BACKGROUND
one of the major reasons that insects are the most diverse and abundant animals on earth is due to their coevolution with plants and the myriad strategies they have evolved to successfully feed on them  <cit> .
only recently have we come to appreciate that microbial endosymbionts of phytophagous insects have played a important role in this success  <cit> , for example by providing essential metabolites and vitamins  <cit> , breaking down cell wall components, such as lignocellulose  <cit> , recycling nitrogenous waste  <cit>  and detoxifying plant secondary metabolites  <cit> .
maternally transmitted intracellular symbionts are extremely common in herbivorous insects  <cit> .
obligate nutritional symbionts are usually found within specialized host-derived organs called bacteriomes and they often exhibit co-speciation with their host lineages, indicative of an ancient association stabilized by strict vertical transmission from mother to offspring  <cit> .
in addition, many insects harbour facultative heritable endosymbionts that are not necessary for the development and reproduction of the host  <cit> .
these symbionts have evolved diverse strategies to persist in their hosts, including manipulating reproduction, for example by inducing parthenogenesis  <cit> .
other facultative symbionts increase host fitness under certain conditions, and it is in this regard that they are potentially important in mediating plant-insect interactions  <cit> .
for example, facultative inherited symbionts of pea aphids have been implicated in facilitating the colonization of novel host plants  <cit> .
gut microbes also play critical roles in plant-insect interactions.
some herbivorous insects are associated with essential communities of microbes found within gut chambers   <cit>  or crypts   <cit> .
several posthatch transmission mechanisms have evolved to ensure transmission of gut associates from generation to generation, such as egg-smearing  <cit> , coprophagy  <cit>  and capsule-mediated transmission  <cit> .
in addition, some true bugs acquire their gut microbes de novo every generation from the environment  <cit> .
gut bacteria can affect a herbivore’s host range.
for example, when the symbiont capsule from a stinkbug pest of soybean, megacopta punctatissima, is exchanged with a non-pest species, m. cribraria, there is an increase in fitness of this species on soybean and a decrease in fitness of the pest species on soybean  <cit> .
this implies that the obligate symbiont dictates the pest status of the host.
since some of the major lineages of gut symbionts have only recently been discovered and characterized, we are still in early days in our understanding of how associated microbial communities are able to shape plant-insect interactions  <cit> .
there are many examples of nutritional symbiosis among phytophagous hymenopterans.
xylophagous woodwasps and horntails rely on a symbiotic fungus for cellulose-digestion and/or nutrition during larval stages  <cit>  and woodwasps have also been found to be associated with cellulose degrading bacteria  <cit> .
leaf-cutter ants have also formed a symbiotic relationship with fungi, in which the ants cultivate and consume a mutualistic fungus on a substrate of foraged leaf fragments  <cit> .
the honeybee, apis mellifera, is known to be associated with a distinct microbiota  <cit> , that is thought to be important for both bee health and nutrition  <cit> , including pollen coat digestion.
arboreal herbivorous ants that subsist mainly on a nutrient-poor diet of sugary plant exudates and hemipteran honeydew secretions harbour gut symbionts, which aid in nutrition.
these symbiotic gut microbes include bacteria that are related to nitrogen-fixing root-nodule bacteria  <cit> .
carpenter ants in the genus camponotus have an obligate endosymbiont, the gammaproteobacterium blochmannia, which is found in host-derived bacteriomes  <cit> .
sequencing of the blochmannia genome suggests that this symbiont provides its host with essential amino acids  <cit> .
there is also evidence that blochmannia plays a role in nitrogen recycling by encoding urease  <cit> .
many insects have independently evolved the ability to feed from within plant issues, for example, as seed-feeders, gallers, or leaf-miners.
this feeding style permits the larval stage access to internal plant tissues with relatively high nutrient content and low defence response, and often involves complex physiological and morphological modifications of host plant tissue, including differentiation of additional tissues , in situ up-regulation and synthesis of proteins and sugars, translocation of nutrients to the insect feeding site and the formation of green islands   <cit> .
however, the mechanisms controlling these complex modifications are not well understood; it remains an open question whether symbiotic microbes might have a role in these systems.
an interesting study recently implicated bacterial symbionts in insect endophytophagy.
feeding by leaf-mining phyllonorycter blancardella caterpillars prevents leaf senescence, resulting in characteristic islands of green tissue.
these green islands are associated with increased levels of plant hormones  <cit> , including cytokinins similar to those used by bacteria to manipulate plant physiology  <cit> .
when leaf-miners were treated with antibiotics, the green-island phenotype failed to appear, suggesting that bacterial symbionts of p. blancardella might be involved in manipulation of the plant  <cit> .
seed chalcid wasps of the genus megastigmus  provide an interesting system to explore the role of microbes in nutrition and host manipulation of endophytophagous insects.
the genus megastigmus contains  <dig> described species, of which more than  <dig> are tree and shrub seed feeders; the remaining species are thought to be mainly parasitoids of gall insects  <cit> .
seed infesting species of megastigmus undergo their development within the seeds of plants, obtaining nourishment from the developing embryo and storage reserves within the megagametophyte  <cit> .
the best-studied species, m. spermotrophus, is a major pest of douglas-fir .
this insect has the ability to manipulate the seed development of douglas-fir for its own reproductive success  <cit> .
first, m. spermotrophus can re-direct unfertilized ovules that normally abort to continue developing.
ovules do not redirect resources back to the mother plant, but instead feed the insect  <cit> .
second, the developing larva acts like a ‘surrogate’ embryo, causing the continued accumulation of storage reserves in the megagametophyte, which provides nourishment for the larva  <cit> .
the re-direction of unfertilized ovule development by the presence of the parasite can be partially explained by changes in seed hormone levels, especially cytokinins  <cit> .
it is suspected that all megastigmus species infesting pinaceae hosts can manipulate seed development  <cit> .
do megastigmus wasps contain bacterial associates, and if so could they play an important role in the endophytophagous lifestyle of the host?
in this paper, we used two approaches to characterize the microbial symbionts of megastigmus, with the long-term goal of understanding their role in host nutrition and manipulation.
using symbiont-specific primers we screened a large sample of sexual megastigmus species and two parasitoids of m. spermotrophus for common insect facultative heritable endosymbionts  <cit> .
we also used 16s rrna bacterial amplicon pyrosequencing to perform an unbiased and in-depth survey of the microbes associated with different developmental stages of m. spermotrophus , douglas-fir ovules and the parasitoid eurytoma sp.
there have not been any studies on the microbial associates of megastigmus except for a recent study that showed that thelytokous parthenogenesis in megastigmus is caused by the reproductive parasite wolbachia  <cit> .
RESULTS
common heritable endosymbiont infections in megastigmus
three species tested positive in our inherited symbiont screens, with infection frequencies ranging from 33–100% .
megastigmus milleri harbours a strain of rickettsia from the bellii clade  .
megastigmus amicorum and m. bipunctatus harbour a strain of rickettsia that is allied with r. felis, i.e.
in the ‘transitional’ group  <cit> .
rickettsia citrate synthase sequences from these two hosts were identical .
these two hosts also harboured supergroup a wolbachia infections  .
m. amicorum collected from different host plants and locations  were 2% divergent in mitochondrial coi  and infected with different wolbachia strains.
there was no significant difference in the frequency of infection in males and females, nor did we find an association between wolbachia and rickettsia in coinfected species .
arsenophonus, cardinium, and spiroplasma were not detected in megastigmus samples screened using pcr with symbiont-specific primers.table  <dig>
megastigmus
spp.
and parasitoids screened for common heritable symbionts using pcr
species
host plant
year
location
n
sample type
wolbachia
positive
rickettsia
positive
m. schimitscheki
cedrus atlantica
m. schimitscheki
cedrus atlantica
m. schimitscheki
cedrus atlantica
m. schimitscheki
cedrus atlantica
m. schimitscheki
cedrus atlantica
m. schimitscheki
cedrus libani
m. rafni
abies alba
m. rafni
abies alba
m. rafni
abies alba
m. rafni
abies alba
m. rafni
abies nordmanniana
m. rafni
abies grandis
m. rafni
abies grandis
m. milleri
abies grandis
m. milleri
abies grandis
m. spermotrophus
pseudotsuga menziesii
m. spermotrophus
pseudotsuga menziesii
m. watchli
cupressus sempervirens
m. watchli
cupressus sempervirens
m. watchli
cupressus sempervirens
m. watchli
cupressus sempervirens
m. bipuncatatus
juniperus sabina
m. bipuncatatus
juniperus sabina
m. bipuncatatus
juniperus sabina
m. amicorum
juniperus phoenicea
m. amicorum
juniperus phoenicea
m. amicorum
juniperus phoenicea
m. amicorum
juniperus oxycedrus
m. amicorum
juniperus oxycedrus
m. amicorum
juniperus oxycedrus
parasitoids of m. spermotrophus
eurytoma sp.
mesopolobus sp.
these species did not host arsenophonus, cardinium, or spiroplasma.
spiroplasma was identified from eurytoma sp.
using 16s rrna pyrosequencing.
maximum likelihood phylogeny for
rickettsia
citrate synthase sequence constructed using the tamura 3-parameter plus gamma distributed rates among sites model of nucleotide substitution.
the sequences generated by this study are highlighted in red.
numbers next to the nodes indicate percentage of bootstrap support from  <dig> bootstrap replicates.
nodes without numbers received less than 65% bootstrap support.
concatenated maximum likelihood phylogeny for
wolbachia
coxa, ftsz and gatb sequence constructed using the tamura 3-parameter plus gamma distributed rates among sites model of nucleotide substitution.
sequences generated by this study are red and sequences previously obtained from parthenogenetic megastigmus are green  <cit> .
numbers next to the nodes indicate percentage of bootstrap support from  <dig> bootstrap replicates.
nodes without numbers received less than 65% bootstrap support.
microbial associates of m. spermotrophus
16s rrna bacterial amplicon pyrosequencing of m. spermotrophus , adult eurytoma sp.
and p. menziesii ovules generated  <dig>  raw reads with an average length of 422 bp  .
quality and chimera filtering removed approximately 27% of the reads.
the assignment of operational taxonomic units  resulted in  <dig> unique bacterial clusters after the removal of singletons.
a total of  <dig> otus were assigned to the genus level.
the average sequencing depth was  <dig>  sequences per sample .
rarefaction analysis showed that for most of the m. spermotrophus samples the number of observed otus no longer exponentially increased after an approximate sampling depth of  <dig>  sequences  and the average number of observed species was 60 ±  <dig> and the average chao <dig> species diversity estimate was 71 ±  <dig>
fifteen major otus form the core bacterial microbiome of m. spermotrophus, i.e.
having a total relative abundance of  <dig> % or greater .
these otus are from five bacterial classes: betaproteobacteria, gammaproteobacteria, actinobacteria, firmicutes and alphaproteobacteria.
over 60% of the sequences from the m. spermotrophus samples were assigned to the genus ralstonia spp.
.
other major otus were assigned to the genera acinetobacter and corynebacterium representing  <dig> % and  <dig> % of total relative abundance, respectively.
further investigation using blast searches against the ribosomal database project  and genbank’s 16s ribosomal rna sequence database revealed that all but one of the major otus not assigned to the genus level were actually acinetobacter, corynebacterium, or ralstonia.
the unknown firmicutes is most closely related to turicibacter, a strictly anaerobic gram-positive bacteria in the family erysipelotrichaceae  <cit> ; this otu represents  <dig> % of the total relative abundance of the 16s rrna sequences in the m. spermotrophus samples.table  <dig>
major bacterial otus associated with
m. spermotrophus
based on 16s rrna amplicons from pyrosequencing
phylum
class
order
family
genus
percent total relative abundance
ralstonia
acinetobacter
corynebacterium
ralstonia
ralstonia
corynebacterium
acinetobacter
anaerococcus
anaerococcus
the relative abundance of the major otus from the different developmental stages of m. spermotrophus was mostly conserved , and there was no difference in the core microbiomes of the different developmental stages, based on principle coordinate analysis of weighted or unweighted unifrac phylogenetic distances .
the total relative abundance of otus from the class betaproteobacteria  ranged from  <dig>  -  <dig> %.
one female sample contained only a very small proportion of otus assigned to the class gammaproteobacteria  while the total relative abundance of gammaproteobacteria ranged from  <dig>  -  <dig> % in the remaining samples.
the total relative abundance of all otus within the class actinobacteria  ranged from  <dig>  -  <dig> %.figure  <dig>
relative abundance of major bacterial otus associated with larvae, pupae and adult
m
.
spermotrophus
based on  <dig> rrna sequence from pyrosequence.
unknown classes are coloured grey.
a maximum likelihood phylogeny for ralstonia was created using 16s rrna sequence from the most abundant ralstonia otu in the pyrosequencing data set .
strong bootstrap support  clusters the ralstonia isolated from m. spermotrophus with the human pathogen r. pickettii .figure  <dig>
maximum likelihood phylogeny for
ralstonia
16s rrna sequence constructed using the tamura-nei with invariant sites and gamma distributed rate among sites model of nucleotide substitution.
numbers next to the nodes indicate percentage of bootstrap support from  <dig> bootstrap replicates.
nodes without numbers received less than 65% bootstrap support.
ovule samples were dominated by chloroplast rrna ; the remaining otus included ralstonia  and acinetobacter .
the eurytoma parasitoid samples were dominated by one otu, which is allied with inherited spiroplasma in the ixodetis group  , .
the remaining otus were ralstonia.
discussion
common heritable endosymbiont infections in megastigmus
we found three sexual megastigmus species infected with rickettsia, and two of these same species infected with wolbachia.
none of the species was infected with arsenophonus, spiroplasma, or cardinium.
from this patchy distribution , we can likely conclude that none of these inherited symbionts are essential in host nutrition and/or manipulation.
it is not surprising that wolbachia was detected, as it is the most common intracellular bacterial symbiont of insects  <cit> .
wolbachia are transmitted maternally, in the egg cytoplasm, and many strains have evolved strategies to increase the frequency of infected female hosts in the population.
reproductive manipulating strains of wolbachia have been show to either cause cytoplasmic incompatibility or distort sex ratios by killing males or inducing parthenogenetic reproduction  or feminization  <cit> .
parthenogenesis-inducing wolbachia are common in hymenoptera and have been characterized in several parasitoid  <cit>  and cynipid gall wasps  <cit> .
a recent study implicated wolbachia in parthenogenetic reproduction in megastigmus, with 10/ <dig> asexual species infected  <cit> .
treating m. pinsapinis with the antibiotic tetracycline restored the production of males, strongly suggesting that wolbachia is the causative agent of thelytoky in asexual megastigmus.
no sexual megastigmus species were infected with wolbachia in the boivin et al.
study  <cit> ; however, we found infections in m. amicorum and m. bipunctatus.
the wolbachia strains that we identified from sexual megastigmus are closely allied with those in asexual megastigmus.
it would be interesting to determine if parthenogenesis-induction in megastigmus is due to the host or the particular wolbachia strain.
rickettsia infections were discovered in three species.
bacteria in the genus rickettsia are well known for being insect-vectored vertebrate pathogens, such as the causal agents of rocky mountain spotted fever  and typhus .
however, recent surveys have uncovered many rickettsia that are vertically transmitted symbionts of diverse arthropods, most of which do not feed on vertebrates  <cit> .
some rickettsia symbionts have been shown to distort host sex ratios via male-killing  <cit>  or parthenogenesis-induction  <cit> .
the presence of rickettsia and wolbachia in males likely rules out sex ratio distortion in our study.
alternatively, facultative symbionts may benefit their hosts under some circumstances.
for example, some wolbachia and rickettsia increase host fitness by providing protection against natural enemies  <cit> .
phylogenetic analysis shows that closely related rickettsia and wolbachia infect distantly related megastigmus .
this provides strong evidence of horizontal transmission over evolutionary timescales, and is a common pattern in facultative inherited symbionts of insects  <cit> .
in most cases, it is not known how inherited symbionts colonize novel hosts; shared hosts and shared natural enemies have both been implicated  <cit> .
interestingly, for some inherited symbionts, horizontal transmission over ecological timescales may be quite common  <cit> .
it would be useful to sequence more rapidly evolving rickettsia genes, to determine if there was very recent transmission between m. amicorum and m. bipunctatus.
since both these species develop in junipers, we could speculate that horizontal transmission occurs via shared host plants; the boivin et al.
study of wolbachia in asexual megastigmus also found evidence for such host-plant-mediated transmission  <cit> .
plant-mediated transmission may be an important and underappreciated way for symbionts to colonize hosts.
indeed, a recent study showed that an inherited rickettsia in the sweet potato whitefly can be transmitted via phloem  <cit> .
two strains of arsenophonus that infect planthoppers are transmitted both transovarially and via plants, and both have been implicated in plant disease  <cit> .
however, as far as we are aware, interspecific transmission via plants has not yet been demonstrated in any inherited symbionts.
microbial associates of m. spermotrophus
our estimate of m. spermotrophus microbial species richness  fell within the range of other studies of insect microbiomes.
pollenivorous and predacious hymenoptera  harbour distinct bacterial communities with the lowest level of species richness , while termites harbour the highest species diversity , based on a recent meta-analysis  <cit> .
a recent study estimated the diversity of bacteria associated with parasitoid wasps from the genus nasonia ranged from  <dig> to  <dig> bacterial otus  <cit> .
pyrosequencing has been show to detect a greater number of otus compared to traditional methods, such as 16s rrna clone sequencing  <cit> .
this might explain why the estimated bacterial diversity associated with m. spermotrophus is comparably high because the nasonia study and many previous insect microbiome surveys were done using 16s rrna clone sequencing.
despite a relatively high overall richness, only fifteen major otus are present with a total relative abundance of  <dig> % or greater.
the core bacterial community of m. spermotrophus can thus be considered to have a somewhat low diversity, characterized by bacterial otus that are commonly found associated with insect guts.
the major otus associated with m. spermotrophus can be grouped into five distinct phylotypes: betaproteobacteria , gammaproteobacteria , actinobacteria , firmicutes  and alphaproteobacteria .
most of these otus are related to bacteria that have been previously reported in insect guts, with acinetobacter and corynebacterium especially common .
all of the major otus identified below the order level are bacteria that commonly occur in the environment, such as in soil  <cit>  and in the rhizospere  <cit> .
similar results are commonly found with microbial associates of insects.
for example, the microbial symbionts of tetraponera ants are closely related to nitrogen-fixing root nodule bacteria  <cit> .
the giant mesquite bug, thasus neocalifornicus acquires an important mutualistic gut symbiont de novo every generation from the soil  <cit> .
the presence of the same major otus in m. spermotrophus in ovule and even eurytoma samples provides clues to the distribution and transmission of the megastigmus microbiome; it suggests that it is derived from the environment, which, for the developing wasp, is the ovule.
acinetobacter and corynebacterium have been previously cultured from within surface-sterilized seeds and ovules  <cit> .
the m. spermotrophus microbiome appears to be highly conserved across development, as demonstrated by the unifrac analysis, with all of the samples tightly grouped.
this contrasts with a recent survey of microbial associates of three nasonia species that found that bacterial species richness increased with development  <cit> .
like most higher hymenoptera, the larvae of m. spermotrophus have a blind digestive system with the midgut and hind gut only uniting during the last larval instar.
prior to pupation all of the built-up wastes are voided in a fecal pellet, termed the meconium  <cit> .
during metamorphosis the larval midgut epithelium is discarded and replaced by a new pupal epithelium  <cit> .
if these bacteria are associated with the gut, how m. spermotrophus maintains its major associates throughout development is not known.
some insects, like true bugs, termites and cockroaches, have crypts or paunches associated with the gut that are thought to enhance persistence of the microbiota  <cit> .
this physiological feature is not well characterized in the hymenoptera, with the exception of some ants  <cit> .
a single otu assigned to the genus ralstonia comprised over 55% of all sequences from the m. spermotrophus samples.
the high abundance and persistence of ralstonia throughout host development is a strong indicator that this bacterium is an important associate of m. spermotrophus.
ralstonia was also found to be associated with douglas-fir ovules and the parasitoid eurytoma.
the genus ralstonia contains species from ecological diverse niches, such as the plant pathogen r. solanacearum, the opportunistic human pathogen r. pickettii and the environmental isolate r. eurytropha  <cit> .
a maximum likelihood phylogeny placed m. spermotrophus associated ralstonia in a cluster with the human pathogen r. pickettii .
to our knowledge, this is the first report of ralstonia being a very abundant and potentially important component of an insect microbiome, although ralstonia spp.
have been previously reported from microbial surveys of insects, including the cotton bollworm , bartonella-positive fleas  <cit> , an omnivorous carabid beetle  <cit>  and the potato psyllid   <cit> .
recently, husnik et al.
also report the horizontal transfer of one ralstonia gene into the genome of the mealybug planococcus citri  <cit> .
also, r. oxalatica was isolated from the alimentary canal of an indian earthworm  <cit> .
a recent meta-analysis of 16s clone-library studies of insect associated microbes found that betaproteobacteria contributed over 50% to all sequences from hymenoptera  <cit> .
the most common bacterial phylotype identified from solitary bee species, was a betaproteobacteria from the genus burkholderia  <cit> , which is closely related to ralstonia.
burkholderia spp.
have also been identified as important mutualists of some phytophagous true bugs , where they reside in gut crypts  <cit> .
the developing m. spermotrophus larva feeds on megagametophyte tissue, which contains all of the seed storage reserves, primarily in the form of starch, triacylglycerols, and nitrogen rich proteins  <cit> .
therefore, ralstonia and other microbial associates of m. spermotrophus would not likely play a role in supplementing this already rich diet with missing essential nutrients but instead may play a role in nutrient recycling.
parasitism by m. spermotrophus results in the formation of a nutrient sink, in which the larva and associated microbes are nourished by storage reserves of the megagametophyte.
the reserves are intended to provide nourishment for the developing seedling or to be re-absorbed by the mother plant in the event of megagametophyte abortion.
in loblolly pine, more than half of the nitrogen in megagametophytes comes from the amino acid arginine  <cit> .
insects use the enzyme arginase to hydrolyze arginine into ornithine and urea  <cit> .
excretion of urea would result in the substantial loss of nitrogen, especially since larvae must undergo extended periods of diapause.
very few insects are known to produce urease, the enzyme required to convert urea into ammonium for subsequent amino acid biosynthesis  <cit> .
we speculate that ralstonia or other microbial associates of m. spermotrophus might play an important role in nitrogen recycling by producing urease or other key enzymes missing from the host genome.
many insect symbionts have been suggested to promote increased availability of nitrogen in a variety of ways  <cit> .
for example, blochmannia and blattabacterium, the obligate nutritional symbionts of carpenter ants and cockroaches, respectively, use ureases to recycle nitrogen from urea  <cit> .
nitrogen recycling by symbionts has also been shown to be important during diapause in the shield bug, parastrachia japonensi  <cit> .
it is also tempting to speculate that ralstonia could potentially play a role in plant manipulation.
another ralstonia species, r. taiwanensi, has been shown to be capable of nodulating and fixing nitrogen in mimosa spp.
<cit> , which implies an ability to manipulate plant physiology.
alternatively, ralstonia may not be a key associate of megastigmus species in general, but rather a microbe that is found in the seed environment that encodes enzymes required for the catabolism of seed storage molecules or other essential pathways required for the seed feeding lifestyle of m. spermotrophus.
now that ralstonia has been identified as a likely symbiont of m. spermotrophus, further targeted surveys using ralstonia-specific pcr primers would be helpful in determining its prevalence in other populations of m. spermotrophus, in other megastigmus species, and in associated plants.
the development of strain-specific markers for fluorescence in situ hybridization would also be useful for localizing ralstonia on or within m. spermotrophus and the ovule, and following its transmission throughout its life cycle.
it would also be interesting to examine ralstonia’s role in nitrogen recycling, for example by identifying and following the expression of ureases and other key enzymes during m. spermotrophus development.
CONCLUSIONS
in this study two different approaches were used to survey megastigmus for microbial symbionts.
the directed pcr screens identified the presence of two common heritable symbionts, wolbachia and rickettsia; these are not likely distorting sex ratios in the sexual megastigmus species surveyed in this study.
pyrosequencing was used to characterize the core microbiome of the douglas-fir seed chalcid, m. spermotrophus, which is dominated by ralstonia, a microbe that has not been previosly characterized as an important microbial associated of an insect.
interestingly, ralstonia was also present in ovule and eurytoma samples, indicating its prevalence within the niche of the ovule and potential horizontal transmission route from host to parasitoid.
this initial characterization of microbial associates of megastigmus did not provide any insight into the potential involvement in host manipulation, although the maintenance of a consistent microbiome from larvae to adult suggests that microbes may be vital to the development and reproduction of m. spermotrophus.
many new questions are inspired by these findings, such as, how is the microbiome of m. spermotrophus maintained and transmitted?
how widespread is the association with ralstonia?
what is the effect of heritable symbionts in sexual megastigmus?

 one of the major reasons that insects are the most diverse and abundant animals on earth is due to their coevolution with plants and the myriad strategies they have evolved to successfully feed on them. only recently have we come to appreciate that microbial endosymbionts of phytophagous insects have played a important role in this success, for example by providing essential metabolites and vitamins, breaking down cell wall components, such as lignocellulose, recycling nitrogenous waste and detoxifying plant secondary metabolites. maternally transmitted intracellular symbionts are extremely common in herbivorous insects. obligate nutritional symbionts are usually found within specialized host-derived organs called bacteriomes and they often exhibit co-speciation with their host lineages, indicative of an ancient association stabilized by strict vertical transmission from mother to offspring. in addition, many insects harbour facultative heritable endosymbionts that are not necessary for the development and reproduction of the host. these symbionts have evolved diverse strategies to persist in their hosts, including manipulating reproduction, for example by inducing parthenogenesis. other facultative symbionts increase host fitness under certain conditions, and it is in this regard that they are potentially important in mediating plant-insect interactions. for example, facultative inherited symbionts of pea aphids have been implicated in facilitating the colonization of novel host plants. gut microbes also play critical roles in plant-insect interactions. some herbivorous insects are associated with essential communities of microbes found within gut chambers or crypts. several posthatch transmission mechanisms have evolved to ensure transmission of gut associates from generation to generation, such as egg-smearing, coprophagy and capsule-mediated transmission. in addition, some true bugs acquire their gut microbes de novo every generation from the environment. gut bacteria can affect a herbivore’s host range. for example, when the symbiont capsule from a stinkbug pest of soybean, megacopta punctatissima, is exchanged with a non-pest species, m. cribraria, there is an increase in fitness of this species on soybean and a decrease in fitness of the pest species on soybean. this implies that the obligate symbiont dictates the pest status of the host. since some of the major lineages of gut symbionts have only recently been discovered and characterized, we are still in early days in our understanding of how associated microbial communities are able to shape plant-insect interactions. there are many examples of nutritional symbiosis among phytophagous hymenopterans. xylophagous woodwasps and horntails rely on a symbiotic fungus for cellulose-digestion and/or nutrition during larval stages and woodwasps have also been found to be associated with cellulose degrading bacteria. leaf-cutter ants have also formed a symbiotic relationship with fungi, in which the ants cultivate and consume a mutualistic fungus on a substrate of foraged leaf fragments. the honeybee, apis mellifera, is known to be associated with a distinct microbiota, that is thought to be important for both bee health and nutrition, including pollen coat digestion. arboreal herbivorous ants that subsist mainly on a nutrient-poor diet of sugary plant exudates and hemipteran honeydew secretions harbour gut symbionts, which aid in nutrition. these symbiotic gut microbes include bacteria that are related to nitrogen-fixing root-nodule bacteria. carpenter ants in the genus camponotus have an obligate endosymbiont, the gammaproteobacterium blochmannia, which is found in host-derived bacteriomes. sequencing of the blochmannia genome suggests that this symbiont provides its host with essential amino acids. there is also evidence that blochmannia plays a role in nitrogen recycling by encoding urease. many insects have independently evolved the ability to feed from within plant issues, for example, as seed-feeders, gallers, or leaf-miners. this feeding style permits the larval stage access to internal plant tissues with relatively high nutrient content and low defence response, and often involves complex physiological and morphological modifications of host plant tissue, including differentiation of additional tissues, in situ up-regulation and synthesis of proteins and sugars, translocation of nutrients to the insect feeding site and the formation of green islands. however, the mechanisms controlling these complex modifications are not well understood; it remains an open question whether symbiotic microbes might have a role in these systems. an interesting study recently implicated bacterial symbionts in insect endophytophagy. feeding by leaf-mining phyllonorycter blancardella caterpillars prevents leaf senescence, resulting in characteristic islands of green tissue. these green islands are associated with increased levels of plant hormones, including cytokinins similar to those used by bacteria to manipulate plant physiology. when leaf-miners were treated with antibiotics, the green-island phenotype failed to appear, suggesting that bacterial symbionts of p. blancardella might be involved in manipulation of the plant. seed chalcid wasps of the genus megastigmus provide an interesting system to explore the role of microbes in nutrition and host manipulation of endophytophagous insects. the genus megastigmus contains described species, of which more than are tree and shrub seed feeders; the remaining species are thought to be mainly parasitoids of gall insects. seed infesting species of megastigmus undergo their development within the seeds of plants, obtaining nourishment from the developing embryo and storage reserves within the megagametophyte. the best-studied species, m. spermotrophus, is a major pest of douglas-fir. this insect has the ability to manipulate the seed development of douglas-fir for its own reproductive success. first, m. spermotrophus can re-direct unfertilized ovules that normally abort to continue developing. ovules do not redirect resources back to the mother plant, but instead feed the insect. second, the developing larva acts like a ‘surrogate’ embryo, causing the continued accumulation of storage reserves in the megagametophyte, which provides nourishment for the larva. the re-direction of unfertilized ovule development by the presence of the parasite can be partially explained by changes in seed hormone levels, especially cytokinins. it is suspected that all megastigmus species infesting pinaceae hosts can manipulate seed development. do megastigmus wasps contain bacterial associates, and if so could they play an important role in the endophytophagous lifestyle of the host? in this paper, we used two approaches to characterize the microbial symbionts of megastigmus, with the long-term goal of understanding their role in host nutrition and manipulation. using symbiont-specific primers we screened a large sample of sexual megastigmus species and two parasitoids of m. spermotrophus for common insect facultative heritable endosymbionts. we also used 16s rrna bacterial amplicon pyrosequencing to perform an unbiased and in-depth survey of the microbes associated with different developmental stages of m. spermotrophus, douglas-fir ovules and the parasitoid eurytoma sp. there have not been any studies on the microbial associates of megastigmus except for a recent study that showed that thelytokous parthenogenesis in megastigmus is caused by the reproductive parasite wolbachia.  common heritable endosymbiont infections in megastigmus three species tested positive in our inherited symbiont screens, with infection frequencies ranging from 33–100%. megastigmus milleri harbours a strain of rickettsia from the bellii clade. megastigmus amicorum and m. bipunctatus harbour a strain of rickettsia that is allied with r. felis, i.e. in the ‘transitional’ group. rickettsia citrate synthase sequences from these two hosts were identical. these two hosts also harboured supergroup a wolbachia infections. m. amicorum collected from different host plants and locations were 2% divergent in mitochondrial coi and infected with different wolbachia strains. there was no significant difference in the frequency of infection in males and females, nor did we find an association between wolbachia and rickettsia in coinfected species. arsenophonus, cardinium, and spiroplasma were not detected in megastigmus samples screened using pcr with symbiont-specific primers.table megastigmus spp. and parasitoids screened for common heritable symbionts using pcr species host plant year location n sample type wolbachia positive rickettsia positive m. schimitscheki cedrus atlantica m. schimitscheki cedrus atlantica m. schimitscheki cedrus atlantica m. schimitscheki cedrus atlantica m. schimitscheki cedrus atlantica m. schimitscheki cedrus libani m. rafni abies alba m. rafni abies alba m. rafni abies alba m. rafni abies alba m. rafni abies nordmanniana m. rafni abies grandis m. rafni abies grandis m. milleri abies grandis m. milleri abies grandis m. spermotrophus pseudotsuga menziesii m. spermotrophus pseudotsuga menziesii m. watchli cupressus sempervirens m. watchli cupressus sempervirens m. watchli cupressus sempervirens m. watchli cupressus sempervirens m. bipuncatatus juniperus sabina m. bipuncatatus juniperus sabina m. bipuncatatus juniperus sabina m. amicorum juniperus phoenicea m. amicorum juniperus phoenicea m. amicorum juniperus phoenicea m. amicorum juniperus oxycedrus m. amicorum juniperus oxycedrus m. amicorum juniperus oxycedrus parasitoids of m. spermotrophus eurytoma sp. mesopolobus sp. these species did not host arsenophonus, cardinium, or spiroplasma. spiroplasma was identified from eurytoma sp. using 16s rrna pyrosequencing. maximum likelihood phylogeny for rickettsia citrate synthase sequence constructed using the tamura 3-parameter plus gamma distributed rates among sites model of nucleotide substitution. the sequences generated by this study are highlighted in red. numbers next to the nodes indicate percentage of bootstrap support from bootstrap replicates. nodes without numbers received less than 65% bootstrap support. concatenated maximum likelihood phylogeny for wolbachia coxa, ftsz and gatb sequence constructed using the tamura 3-parameter plus gamma distributed rates among sites model of nucleotide substitution. sequences generated by this study are red and sequences previously obtained from parthenogenetic megastigmus are green. numbers next to the nodes indicate percentage of bootstrap support from bootstrap replicates. nodes without numbers received less than 65% bootstrap support. microbial associates of m. spermotrophus 16s rrna bacterial amplicon pyrosequencing of m. spermotrophus, adult eurytoma sp. and p. menziesii ovules generated raw reads with an average length of 422 bp. quality and chimera filtering removed approximately 27% of the reads. the assignment of operational taxonomic units resulted in unique bacterial clusters after the removal of singletons. a total of otus were assigned to the genus level. the average sequencing depth was sequences per sample. rarefaction analysis showed that for most of the m. spermotrophus samples the number of observed otus no longer exponentially increased after an approximate sampling depth of sequences and the average number of observed species was 60 ±  and the average chao species diversity estimate was 71 ±  fifteen major otus form the core bacterial microbiome of m. spermotrophus, i.e. having a total relative abundance of % or greater. these otus are from five bacterial classes: betaproteobacteria, gammaproteobacteria, actinobacteria, firmicutes and alphaproteobacteria. over 60% of the sequences from the m. spermotrophus samples were assigned to the genus ralstonia spp.. other major otus were assigned to the genera acinetobacter and corynebacterium representing % and % of total relative abundance, respectively. further investigation using blast searches against the ribosomal database project and genbank’s 16s ribosomal rna sequence database revealed that all but one of the major otus not assigned to the genus level were actually acinetobacter, corynebacterium, or ralstonia. the unknown firmicutes is most closely related to turicibacter, a strictly anaerobic gram-positive bacteria in the family erysipelotrichaceae; this otu represents % of the total relative abundance of the 16s rrna sequences in the m. spermotrophus samples.table major bacterial otus associated with m. spermotrophus based on 16s rrna amplicons from pyrosequencing phylum class order family genus percent total relative abundance ralstonia acinetobacter corynebacterium ralstonia ralstonia corynebacterium acinetobacter anaerococcus anaerococcus the relative abundance of the major otus from the different developmental stages of m. spermotrophus was mostly conserved, and there was no difference in the core microbiomes of the different developmental stages, based on principle coordinate analysis of weighted or unweighted unifrac phylogenetic distances. the total relative abundance of otus from the class betaproteobacteria ranged from - %. one female sample contained only a very small proportion of otus assigned to the class gammaproteobacteria while the total relative abundance of gammaproteobacteria ranged from - % in the remaining samples. the total relative abundance of all otus within the class actinobacteria ranged from - %.figure relative abundance of major bacterial otus associated with larvae, pupae and adult m. spermotrophus based on rrna sequence from pyrosequence. unknown classes are coloured grey. a maximum likelihood phylogeny for ralstonia was created using 16s rrna sequence from the most abundant ralstonia otu in the pyrosequencing data set. strong bootstrap support clusters the ralstonia isolated from m. spermotrophus with the human pathogen r. pickettii.figure maximum likelihood phylogeny for ralstonia 16s rrna sequence constructed using the tamura-nei with invariant sites and gamma distributed rate among sites model of nucleotide substitution. numbers next to the nodes indicate percentage of bootstrap support from bootstrap replicates. nodes without numbers received less than 65% bootstrap support. ovule samples were dominated by chloroplast rrna; the remaining otus included ralstonia and acinetobacter. the eurytoma parasitoid samples were dominated by one otu, which is allied with inherited spiroplasma in the ixodetis group,. the remaining otus were ralstonia. discussion common heritable endosymbiont infections in megastigmus we found three sexual megastigmus species infected with rickettsia, and two of these same species infected with wolbachia. none of the species was infected with arsenophonus, spiroplasma, or cardinium. from this patchy distribution, we can likely conclude that none of these inherited symbionts are essential in host nutrition and/or manipulation. it is not surprising that wolbachia was detected, as it is the most common intracellular bacterial symbiont of insects. wolbachia are transmitted maternally, in the egg cytoplasm, and many strains have evolved strategies to increase the frequency of infected female hosts in the population. reproductive manipulating strains of wolbachia have been show to either cause cytoplasmic incompatibility or distort sex ratios by killing males or inducing parthenogenetic reproduction or feminization. parthenogenesis-inducing wolbachia are common in hymenoptera and have been characterized in several parasitoid and cynipid gall wasps. a recent study implicated wolbachia in parthenogenetic reproduction in megastigmus, with 10/ asexual species infected. treating m. pinsapinis with the antibiotic tetracycline restored the production of males, strongly suggesting that wolbachia is the causative agent of thelytoky in asexual megastigmus. no sexual megastigmus species were infected with wolbachia in the boivin et al. study; however, we found infections in m. amicorum and m. bipunctatus. the wolbachia strains that we identified from sexual megastigmus are closely allied with those in asexual megastigmus. it would be interesting to determine if parthenogenesis-induction in megastigmus is due to the host or the particular wolbachia strain. rickettsia infections were discovered in three species. bacteria in the genus rickettsia are well known for being insect-vectored vertebrate pathogens, such as the causal agents of rocky mountain spotted fever and typhus. however, recent surveys have uncovered many rickettsia that are vertically transmitted symbionts of diverse arthropods, most of which do not feed on vertebrates. some rickettsia symbionts have been shown to distort host sex ratios via male-killing or parthenogenesis-induction. the presence of rickettsia and wolbachia in males likely rules out sex ratio distortion in our study. alternatively, facultative symbionts may benefit their hosts under some circumstances. for example, some wolbachia and rickettsia increase host fitness by providing protection against natural enemies. phylogenetic analysis shows that closely related rickettsia and wolbachia infect distantly related megastigmus. this provides strong evidence of horizontal transmission over evolutionary timescales, and is a common pattern in facultative inherited symbionts of insects. in most cases, it is not known how inherited symbionts colonize novel hosts; shared hosts and shared natural enemies have both been implicated. interestingly, for some inherited symbionts, horizontal transmission over ecological timescales may be quite common. it would be useful to sequence more rapidly evolving rickettsia genes, to determine if there was very recent transmission between m. amicorum and m. bipunctatus. since both these species develop in junipers, we could speculate that horizontal transmission occurs via shared host plants; the boivin et al. study of wolbachia in asexual megastigmus also found evidence for such host-plant-mediated transmission. plant-mediated transmission may be an important and underappreciated way for symbionts to colonize hosts. indeed, a recent study showed that an inherited rickettsia in the sweet potato whitefly can be transmitted via phloem. two strains of arsenophonus that infect planthoppers are transmitted both transovarially and via plants, and both have been implicated in plant disease. however, as far as we are aware, interspecific transmission via plants has not yet been demonstrated in any inherited symbionts. microbial associates of m. spermotrophus our estimate of m. spermotrophus microbial species richness fell within the range of other studies of insect microbiomes. pollenivorous and predacious hymenoptera harbour distinct bacterial communities with the lowest level of species richness, while termites harbour the highest species diversity, based on a recent meta-analysis. a recent study estimated the diversity of bacteria associated with parasitoid wasps from the genus nasonia ranged from to bacterial otus. pyrosequencing has been show to detect a greater number of otus compared to traditional methods, such as 16s rrna clone sequencing. this might explain why the estimated bacterial diversity associated with m. spermotrophus is comparably high because the nasonia study and many previous insect microbiome surveys were done using 16s rrna clone sequencing. despite a relatively high overall richness, only fifteen major otus are present with a total relative abundance of % or greater. the core bacterial community of m. spermotrophus can thus be considered to have a somewhat low diversity, characterized by bacterial otus that are commonly found associated with insect guts. the major otus associated with m. spermotrophus can be grouped into five distinct phylotypes: betaproteobacteria, gammaproteobacteria, actinobacteria, firmicutes and alphaproteobacteria. most of these otus are related to bacteria that have been previously reported in insect guts, with acinetobacter and corynebacterium especially common. all of the major otus identified below the order level are bacteria that commonly occur in the environment, such as in soil and in the rhizospere. similar results are commonly found with microbial associates of insects. for example, the microbial symbionts of tetraponera ants are closely related to nitrogen-fixing root nodule bacteria. the giant mesquite bug, thasus neocalifornicus acquires an important mutualistic gut symbiont de novo every generation from the soil. the presence of the same major otus in m. spermotrophus in ovule and even eurytoma samples provides clues to the distribution and transmission of the megastigmus microbiome; it suggests that it is derived from the environment, which, for the developing wasp, is the ovule. acinetobacter and corynebacterium have been previously cultured from within surface-sterilized seeds and ovules. the m. spermotrophus microbiome appears to be highly conserved across development, as demonstrated by the unifrac analysis, with all of the samples tightly grouped. this contrasts with a recent survey of microbial associates of three nasonia species that found that bacterial species richness increased with development. like most higher hymenoptera, the larvae of m. spermotrophus have a blind digestive system with the midgut and hind gut only uniting during the last larval instar. prior to pupation all of the built-up wastes are voided in a fecal pellet, termed the meconium. during metamorphosis the larval midgut epithelium is discarded and replaced by a new pupal epithelium. if these bacteria are associated with the gut, how m. spermotrophus maintains its major associates throughout development is not known. some insects, like true bugs, termites and cockroaches, have crypts or paunches associated with the gut that are thought to enhance persistence of the microbiota. this physiological feature is not well characterized in the hymenoptera, with the exception of some ants. a single otu assigned to the genus ralstonia comprised over 55% of all sequences from the m. spermotrophus samples. the high abundance and persistence of ralstonia throughout host development is a strong indicator that this bacterium is an important associate of m. spermotrophus. ralstonia was also found to be associated with douglas-fir ovules and the parasitoid eurytoma. the genus ralstonia contains species from ecological diverse niches, such as the plant pathogen r. solanacearum, the opportunistic human pathogen r. pickettii and the environmental isolate r. eurytropha. a maximum likelihood phylogeny placed m. spermotrophus associated ralstonia in a cluster with the human pathogen r. pickettii. to our knowledge, this is the first report of ralstonia being a very abundant and potentially important component of an insect microbiome, although ralstonia spp. have been previously reported from microbial surveys of insects, including the cotton bollworm, bartonella-positive fleas, an omnivorous carabid beetle and the potato psyllid. recently, husnik et al. also report the horizontal transfer of one ralstonia gene into the genome of the mealybug planococcus citri. also, r. oxalatica was isolated from the alimentary canal of an indian earthworm. a recent meta-analysis of 16s clone-library studies of insect associated microbes found that betaproteobacteria contributed over 50% to all sequences from hymenoptera. the most common bacterial phylotype identified from solitary bee species, was a betaproteobacteria from the genus burkholderia, which is closely related to ralstonia. burkholderia spp. have also been identified as important mutualists of some phytophagous true bugs, where they reside in gut crypts. the developing m. spermotrophus larva feeds on megagametophyte tissue, which contains all of the seed storage reserves, primarily in the form of starch, triacylglycerols, and nitrogen rich proteins. therefore, ralstonia and other microbial associates of m. spermotrophus would not likely play a role in supplementing this already rich diet with missing essential nutrients but instead may play a role in nutrient recycling. parasitism by m. spermotrophus results in the formation of a nutrient sink, in which the larva and associated microbes are nourished by storage reserves of the megagametophyte. the reserves are intended to provide nourishment for the developing seedling or to be re-absorbed by the mother plant in the event of megagametophyte abortion. in loblolly pine, more than half of the nitrogen in megagametophytes comes from the amino acid arginine. insects use the enzyme arginase to hydrolyze arginine into ornithine and urea. excretion of urea would result in the substantial loss of nitrogen, especially since larvae must undergo extended periods of diapause. very few insects are known to produce urease, the enzyme required to convert urea into ammonium for subsequent amino acid biosynthesis. we speculate that ralstonia or other microbial associates of m. spermotrophus might play an important role in nitrogen recycling by producing urease or other key enzymes missing from the host genome. many insect symbionts have been suggested to promote increased availability of nitrogen in a variety of ways. for example, blochmannia and blattabacterium, the obligate nutritional symbionts of carpenter ants and cockroaches, respectively, use ureases to recycle nitrogen from urea. nitrogen recycling by symbionts has also been shown to be important during diapause in the shield bug, parastrachia japonensi. it is also tempting to speculate that ralstonia could potentially play a role in plant manipulation. another ralstonia species, r. taiwanensi, has been shown to be capable of nodulating and fixing nitrogen in mimosa spp., which implies an ability to manipulate plant physiology. alternatively, ralstonia may not be a key associate of megastigmus species in general, but rather a microbe that is found in the seed environment that encodes enzymes required for the catabolism of seed storage molecules or other essential pathways required for the seed feeding lifestyle of m. spermotrophus. now that ralstonia has been identified as a likely symbiont of m. spermotrophus, further targeted surveys using ralstonia-specific pcr primers would be helpful in determining its prevalence in other populations of m. spermotrophus, in other megastigmus species, and in associated plants. the development of strain-specific markers for fluorescence in situ hybridization would also be useful for localizing ralstonia on or within m. spermotrophus and the ovule, and following its transmission throughout its life cycle. it would also be interesting to examine ralstonia’s role in nitrogen recycling, for example by identifying and following the expression of ureases and other key enzymes during m. spermotrophus development.  in this study two different approaches were used to survey megastigmus for microbial symbionts. the directed pcr screens identified the presence of two common heritable symbionts, wolbachia and rickettsia; these are not likely distorting sex ratios in the sexual megastigmus species surveyed in this study. pyrosequencing was used to characterize the core microbiome of the douglas-fir seed chalcid, m. spermotrophus, which is dominated by ralstonia, a microbe that has not been previosly characterized as an important microbial associated of an insect. interestingly, ralstonia was also present in ovule and eurytoma samples, indicating its prevalence within the niche of the ovule and potential horizontal transmission route from host to parasitoid. this initial characterization of microbial associates of megastigmus did not provide any insight into the potential involvement in host manipulation, although the maintenance of a consistent microbiome from larvae to adult suggests that microbes may be vital to the development and reproduction of m. spermotrophus. many new questions are inspired by these findings, such as, how is the microbiome of m. spermotrophus maintained and transmitted? how widespread is the association with ralstonia? what is the effect of heritable symbionts in sexual megastigmus? 
we characterized the bacterial symbionts of megastigmus, a lineage of seed-parasitic chalcid wasps, with the goal of identifying microbes that might play an important role in aiding development within seeds, including supplementing insect nutrition or manipulating host trees. douglas-fir ovules and the parasitoid eurytoma sp. the success of herbivorous insects has been shaped largely by their association with microbes. the bacterial community associated with m. spermotrophus remained constant throughout wasp development and was dominated by a single otu – a strain of ralstonia, in the betaproteobacteria, comprising over 55% of all bacterial otus from megastigmus samples. we also performed culture independent surveys of larvae, pupae, and adults of m. spermotrophus using pyrosequencing. were also surveyed using pyrosequencing to help elucidate possible transmission mechanisms of the microbial associates of m. spermotrophus. the m. spermotrophus microbiome is dominated by five bacterial otus, including lineages commonly found in other insect microbiomes and in environmental samples. little is known about the microbial associates of seed-parasitic insects. the developing wasp larva feeds on megagametophyte tissue, which contains the seed storage reserves and is especially rich in nitrogen. males and females were infected at similar frequencies, suggesting that these bacteria do not distort sex ratios. this is the first report of ralstonia being an abundant and potentially important member of an insect microbiome, although other closely-related betaproteobacteria, such as burkholderia, are important insect symbionts. this major pest of douglas-fir is the best-studied megastigmus, and was previously shown to manipulate its tree host into redirecting resources towards unfertilized ovules. future studies using ralstonia-specific markers will determine its distribution in other megastigmus species, its mode of transmission, and its role in wasp nutrition.
 the success of herbivorous insects has been shaped largely by their association with microbes. seed parasitism is an insect feeding strategy involving intimate contact and manipulation of a plant host. little is known about the microbial associates of seed-parasitic insects. we characterized the bacterial symbionts of megastigmus, a lineage of seed-parasitic chalcid wasps, with the goal of identifying microbes that might play an important role in aiding development within seeds, including supplementing insect nutrition or manipulating host trees. we screened multiple populations of seven species for common facultative inherited symbionts. we also performed culture independent surveys of larvae, pupae, and adults of m. spermotrophus using pyrosequencing. this major pest of douglas-fir is the best-studied megastigmus, and was previously shown to manipulate its tree host into redirecting resources towards unfertilized ovules. douglas-fir ovules and the parasitoid eurytoma sp. were also surveyed using pyrosequencing to help elucidate possible transmission mechanisms of the microbial associates of m. spermotrophus.  three wasp species harboured rickettsia; two of these also harboured wolbachia. males and females were infected at similar frequencies, suggesting that these bacteria do not distort sex ratios. the m. spermotrophus microbiome is dominated by five bacterial otus, including lineages commonly found in other insect microbiomes and in environmental samples. the bacterial community associated with m. spermotrophus remained constant throughout wasp development and was dominated by a single otu – a strain of ralstonia, in the betaproteobacteria, comprising over 55% of all bacterial otus from megastigmus samples. this strain was also present in unparasitized ovules.  this is the first report of ralstonia being an abundant and potentially important member of an insect microbiome, although other closely-related betaproteobacteria, such as burkholderia, are important insect symbionts. we speculate that ralstonia might play a role in nutrient recycling, perhaps by redirecting nitrogen. the developing wasp larva feeds on megagametophyte tissue, which contains the seed storage reserves and is especially rich in nitrogen. future studies using ralstonia-specific markers will determine its distribution in other megastigmus species, its mode of transmission, and its role in wasp nutrition. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords burkholderiaendophytophagygallsmicrobiomeralstoniarickettsiaseed parasitismsymbiosiswolbachiaissue-copyright-statement© the author 2014 