects revisited
G. Lushai1,2*, O. Markovitch2 and H.D. Loxdale3
1Agriculture and Agri-Food Canada, Eastern Cereal and Oilseed Research
Centre, Ottawa, Ontario, K1A OC6, Canada: 2Southampton University,
School of Biological Sciences, Biodiversity and Ecology Division, Bassett
Crescent East, Southampton, SO16 7PX, UK: 3Plant and Invertebrate
Ecology Division, IACR-Rothamsted, Harpenden, Hertfordshire,
AL5 2JQ, UK

Abstract

Elucidation of the genetic variability of a model insect species, the grain aphid,
Sitobion avenae (Fabricius), a predominantly asexual herbivore within the
temperate agro-ecosystem tested, was initiated using molecular DNA markers
(RAPDs). This revealed genetic profiles that appeared related to host adaptation at
the specific level amongst the natural populations colonizing different grasses and
cereals (Poaceae) within the same geographic location. These profiles were
recorded either as ‘specialist’ genotypes found on specific grasses, or as ‘generalist’
genotypes colonizing several host types including cultivated cereals or native
grasses. These findings are compared with analogous systems found amongst
insect species, including at a higher trophic level, i.e. interactions between
hymenopterous aphid parasitoids. As the aphids and their respective plant hosts
occur in the same geographical region at the same time, this appears to be a rare
example of the evolutionary transition leading to sympatric speciation in insects.
Hence, this study highlights the importance of understanding not only the
demographic parameters to genetic diversity, but also the more intricate
correlation of genetic diversity to host types in agricultural environments.

Introduction fig. 12.4). The entire panoply of evolutionary events form a

spectrum, starting with ‘ecotypes’ (e.g. Thomas & Singer,
By definition, species are considered as reproductively
1998) and ‘biotypes’ (e.g. Eastop, 1973) at one extreme,
isolated groups of individuals maintaining genetic identity
passing through races/strains, sub- and sibling species, and
within their respective ecosystems (Mayr, 1963; Claridge et
culminating with speciation itself (Avise, 1977, 1994, 2000).
al., 1997a). However, whilst selection may be the mechanism
Many of the terms used for the various stages of the
of evolution, adaptive radiation is the means by which this is
evolutionary process are imprecisely defined, often because
accomplished, as populations diverge and specialize to fill
their occurrence has only been noted recently. Whatever
ecological niches (Futuyma & Peterson, 1985; Thompson,
their precise meaning, such as the ambiguous term ‘biotype’,
1994). Specialization is the ‘cutting edge’ of evolution and its
sexual individuals within specialized populations may show
importance lies in the fact that competition is reduced not
assortative mating and reproductive isolation from the
only at an interspecific level (e.g. Jeffries & Lawton, 1984),
original founder population (Via, 1999; Via et al., 2000),
but also probably at an intraspecific level (e.g. host-related
whilst some asexuals may also evolve into apparently host-
biotypes of the brown plant hopper, Nilaparvata lugens (Stål)
adapted forms (De Barro et al., 1995a) or exist as largely
(Hemiptera: Delphacidae); Claridge et al., 1997b, see their
isolated genetic pools (Simon et al., 1999). Such isolation is
initially achieved due to genetic variants inducing
*Fax: +(613) 759-1701 behavioural and/or some degree of life-cycle asynchrony
E-mail: lushaig@em.agr.ca (Henry, 1994; Feder et al., 1998). This view of ecological
160 G. Lushai et al.

systems is very dependent on the empirical method of
measuring the population in question (Claridge et al., 1997a)
and clearly markers which show enhanced levels of genetic
variation are likely to reveal greater ecological resolution
(Lushai & Loxdale, 2002).
The advent of molecular markers in ecological studies
(Carvalho, 1998; Loxdale & Lushai, 1998, 1999) has brought
about a revolution in the understanding of the intricacy of
biological systems, the resolution of which has increased
using DNA-based technology. In insects, because of their
huge diversity and generally fast rates of reproduction, it
may be assumed that they would provide many examples of
intraspecific adaptations leading to speciation. In an
agricultural context, such radiations are important as pests
and beneficial insects (e.g. predators, parasitoids and
pollinators) adapt to new hosts with potentially significant
economic implications. Examples of such adaptations are to
be found in the following Orders of insects: Diptera (e.g.
Tephritidae, Feder et al., 1998), Hemiptera (e.g. Aphididae,
Powers et al., 1989; Aleyrodidae, Legg, 1996; Delphacidae,
Claridge et al., 1997b), Hymenoptera (e.g. Braconidae,
Atanassova et al., 1998; Apidae, Scholl et al., 1992),
Lepidoptera (e.g. Noctuidae, Pashley, 1993) and Coleoptera
(e.g. Chrysomelidae, Jacobson & Hsiao, 1983).
In the present article, further empirical evidence is
presented to show that genotypes of a cereal herbivore, the
grain aphid, Sitobion avenae (Fabricius) (Hemiptera:
Aphididae) colonizing different grass and cereal hosts
(Poaceae), can be discriminated within a local population
using DNA markers. The results are related to other
examples of sympatric host based adaptation in insects. In
addition, mention is made of the relevance of this study and
the usefulness of the present experimental approach to the
accurate identification of pest and beneficial insects in
sustainable agricultural practices such as integrated pest
management (IPM).
secondary replication following colony formation. Note:
general field or suction trap collections with high sample
numbers often unavoidably exhibit this form of pseudo-
replication.
Molecular markers: DNA extraction and RAPD–PCR
‘fingerprint’ analysis
DNA was extracted from single aphids as described by
De Barro et al. (1995a). Samples were homogenized in 300 l
TEN (250 mM NaCl, 10 mM EDTA, 50 mM Tris HCl, pH 8.0)/
2% SDS and 30 l of 1 mg ml1 proteinase-K and incubated
overnight at 37°C. Following this, organic waste was
removed with phenol/chloroform and DNA was
precipitated using 1 ml of ice cold absolute ethanol. The
DNA pellet was washed twice with 1 ml of 70% ethanol,
vacuum dried and resuspended in 40 l of TE (100 mM Tris,
10 mM EDTA, pH 8.0)/HPLC H2O (1:1). The DNA
concentration was determined using calibrations obtained
by fluorometry with 2 l of template DNA mixed with
2 ml TNE (10 mM Tris, pH 8.0, 0.1 M NaCl, 1m M
EDTA,)/Hoechst stain (100 ng ml1) in a cuvette and a DNA
fluorometer (Hoeffer, TK0 100). DNA was diluted to ~100 ng
l1 using HPLC H2O ready for PCR (polymerase chain
reaction; Saiki et al., 1988).
DNA was ‘fingerprinted’ using random amplified
polymorphic DNA–PCR (RAPD–PCR) techniques (Loxdale
& Lushai, 1998). The RAPD-PCR protocol differed from De

Materials and methods

Sitobion avenae were collected near Southampton,
southern England from 4  4 (2 m2) Latin-square plots of
winter wheat, Triticum aestivum L., barley, Hordeum vulgare
L., cocksfoot grass, Dactylis glomerata L. and Yorkshire fog
grass, Holcus lanatus L. (all Poaceae) (fig. 1 plot dimensions
and layout). Each plant host was grown in seed trays (30 
15 cm) in outdoor experimental plots (Chilworth:
Southampton University experimental garden) and watered
using an irrigation system. Placing the trays on cleared soil
covered with black heavy-duty polythene sheeting
prevented weed colonization. From January to December of
a single year (1996), all plants were examined weekly for
aphid colonization. This was increased to twice weekly
during the May–June growing season. All aphids were
collected and identified and individual S. avenae alatae were
then cultured on wheat for 10 days to screen for
hymenopterous parasitism and fungal infection. This was
as a precaution against amplifying foreign DNA; however
no alatae were found with such infections. The number (n)
of aphid samples tested electrophoretically per plant host
and representing the original winged colonizers were as
follows: wheat, n = 73; barley, n = 57; cocksfoot, n = 60 and
Yorkshire fog, n = 16. These aphid numbers represent all the Fig. 1. The size and host planting allocation of the 4  4 Latin
individual colonization events that occurred in the plot square experimental plot. W, wheat; B, barley; C, cocksfoot and
during the year of collection and are not indicative of H, Holcus lanatus (Yorkshire fog).
 Host-based variation in aphids 161

Barro et al. (1995a) in that 1 unit of Appligene Taq DNA
polymerase and 2.5 l 10 Appligene reaction buffer were
used. All RAPD amplifications were primed using the OPF-
12, 10-mer (5 ACGGTACCAG 3) (Operon Technologies,
Inc., California, USA) in an Omnigene thermal cycler
(Hybaid). PCR reactions were performed as follows:
denaturation step of 94 C/2 min for one cycle; 40 cycles of
denaturation at 92 C/1 min; annealing at 35 C/1 min;
extension at 72 C/1 min and final extension at 72 C/6 min
for one cycle. After amplification, the samples were run on a
1.5% agarose gel (5 V cm1), then stained with ethidium
bromide and visualized under UV light. A 100 base pair (bp)
ladder (Gibco-BRL) was used as a standard molecular.
Individual DNA extracts were amplified several times to test
for and confirm reproducibility.
Statistical analysis
From the DNA profile of the RAPD-primer used, a binary
matrix was constructed by verifying bands at a particular
molecular length (bp) as being present, ‘1’, or absent, ‘0’. The
banding profiles allowed typing of the aphid clones into
groups or genotypes. These were typed for all the clones
collected on each of the four host plants (fig. 2). Counts of
each aphid genotype were summed over the four replicate
host plant treatments and expressed as proportions of the

Fig. 2. Stacked-bar graph indicating the 36 Sitobion avenae genotypes resolved using RAPD–PCR. The frequencies of the aphid genotypes
are listed along with the host they were collected from and associated with a dendrogram clustering aphid genotypes into four potential
host-related clades based on genetic similarity produced by an Euclidean matrix. , wheat; □, barley; □, cocksfoot; □, Yorkshire fog.
162 G. Lushai et al.
total number of aphids of each genotype sampled, i.e. Pij =
Cij / sum over j(Cij) (S. Clark, personal communication).
Therefore there were 36 clones described (i = clone 1–36);
four plant hosts, (j = 1–4), whilst Cij are counts and Pij are
proportions. A matrix of similarities between genotypes was
computed from the proportions using a Euclidean matrix
with the data range set to 100 for all four host plants. This
matrix was analysed using single-link hierarchical cluster
analysis to produce a dendrogram for the genotypes. This
dendrogram is schematically linked to a cumulative bar
chart, emphasizing the relationship of the grouped
genotypes to their sample number and specificity on certain
host plants (fig. 2).
Results
RAPD analysis of individual winged S. avenae colonizers
caught throughout a year on the experimental plants
revealed the presence of several novel genotypes on the
different hosts (fig. 2). As seen in the dendrogram, there are
three main branches, one generally clustering aphids caught
on agricultural crops (wheat and barley) and the other two
separating the insects colonizing the native grasses,
cocksfoot and Yorkshire fog. The crop-based clades can be
approximately resolved into the two crop varieties. Hence in
effect there are four clades of genetically similar aphids. The
first three clades comprise some genotypes of S. avenae that
colonize more than one host type: the first ten genotypes (n
= 111) form clade I, the next 11 genotypes (n = 41) clade II,
the next nine genotypes (n = 47) clade III, whilst the last six
genotypes (n = 7) form clade IV, which were exclusive to
Yorkshire fog.
A genotype found on a single host is described here as
monophagous ‘specialist’, and if on more than one host,
oligophagous ‘generalist’. Hence, five genotypes were
monophagous on wheat (n = 18), nine on barley (n = 29),
seven on cocksfoot (n = 38) and six on Yorkshire fog (n = 7),
making a total of 27 genotypes (n = 92). Oligophagy was
demonstrated by nine genotypes (n = 114). Therefore, almost
twice the numbers of genotypes were monophagous.
However, although fewer genetically distinct groups
represented oligophagy, the sum of individuals was similar
to the more genetically diverse monophages. Clearly the
numbers of alate colonizers, if not genotype, are evenly
distributed in terms of host preference.
Discussion
An earlier study involving the same RAPD markers used
here to investigate the genetic variability of populations
within and between different habitats at the same
geographic location, and at different times of the year, also
revealed the existence of specific genotypes of S. avenae (De
Barro et al., 1995a). As far as plant host types were
concerned, this was a simpler study involving the screening
of aphid genotypes on two hosts, wheat or cocksfoot, and it
was noted that the host-based stratification tended to break
down as the field season progressed (De Barro et al., 1995a).
Microsatellite analysis of these same populations further
resolved the genetic sub-structuring (Sunnucks et al., 1997),
and showed similar host selection. Cross analysis with
mtDNA revealed that many individuals of the aphid
genotypes found exclusively on cocksfoot appeared to have
introgressed with a holocyclic sister species, the blackberry
grain aphid, Sitobion fragariae (Walker) (Aphididae).
Vanlerberghe-Masutti & Chavigny (1998) utilized RAPD
markers to show host-based genetic differentiation in the
aphid, Aphis gossypii Glover (Aphididae), and were able to
distinguish populations from cucurbit and non-cucurbit
hosts. The data presented here, also using RAPDs, provides
further evidence in support of host-based stratification and
suggest that this phenomenon is much more widespread,
across several host niches, than previously realized. Perhaps
the fact that RAPDs reveal host-related genetic differentia-
tion of aphids is because these markers often contain
microsatellite regions (Ender et al., 1996), that are scattered at
random throughout the genome (Queller et al., 1993) and are
possibly linked by ‘hitchhiking’ (Maynard Smith & Haigh,
1974; Wiehe, 1998) to genes under selection. In contrast,
other markers such as ribosomal DNA (IGS, intergenic
spacer) markers which are confined to the distal end of
chromosomes, have failed to show host-related genotypes in
the peach–potato aphid, Myzus persicae (Sulzer) (Aphididae),
when populations from different hosts were tested (Fenton
et al., 1998). Biotype divergence in the greenbug, Schizaphis
graminum (Rondani) (Aphididae) has, however, recently
been reported by phylogenetic analysis of the mt DNA
cytochrome oxidase subunit I (COI) (Shufran et al., 2000).
The data presented here also show that there are more
specialist than generalist genotypes (as defined earlier),
whereas numbers of individuals in both categories were
similar. This may be an indication of a useful ecological
trend, suggesting a balance between these evolutionary
strategies. Hence an experimental design involving several
host vs. genotype interactions as carried out here could
point the way towards dissecting the underlying forces
driving ecosystem dynamics, and the trade-offs inherent in
the system between these selective regimes.
The significance of smaller sample numbers, e.g. as
represented by genotypes on Yorkshire fog, is difficult to
establish at this stage. However, it is important to note that
these exclusive genotypes were collected during four
months of the full year (June to September) and no similar
genotypes were identified on the other adjacent hosts. Far
from being insignificant and statistically inconsequential, it
is exactly this form of rare genotype that has been
considered an evolutionary ‘Red Queen-pacemaker’ (W.D.
Hamilton, personal communication). Such a mechanism
operating in these populations could maintain an interaction
between a parasite and its host, even in populations where
sexual recombination was rare (cf. Vrijenhoek, 1998; see
Delmotte et al. (2001) for evidence of occasional sexual
leakage between asexual and sexual aphid life-cycle types).
Improving our understanding of pest epidemiology may
need to include as rigorous screening for rare infrequent
genotypes as for the more common genotypes found
distributed among native and agricultural host plants. This
is because any one of these genotypes may gain selective
advantage at a given point in time and along with other
consequences, may carry plant diseases from reservoir hosts
in to crops.
Sitobion avenae genotypes from wheat and cocksfoot have
not only been genetically typed as here, but have also been
correlated against several ḿeasures of fitness in reciprocal
host transfer experiments (De Barro et al., 1995b), an
approach which supports the existence of host-adapted
races of the aphid. Studies with winged morphs of the same
aphid species show a strong influence of the previous host
 Host-based variation in aphids
environment on the subsequent selection of plant host by the
aphid (Lushai et al., 1997). Therefore, these host ‘ecotypes’
are indicative of the dynamic processes leading to
evolutionary change in the agricultural ecosystem (Mopper
& Strauss, 1998), i.e. biotype, strain or race formation, etc.,
leading to sympatric speciation (see Via, 1999, 2001; Via et al.,
2000). Parallel examples are known in other insects such as
the fruit fly, Rhagoletis pomonella (Walsh) (Diptera:
Tephritidae) which has host races on apple, Malus pumila
and hawthorn, Crataegus spp. (Rosaceae) (Feder et al., 1998)
and the larch budworm moth, Zeiraphera diniana Guenée
(Lepidoptera: Tortricidae) which has evolved races
sympatrically on larch, Larix decidua and pine, Pinus spp.
(Pinaceae) (Emelianov et al., 1995). Similar host-based
genetic variation has been observed at a further trophic level
within sympatric populations of the sexually reproducing
primary parasitoid, Aphidius ervi Haliday sensu lato
(Hymenoptera: Braconidae) attacking sympatric field
populations of the pea aphid, Acyrthosiphon pisum (Harris)
(Aphididae) and the nettle aphid, Microlophium carnosum
(Buckton) (Aphididae) in the UK (Atanassova et al., 1998; see
also Pennacchio et al., 1994). All these examples emphasize
the biological and genetic complexity of natural systems.
In conclusion, further evidence is presented that host-
preferring genotypes occur in cereal aphids, here in a simply
managed trial plot. Furthermore, in cereal aphids, multi-
generation field and laboratory experiments clearly
demonstrate that such host-associated forms may be
explained partly by genetic variation in preference as well as
performance on particular plant hosts. Many new pure and
applied research challenges arise from this and other such
studies showing a lack of genetic homogeneity within
apparent ’species’ populations and the existence of host-
adapted forms. On a more practical level, concern has been
expressed by agriculturists that uncultivated land areas may
act as refugia for important crop pests such as aphids, as is
true for their biological control agents (Vorley & Wratten,
1987; Thomas et al., 1991; Hopper et al., 1993; Corbett &
Rosenheim, 1996). Our study demonstrates that the
appropriate application of molecular techniques allows fresh
insights into understanding the subtle natural biological
interactions that occur within an agricultural ecosystem,
essential if pests are to be controlled and their biological
control agents effectively managed and manipulated.
Acknowledgements
The authors thank Suzanne Clark of the Biomathematics
Unit, IACR-Rothamsted for kindly analysing the aphid data
and Owain Edwards and an anonymous reviewer for critical
appraisal of the manuscript. This work was supported by the
Biotechnology and Biological Sciences Research Council,
BBSRC, UK (ref: AO1666) and Department of Environment,
Food and Rural Affairs, DEFRA, UK (H.D.L.). IACR-
Rothamsted receives grant-aided support from the BBSRC.
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(Accepted 25 October 2001)
© CAB International, 2002