Insect endosymbionts: manipulators of insect herbivore trophic ...

Protoplasma (2010) 244:25–51 DOI 10.1007/s00709-010-0156-2

REVIEW ARTICLE

Insect endosymbionts: manipulators of insect herbivore trophic interactions? Emily L. Clark & Alison J. Karley & Stephen F. Hubbard

Received: 22 April 2010 / Accepted: 22 April 2010 / Published online: 21 May 2010 # Springer-Verlag 2010

Abstract Throughout their evolutionary history, insects have formed multiple relationships with bacteria. Although many of these bacteria are pathogenic, with deleterious effects on the fitness of infected insects, there are also numerous examples of symbiotic bacteria that are harmless or even beneficial to their insect host. Symbiotic bacteria that form obligate or facultative associations with insects and that are located intracellularly in the host insect are known as endosymbionts. Endosymbiosis can be a strong driving force for evolution when the acquisition and maintenance of a microorganism by the insect host results in the formation of novel structures or changes in physiology and metabolism. The complex evolutionary dynamics of vertically transmitted symbiotic bacteria have led to distinctive symbiont genome characteristics that have profound effects on the phenotype of the host insect. Symbiotic bacteria are key players in insect–plant interactions influencing many aspects of insect ecology and playing a key role in shaping the diversification of many

Handling Editor: David Robinson E. L. Clark (*) : A. J. Karley : S. F. Hubbard Environment Plant Interactions Programme, Scottish Crop Research Institute, Invergowrie DD2 5DA, Scotland, UK e-mail: [email protected] E. L. Clark : S. F. Hubbard Division of Plant Sciences, University of Dundee at SCRI, SCRI, Invergowrie DD2 5DA, Scotland, UK S. F. Hubbard School of Biology, University of St Andrews, St Andrews KY16 9TS, Scotland, UK

insect groups. In this review, we discuss the role of endosymbionts in manipulating insect herbivore trophic interactions focussing on their impact on plant utilisation patterns and parasitoid biology. Keywords Endosymbiont . Facultative . Herbivore . Insect . Obligate . Parasitoid

Abbreviations SOPE Sitophilus oryzae primary endosymbiont SZPE Sitophilus zeamais primary endosymbiont PAXS Pea aphid X-type symbiont PGRPs Peptidoglycan recognition proteins Jak/ Janus kinase/signal transducers and activators of STAT transcription proteins APSE- Bacteriophage 1, Acyrthosiphon pisum secondary 1 endosymbiont APSE- Bacteriophage 3, Acyrthosiphon pisum secondary 3 endosymbiont CI Cytoplasmic incompatibility T3SSs Type 3 secretion systems UV-B Ultraviolet B light

Introduction Symbiotic bacteria are key players in insect–plant interactions influencing many aspects of insect ecology. A large number of herbivorous insects harbour symbiotic bacteria (Fig. 1) and as a consequence are able to thrive on nutrient poor plant tissues. The gut symbiotic bacteria and protists of wood-feeding insects, for example, play a role in

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enzymatic degradation of cellulose (Tokuda and Watanabe 2007; Carpenter et al. 2010), and the intracellular symbiont Buchnera aphidicola synthesises essential amino acids for its aphid (Hemiptera; Aphididae) host (Douglas 1998; Gündüz and Douglas 2009). In addition, symbiotic bacteria can influence the fitness of herbivorous insects in more subtle ways by, for example, increasing resistance to hymenopteran parasitism (Oliver et al. 2003) and broadening the range of thermal tolerance (Montllor et al. 2002). Some insects form very stable relationships with intracellular endosymbiotic bacteria leading to the formation of novel metabolic pathways and specialised structures for housing endosymbionts. The origin of the association between some endosymbionts and their hosts can be traced back to changes in the life history of the host. The weevil (Coleoptera; Curculionidae) species Sitophilus linearis, for example, is the only aposymbiotic (= without symbionts) species in the Dryophthoridae weevil family, and its lack of endosymbiotic bacteria can be associated with a switch from feeding on nutritionally poor monocotyledons such as cereals to the nutritionally balanced dicotyledon tamarind seed (Delobel and Grenier 1993). Co-diversification of insects and endosymbiotic bacteria into novel niches highlights the extent to which endosymbiont bacteria can be extremely influential in shaping insect ecology.

The symbiont bacteria of insects are commonly divided into two categories. The first category includes obligate endosymbionts, often referred to as primary endosymbionts. These are located in the cytoplasm of hypertrophied cells specialised for endosymbiosis called mycetocytes (also referred to as bacteriocytes) which reside in a specialised organ called a mycetome (also referred to as a bacteriome; Mclean and Houk 1973; Buchner 1965). The most familiar example of a primary endosymbiont is B. aphidicola, the primary symbiont of aphids, though there are many other examples of primary symbioses in herbivorous insects including sharpshooters (Hemiptera; Cicadellidae) and Sulcia muelleri and psyllids (Hemiptera; Psyllidae) and Carsonella ruddii (Fig. 1). The primary symbioses of insects are ancient, 160–180 Ma in the case of B. aphidicola (Munson et al. 1991; Moran et al. 1993), and more than 260 Ma for S. muelleri (Moran et al. 2005a), and over evolutionary time, the insect and bacteria have become completely dependent on each other. In the absence of B. aphidicola, the development of nymphs and the reproductive output of adult aphids are reduced (Douglas 1992, 1996, 1998). In return for a stable niche and provision of nutrients, the primary symbionts play a functional role in the physiology of the host by synthesising essential nutrients which are missing from the

Fig. 1 Maximum likelihood phylogeny based on available database sequences for the 16S gene to illustrate the diversity of insect symbionts and the major bacterial classes in which they are found.

Primary obligate endosymbionts are highlighted in blue. Sequences are preceded by their accession number in the NCBI database

Insect endosymbionts: manipulators of insect herbivore trophic interactions?

diet of the host insect (Douglas 1998, 2009; McCutcheon et al. 2009a). The second category of symbiont bacteria have a more facultative relationship with their insect hosts and are referred to as secondary symbionts. Unlike the primary endosymbionts, they are not restricted to one cell type and are not essential for the survival of the host insect. Secondary symbionts have been found in a variety of cell types including the reproductive organs, the gut and the haemolymph (Griffiths and Beck 1973; McLean and Houk 1973; Fukatsu et al. 2000). They are also found in localised concentrations in the secondary mycetocytes, a cell type similar to and in close proximity with the B. aphidicola containing primary mycetocytes in the aphid haemocoel (Hinde 1971; Fukatsu et al. 2000). Examples of secondary symbionts include three types characterised in aphids, namely, Serratia symbiotica, Hamiltonella defensa and Regiella insecticola (Fukatsu et al. 2000; Chen et al. 2000; Darby et al. 2001). The three types of secondary symbiont in aphids are members of the Enterobacteriaceae (Moran et al. 2005b). S. symbiotica is a symbiotic Serratia species whilst H. defensa and R. insecticola are sister groups to one another with a closer evolutionary relationship to Photorhabdus species (Moran et al. 2005b). The diversity of secondary endosymbionts associated with the pea aphid (Acyrthosiphon pisum) has made it a model for studying many aspects of facultative symbioses in insects, although there are many other examples of insect secondary symbionts including the Arsenophonus species found in psyllids and other arthropods (Dale et al. 2006; Hansen et al. 2007) (Fig. 1).

Co-diversification of primary symbiotic bacteria with herbivorous insects The fossil record suggests that the origin of the primary endosymbiont of insects is ancient, and molecular phylogenetic studies have built on information from the fossil record to shed light on the origin, transmission routes and diversification of many primary endosymbiotic bacteria (Munson et al. 1991; Baumann et al. 1995a; Moran et al. 2005a). The primary symbiont of aphids, B. aphidicola, is maternally transmitted via the ovaries to the developing embryos (Buchner 1965; Hinde 1971), but transmission routes vary between insect groups. In mealybugs (Homoptera; Pseudococcidae), for example, the symbiont is transferred via the eggs (von Dohlen et al. 2001), and there is considerable phylogenetic evidence to suggest that transmission of the primary insect symbionts is strictly vertical as co-diversification of primary symbionts and their insect hosts has been demonstrated repeatedly for numerous insect groups.

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The first example of co-diversification of primary symbiont and host insect came from aphids and B. aphidicola (Munson et al. 1991; Baumann et al. 1995a). It is now known that the primary symbionts of the mealybug Tremblaya princeps (Hemiptera; Pseudococcidae; Downie and Gullan 2005), of sharpshooters (S. muelleri; Moran et al. 2005a), of psyllids (C. ruddii; Thao et al. 2000a, b), of weevils in the genus Nardonella (Lefèvre et al. 2004) and of cockroaches (Blattaria; Blattabacterium species; Clark et al. 2001), all show considerable co-diversification with their insect host. Even within insect groups, there is strong evidence for strict vertical transmission and codiversification between insect and symbiont. The primary symbiont of whitefly (Hemiptera; Aleyrodidae) Portiera aleyrodidarum, for example, shows evolutionary congruence from a single bacterial infection in a common ancestor of whiteflies and subsequent co-speciation with a major subdivision of endosymbiont clades occurring between two whitefly subfamilies (Thao and Baumann 2004). At an even finer scale, the aphid genera Uroleucon (Clark et al. 2000) and Brachycaudus (Jousselin et al. 2009) show considerable co-diversification of B. aphidicola within close relatives in each aphid genus. Although insects and their primary symbiotic bacteria have diversified in parallel, there is little evidence to suggest that changes in the primary symbiont genome mirror exactly diversification of their insect hosts. Extreme genome stability is characteristic of the genomes of primary symbionts. B. aphidicola genomes from unrelated aphid species, for example, show no gene acquisitions or chromosome rearrangements in the past 50–70 Ma, indicating the preservation of genome stability of B. aphidicola through several episodes of aphid speciation (Funk et al. 2001; Tamas et al. 2002). Consequently, given that the evolutionary history of B. aphidicola spans a period including many evolutionary shifts in the diet and life cycle of its aphid hosts, the ecological diversity of aphids is unlikely to be explained by the genetic diversity of B. aphidicola (Funk et al. 2001; Tamas et al. 2002). Small changes in the genome of the primary symbiont can, however, influence insect fitness and are therefore causes of and subject to selective pressures. For example, a point mutation in the small heat shock gene (ibpA) can influence thermal tolerance and reach relatively high frequencies in aphid populations (Dunbar et al. 2007). However, the lack of evidence for horizontal transmission coupled with the highly obligate functional nature of primary symbionts suggests that primary symbionts persist due to their contribution to nutrition rather than their infection capacity or other benefits to host fitness (Wernegreen and Moran 2001). The nutritional requirements of different aphid species vary and are influenced by variation in the biosynthetic

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pathways for amino acid synthesis determined by the capabilities of their B. aphidicola genome (Moran et al. 2008). Consequently, the contribution of primary symbionts to the host phenotype are nutritionally based rather than the wide-ranging effects on insect fitness that are exhibited by endosymbionts such as Wolbachia pipientis in Drosophila (Diptera; Drosophilidae), which can be transmitted horizontally as well as vertically with major consequences for the host phenotype (Huigens et al. 2000). The secondary symbionts of herbivorous insects can also be transmitted horizontally as well as vertically, unlike primary symbionts, also influencing the phenotype of the host insect. As a consequence, secondary symbionts often have a more facultative and dynamic role to play in influencing insect ecology and evolutionary diversification.

Transmission patterns of secondary symbiotic bacteria in herbivorous insects Secondary symbionts are found in a variety of unrelated taxa (Darby et al. 2001; Russell et al. 2003), and they do not exhibit the same high level of co-diversification observed between primary endosymbionts and their insect hosts. For example, there is no evolutionary congruence between psyllids and their secondary symbionts (Thao et al. 2000a) or between the insect host and several types of the arthropod secondary symbiont Rickettsia (Weinert et al. 2009). Some evidence suggests that S. symbiotica may have co-diversified with aphids in the subfamily Lachninae (Lamelas et al. 2008; Burke et al. 2009), but secondary symbionts appear to be distributed somewhat erratically within and between insect taxa and are not consistently associated with any species of herbivorous insects (Tsuchida et al. 2002; Haynes et al. 2003; Hansen et al. 2007). The labile distribution of secondary symbionts across the insect genera could be a consequence of repeated horizontal transmission (Sandström et al. 2001). Secondary symbionts were, according to phylogenetic analysis, acquired independently in a wide variety of herbivorous insects, indicating they can be transmitted horizontally as well as vertically (Thao et al. 2000a; Sandström et al. 2001; Russell et al. 2003). In addition, the lack of genetic divergence of secondary symbionts within and between herbivorous insect species suggests that horizontal transmission across taxa occurred far more recently in evolutionary time than the origin of ancient primary symbioses (Fukatsu et al. 2000; Russell et al. 2003). To date, however, all attempts to demonstrate horizontal transmission of secondary symbionts via an ecological route using laboratory experiments have been unsuccessful. Attempts to establish horizontal transmission between sympatric infected and uninfected aphid lines on

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the same host plant were unsuccessful as were experiments using aphid parasitoids to transmit the bacteria (Chen et al. 2000). Transfer of secondary symbionts to insect hosts via artificial diets and microinjection have, however, been achieved in the laboratory, suggesting that horizontal transmission by such means is possible (Pontes and Dale 2006). Current opinion suggests that occasional horizontal transmission events are essential for the establishment of facultative secondary infection in wild populations of herbivorous insects, which are then maintained by high levels of vertical transmission and account for the multiple evolutionary origins of secondary symbionts across insect taxa (Sandström et al. 2001; Russell et al. 2003). However, in natural populations, fluctuating selection pressures will constantly be imposed by environmental variables, including pressure from predators and parasitoids, fluctuations in temperature or the nutritional provision of plants. The evolutionary relationship between the secondary symbionts and their insect hosts is therefore far more dynamic than that of the obligate primary symbionts, a fact which is reflected in the genomes of the two categories of symbiont. In contrast to most obligate symbionts, the aphid secondary symbiont, H. defensa, has a very dynamic genome which contains virulence and toxin-encoding genes and exhibits recombination, uptake of genes mediated by a bacteriophage and horizontal gene transfer (Degnan and Moran 2008a, b; Oliver et al. 2010). The obligate nature and lack of horizontal transmission in the primary symbionts has meant that the characteristics of their genomes are very different from those of the secondary symbionts leading to profound differences their evolutionary dynamics and influences on the fitness and ecology of herbivorous insects.

Key genetic characteristics of primary endosymbionts There are several important aspects of the genomics and evolution of the symbiotic bacteria in insects, which have been comprehensively reviewed in Moran et al. (2008) and are summarised here. As mentioned previously, the primary symbioses of insects are ancient, and over evolutionary time, the symbiotic relationship between the insect and bacterium has become obligate (Moran 2003; Moran et al. 2005a). The genomes of the primary symbionts have undergone significant reduction; the genome size of B. aphidicola in the pea aphid (A. pisum) is 657 kbp (Shigenobu et al. 2000) and in the cedar aphid (Cinara cedri) is 416 kbp (Pérez-Brocal et al. 2006). Primary symbionts of other herbivorous insects have even smaller genomes such as S. muelleri (246 kbp; McCutcheon and Moran 2007), C. ruddii (159 kbp; Nakabachi et al. 2006)


and the symbiont of cicadas (Hemiptera; Cicadidae) Hodgkinia cicadicola (144 kbp), which is the smallest (McCutcheon et al. 2009b; Table 1). They are much smaller than the genomes of free-living γ-proteobacteria (one seventh the size of Escherichia coli 4,639 kbp; Blattner et al. 1997), and as a consequence, they are viable only in their specialised niche (Sasaki et al. 1991). Such reduction in size is characteristic of the genomes of primary symbiont bacteria, which exhibit significant gene loss, large deletions, a rich adenine and thymine content and elevated rates of evolution followed by long periods of stasis (Moran 2003; Moran et al. 2008, 2009). B. aphidicola exhibits a mutation rate ten times higher than any other bacteria investigated to date (Moran et al. 2009), and the same characteristics are observed in S. muelleri and Baumannia cicadellinicola the co-symbiont bacteria of sharpshooters (McCutcheon and Moran 2007). In addition, the primary symbionts are remarkably polyploid, varying from tens to hundreds of genomes per cell (Komaki and Ishikawa 1999).

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In recent years, advancements in genomic techniques have shed light on many of the interesting aspects of the genomes of symbiotic bacteria. Loss of genes for biosynthesis of cell surface components and non-essential amino acids, regulator genes and genes involved in defence of the cell that are essential for a free-living life and the maintenance of genes for replication, transcription and translation and multiple genes for biosynthesis of essential amino acids have occurred in the B. aphidicola genome (Shigenobu et al. 2000; Pérez-Brocal et al. 2006), highlighting the mutual dependence between the aphid and its primary symbiont. Given the high levels of genome reduction observed in primary symbiotic bacteria, there has been considerable investigation into the dynamics of gene loss and what its evolutionary consequences may be (Moran et al. 2003a, 2005c, 2008, 2009). Since B. aphidicola and many of the other primary symbionts, including B. cicadellinicola and the Camponotus (Carpenter) ant (Hymenoptera; Formicidae) symbiont Blochmannia

Table 1 Genome sizes of facultative, obligate and free living bacteria in insects showing average percentage reduction in genome size associated with each bacterial life style Insect sub-order

Insect species

Symbiont

Role

Sternorrhyncha Aphids Aphids Aphids Aphids Aphids Aphids Psyllids Cicadas Cicadas Sharpshooters

Schizaphis graminum Acyrthosiphon pisum Baizongia pistaciae Cinara cedri Acyrthosiphon pisum Acyrthosiphon pisum Homalodisca coagulata Diceroprocta semicincta Diceroprocta semicincta Homalodisca vitripennis

Buchnera aphidicola Buchnera aphidicola Buchnera aphidicola Buchnera aphidicola Hamiltonella defensa Regiella insecticola Carsonella ruddii Hodgkinia cicadicola Sulcia muelleri Sulcia muelleri

Homalodisca vitripennis

Baumannia cicadellinicola

Obligate 641,454 Tamas et al. (2002) Obligate 640,681 Shigenobu et al. (2000) Obligate 617,838 van Ham et al. (2003) Obligate 422,434 Pérez-Brocal et al. (2006) Facultative 2,110,331 Degnan et al. (2009a) Facultative 2,035,106 Degnan et al. (2010) Obligate 159,662 Nakabachi et al. (2006) Obligate 143,795 McCutcheon et al. (2009b) Obligate 276,984 McCutcheon et al. (2009a) Obligate 245,530 McCutcheon and Moran (2007) Obligate 686,192 Wu et al. (2006)

Sharpshooters Apocrita Carpenter Ants Carpenter Ants Dictyoptera Cockroaches Cockroaches Cyclorrapha Tsetse flies Tsetse flies Brachycera Fruit flies Free-living γproteobacteria

Camponotus floridanus Blochmannia floridanus Camponotus pennsylvanicus Blochmannia pennsylvanicus

Obligate Obligate

Genome size

Reference

705,557 Gil et al. (2003) 791,654 Degnan et al. (2005)

Periplaneta americana Blattella germanica

Blattabacterium sp. BPLAN Obligate Blattabacterium str. Bge Obligate

636,994 Sabree et al. (2009) 636,850 López-Sánchez et al. (2009)

Glossina brevipalpis Glossina morsitans morsitans

Wigglesworthia glossinidia Sodalis glossinidius

Obligate 697,724 Akman et al. (2002) Facultative 4,171,146 Toh et al. (2006)

Drosophila melanogaster

Wolbachia pipientis Pseudomonas aeruginosa Escherichia coli

Facultative 1,267,782 Wu et al. (2004) 6,264,403 Stover et al. (2000) 4,639,221 Blattner et al. (1997)

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floridanus, are members of the Enterobacteriaceae, a family of well-studied γ-proteobacteria, which includes E. coli, they provide good models to reconstruct the process of evolutionary gene loss against a wealth of available genomic information (Moran et al. 2003a). The close relationship of the primary symbionts to model organisms such as E. coli has meant that recently developed system level approaches have been applied to the primary symbionts to investigate key biosynthetic pathways involved in the symbiosis (e.g. Thomas et al. 2009). Interestingly, recent genome sequencing of some examples of secondary symbiont bacteria revealed that they exhibit to a lesser extent the genome characteristics of primary endosymbionts, including elevated sequence evolution, lower GC bias, gene loss and a reduction in genome size (Table 1) due to an intracellular lifestyle and prolonged vertical transmission (Wu et al. 2004; Degnan et al. 2009, 2010). Consequently, the significant reduction in genome size from free-living bacteria to secondary endosymbionts and thence to obligate endosymbionts provides a clear illustration of the evolutionary consequences of symbiosis for bacteria (Fig. 2). Interestingly, the vertically transmitted symbiont Ishikawaella capsulata of Plataspidae stinkbugs (Hemiptera; Pentatomidae) also shows elevated levels of sequence evolution, gene loss and AT bias, but is extracellular (Hosokawa et al. 2006), indicating that the population genetic attributes of vertically transmitted endosymbionts are likely to be responsible for their singular genome characteristics rather than their intracellular lifestyle (Hosokawa et al. 2006).

Problems faced by symbiotic bacteria The primary symbiont of aphids, B. aphidicola, is maternally transmitted via the ovaries from one generation to the next (Buchner 1965; Hinde 1971). Only a small proportion

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of B. aphidicola are transmitted transovarially to the embryo via a stream of bacteria from a single mycetocyte in the adult at stage 7 of embryo development immediately before anatrepsis (Miura et al. 2003; Wilkinson et al. 2003), resulting in a bottleneck effect following each transmission event (Wernegreen and Moran 2001). Asexual reproduction, with repeated population bottlenecks, leads to an accumulation of deleterious mutations, which are a major factor in genome reduction and a loss of fitness, a phenomenon termed Muller’s ratchet (Felsenstein 1974). It is argued that because each aphid contains small sub-populations of B. aphidicola in each mycetocyte and selection acts at the host level, deleterious mutations will accumulate in the smaller sub-populations, which will be driven to extinction sparing the larger populations (Pettersson and Berg 2007). However, repeated population bottlenecks enable selection to act on very small changes that can cause differences in fitness, exacerbating the effects of genetic drift and leading to an accumulation of deleterious mutations, decreased selection to maintain gene functionality and irreversible loss of fitness (Moran 1996; Funk et al. 2001; Wernegreen 2002; Mira et al. 2001; Fares et al. 2005). A particular problem caused by the buildup of deleterious mutations in primary endosymbionts is low stability of proteins (van Ham et al. 2003). The chaperonin protein GroEL has been suggested as a compensatory mechanism for two types of primary endosymbiont, B. aphidicola and Blattabacterium species, in which amino acid substitutions in functionally important domains have been detected that have become fixed by positive selection (Fares et al. 2002, 2005). Mutations occurring in functionally important regions may optimise the ability of GroEL to bind and prevent inappropriate folding of proteins which are conformationally damaged as a consequence of the accumulation of mildly deleterious mutations (Fares et al. 2002, 2005). Reduction of primary symbiont genomes is a gradual process occurring at a rate of approximately one gene per 5–10 Ma (Tamas et al. 2002). Strong host level selection means that gene losses are unlikely to result in the extinction of the symbiont (Pettersson and Berg 2007), but if their functional role is compromised, there could be profound consequences for the insect host.

Effects of multiple infection with symbiotic bacteria

Fig. 2 The genome size of obligate symbiotic bacteria is significantly reduced compared to facultative symbionts and free-living bacteria (values are mean based on data in Table 1 ±95% confidence limits)

Herbivorous insects are dependent on their primary symbiotic bacteria for development and reproduction, and if these are experiencing irreversible fitness decline, reliance on obligate symbiosis may represent an evolutionarily unstable situation. Infection with multiple symbiont types has been suggested as a possible compensatory mechanism to remedy the problems faced by obligate endosymbionts that


could lose essential metabolic capabilities, or even be driven to extinction. As mentioned previously, the role of B. aphidicola is largely nutritional; it provides the aphid with essential amino acids, which are scarce in the phloem sap upon which it feeds (Douglas 1992, 1998, 2003; Wilkinson and Douglas 1995a, b; Sandström and Pettersson 1994; Sandström and Moran 1999; Gündüz and Douglas 2009). Thus, maintenance of genes for synthesis of essential amino acids allows the symbiosis to continue. This is, however, not the case for C. ruddii whose genome has become so reduced that it has lost at least half the pathways for biosynthesis of amino acids, which represents its proposed symbiotic function (Tamames et al. 2007). In sharpshooters, there is evidence of convergent evolution of the metabolic roles in the co-resident symbionts S. muelleri and B. cicadellinicola and in cicadas between S. muelleri and H. cicadicola (McCutcheon et al. 2009a). In both cases, one symbiont can provide a proportion of the amino acids and nutrients required by the host with the other making up the shortfall, indicating that mutualistic relationships between two symbiont types may be evolutionarily stable over millions of years (McCutcheon et al. 2009a). Functional replacement of symbiotic bacteria by other bacteria and metabolic interdependence of endosymbionts might be key in shaping trophic interactions involving insect herbivores. Selective elimination techniques in the pea aphid were used to show that the secondary symbiont S. symbiotica can compensate for the loss of the primary symbiont B. aphidicola by physiologically and cytologically taking over its symbiotic niche (Koga et al. 2003). After elimination of B. aphidicola by antibiotic injection, S. symbiotica infected the cytoplasm of the mycetocytes which would normally house B. aphidicola (Koga et al. 2003). Replacement of B. aphidicola by S. symbiotica suggests that evolutionary routes to novel obligate endosymbioses could exist via endosymbiotic associations with facultative bacteria (Koga et al. 2003). Amelioration in the mycetocyte reducing effect of heat stress on pea aphids was, for example, explained by S. symbiotica effectively rescuing B. aphidicola (Montllor et al. 2002). The positive effects of S. symbiotica might be caused by the delivery of protective metabolites to B. aphidicola after heat exposure (Burke et al. 2010). In addition, the size of the B. aphidicola genome in C. cedri has been significantly reduced compared to other aphid species (Table 1) and has lost the genes for synthesis of some essential micronutrients, including tryptophan (Pérez-Brocal et al. 2006). Consequently, in order to compensate for this loss C. cedri also harbours large numbers of the secondary symbiont S. symbiotica, which can synthesise the missing micronutrients (Gómez-Valero et al. 2004; Gosalbes et al. 2008). A long-term evolutionary relationship has been suggested between S. symbiotica and aphids in the genus Cinara

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and subfamily Lachninae, in which a clade of S. symbiotica associated with Cinara species has been established separately from S. symbiotica associated with other aphid subfamilies in the family Aphididae (Lamelas et al. 2008). S. symbiotica in the clade associated with the Lachninae subfamily showed faster evolution of the protein coding atpD gene and were similar in size and shape to B. aphidicola (Lamelas et al. 2008), suggesting that functional replacement of B. aphidicola by S. symbiotica might be occurring in Lachninae aphids. However, some Cinara species within the clade lacked any symbiont other than B. aphidicola, suggesting that the requirement for S. symbiotica is not universal (Burke et al. 2009). Moran and Baumann (1994) first suggested that symbiont replacement might have occurred in aphids, and subsequently, Fukatsu and Ishikawa (1996) found that B. aphidicola had been lost in the aphid genus Hamiltonaphis and replaced with a yeast-like symbiont. In fact, there is evidence to suggest that symbiont replacement has occurred in many insects. Members of the Dryophthoridae family of weevils, for example, harbour one of the three different clades of γ-3 proteobacteria, and phylogenetic evidence suggests that complete replacement of one symbiont by another has occurred over evolutionary time (Lefèvre et al. 2004). The genus Nardonella forms the ancestral clade (R-clade) which exhibits the characteristics commonly observed in obligate endosymbionts, including genome reduction, high GC content, rapid rates of evolution and strict host–symbiont co-diversification (Lefèvre et al. 2004). The other two symbiont clades (S- and D-clades) are closer to free living bacteria and appear to have been established more recently than Nardonella. The D-clade of weevil symbionts includes the Sitophilus oryzae primary endosymbiont (SOPE) and Sitophilus zeamais primary endosymbiont (SZPE) symbionts of the cereal weevils S. oryzae and S. zeamais, respectively, which are closely allied to the Sodalis secondary symbionts of tsetse flies (Toju et al. 2010). Competition between the S- and D-clades of endosymbionts and the ancestral R-clade could have led to displacement of Nardonella in some modern Dryophthoridae weevil species (Lefèvre et al. 2004). The SOPE and SZPE symbionts in the recently derived D-clade of weevil symbionts provide an example of the first stages of becoming an obligate endosymbiont as they exhibit a relatively larger genome size and lower AT bias (Moya et al. 2009). Curculio weevils harbour ‘Candidatus Curculioniphilus buchneri’ an additional clade of primary symbiont, which occurs together with a secondary symbiont in the chestnut weevil, Curculio sikkimensis, which could be a possible candidate for symbiont replacement in weevils (Toju et al. 2010). Interestingly, displacement events could also be associated with changes in host life history strategy. In the Sitophilus weevil species, association with one of the

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secondary endosymbiont clades occurred at the same time as a switch from the ancestral method of stem feeding to a novel method of sap feeding (Lefèvre et al. 2004). Novel symbioses formed by the changing dynamics of insect endosymbiotic bacteria can facilitate insect diversification into new niches and enable survival in a highly variable environment.

Survival of insect symbionts in changing environments Several strategies are employed by facultative symbionts to facilitate their maintenance in host populations. The environment experienced by endosymbiotic bacteria varies according to variation in host diet, life cycle stage of the host and the location of the symbiont within the host (Estes et al. 2009). There are several interesting symbioses in insects that have evolved to ensure the survival of the symbiotic bacteria when the host environment changes. One example is that of the olive fruit fly (Bactrocera oleae; Diptera; Tephritidae) endosymbiotic bacterium ‘Candidatus Erwinia dacicola’, which can switch between an intracellular or extracellular lifestyle according to the life cycle stage of the insect host (Estes et al. 2009). ‘Candidatus Erwinia dacicola’ is found in the digestive system of all life stages of olive fruit fly but resides intracellularly in the midgut of the larvae and extracellularly in the foregut and ovipositor diverticulum of adult flies (Estes et al. 2009). It is one of only a few non-pathogenic endosymbionts that can switch from an intracellular to an extracellular life style during different phases of the host life cycle in order to facilitate its survival in a holometabolous insect (Estes et al. 2009). Vertically transmitted endosymbionts must be able to survive the breakdown and rebuilding of host tissues that occurs in holometabolous insect development, and they consequently have a larger genome and are more closely related to free living bacteria than the primary intracellular endosymbionts of other insects, such as aphids, that provide a more constant environment (Estes et al. 2009). The endosymbiont of the olive fruit fly provides an example of the extent to which host physiology and life history influence endosymbiont characteristics and the dynamic nature of symbiotic evolution in developing means to overcome transmission barriers imposed by the host. Another interesting symbiosis has evolved in mealybugs (Hemiptera; Pseudococcidae), which exhibit a peculiar nested formation of the endosymbiotic system (Kono et al. 2008). Mealybugs package intracellular symbionts, within their large ovoid bacteriomes, into mucus-filled spheres, which occupy most of the cytoplasm and surround the host cell nucleus (von Dohlen et al. 2001; Kono et al. 2008). The symbiotic spheres consist of the primary β-type proteobacteria symbiont T. princeps (Thao et al. 2002) and

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the secondary γ-type proteobacteria living symbiotically within the primary β-proteobacteria (von Dohlen et al. 2001; Kono et al. 2008). The nested life style of the γproteobacteria helps to facilitate their transmission. The host cells of the gravid female mealybug break down and the freed spheres cluster around the nutrient plasma cord connecting the nurse cells to the oocytes anterior pole, and the β-proteobacteria spheres penetrate the oocyte, facilitating transmission of both type of symbionts into the oocyte and consequently the next generation (von Dohlen et al. 2001). The intracellularisation of the γ-proteobacteria by βproteobacteria has been suggested as a compensatory mechanism which facilitates the exchange of genes or gene products between the two types of bacteria to slow or reverse the genome reduction caused by their endosymbiotic life cycle (von Dohlen et al. 2001). The βproteobacteria have experienced a strong co-evolution with their host from an evolutionary origin within a common ancestor of extant mealybugs (Thao et al. 2002; Kono et al. 2008), but they do not exhibit the same AT bias, gene loss or accelerated evolution of the obligate endosymbiont bacteria of insects such as B. aphidicola (Baumann et al. 2002). Consequently, co-existence with the γ-proteobacteria might provide a solution to the problems posed by an endosymbiotic lifestyle (Kono et al. 2008). The genes retained in the T. princeps genome code for essential functions such as DNA transcription, translation and replication and cell division (Baumann et al. 2002). Interestingly, however, genes for amino acid and purine ribonucleotide biosynthesis and the pentose phosphate pathway are also maintained (Baumann et al. 2002), indicating that retention of the essential nutritional capacity of T. princeps could be dependent on the presence of the secondary symbiont bacteria. The dual symbiosis found in mealybugs highlights the extent to which interactions between symbiotic bacteria can influence the phenotype of the host insect to the extent that they become crucial in shaping insect herbivore trophic interactions.

Endosymbionts and plant utilisation by insect herbivores The suitability of a plant as a food source and habitat for insect herbivores is determined by a number of plant factors that include attraction or deterrence by volatile chemical cues, differences in nutritional quality and differences in physical and chemical defences that alter palatability or cause toxicity (Renwick 2001; Schoonhoven et al. 2005). In addition, variation in the seasonal availability of the plant resource is a major determinant of host plant use by insects (e.g. Martel et al. 2001). The range of plant species acceptable as a host is generally small for most phytoph-


agous insects, reflecting competition for food resources, for suitable oviposition sites and for enemy-free living space (Schoonhoven et al. 2005). Situations where closely related individuals exhibit differential acceptance of and performance on host plants can lead to ecological specialisation that might eventually result in assortative mating and sympatric speciation (Caillaud and Via 2000). The degree of ecological specialisation and reproductive isolation of individuals on different host plants will be dependent on the genetic control of insect fitness traits and traits that influence mating and any trade-offs between these traits (Via and Hawthorne 2002). In addition to insect genotype, differential expression of phenotypic plasticity in insect fitness traits on diverse host plants could also facilitate the formation of insect races associated with particular plant types (Gorur et al. 2005). Insect endosymbionts provide a source of genetic and functional variation in insect herbivores and the presence of titre and functioning of symbionts can have profound effects on insect fitness that could influence plant choice and patterns of plant utilisation.

Obligate endosymbionts and the nutritional basis for host plant affiliation Plant tissues are generally nitrogen-poor relative to the protein requirements of insect tissues (Mattson 1980) and frequently provide insect herbivores with an imbalanced supply of particular essential nutrients, such as the low essential amino acids and lipid concentrations in phloem sap (Douglas 2003). The formation of obligate nutritional endosymbioses with microbial partners that can supplement the nutrient-poor plant diet has been critical to the success of insect herbivores (Buchner 1965; Douglas 2009). However, differences in host plant nutritional quality between plant species (Wilkinson et al. 2001, 2003; Sandström and Moran 1999), between ecotypes or cultivars within a species (Karley et al. 2008; Weibull et al. 1990), between individual plants of the same genotype (Gunduz and Douglas 2009) and during plant development and maturation (Karley et al. 2002; Fry et al. 2009) can significantly alter insect herbivore fitness. Host nutritional physiology and quality for herbivores is additionally modified by a variety of external biotic and abiotic factors that can range from mycorrhizal infection or nutrient supply (e.g. Gange et al. 1999) to attack by multiple herbivore types (Johnson et al. 2009). Thus, by determining the degree to which the insect partner relies on the plant for certain essential nutrients, it is possible that obligate endosymbionts can influence the ability of the insect partner to utilise different plant types. Genomic, meta-genomic and post-genomic approaches to study the function of microbes harboured by insects have

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improved significantly our understanding of the contribution of endosymbionts to insect herbivore nutrition. An array of studies in a number of insect herbivore species has highlighted the potential for endosymbionts to provide the insect host with essential nutrients and to contribute to energy metabolism and nitrogen recycling. The severely reduced genome of primary endosymbionts (Baumann 2005), by comparison with free-living relatives, has resulted in only a subset of the genes required for independent life being retained (Moran et al. 2008). However, genes for the synthesis of essential amino acids, fatty acids, vitamins and cofactors and for energy metabolism, sulphate reduction and urea breakdown and recycling have been variously retained in the genomes of endosymbionts associated with aphids (Shigenobu et al. 2000; Tamas et al. 2002; van Ham et al. 2003), psyllids and cicadas (Nakabachi et al. 2006; McCutcheon and Moran 2007; McCutcheon et al. 2009a, b), carpenter ants (Gil et al. 2003), cockroaches (Sabree et al. 2009), mealybugs (Baumann et al. 2002) and weevils (Rio et al. 2003). Frequently, genomic studies are supported by physiological evidence that endosymbiont metabolic capacity is essential for insect host functioning, as has been demonstrated for provision of essential amino acids and the vitamins riboflavin and pantothenate to the aphid host by B. aphidicola (Bermingham and Wilkinson 2010; Wilkinson et al. 2001; Gündüz and Douglas 2009; Sasaki and Ishikawa 1995; Nakabachi and Ishikawa 1999) and for provision of vitamins and aromatic amino acids to the weevil host by Nardonella (Wicker and Nardon 1982; Wicker 1983). Obligate endosymbionts might influence plant choice within a particular insect group in two ways. Firstly, genetic variation within an endosymbiont group might lead to differences in nutrient biosynthesis capacity, through loss of genes and their associated function, leading to differential reliance between insects on plant supply of essential nutrients. Secondly, transcriptional and post-transcriptional mechanisms might regulate endosymbiont nutrient provision in response to host insect nutrient status resulting from variation in plant quality. The increasing availability of genomic information has revealed genetic variation within an insect group in symbiont capacity to provide essential nutrients. Comparison of sequence data for the aphid endosymbiont B. aphidicola derived from A. pisum, C. cedri, Baizongia pistacea and Schizaphis graminum indicates differences between aphid species in B. aphidicola biosynthetic capacity for essential nutrients, with loss of genes for tryptophan and riboflavin synthesis in C. cedri (Shigenobu et al. 2000; Pérez-Brocal et al. 2006), for biotin synthesis in A. pisum and S. graminum and for arginine biosynthesis in B. pistacea (van Ham et al. 2003). Loss of biosynthetic capacity is likely to place pressure on the insect to select host plants that can supply the

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nutrient shortfall, as has been speculated for sulphur nutrition in some strains of B. aphidicola. The complete pathway for sulphate reduction has been lost from B. aphidicola harboured by B. pistacea and S. graminum but not A. pisum (van Ham et al. 2003), suggesting that the former two aphid species need to exploit alternative sources of organic sulphur compounds, either in the plant diet or from gut bacteria that can reduce sulphur (Zientz et al. 2004). Indeed, nutritional enhancement of phloem quality induced by feeding of S. graminum on grasses (Sandström et al. 2000) might replace the shortfall in B. aphidicola biosynthetic capacity (Tamas et al. 2002; Moran et al. 2005c). Similarly, loss of genes encoding arginine biosynthesis in the Blochmannia endosymbiont of Camponotus ants might reflect the fact that dietary supply of this essential amino acid is not limiting in this omnivorous insect (Zientz et al. 2004). However, retention in the aphid herbivore Cinara tujafilina of the plasmid-borne genes for synthesis of the essential amino acid tryptophan and loss of these genes in the related species C. cedri does not appear to lead to differential selection of plant species (Gil et al. 2006; Pérez-Brocal et al. 2006), suggesting that the tryptophan biosynthetic capacity of the primary endosymbiont does not directly influence the plant utilisation patterns of these two aphid species. In aphids, transfer to plasmids of genes encoding essential nutrient synthesis, such as the trpEG and leuABCD genes encoding key enzymes in tryptophan and leucine synthesis, could reflect a mechanism to control gene amplification and therefore production of these essential amino acids (Baumann et al. 1995b, 1999; Moran et al. 2003b). However, evidence that this provides a mechanism to respond to variation in plant nutritional quality is limited. The copy number of these plasmid-borne genes relative to chromosomal genes can vary significantly between and within aphid species (Birkle et al. 2002, 2004; Thao et al. 1998; Moran et al. 2003a), but in A. pisum, this did not relate either to rate of tryptophan synthesis (Birkle et al. 2002) or to aphid performance on Vicia faba plants providing low levels of dietary tryptophan (Birkle et al. 2004). Interpretation of plasmid gene copy number is confounded by the widespread presence of inactivated trpEG pseudogenes in a number of aphid species (Baumann et al. 1997; Wernegreen and Moran 2000). The presence of trpEG pseudogenes in Diuraphis species was associated with reduced tryptophan concentration in insect tissues (Wernegreen and Moran 2000), but the widespread existence of pseudogenes in a number of aphid species could suggest that aphids are decreasingly reliant on symbiont provision of this nutrient (Wernegreen and Moran 2000). Indeed, the increased phloem transport of amino acids induced by feeding in aphid species such as Diuraphis noxia and S. graminum (Telang et al. 1999; Sandström et al.

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2000) might reflect reduced aphid dependence on B. aphidicola-derived essential nutrients (Sandström et al. 2000). Transcriptomic studies indicate that control of nutrient provisioning is unlikely to occur at the level of endosymbiont gene expression. In aphids, the level of B. aphidicola gene expression appears to be controlled by spatial organisation of the genome, and the most highly expressed B. aphidicola genes are those providing essential cell functions (e.g. ribosome and chaperone proteins and proteins involved in cell division) and those involved in essential amino acid synthesis (Viñuelas et al. 2007). Expression of B. aphidicola genes with biosynthetic function is relatively unresponsive to variation in environmental factors such as aphid diet quality and heat shock, although this can vary to some extent between aphid species and treatments (Wilcox et al. 2003; Moran et al. 2003b, 2005c; Wilson et al. 2006; Reymond et al. 2006). This lack of variation in the level of gene expression probably reflects extensive loss of transcriptional regulator genes in the B. aphidicola genome (Moran and Degnan 2006). A notable exception is the induction of the metE gene in S. graminum in response to changes in dietary amino acid supply. The metE gene forms part of the methionine synthesis pathway and its transcriptional regulator metR is still retained in the B. aphidicola genome of S. graminum (Moran et al. 2005c). Transcriptional control of methionine synthesis might enable symbiont provision to be co-ordinated with aphid-induced changes in phloem amino acid supply (Sandström et al. 2000) or might reflect the inability of B. aphidicola in S. graminum to provide organic sulphur compounds (Tamas et al. 2002; Moran et al. 2005c). A general lack of transcriptional control of endosymbiont genes has also been observed for Blochmannia harboured by Camponotus floridanus ants (Stoll et al. 2009). Thus, genomic and transcriptomic studies indicate limited control at the level of gene expression over nutrient provisioning to the insect host. Instead, mechanisms controlling flux of substrates and nutrients from insect tissues to mycetocytes across the mycetocyte and bacterial cell membranes might regulate symbiotic function (Moran et al. 2003b). Analysis of the metabolic contributions and requirements of obligate endosymbionts indicates that they are highly dependent on their insect hosts for supply of carbon and nitrogen substrates (Zientz et al. 2004) and are intolerant of large perturbations in substrate supply due to lack of functional gene redundancy associated with genome reduction (Thomas et al. 2009). This is corroborated by comparative genome analysis of A. pisum and B. aphidicola, which indicates that the two organisms have highly complementary metabolic capacities, including for synthesis of several amino acids (Wilson et al. 2010) and for purine synthesis and recycling (Ramsey et al. 2010). Regulation of endosymbiont replication and the number of


endosymbiont cells or genomes within the insect is thought to be a primary determinant of levels of gene expression per insect in aphids and carpenter ants (Stoll et al. 2009; Bermingham et al. 2009) and potentially also in mealybugs (Kono et al. 2008). Although transcriptional studies provide limited evidence for up- or downregulation of symbiont metabolism in response to fluctuations in insect diet, physiological studies indicate that plant quality can influence endosymbiont function and its ability to satisfy the nutritional needs of the insect. Phloem sap generally contains inadequate amounts of at least one of the essential amino acids required to support aphid growth (Gündüz and Douglas 2009). For A. pisum on V. faba, this deficiency is readily compensated by the biosynthetic capacity of B. aphidicola (Gündüz and Douglas 2009). However, plant speciesspecific or genotype-specific profiles of essential nutrients can have a significant impact on insect growth and symbiont function. Low phloem concentrations of amino acids in Lamium purpureum have a negative impact on B. aphidicola function, particularly the ability to provide the essential amino acid threonine and leads to poor performance of Aphis fabae on Lamium compared to other plant species (Wilkinson et al. 2001; Chandler et al. 2008). Upregulation of B. aphidicola-derived chaperonin in Myzus persicae in response to particular plant species (Francis et al. 2006) and plant stress (An Nguyen et al. 2007) also indicates that the endosymbiont genome can respond to plant chemistry either directly or through plant-mediated effects on insect performance. Obligate endosymbionts that have a long coevolutionary history with their insect hosts are vulnerable to loss of symbiotic function as mutations accumulate as a result of gene loss and limited functional gene redundancy (Moran and Degnan 2006). C. ruddii, an obligate endosymbiont of the psyllid Pachpsylla venusta, possesses the smallest known bacterial endosymbiont genome (160 kb), which lacks functional genes for many proteins essential for viability and for carrying out symbiotic function, including genes for provision of the essential amino acids histidine, threonine and phenylalanine (Nakabachi et al. 2006; Tamames et al. 2007). In cases where there are apparently no alternative symbionts to fulfil the missing functions, choice of host plant species and quality might be crucially important for ensuring adequate insect nutrition. Loss of symbiont function creates an opportunity for invasion of the insect by facultative symbionts that have nutrient provision capacity and can either complement or replace the obligate endosymbiont. Acquisition by insect herbivores of novel endosymbionts with divergent nutrient synthesis capacities might cause a shift in the feeding niche of the insect herbivore, as has been postulated for the switch from chewing to sap-feeding in recent lineages of Sitophilus

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weevils (Lefèvre et al. 2004). Similarly, a shift to a novel diet with enhanced nutritional quality might explain loss of ancestral endosymbionts (Lefèvre et al. 2004). Evidence that consortia of symbionts with complementary metabolic capabilities can evolve to overcome loss of symbiont function comes from identification of multiple obligate endosymbionts within a single insect. The complementary biosynthetic capacities of the bacterial endosymbionts S. muelleri and B. cicadellinicola in the psyllid Homalodisca coagulata (the glassy-winged sharpshooter), determined by genome analysis, indicates that S. muelleri provides eight of the essential amino acids to the insect and B. cicadellinicola provides a further two essential amino acids in addition to vitamins and cofactors that S. muelleri cannot provide (Wu et al. 2006; McCutcheon and Moran 2007; McCutcheon et al. 2009a). In cicadas, which posses S. muelleri but not B. cicadellinicola, synthesis of the two essential amino acids that S. muelleri cannot provide is performed by H. cicadicola, although H. cicadicola and B. cicadellinicola have divergent capacities for synthesising vitamins (McCutcheon et al. 2009a). The intriguing ‘nested’ relationship between the two obligate bacterial endosymbionts of Planococcus citri (citrus mealybug; von Dohlen et al. 2001) raises interesting questions about the interdependence and regulation of the metabolic capacities of the two microbial partners and how this is coordinated with insect host metabolism. The loss of tryptophan synthesis capacity in B. aphidicola associated with the cedar aphid C. cedri (Pérez-Brocal et al. 2006) can be compensated by the facultative symbiont S. symbiotica (Gosalbes et al. 2008) and highlights the potential for facultative symbionts to influence insect herbivore nutrition.

Facultative endosymbionts and enemy-free space Heritable facultative endosymbionts of insects include mutualists and symbionts with unknown function and reproductive manipulators (Moran et al. 2008). A number of facultative endosymbionts belonging to different bacterial groups have been described from phloem-feeders, weevils and psyllids (Moran et al. 2008; Fig. 1). The lack of facultative symbionts in certain insect herbivore groups such as Camponotus species (Sauer et al. 2000) has been postulated to reflect diversification in the insect diet, particularly in the adult stadium, after establishment of the ancestral symbiosis (Zientz et al. 2004), indicating a role for facultative symbionts in the evolution of the nutritional endosymbiosis. Facultative endosymbionts have been detected in a wide range of aphid species over large geographic areas (e.g. Russell et al. 2003; Tsuchida et al. 2002, 2006). The γ-proteobacteria H. defensa, S. symbiotica and R. insecticola have been the focus of a number of

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E.L. Clark et al.

studies to demonstrate their influence on aphid performance in relation to aphid nutrition and plant use (Table 2). Physiological studies of pea aphids on chemically defined diets of different sucrose and amino acid contents did not identify any consistent association between aphid performance and infection with H. defensa, S. symbiotica or R. insecticola, indicating that the presence and impact of facultative endosymbionts is not simply related to aphid nutrition (Douglas et al. 2006). However, S. symbiotica is able to synthesise certain essential amino acids, including tryptophan, and can at least partially replace loss of B. aphidicola function in A. pisum and C. cedri (Koga et al. 2003; Koga et al. 2007; Pérez-Brocal et al. 2006; GómezValero et al. 2004; Gosalbes et al. 2008). By contrast, H. defensa cannot synthesise eight of the essential amino acids and, along with the host aphid, is dependent on B. aphidicola for these nutrients (Degnan et al. 2009). One could speculate that facultative symbionts with the capacity to supply the insect with essential nutrients might enable insect herbivores to feed on nutritionally poor plants. Conversely, symbionts that compete with the insect for nutrients supplied by the obligate endosymbiont might

force the insect to focus on plants of high nutritional quality. In fact, there is increasing evidence that the effect of facultative endosymbionts on plant utilisation by herbivores is a complex interaction between aphid and endosymbiont genotype and acquired defence against natural enemies. Host plant specialisation has been studied extensively in the pea aphid, A. pisum, and the black bean aphid, A. fabae, and there is strong evidence that aphid genotype determines pea aphid specialisation on alfalfa and red clover in New York State (Via 1999; Caillaud and Via 2000; Hawthorne and Via 2001), and A. fabae specialisation on V. faba and Tropaeolum majus (Douglas 1997; Tosh et al. 2001, 2004; Gorur et al. 2005). Distinct genetic clusters of pea aphid lineages are associated with clover, alfalfa and pea/bean plants in France (Via 1999; Simon et al. 2003; Frantz et al. 2006) and exhibit differentiation in phenotypic characteristics such as colour morph, dispersal morph, reproductive mode and male phenotype (Leonardo and Mondor 2006; Frantz et al. 2009), which are themselves likely to influence further genetic differentiation associated with each host plant. In contrast to the distinct aphid genetic differentiation

Table 2 Plant affiliation and association with facultative endosymbionts of insect herbivores Insect species

Facultative symbiont

Plant species

Acyrthosiphon pisum

Regiella insecticola

Trifolium pratense (red clover) High frequency on red clover in France, UK


Trifolium repens (white clover) High frequency on white clover in California, Japan


Trifolium spp.

Improved performance on clover compared to alfalfa and vetch

Hamiltonella defensa

Medicago sativa (alfalfa)

Higher frequency of symbiont on alfalfa and Lotus

Lotus pedunculatus (bird’s foot trefoil)

Observation

Serratia symbiotica Rickettsia

Vicia pisum (pea)

High frequency on pea and bean

Rickettsia

Vicia faba (faba bean)

Reduced fecundity on bean and clover

Acyrthosiphon pisum A. kondoi

Rickettsia

Vicia pisum (pea)

Serratia symbiotica

Bemisia tabaci

Rickettsia Arsenophonus

Vicia faba (faba bean) Medicago sativa (alfalfa) Lathyrus odorata (sweet pea) Salvia officinalis (sage)

Variable effects on aphid fitness depending on aphid genotype and plant species

Vicia faba (faba bean)

High frequency in Q biotypes on sage

References

Simon et al. 2003 Ferrari et al. 2004 Frantz et al. 2009 Leonardo and Muiru 2003 Tsuchida et al. 2004 Leonardo and Muiru 2003 Tsuchida et al. 2004 (but see Leonardo 2004 and Ferrari et al. 2007) Simon et al. 2003 Ferrari et al. 2004 Frantz et al. 2009 (but see Darby and Douglas 2003) Simon et al. 2003 Ferrari et al. 2004 Frantz et al. 2009 Simon et al. 2007 Chen et al. 2000 Chen et al. 2000

Chiel et al. 2007


associated with host plant species, there was close genetic allegiance between B. aphidicola strains in aphids on pea and alfalfa, but divergence on clover (Simon et al. 2003). Furthermore, specialisation of pea aphid on white clover in California was associated with the presence of the facultative symbiont R. insecticola; these clones showed greater fecundity on white clover than R. insecticola-free clones and greater mortality when transferred to alfalfa (Leonardo and Muiru 2003). R. insecticola occurs at higher frequency in pea aphids collected from clover compared to alternative host plants in populations from California, France, the UK and Japan (Leonardo and Muiru 2003; Simon et al. 2003; Ferrari et al. 2004; Tsuchida et al. 2004). Experimental manipulation of R. insecticola infection in a single aphid genotype indicated that this symbiont was responsible for enhanced aphid fecundity on clover compared to vetch (Tsuchida et al. 2004). However, more extensive study of manipulated R. insecticola infections in a number of pea aphid genotypes revealed that specialisation on clover compared to alfalfa or bean was determined by aphid genetic background (Leonardo 2004), or an interaction between aphid and bacterial genotypes (Ferrari et al. 2007), rather than presence of R. insecticola alone. The influence of aphid genotype on symbiont fitness consequences is increasingly recognised as a factor in the outcome of the aphid–endosymbiont interaction (Koga et al. 2007). Maintenance of high frequencies of R. insecticola in pea aphid specialists on clover is not simply related to aphid fitness of nutrition and therefore must be linked to as yet unidentified aphid traits or trait trade-offs. One possibility is that R. insecticola offers protection against particular natural enemy pressures associated with clover. Regiella has been associated with increased aphid resistance to the fungal pathogen Pandora in pea aphid (Scarborough et al. 2005) and to the parasitoid wasp (Hymenoptera: Braconidae) Aphidius colemani in M. persicae and A. fabae (Vorburger et al. 2009). Conversely, low frequencies of R. insecticola associated with aphid specialists on non-clover plant species might reflect selection of enemy-free space that reduces the need for aphids to retain an otherwise costly facultative symbiont. An example of this mechanism might be the utilisation of L. purpureum by A. fabae despite poor aphid performance on this host compared to alternative host plant species (Wilkinson et al. 2001). Low phloem amino acid concentrations in this plant species appear to impair aphid control over bacterial numbers (Chandler et al. 2008), and densities of R. insecticola and H. defensa in A. fabae are elevated on Lamium (Wilkinson et al. 2001). These observations would suggest that there is strong selection against A. fabae using Lamium as a host. However, there is some evidence that Lamium provides the aphid with enemy-free space in natural vegetation (Raymond et al. 2000). Thus, occasional use of Lamium as

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an enemy-free refuge might select against high frequencies of R. insecticola (and H. defensa) in A. fabae populations in natural mixed-species vegetation due to the fitness costs incurred by harbouring these symbionts. Affiliation of A. pisum genotypes with alfalfa and Lotus has been linked to the presence of the facultative endosymbiont H. defensa, whilst genotypes on pea and bean harboured S. symbiotica and Rickettsia infections (Simon et al. 2003; Ferrari et al. 2004; Frantz et al. 2009), although other studies have found no association between H. defensa presence and host plant range (Darby and Douglas 2003). Common co-infections include S. symbiotica and Rickettsia on pea and H. defensa and R. insecticola on alfalfa (Simon et al. 2003). The mechanistic basis of the association between these two facultative endosymbionts and host plant use by aphids is not known. Serratia had variable impacts on A. pisum fecundity depending on aphid genotype and host plant species (Chen et al. 2000). However, the fecundity of A. pisum and A. kondoi harbouring Rickettsia was generally reduced independently of aphid genotype on pea and bean (Chen et al. 2000; Simon et al. 2007), with neutral or small negative effects on alfalfa and sweet pea (Chen et al. 2000). This is surprising given the high frequency of Rickettsia in aphid clones on pea and bean and indicates fitness trade-offs associated with harbouring this endosymbiont. By contrast, Rickettsia had no apparent effect on performance of whitefly B biotype (Chiel et al. 2009a), which frequently harbours this endosymbiont (Chiel et al. 2007). The impact of symbiont presence on performance of the Q biotype of Bemisia tabaci is unknown but, of the four symbiont types detected in B. tabaci from a range of collection plants, Q biotype insects collected from sage only harboured Rickettsia and Arsenophonus (Chiel et al. 2007; Table 2). Thus, although various facultative endosymbionts have been associated with patterns of host plant use by insect herbivores (Table 2), this might reflect or be maintained indirectly through differences between host plants in their association with the natural enemies of insect herbivores. Possession of H. defensa, for example, is known to offer variable protection against different parasitoid wasp genotypes in A. pisum (Vorburger et al. 2009) and A. fabae (Vorburger et al. 2010a) and might influence the outcome of superparasitism (Vorburger et al. 2010b). Herbivore predators and parasitoids frequently exploit plant nectar and pollen resources (Wackers et al. 2008) and can exhibit strong preferences for the floral resources of particular plant species (Wackers 2004), which might contribute to differential parasitism or predation pressures associated with different plant types. Aphid honeydew can be utilised by parasitoid wasps as an alternative food source (Wackers et al. 2008), but the quality of honeydew varies significantly between aphids and their food plants (Wackers et al. 2008),

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suggesting that plant choice by insect herbivores might indirectly influence the performance of their natural enemies. The potential has yet to be explored for the host plant to influence symbiont-mediated protection against natural enemies, either directly through changes in herbivore nutritional status and fitness or indirectly through the supply and quality of plant and honeydew resources.

Symbiotic bacteria and parasitoid biology Many and perhaps the majority of insect species are attacked by one or more species of insect parasitoids (Godfray 1994). Parasitoids are probably the most important natural enemies of insects, more so than predators or pathogens (Hawkins et al. 1997), are ubiquitous in the majority of natural communities, with some exceptions, and have a singular biology and a variety of life history types which has made them objects of intense research focus. Much of this has been directed towards determining their utility as biological control agents against field and glasshouse pests, but there has been a strong complementary strand of research which has used insect parasitoid– host associations as model systems for the study of the evolution of characteristics such as sex ratios, sex determination mechanisms, foraging strategies, host selection and utilisation and the relationships between insects and microorganisms. This last field has seen explosive growth recently, and there is little sign that this is abating. Whilst symbiotic associations between eukaryotes and microbes are relatively common, we have very little knowledge of the selective pressures which have determined the prevalence of most species of insect endosymbionts.

Host fitness effects The effects of B. aphidicola on aphid fitness are well documented, and key components of fitness, such as embryo production, are directly dependent on the presence of B. aphidicola. When an aphid host has been parasitised host resources, many of them provided by B. aphidicola, are diverted to sustain the growth of the parasitoid immature. Cloutier and Douglas (2003) showed that parasitised aphids, containing a growing parasitoid larva, had more mycetocytes with a greater overall biomass, as well as embryos of lower mass, when compared to unparasitised hosts. Moreover, clone-specific susceptibility to parasitoid attack revealed that mothers from more vulnerable clones had embryos which were significantly smaller than those which were more resistant. Such observations make it unsurprising that symbiont-mediated resistance to parasitoids also carries a general fitness cost to the aphid host.

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Using the pea aphid–Aphidius ervi association as an experimental model, Gwynn et al. (2005) showed that there was a positive relationship between aphid fecundity and their susceptibility to parasitoid attack, suggesting a general physiological cost to carrying a resistance-conferring bacterial infection, which in turn indicates a basic life history trade-off in which the aphid can invest metabolic resources in either reproduction or resistance to parasitoids, but which is affected by the presence of symbiont infection. A similar effect was observed by Fytrou et al. (2006) using an experimental model consisting of the parasitoid Leptopilina heterotoma and its host Drosophila simulans. Here, infection with Wolbachia, which could infect either the host, the parasitoid, neither or both, had a complex effect on the ability of the host to mount an immune response, as well as on the parasitoid to overcome the response. An uninfected parasitoid attacking an infected host encountered a relatively weak immune response, but an infected parasitoid attacking an uninfected host encountered a strong immune response. The strength of the immune response (measured as parasitoid eggs or larvae killed) when host and parasitoid were both either infected or uninfected was not significantly different between these two treatments, indicating that the fitness costs to both host and parasitoid of Wolbachia infection are about the same, which would be expected if the association were evolutionarily stable and other life history parameters about the same.

Effects on parasitoid reproduction and behaviour Vertically transmitted endosymbiotic bacteria, such as Wolbachia, Cardinium and Rickettsia, modify host reproduction in several ways to facilitate their own spread and in situations where simple fitness effects are extended into actual manipulation the consequences can be subtle and complex. Pannebakker et al. (2007) demonstrated a B. aphidicola-like association in Asobara tabida, a parasitoid of Drosophila larvae, which is naturally infected with three strains of Wolbachia. In most species where it occurs, Wolbachia acts as a reproductive parasite, but in this case, Wolbachia is an obligate endosymbiont; female wasps which are cured of the infection with antibiotics fail to mature eggs and consequently have no fitness; a particular strain of Wolbachia, which is normally required for the completion of oogenesis, manipulates the process of apoptosis (programmed cell death) during egg maturation. Apoptosis has a normal role to play in oogenesis in this species and is an essential general part of insect oogenesis, but in wasps cured of the infection with antibiotics (aposymbiotic individuals), there was a considerable increase in the apoptosis of nurse cells in mid-stage egg


chambers that did not occur in normally symbiotic wasps. Pannebakker et al. (2007) were not able to conclude whether this function is under the control of the host or the symbiont, but Wolbachia is certainly playing some role in mediating apoptotic pathways in parasitoid egg development and is doing so without providing the host with any additional fitness advantage. A similar, though not quite obligate, association between a symbiont and a parasitoid exists between Encarsia hispida, a small parasitoid of insects in the Homoptera, and Cardinium, with which field populations of E. hispida, are naturally infected. E. hispida reproduces by thelytokous parthenogenesis (the production of diploid female offspring without fertilisation), and when wasps were cured of the Cardinium infection by antibiotic treatment, only male offspring were produced, which Giorgini et al. (2009) showed were diploid. Diploid males are unusual generally within the Hymenoptera, but have not before been associated with a symbiont infection in this group of parasitoids, where the greatest extent of symbiont-induced parthenogenesis in parasitoids occurs, and this result suggests that Cardinium feminises unfertilised diploid male eggs which then become fully functional female wasps. Wolbachia-induced parthenogenesis is taken to occur because of the influence of Wolbachia on ploidy levels (Huigens and Stouthamer 2003), where female development occurs without any direct effect of the symbiont on the sex determination system. What Giorgini et al. (2009) have clearly demonstrated is that this is not the case for Cardinium and that the symbiont must be directly interacting with the sex determination system of the parasitoid by an as yet undescribed mechanism. Cardinium has also been shown to manipulate the actual oviposition choices of parasitoid females in ways which ensure its own transmission. Kenyon and Hunter (2007), working with an experimental model consisting of the wasp Encarsia pergandiella infected with Cardinium, showed that the behavioural changes induced by the symbiont were sufficient to allow its invasion and establishment in sexual populations of its host. E. pergandiella is an autoparasitoid (sometimes called a heteronomous hyperparasitoid), where in sexual strains female eggs are laid in homopteran nymph hosts and male eggs are laid as hyperparasitoids of female parasitoid pupae developing within the primary host and eggs laid in the ‘wrong’ host type do not normally complete their development. Kenyon and Hunter (2007) investigated the way in which Cardinium appeared to manipulate host behaviour in a population of E. pergandiella with thelytokous parthenogenesis so that eggs were laid into the correct host type. In an experiment where infected and uninfected wasps were offered equal numbers of hosts suitable for the development of male and female eggs respectively, wasps which had been cured of their infection exhibited behaviour

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similar to that of unmated sexual wasps, allocating their eggs to hosts suitable for male eggs, and infected thelytokous wasps divided their eggs almost exactly evenly between the two host types, as if they had been mated sexual females.

The effect of multiple symbiont infections within parasitoids Multiple infections of symbionts can occur as different strains of the same species of symbiont within the same host individual, or communities of different symbiont species in a single host. Mouton et al. (2004) used the parasitic wasp A. tabida, which is a natural host of three different strains of Wolbachia, two of which are facultative and cause cytoplasmic incompatibility and the third of which is obligate and needed for the host to complete oogenesis. Different lines of the wasp were infected with different sub-groupings of the symbiont strains, and various indicators of host fitness were measured. In general, multiple infections caused loss of fitness over single infections in terms of weight loss and reduced lifespan, although some fitness indicators were unaffected. The marginal loss in fitness resulting from multiple infection was slight and probably not sufficient to promote strong selection against multiple infection in natural populations of A. tabida, and generally the physiological costs appeared to be more or less dependent upon the total bacterial density, itself a function of the number of co-infecting strains. Interestingly, in multiple infections, the obligate strain had lower fitness when present as a single infection than when present as part of a multiple infection with either of the two facultative strains, suggesting a positive synergistic interaction between facultative and obligate strains. Oliver et al. (2006) investigated the effect of multispecies infections (superinfections) of secondary symbionts in the pea aphid. Using the symbionts H. defensa and S. symbiotica, both of which have been shown to confer enhanced resistance to the insect parasitoid A. ervi, they showed that a double infection of both symbionts conferred greater resistance to parasitism than either single infection. Double infections are rare in natural populations of A. pisum in Utah, where this study was carried out, which suggests that there are severe fitness costs, probably reductions in fecundity, which mitigate against the establishment of multiple infections in spite of the fitness benefits conferred. In addition, the spatially densitydependent nature of insect parasitism would, through the existence of a partial refuge effect where a proportion of hosts escape parasitism, diminish selection in favour of multiple infections because of the absence of the stimulus to symbiont proliferation which is caused by parasitism.

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The role of parasitoids in horizontal transmission of symbionts We have stated earlier that insect parasitoids are one obvious possible route by which bacteria can be transferred between different host taxa, and the mechanisms by which this can be achieved are themselves varied and in many ways analogous to the mechanisms by which aphids transfer pathogenic viruses between host plants. The simple ‘dirty needle’ effect is perhaps the most obvious, but it has not been experimentally demonstrated that this occurs naturally. A theoretical study of the possibility (Preedy et al. 2010) developed a mathematical model of contact spread infection to investigate the separate influences of horizontal and vertical transmission which suggested that the transient and longterm dynamics exhibited under contact spread infection may be highly complex. Horizontal transmission has a stabilising effect on the system, but vertical transmission can destabilise it to the point of chaotic fluctuations in population levels. This is in contrast to earlier conclusions reached by Darby and Douglas (2003), also using mathematical models, which suggested that a 98% vertical transmission rate combined with 3% horizontal transmission would be sufficient to maintain the naturally occurring stable level of infection with H. defensa of 37% in the pea aphid. Horizontal transmission of CI-inducing Wolbachia was experimentally demonstrated by Heath et al. (1999), where the resulting infection proved not to be vertically stable and for parthenogenesis-inducing Wolbachia by Huigens et al. (2000) where the infection was vertically transmitted to the offspring of the wasp which had acquired it. For both of the latter studies, the mode of infection was by the sharing of a common food source, such as occurs in superparasitism when two or more larvae are present within the same host and where the infection can be passed from host to parasitoid, or from parasitoid to parasitoid. More recently, Chiel et al. (2009b) showed that infections of Rickettsia and Hamiltonella in the sweet potato whitefly B. tabaci have differential horizontal transmission to their parasitoids Eretmocerus emiratus, E. eremicus and E. pergandiella. Rickettsia was successfully horizontally transmitted from host to Eretmocerus larvae but was not subsequently vertically transmitted within the parasitoid lineage, and Encarsia did not become infected by Rickettsia. Hamiltonella did not establish in any of the parasitoid species, and together the results reinforce the conclusions reached by earlier studies that the likelihood of a horizontally acquired infection becoming evolutionarily stable are extremely small and absolutely dependent on the cytoplasmic and genetic background into which the infection is introduced.

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Secondary symbionts and resistance to parasitism Much of the initial interest in the associations between bacteria and insect parasitoids and their hosts focussed on Wolbachia, an ancient genus of reproductive parasite which may be carried by as many as 75% of all insect species (Werren and Windsor 2000), though such estimates are unreliable and obviously dependent on sampling intensity. Wolbachia is an example of a facultative symbiont whose presence is not required for the nutrition, development and reproduction of the host, in the way that B. aphidicola is required for normal growth and reproduction in aphids, but is a manipulator of the sex ratio of its host, distorting it towards the female by one of four possible mechanisms in order to facilitate its own vertical transmission. There is also substantial evidence that Wolbachia, like some other symbionts, has been horizontally transferred between arthropod lineages in the evolutionary past, and it has been demonstrated experimentally that parasitoids can be agents of such transfer (Heath et al. 1999). More recently, Wolbachia has been shown to confer protection from pathogenic viruses in Drosophila melanogaster (Hedges et al. 2008), a finding which has been anticipated as having possibly important implications for the control of insect pathogens in beneficial insects such as honeybees. Apart from Wolbachia, the majority of recent research on the relationship between parasitoids and the loose group of bacteria known as secondary symbionts has focussed on the role which some species play in mediating host resistance to attack by insect parasitoids. This phenomenon has been most extensively explored in aphids and in the pea aphid A. pisum in particular, where the closely linked physiological and cytological relationship between the wasp larva and its host provides an opportunity for interaction between the host defences, the symbiotic bacterium and the parasitoid. A. ervi is a small parasitic wasp which attacks several common aphid species, including the pea aphid, and it has been demonstrated that different clones of this species vary considerably in their resistance to A. ervi larval development after initial oviposition (Henter and Via 1995; Ferrari et al. 2001. 2004). The three reasonably well characterised endosymbionts of the pea aphid described earlier, H. defensa, S. symbiotica and R. insecticola, have been shown to have very different effects on the ability of the aphid to resist parasitism by A. ervi. Until recently, the evidence suggested that H. defensa and to a lesser extent S. symbiotica were the symbionts which were predominantly responsible for enhancing resistance to parasitoids, and it is now clear that the first does so because it carries a bacteriophage (A. pisum secondary endosymbiont, APSE) which encodes toxins responsible for the defensive function (Oliver et al. 2003, 2009; see below). However, although it had been shown that R. insecticola was important in


resistance to at least one fungal pathogen (Ferrari et al. 2004; Scarborough et al. 2005), there had been little evidence that it had any role in conferring resistance to parasitoids (but see Ferrari et al. 2004). Recently, however, it has been demonstrated that R. insecticola confers very high levels of resistance to parasitoids in a single Australian clone of the peach potato aphid M. persicae (Vorburger et al. 2010a). The role of R. insecticola in resistance was demonstrated by curing the original host strain using antibiotics, resulting in the loss of the resistance function. In addition, the symbiont strain was transferred using micro-injection to three uninfected aphid lines, including one of a different species, A. fabae, and in all cases, resistance was conferred. Evidence for resistance is so far confined to this particular strain of R. insecticola, and there is no indication of any role played by APSE, but together with studies quoted elsewhere in this review, this research indicates a set of rather generalist ecological strategies being employed by these bacteria, with a lack of host specificity which should lead to greater frequencies, if not fixation, in natural populations. That this is not the case represents an important research direction for the future. Oliver et al. (2003) investigated whether secondary symbiont complement determines variation in aphid resistance to parasitism by microinjection of haemolymph containing H. defensa, S. symbiotica or R. insecticola from infected to uninfected pea aphid clones. In a controlled genetic background, secondary symbiont infection promoted resistance to the parasitoid by causing elevated mortality amongst the developing A. ervi larvae. When exposed to A. ervi, aphids harbouring H. defensa or S. symbiotica were less likely to support wasp larval development than aphids lacking either symbiont. In particular, aphids bearing H. defensa were approximately twice as likely to survive parasitism as secondary symbiont-free control aphids. Ferrari et al. (2004) also demonstrated that possession of H. defensa was associated with resistance to A. ervi and its congeneric Aphidius eadyi in 41 clones harbouring unmanipulated secondary symbionts, although the presence of S. symbiotica did not relate to resistance to either parasitoid species. Interestingly, Oliver et al. (2003) did not find any discrimination on the part of the parasitoid against hosts containing the resistance-conferring symbionts, which is surprising given the high cost to parasitoid fitness. One possible explanation for this finding is that it is necessary for a more prolonged association to exist between an aphid clone, one or more secondary symbionts, and a parasitoid before the kind of co-evolutionary relationship which might allow such discrimination becomes established. In the study by Oliver et al. (2003), the parasitoids were maintained throughout on a symbiont-free aphid line. Even in the presence of H. defensa, variation between aphid clones in their susceptibility to parasitoid attack is

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evident (Ferrari et al. 2001). In a test of the relative importance of the aphid genetic background as opposed to the symbiont infection, Oliver et al. (2005) introduced different H. defensa isolates by microinjection into a common aphid genetic background and monitored resistance to A. ervi. In contrast, a single symbiont isolate was introduced into five separate aphid lineages, each with a different genetic background. The results showed that multiple H. defensa isolates could confer resistance to A. ervi irrespective of genetic background of the A. pisum clone into which they were injected, suggesting that the symbiont determined the level of resistance and not the aphid genotype. Reflecting on the results obtained in the earlier study by Oliver et al. (2003), this would suggest that for the possibility of parasitoid discrimination against symbiont infection to occur, a co-evolutionary relationship between particular parasitoid and symbiont lineages is sufficient, irrespective of aphid genetic background, which should increase the frequency of the resistance phenotype in natural populations. Vorburger et al. (2009), in a more recent study of the effects of genetic variation in the black bean aphid A. fabae and its parasitoid Lysiphlebus fabarum, showed that, in general, aphid clones which were infected with H. defensa enjoyed a greatly reduced susceptibility to parasitism, as in other cases, but no clone-specific genotype specificity with the parasitoid. However, clones infected with the bacterium showed strong genotype specific associations with the parasitoid, suggesting strongly that the fundamental ecological and evolutionary association exists between the symbiont and the parasitoid and not between the host and the parasitoid, supporting the conclusions drawn by Oliver et al. (2005), and also suggesting that any aphid species infected with H. defensa is likely to enjoy enhanced resistance to parasitoids. Cloutier and Douglas (2003) proposed a model of host regulation in which wasp venom and teratocytes (giant cells derived from the embryonic serosa of the parasitoid) function in tandem to redirect nutritional resources from the aphid embryos to the developing parasitoid larva with the implication that bacterial symbiosis may be promoted in parasitised aphids. The model was supported by experimental evidence which showed that the biomass of primary mycetocytes and number of secondary mycetocytes increased in aphids bearing young parasitoid larvae, but the biomass of aphid embryos was lower than in unparasitised aphids. They suggested that parasitoid infection was likely to promote proliferation of B. aphidicola cells and inhibit death of mycetocytes (Cloutier and Douglas 2003). Miao et al. (2004) examined the effect of eliminating B. aphidicola, using the antibiotic rifampicin, on parasitoid development and reproduction and found that, in the absence of B. aphidicola, the larval size, growth rate, rate of emergence and number of progeny were significantly reduced.

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Notwithstanding the conclusions reached by Oliver et al. (2005), the interaction between host genotype and symbiont complement could be affected by other environmental variables which could influence aphid resistance to parasitoids. For example, Bensadia et al. (2006) examined the susceptibility to parasitism of three pea aphid clones containing H. defensa under heat stress and found that resistant clones became highly susceptible at temperatures above 25°C, indicating that environmental variables are important in the aphid–endosymbiont–parasitoid interaction. A similar result found in Wolbachia, which are killed by exposure to high temperatures, suggests there may be an analogous effect on the secondary endosymbionts of aphids and that the change in patterns of host resistance is due to temperature-induced bacterial mortality (Werren 1997). Bensadia et al. (2006) suggested that the heightened immune response, attributed to the bacteria, broke down at high temperatures accounting for the observed decrease in resistance. More recently, in an intriguing study of the effects of both temperature and UV-B radiation on symbiont-mediated resistance to parasitoids, Guay et al. (2009) were able to show that not only did increasing temperature reduce the resistance conferred by H. defensa, as before, but that superinfected clones, which harboured not only H. defensa but also a newly identified facultative symbiont of pea aphid, designated pea aphid X-type symbiont, retained high levels of resistance to parasitism under heat stress and even when not heat stressed showed much higher resistance levels than those conferred by H. defensa alone, suggesting a strong synergy between the two symbionts.

life cycle. This association was explored further by Degnan and Moran (2008b) who carried out a multi-locus analysis of five phage and ten bacterial loci for strains from various aphid species and found that, whilst the H. defensa chromosome was essentially clonal, the phage chromosomes exhibited significant recombination. They suggested that genetic recombination within the phage, together with sexual transmission of both phage and symbiont, would facilitate the proliferation of resistance conferring genes amongst symbiont lineages, and they speculated that it is likely that many strains of H. defensa will have lost the bacteriophage over time, which they showed brings about diminished protection against parasitoids, and suggests that genes encoded by the phage are critical for the expression of the defensive phenotype. This conclusion was reinforced by Oliver et al. (2009) who showed that another toxinencoding bacteriophage (APSE-3) is required to induce the parasitoid resistant phenotype. Using A. pisum parasitised by A. ervi, they demonstrated that, in a controlled host genetic background, the presence of the phage is required to produce the resistant phenotype, which confers protection which is up to 10-fold higher than aphid lines infected with H. defensa which do not carry the phage (Fig. 3). The ecological significance of these results is considerable because they suggest that any trait encoded by a bacteriophage which is of benefit to the host may lead to the rapid spread of an associated bacterial symbiont into host populations, and the fluidity of the relationships between the phage, the symbiont and its host will increase the rapidity with which the aphid host and its symbionts can respond to selective pressures imposed by parasitic wasps.

The APSE bacteriophage association

Ecology of secondary symbionts in natural populations

Just over 10 years ago, van der Wilk et al. (1999) described an endosymbiont-associated bacteriophage, which they named bacteriophage 1, A. pisum secondary endosymbiont (APSE-1) and which they assigned to the family Podoviridae based on morphology and similarities in genome organisation, but to which they did not ascribe any function. It was not for a further 5 years that Moran et al. (2005d) were able to obtain sequence data for the secondary symbiont H. defensa and an associated bacteriophage APSE-2, similar to APSE-1, but which carried a gene which encoded cytolethal distending toxin (cdtB), which has been recorded from several mammalian pathogens and which acts by breaking the eukaryotic cell cycle. Moran et al. (2005d) proposed that the toxin encoding genes carried by the phage are responsible for the previously observed capacity of the endosymbiont to confer protection to the aphid host against parasitic wasps and that the phage itself is an obligate component of the H. defensa

There is almost no information regarding the population dynamics and community ecology of symbionts in natural populations. Hansen et al. (2007) conducted a Californiawide survey of the eucalyptus psyllid Glycaspis brimblecombei to describe the secondary symbiont distribution from 380 individuals taken from 19 separate populations. A single symbiont was present across these populations at an average infection rate of 40%. Between populations, the infection ranged from 0% to 75%, and most interestingly, the rate of infection within a population was strongly positively related to the rate of parasitism by the solitary endoparasitic wasp Psyllaphaegus bliteus. This could either be interpreted as the symbiont conferring resistance to parasitism, or susceptibility, and these alternatives cannot be distinguished from the data in Hansen et al. (2007). Oliver et al. (2008) addressed the question of why H. defensa does not evolve to fixation in natural populations and remains at intermediate frequencies. They studied the


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Fig. 3 Effect of APSE presence on the percentage of A. pisum successfully parasitised by the parasitoid wasp A. ervi. a Aphid line 5A is uninfected with H. defensa, line A1A+ → 5A is the same A. pisum clone infected with H. defensa carrying bacteriophage APSE-3 and line A1A− → 5A is the same A. pisum clone infected with the

same strain of H. defensa but lacking phage APSE-3. b Effect of APSE on percentage of different A. pisum clonal lineages successfully parasitised by A. ervi. Numbers above columns refer to the total number of aphids counted as alive or parasitised. ***P