BACKGROUND
symbiotic communities of eukaryotic organisms are known to influence host developmental programs  <cit>  and also to shape immune response against pathogens  <cit> .
interestingly, some genes/pathways  have a pleiotropic role in immunity and development, and could play a major role in the maintenance of a specific bacterial community.
for instance, the homeobox gene caudal is involved in the formation of the antero-posterior body axis of drosophila, but also in the regulation of the commensal gut microbiota  <cit> .
in the squid-vibrio association, it has recently been shown that the regulation of a peptidoglycan recognition protein , classically involved in innate immunity, plays a role in the activation of the apoptotic process initiating the morphogenetic changes of the symbiont-harboring organ  <cit> .
the generality of the interplay between immunity and development during symbiosis is currently unknown.
wolbachia  is among the most abundant intracellular bacteria.
it infects both arthropods and nematodes, and is known to be a master manipulator of host biology  <cit> .
wolbachia is generally a facultative reproductive parasite in arthropods, and invades the host population by inducing cytoplasmic incompatibility, male-killing, feminization or thelytokous parthenogenesis  <cit> .
another extended phenotype due to the presence of wolbachia is observed in the parasitoid wasp asobara tabida , in which aposymbiotic females exhibit a strong developmental defect.
surprisingly, wolbachia has become necessary for egg production in this wasp, since aposymbiotic females are unable to produce viable offspring  <cit> .
interestingly, a. tabida is the only member of the genus asobara to be dependent on wolbachia for oogenesis, which suggests that the dependence has evolved recently, and makes it possible to study the molecular mechanisms underlying this transition.
in addition, polymorphism of the ovarian phenotype is observed in natural populations after the elimination of wolbachia: some aposymbiotic females do not produce eggs, whereas others produce a few eggs that die prematurely  <cit> .
this polymorphism constitutes a tool to better understand the influence of these molecular mechanisms on the severity of the ovarian phenotype and on the evolution of dependence.
at a mechanistic level, cytological analysis of the ovarian phenotype has begun to shed light on the mechanisms underlying dependence in a. tabida.
indeed, eliminating wolbachia triggers programmed cell death  in the egg chambers within the ovaries of a. tabida females  <cit> .
as egg production is tightly controlled by two main apoptotic checkpoints during oogenesis  <cit> , the deregulation of pcd in aposymbiotic wasps must result in female inability to complete oogenesis.
because pcd is frequently involved in infection processes by bacterial pathogens  <cit> , it has been hypothesised that a mechanism underlying the maintenance of wolbachia at the individual level may have given rise to the evolution of dependence through its pleiotropic role in immunity and development  <cit> .
this hypothesis is supported by recent findings showing that consequences of wolbachia infection in insects may extend far beyond the classical effect on reproduction, by impacting host physiology and immunity.
wolbachia could play a role as a nutritional mutualist, by influencing iron utilization by its drosophila hosts  <cit> .
wolbachia infection has also been shown to generate oxidative stress in one aedes aegypti cell line, which reacts by the over-expression of host antioxidant genes  <cit> .
interestingly, reactive oxygen species  are known to play a major role in immunity as a first line of defence  <cit>  but also as a mechanism insuring microbe homeostasis  <cit> .
finally, wolbachia is known to confer resistance against rna viral infection in d. melanogaster and d. simulans  <cit> , and against various pathogens in the mosquito a. aegypti, notably by priming the innate immune system  <cit> .
to summarize, increasing evidence is emerging on the phenotypic effects of wolbachia infection on host physiology and immunity  <cit> .
however, few studies have attempted to describe the molecular mechanisms underlying these phenotypic effects in natural systems  <cit> .
the objective of this paper is to clarify the effect of wolbachia on gene expression in a particular symbiotic association in which wolbachia affects developmental processes, through its effect on wasp oogenesis.
for that purpose, we used both global and dedicated transcriptomic approaches.
even though a. tabida is a model system in host/parasitoid and host/wolbachia interactions, no genetic data were available for this parasitoid wasp.
thus, the first aim of this study was to build a reference transcriptome based on several tissues  and physiological conditions .
by sequencing  <dig> cdna libraries , we provide here the first large-scale, genetic information on this wasp.
the second aim of the study was to better understand how dependence arose in this particular species by deciphering the molecular mechanisms underlying this evolutionary transition.
an overview of functions that could be differentially expressed in response to symbiosis was outlined through in silico analyses on ovaries est libraries  and in vitro subtractions .
then, we focused on candidate genes involved in immunity , programmed cell death and oogenesis; functions which could play a major role in the control of ovarian phenotype through pleiotropy.
using quantitative real-time pcr, we thus characterized the effect of symbiosis on host gene expression in both males and females, in two populations exhibiting extreme ovarian phenotypes.
methods
biological system
ecology
asobara tabida  is a solitary endoparasitoid laying its eggs into the first or second instar larvae of drosophila species.
after drosophila pupation, the parasitoid becomes an ectoparasite, and consumes its host before it itself pupates prior to emerging.
a. tabida is naturally infected by three strains of the intracellular bacterium wolbachia : watab <dig> and watab <dig> induce cytoplasmic incompatibility, and only watab <dig> is required for oogenesis completion  <cit> .
polymorphism of ovarian phenotype in populations
after wolbachia removal, the ovarian phenotype displays a high level of intra-species variation: whereas uninfected females of the pi strain  produce no eggs, uninfected females of the na strain  produce a small number of aborting eggs  <cit> .
in this study, we used the na strain and a pi-derived strain .
pi <dig> was obtained by moderate antibiotic treatment, and contains only the obligatory wolbachia strain watab <dig>  <cit> .
the lines are stable, and have been maintained by regular sib-matings without antibiotic treatment for about  <dig> generations.
when comparing na and pi <dig> aposymbiotic individuals, three things must be kept in mind.
any differences observed may be explained by the host genotype, whether they are directly linked to the ovarian phenotype or not.
because na is triply infected whereas pi <dig> is singly infected, differences could also be due to the presence or absence of watab <dig> and watab <dig>   na and pi <dig> symbiotic individuals have differing bacterial community compositions due to the moderate antibiotic treatment of pi <dig>  <cit> .
general procedures
rearing
wasps were allowed to parasite wolbachia-free d. melanogaster.
insects were reared on axenic medium  <cit>  and maintained under controlled conditions .
young adults  were collected and anesthetized on ice before being dissected in a drop of pbs and/or stored until use at -80°c.
antibiotic treatment
because we were interested in determining the effect of symbiosis, we performed antibiotic treatments to produce wolbachia-free  wasps.
even though antibiotics could also affect host gene expression directly  or indirectly , antibiotic treatment is the only efficient method to eliminate wolbachia from a. tabida.
aposymbiotic females are sterile, and so it is impossible to establish and maintain aposymbiotic lines.
hence, antibiotic treatments had to be administered just before the experiment to obtain aposymbiotic wasps, as described in  <cit> .
briefly, rifampicin 2%  was added to the axenic nutritive medium to reach a final concentration of  <dig> mg/g of standard diet.
seventy d. melanogaster eggs were deposited in this medium, and allowed to be parasitized by three female wasps.
the developing drosophila thus transferred the antibiotic to each of the endoparasitoid wasp larvae, rendering them aposymbiotic.
as a control, the same procedure was performed without the antibiotic treatment.
bacterial challenge
because we were interested in identifying immunity-related genes, we performed a challenge by the intracellular bacteria salmonella typhimurium  to enhance the immune response of a. tabida .
bacteria were prepared from a  <dig> h-culture initially started with a 1/ <dig> dilution of an overnight culture .
bacteria were rinsed twice and concentrated in  <dig> ml of fresh lb medium.
immune challenge was performed by injecting  <dig>  nl of the mother solution  in the thorax of young  females .
as a control,  <dig>  nl of fresh lb medium was injected as described above.
individuals were collected 3h, 6h and 12h after challenge , and stored until use at -80°c.
constitution of a reference transcriptome and comparison of gene expression profiles between est libraries
preparation of a normalized library for cdna sequences acquisition
in order to build a transcriptome of reference for a.tabida, we constructed a normalized library  based on both whole females  and ovaries , in various physiological conditions .
to limit host genetic variability, only the pi <dig> strain was used for the library preparation.
the normalized library was constructed by evrogen  from an equimolar proportion of total rna prepared from aposymbiotic ovaries, symbiotic ovaries, and 3h-, 6h-, 12h-challenged symbiotic females.
total rna samples were used for ds cdna synthesis using the smart approach  <cit> .
smart-prepared, amplified cdna was then normalized using the dsn normalization method  <cit> .
normalization included cdna denaturing/re-association, treatment by duplex-specific nuclease   <cit>  and amplification of normalized fraction by pcr.
normalized cdna was purified using qiaquick pcr purification kit , digested with restriction enzyme sfi <dig>  purified , and ligated into pal  <dig>  vector  for escherichia coli transformation.
preparation of est libraries for in silico comparisons between symbiotic and aposymbiotic ovaries
in order to increase the number of transcripts from the ovaries and to determine the influence of symbiosis on host gene expression, we constructed est libraries on aposymbiotic  and symbiotic  ovaries .
total rna was extracted from a large number of ovaries  as described in  <cit> , and treated with dnase , following the manufacturer’s instructions.
tissue libraries were prepared using creator smart cdna library construction kit , following the manufacturer’s instructions.
cdna was digested by sfi <dig>  purified , and ligated into pdnrlib vector for e. coli transformation.
preparation of suppression subtractive hybridizations  libraries for in vitro comparisons
because in silico comparisons of est libraries can be limited by the depth coverage, we also used a complementary technique to compare gene expression by directly screening differentially-expressed transcripts through ssh.
in order to better understand the influence of ovarian phenotype, we performed sshs between aposymbiotic  and symbiotic  ovaries in two populations exhibiting extreme phenotypes .
total rna was extracted from a large number of ovaries  and treated with dnase , following the manufacturer’s instructions.
amplified ds cdna was prepared using a smart approach  <cit> .
smart oligo ii oligonucleotide  and cds primer were used for first-strand cdna synthesis.
smart-amplified cdna samples were further digested by rsai endonuclease.
subtractive hybridizations were performed using the ssh method in both directions  as described in  <cit>  using the pcr-select cdna subtraction kit .
in order to reduce the number of false-positive clones in the ssh-generated libraries, the mos procedure  was performed by evrogen  for ssh2s a-s, as described in  <cit> .
purified subtracted cdnas from ssh1s a-s were cloned into the pcr  <dig>  topo vector  and used for e.coli transformation.
<dig> and  <dig> clones , respectively, were selected for further confirmation.
purified cdna from ssh2s a-s were cloned into the pal <dig> vector  and used for e. coli transformation.
<dig> clones for each subtraction were selected for further confirmation.
pcr-amplified inserts from clones representing differentially-expressed gene products were confirmed by differential hybridization using either dig-labeled  or p-32-labeled , subtracted cdna probes.
finally, in order to characterize genes responding to bacterial challenge, we performed sshs between extracts from whole females, challenged or not challenged by s. typhimurium , see above for bacterial challenge procedure.
the preparation of these sshs has been performed by evrogen  with the same procedure as for ssh2s a-s.
est sequencing, data processing and analysis
all clones from the libraries were sequenced using the sanger method , and have been deposited in the genbank database .
a general overview of the expressed sequence tags  data processing is given in figure  <dig>  raw sequences and traces files were processed with phred software  <cit>  in order to eliminate any low quality bases in sequences .
sequence trimming, which includes polya tails/vector/adapter removal, was performed by cross_match.
chimeric sequences were computationally digested into independent ests.
clustering and assembly of the ests were performed with tgicl  <cit>  to obtain putative unique transcripts  composed of contiguous ests  and unique ests .
to do this, a pairwise comparison was first performed using a modified version of megablast .
clustering was done with tclust, which proceeds by a transitive approach .
assembly was done with cap <dig> .
to detect unigene similarities with other species, several blasts  were performed against the following databases: ncbi nr ; e-value <  <dig>  hsp length > 33aa), refseq genomic database , unigene division arthropods , nasonia vitripennis nvit ogs_v <dig>  ) and wolbachia sequences from genbank ; e-value < e-20).
gene ontology annotation was carried out using blast2go software  <cit> .
during the first step , a pool of candidate go terms was obtained for each unigene by retrieving go terms associated with the hits obtained after a blastx search against ncbi nr.
during the second step , reliable go terms were selected from the pool of candidate go terms by applying the score function  of blast2go with permissive annotation parameters .
in the third step of the annotation procedure, the pool of go terms selected during the annotation step was merged with go terms associated with interpro domain .
finally, the annex augmentation step was run to modulate the annotation by adding go terms derived from implicit relationships between go terms  <cit> .
in order to extract the biological processes and molecular functions statistically over-represented in aposymbiotic libraries, we performed a hyper-geometrical test between go terms from the aposymbiotic libraries  and those from the os library, which corresponds to natural physiological conditions.
the p-values were then adjusted using bonferroni’s correction.
to perform a functional enrichment analysis of the unigenes extracted from the ssh, we used the fatigo web tool  <cit>  on the os library.
with respect to the go analysis, levels  <dig> and  <dig> were chosen to describe biological processes, and level  <dig> was chosen to describe molecular functions.
gene expression measurement by quantitative rt-pcr
we sought to determine the effect of symbiosis on the expression of a set of candidate genes involved in immunity, programmed cell death and oogenesis.
for that purpose, we first compared gene expression between symbiotic and aposymbiotic samples, in ovaries  and then in males .
in order to limit the influence of the presence of eggs in symbiotic vs. aposymbiotic ovaries of the pi <dig> strain, only the distal part that does not contain eggs  was dissected in that strain.
because the dependence phenotype is determined by the host genotype  <cit> , we compared gene expression between two populations exhibiting extreme ovarian phenotypes.
total rna was extracted from  <dig> replicates of  <dig> males or  <dig> full /partial  ovaries, as described in  <cit> .
total rna was purified from potential dna contamination by dnase treatment .
first-strand cdna synthesis was performed from  <dig> ng of total rna using the superscript iii enzyme  and oligodt primers, according to the manufacturer’s instructions.
for each biological sample,  <dig> ng of cdna was spotted in duplicate in a 96-well plate .
quantitative pcr was performed using lightcycler lc <dig> system  as follows:  <dig> min at 95°c,  <dig> times ,  <dig> s at 70°c.
a melting curve was recorded at the end of the pcr amplification to confirm that a unique transcript product had been amplified.
the reaction mixture consisted of  <dig>  µm of each primer,  <dig> µl of fast sybr-green master mix , and  <dig> µl of diluted cdna .
primers used for quantitative pcr are summarized in additional file  <dig>  in order to calculate pcr efficiencies, standard curves were plotted using seven dilutions  of a previously amplified pcr product purified using nucleospin extract ii kit .
expression data were estimated by calculating e−cp, where e corresponds to the efficiency of the pcr reaction, and cp to the crossing point  <cit> .
candidate gene expression was normalized by the geometric mean of the expression level of three housekeeping genes , and analyzed by wilcoxon’s test.
the p-values were then adjusted using false discovery rate’s correction .
RESULTS
more than  <dig>  unigenes sequenced in cdna libraries
to construct a major dataset on the transcriptome of a. tabida, ests were generated from several strains and tissues of wasps with different wolbachia-infection and immune-challenge status.
the different combinations represent a total of  <dig> cdna libraries, including  <dig> subtractive suppression hybridization  libraries,  <dig> non-normalized libraries, and one normalized library.
characteristics of these cdna libraries are summarized in figure 2a.
in brief, a total of  <dig>  ests were generated using the sanger sequencing approach.
the average length of these sequences after cleaning was  <dig> ±  <dig> bp.
est assembly was done by tgicl  <cit>  on all est sequences, leading to  <dig>  unique transcripts  composed of contiguous ests  or unique ests .
the average length of these unigenes was  <dig> ±  <dig> bp, for an average depth of  <dig>  .
the average gc content was  <dig> %.
sequences covered around  <dig>  mb vs.  <dig> mb of predicted transcripts in nasonia vitripenis, and  <dig> mb in drosophila.
consequently, this first sequencing data set gives reliable information about the transcriptome of a. tabida.
however, most unigenes were obtained from the normalized library and the ovary libraries .
in addition, the overlap between libraries was low, suggesting that the sampling effort should be increased to perform a transcriptomic analysis at the gene level.
indeed, 60% of the unigenes were defined by a single est .
furthermore, the two aposymbiotic libraries  only partially overlapped , sharing  <dig> unigenes, corresponding to 16% of oa <dig> and 26% of oa <dig>  respectively.
functional annotation was performed on the  <dig>  unigenes using blast against various databases and using the gene ontology procedure .
gene ontology  is a structured vocabulary describing gene products with terms taken from different ontologies, such as the molecular function or biological process resulting from the coordinate action of molecular functions.
we chose high e-value cut-offs because of the ancient divergence between a. tabida and the closest sequenced genomes.
in addition, divergence can be very high for fast-evolving genes like immune effectors.
the principal database sources for the go annotation were uniprotkb , flybase  and mouse genome informatics .
around 70% of the unigenes had blast similarities, mainly against n. vitripennis , apis mellifera , harpegnathos saltator , camponotus floridanus , solenopsis invicta  and tribolium castaneum , with an e-value lower than e- <dig> for more than 55% of the unigenes.
undetectable similarity could correspond to the utr part of the cdna, or to species-specific genes.
around 40% of unigenes were annotated after the blast2go annotation procedure for high scoring pair  over a hit length coverage cut-off of 0%.
we used permissive annotation parameters since our goal was to keep the maximum functional annotation even if it involves only a very short portion of the unigene .
adding interproscan prediction and running the annex augmentation procedure increased the number of unigenes annotated.
while we kept the unigenes/go datatset corresponding to the minimum hsp coverage percentage, the mean number of go terms assigned per unigene was  <dig>  go .
functional analysis of the symbiotic interaction
to determine the effect of wolbachia on host gene expression, we first compared the libraries from aposymbiotic ovaries  to the reference library based on symbiotic ovaries , which represents the natural physiological condition of the wasp.
this analysis was performed in the pi <dig> strain, which exhibits a strong ovarian phenotype.
in total,  <dig> unigenes were present in these three libraries,  <dig> of which occurred only once.
the low sequencing depth made it difficult to detect significant differences at the gene level.
hence, to get a better idea of the biological functions that respond to symbiosis, we extracted all the functional annotations from the unigenes, and performed a function-based analysis .
autophagic  and apoptotic processes  were over-represented in aposymbiotic ovaries.
developmental processes  including female gonad development ) and interspecies interactions between organisms were also over-represented in the aposymbiotic ovaries library.
interestingly, numerous molecular functions over-represented in the aposymbiotic ovaries library were linked to stress regulation  or immune recognition .
go terms represented differently in libraries from aposymbiotic  and symbiotic  ovaries .
the proportion of ests related to each go function is indicated in the oa libraries  and in the reference library .
functions are sorted relative to their a/s ratio, representing the enrichment percentage in the oa library compared to the os library.
an asterisk indicates a function over-represented in both oa <dig> and oa <dig> libraries.
another way of detecting biological functions responding to symbiosis is to directly screen for genes that are differentially expressed after in vitro subtractions between cdna libraries.
we therefore performed two different suppressive subtraction hybridizations  in populations exhibiting extreme ovarian phenotypes after the removal of wolbachia, in order to determine the influence of the ovarian phenotype on gene expression.
the first ssh was carried out on the pi <dig> strain, in which aposymbiotic females do not produce eggs; and the second was carried out on the na strain, in which aposymbiotic females produce a few ‘abnormal’ eggs.
functions over-represented in aposymbiotic ovaries  relative to symbiotic ovaries  were analyzed by the fatigo web tool .
in the pi <dig> strain, genes involved in ferric iron binding were over-represented in aposymbiotic ovaries, whereas those involved in cell cycle regulation and ribosomal machinery were over-represented in the na strain.
interestingly, both in silico and in vitro subtractions between symbiotic and aposymbiotic ovaries highlighted the role of host homeostasis , and the ferritin gene was over-expressed in aposymbiotic individuals in all these comparisons .
go terms represented differently in ssh experiments comparing aposymbiotic ovaries  and symbiotic ovaries  in two different populations.
ssh <dig> was performed in the pi <dig> strain in which females do not produce eggs .
ssh2+mos was performed in the na strain in which females produce a small number of ‘abnormal’ eggs .
suppressive subtraction hybridizations were performed between wasps challenged with s. typhimurium and unchallenged wasps  in order to detect immune genes.
however, the ssh-c was saturated with the antimicrobial peptide hymenoptaecin, and so was not informative.
expression of genes related to immunity , programmed cell death, and oogenesis
previous cytological analyses had shown that the oogenetic defects due to the elimination of wolbachia  <cit>  are associated with an increase in programmed cell death  in the ovaries  <cit> .
in addition to these findings, the global transcriptomics analysis highlighted the fact that removing wolbachia might interfere with signaling pathways related to immunity in its broad sense, including stress regulation.
we used our reference transcriptome to choose unigenes related to these pathways , and studied their expression by qrt-pcr .
unfortunately, it was not possible to study all the genes in these signaling pathways.
hence, we chose those that were the most characteristic of a given pathway and the best annotated using blast.
we first studied their expression in response to wolbachia removal, by comparing symbiotic and aposymbiotic samples, in both ovaries and males.
indeed, the comparison of the two tissue types can provide additional information about the specificity of the process:  gene expression can be observed throughout the male, in which case there is no evidence of apoptotic phenotype or  expression can be specific to the ovaries, in which case an apoptotic phenotype and an oogenetic defect are detected  <cit> .
in the latter case however, the response could also reflect female specificity or any degree of tissue specificity.
as the ovarian phenotype is controlled by the host genotype  <cit> , we finally compared gene expression in response to symbiosis between two different populations with contrasting ovarian phenotypes.
overall, the expression patterns observed in males and ovaries differed considerably in terms of expression level and response to wolbachia removal, highlighting either tissue-specific or sex-specific expression and response.
while most genes displayed a differential response to bacterial infection under at least one condition , the difference in expression was greater than 2-fold  in only one in six of the comparisons, showing that the impact of wolbachia removal on expression was qualitatively important, but quantitatively limited .
as expected, expression was more affected in the ovaries than in the males for both strains .
the fact that expression was affected to a greater extent in pi <dig> than in na ovaries was also expected .
more surprisingly, the same pattern was observed in males , although no clear phenotype has ever been identified in males.
this indicates that the difference in gene expression between pi <dig> and na ovaries was not solely attributable to the ovarian phenotype.
differentially-expressed  genes are those of which the expression, estimated by qrt-pcr, was statistically different under aposymbiotic  and symbiotic  conditions .
de> <dig> corresponds to the number of de genes with an aposymbiotic/symbiotic expression ratio that is greater than  <dig> or smaller than  <dig> .
the pi <dig> strain exhibits a strong ovarian phenotype after wolbachia removal , while the na strain produces a few eggs that fail to develop normally.
if we focus on genes involved in immunity , expression patterns were relatively clear in males.
indeed, na males showed a very limited response to wolbachia removal, whereas pi <dig> males mainly showed an under-expression of these immune genes in symbiotic individuals.
on the opposite, immune genes were mainly over-expressed in symbiotic ovaries of both strains, with however a higher differential expression in pi <dig> ovaries.
this difference could be attributable to the ovarian phenotype, but also to other phenotypic traits controlled by the female genotype.
furthermore, numerous genes involved in immune functions  may also play an important role during the development.
since their transcripts may accumulate during oogenesis, expression results associated with these genes have to be interpreted with caution in aposymbiotic females whose oogenetic process is markedly affected.
curiously, in most of these immune pathways, but particularly the toll and jak-stat pathways, expression profiles depended on the gene being investigated.
indeed, genes upstream in the pathways were mainly over-expressed in symbiotic individuals, whereas downstream effectors, such as anti-microbial peptides and teps, were mainly down-regulated in response to symbiosis.
it is also interesting to note that gene expression was generally much lower in ovaries than in males, suggesting that this tissue may display limited immuno-competency.
in order to study immunity in its broad sense, we also took into account processes involved in the stress response and programmed cell death, as they can also be involved in limiting bacterial infection.
unfortunately, very few genes involved in canonical pathways of apoptosis and autophagy were detected among the libraries, which limited the scope of our investigation.
expression patterns were once again very different in na males and pi <dig> males.
in pi <dig> males, genes involved in stress and programmed cell death were mainly under-expressed in response to symbiosis.
it is difficult to interpret the response of na males to symbiosis, since the very few genes that were differentially regulated were either up or down-regulated within a given pathway.
in the ovaries, where cytological analyses have highlighted apoptotic and autophagic processes in aposymbiotic ovaries , processes associated with pcd were either unchanged in response to symbiosis  or, surprisingly, over-expressed in symbiotic ovaries .
in pi <dig> and na ovaries, genes involved in the stress response  were mainly under-expressed in response to symbiosis, which confirms the trend highlighted by the analyses of est libraries.
wolbachia is known to play a role in oogenesis completion in a. tabida  <cit> , and to restore fertility to the sxlf <dig> d. melanogaster mutant  <cit> .
therefore, we studied the expression of genes known to be involved in sex determination in drosophila  and also in oogenesis and embryogenesis.
expression of sxl and ix was not limited to one sex, as shown by  <cit> , and varied in response to symbiosis in all the populations investigated.
however, the function of sxl in sex determination seems to be restricted to drosophila and could have another role  in other insect species  <cit> .
genes involved in oogenesis and embryogenesis were all over-expressed in symbiotic ovaries, and more significantly so in the pi ovaries.
these findings are thus congruent with the ovarian phenotype of aposymbiotic females .
patterns in gene expression could be explained by the ovarian phenotype’s being related either to a direct role in oogenesis or to mrna storage in the eggs for subsequent embryo development.
discussion
phenotypic effects of wolbachia on host biology are being increasingly reported in arthropod species  <cit> .
furthermore, growing numbers of wolbachia genomes have now been sequenced from strains inducing various phenotypic effects  <cit> , which provides essential information about the biology and evolution of the symbiont.
however, very few studies have focused on the overall response of the host to the presence of wolbachia in natural associations  <cit> .
most studies have focused on host response after stable  <cit>  or transient infection by wolbachia  <cit> , or in cell cultures  <cit> .
the first goal of this work was to generate a first reference transcriptome of a. tabida, a model system both for host/wolbachia  <cit>  and host/parasitoid interactions  <cit> .
the  <dig>  unigenes we isolated from the wasp a. tabida constitute a valuable resource for further genetic studies of these interactions.
for example, the host transcriptional response to parasitoid attack has been studied in d. melanogaster using microarrays  <cit> , but large-scale analyses in wasps are currently lacking.
the genetic information provided here may help to fill this gap.
the second objective was to detect differentially-represented functions in response to symbiosis.
direct analysis of the libraries was limited by the sequencing depth at the gene level, and thus required an analysis based on the go term level.
several genes associated with candidate functions were extracted from the current ests dataset, and were thoroughly studied through qrt-pcr.
the current transcriptomic map can now be used as a backbone for high-throughput sequencing  to provide an accurate global analysis of genes that are differentially expressed in response to symbiosis.
through different approaches, we identified various biological processes that were transcriptionally affected by wolbachia removal.
indeed, almost all the genes we studied using qrt-pcr were differently regulated in male and/or females at least in one population.
the difference in gene expression was generally less than 2-fold, and could not have been detected by microarray analyses.
the influence of wolbachia removal on gene expression was expected in the ovaries, where the absence of wolbachia dramatically alters the ovarian structure.
in males however, the current absence of extended phenotype in response to wolbachia removal would not have suggested changes in gene expression.
as has already been pointed out, these results must be treated with caution.
in aposymbiotic individuals, antibiotic treatment could indeed have directly influenced mitochondrial metabolism  <cit>  and gene expression because of its general cytotoxic effect.
antibiotics could also have indirectly influenced gene expression through the elimination of other bacteria .
we are confident that the variations observed must have been due  to wolbachia infection.
indeed, we would expect the direct effects of antibiotics to affect both strains similarly.
however, we found that  direct effects of the antibiotic treatment may be very limited, as very few genes were differentially regulated in na males,  no gene  was differentially expressed in all comparisons, and  as expected, the pi <dig> strain was more sensitive to wolbachia removal than the na strain.
these results suggest either that changes in gene expression are due to the host genotype in response to wolbachia removal, or that the potential antibiotic effect impacts the expression of genes also involved in the ovarian phenotype.
as variation in dependence phenotype is determined by the host nuclear genotype  <cit> , we studied transcriptional response to symbiosis in two populations with extreme ovarian phenotypes.
however, the comparison between pi <dig> and na populations could have been obscured by their different evolutionary histories and symbiotic status regarding wolbachia strains and other bacteria.
to discard this hypothesis, we subsequently measured the expression of some genes in two strains originating from a same population , but exhibiting different ovarian phenotypes  <cit> .
these strains were genetically related and both triply-infected, and similar patterns were observed as in the comparison between pi <dig> and na ovaries  <cit> .
hence, variation in gene expression in response to symbiosis must be driven by the genetic background associated with the dependence phenotype.
growing evidence shows that the presence of a symbiont can dramatically affect host immunity  <cit> .
for instance, wigglesworthia reduces susceptibility of the tsetse fly to infection by trypanosoma by modulating pgrp-lb  <cit> , and the male-killer spiroplasma weakens antimicrobial expression in d. melanogaster  <cit> .
immuno-modulation by a symbiont could thus be a way of circumventing the host’s immune system and/or to increase host fitness and ability to cope with common pathogens, thus ensuring that the symbiont is maintained within the host.
although wolbachia is hidden in a host-derived vacuole, the transcriptomic analyses presented here suggest that the host organism detects its presence, and that wolbachia may not only adopt an ‘immune-escape’ strategy.
indeed, wolbachia seems to influence host immune system in its broad sense, including both canonical pathways and the stress response to external stimuli.
these observations led us to wonder how wolbachia is detected within the cell, how wolbachia evades the host immune system, and what are the consequences of these manipulations on host cell physiology.
in the present study, most of the canonical immune pgrp receptors were differentially-regulated in the presence of wolbachia, probably through lipoprotein or polysaccharide binding, and the outcome of the interaction tended towards under-expression of immune effectors of the toll, imd and jak-stat pathways.
even when the regulation cascade was too complex to analyze, the expression patterns of most immune genes were modified in response to symbiosis, suggesting that wolbachia may adopt an active strategy of immune evasion in a. tabida.
however, as few immune genes from the toll signaling pathway are also known to play a role in development, expression data have to be interpreted with caution with respect to the important development defect of ovaries in aposymbiotic females.
the regulation appeared to be tissue or sex-specific, immune genes being expressed to a greater extent in males than in ovarian tissues.
wolbachia is mainly concentrated in the ovaries of females, whereas they are spread more widely throughout the male body  <cit> .
hence, modulation of immune pathways could be both gene- and tissue-specific, as shown in the differential immune regulation of bacteriocytes vs. whole body in sitophilus zeamais  <cit> .
the immune response to wolbachia also seems to be host strain-specific, with the pi <dig> strain generally exhibiting a more pronounced pattern than the na strain.
finally, the immune response to wolbachia seems to be host-specific, as drosophila simulans did not repress or induce antimicrobial peptides production  <cit> , whereas the d. melanogaster cell line over-expressed antimicrobial peptides in response to wolbachia infection  <cit> .
similarly, the presence of wolbachia tends to increase immune gene expression in the mosquito hosts when stably introduced  <cit> .
by comparing aposymbiotic and symbiotic tissues of a. tabida, we also highlighted the influence of wolbachia on host immunity in its broad sense, and especially on the regulation of cell homeostasis and the oxidative environment, which are known to play a key role in physiological responses to invasion by pathogens.
indeed, processes involved in the control of the oxidative environment were highlighted both in in silico and in vitro subtractions, and confirmed by qrt-pcr.
given these observations, we further demonstrated the influence of wolbachia on iron homeostasis and oxidative stress regulation in a. tabida  <cit> .
we confirmed the differential expression of ferritin, a protein involved in iron storage and transport, in males, females and ovaries from the pi strain  <cit> .
since the control of iron homeostasis pleiotropically affects apoptotic and oogenetic processes, its perturbation may have played a role in the evolution of dependence  <cit> .
interestingly, ros also interfere with oogenesis in mosquitoes  <cit>  and drosophila  <cit> , probably by controlling apoptotic checkpoints  <cit> .
the influence of wolbachia on iron homeostasis was not restricted to a. tabida, since we demonstrated a similar effect in d. simulans and in an a. aegypti cell line  <cit> .
hence, processes highlighted in an association in which wolbachia induces an extreme phenotype also shed light on more general processes in host/wolbachia interactions.
in the present study, the stress response was not restricted to iron regulation, as other chaperones and enzymes involved in detoxification were also differentially expressed in response to wolbachia symbiosis, in both males and females.
these results suggest a general regulation of the oxidative environment, not solely restricted to the ovaries where the phenotype is observed.
genes involved in the stress response were generally over-expressed in aposymbiotic individuals, suggesting either that wolbachia has a protective effect on host physiology/immunity or that host compensatory mechanisms have been developed to reduce the harmful impact of the presence of wolbachia  <cit> .
interestingly, we observed a differential response in pi <dig> vs. na strains through quantitative rt-pcr, which was confirmed in another population with similar phenotypes  <cit> .
these results suggest that host gene expression has evolved to tolerate the presence of wolbachia, and that the pi <dig> genotype is more sensitive to its presence.
finally, some striking similarities emerge when these results are compared with two other models that have been used in similar studies, but which have radically different extended phenotypes and types of relationships   <cit> .
functions such as oxidative stress regulation  <cit>  and classical immune pathways  <cit>  have already been highlighted, and appear again as being shared between symbiotic associations.
apoptosis has previously been highlighted in a. tabida, owing to the strong cellular phenotype induced by the removal of wolbachia  <cit> , but also appears to be shared by the other associations.
finally, new functions, such as autophagy, have been detected in all three associations, raising the possibility that this pathway also plays a central role in symbiotic interactions.
all these functions are also shared in host-pathogen interactions, suggesting the existence of a common language between bacteria and their hosts, whatever the form their interaction takes.
however, a detailed analysis of these pathways revealed that they may be under- or over-regulated, depending on the symbiotic association.
these differences in gene regulation may reflect different co-evolutionary dynamics , and/or different selective pressures due to symbiont location.
when symbionts are not restricted to specific tissues, deleterious side effects on other traits, e.g.
the response to pathogens or developmental processes modulated by the pleiotropic action of genes, may indeed limit or shape the expression of these pathways.
CONCLUSIONS
in this study, we identified  <dig>  unigenes from the parasitoid wasp a. tabida, which can now facilitate future genetic studies on host/wolbachia and host/parasitoid interactions.
we also highlighted that wolbachia might interfere with the expression of genes involved in development, pcd and immunity, especially through the regulation of oxidative stress.
these results confirm that wolbachia does not only impact its host reproduction, but may also influence more globally the biology and physiology of its hosts with potential unprecedented effects on the evolution of their life history.
authors' contributions
nk was involved in designing the experiments, prepared the libraries, carried out the quantitative pcr analysis, participated in the sequence analysis and drafted the manuscript.
dc was involved in designing the experiments, carried out the est data processing and analysis, and helped with the statistical analysis of expression data.
hh was involved in the design and supervision of the molecular studies.
fg and pw sequenced the libraries.
pm was involved in designing the experiments.
fv conceived and coordinated the study, was involved in its design, and helped to draft the manuscript.
all the authors have read and approved the final manuscript.
competing interests
the authors declare that they have no competing interests.
supplementary material
additional file 1
primers used for quantitative rt-pcr.
click here for file
additional file 2
functions under-represented in wasp ovaries in response to wolbachia infection, biological process level  <dig>  go terms differentially-represented in libraries from aposymbiotic  and symbiotic  ovaries .
the proportion of ests related to each go function is indicated in the oa library  and in the reference library .
biological processes  are sorted relative to their a/s ratio, representing the enrichment percentage in the oa library compared to the os library.
an asterisk indicates functions shared by oa <dig> and oa <dig>
click here for file
additional file 3
expression profiles of genes studied in quantitative rt-pcr quantitative rt-pcr was performed from symbiotic  or aposymbiotic  extracts.
the pi <dig> strain exhibits a strong ovarian phenotype after wolbachia removal , while the na strain produces a few eggs that do not develop normally.
rna was extracted either from  <dig> males or from  <dig> ovaries .
expression of each candidate gene was normalized by the geometric mean of three housekeeping genes.
the mean of  <dig> biological replicates  is shown on the graph.
*: conditions that are significantly different .
click here for file
acknowledgements
we would like to thank two anonymous reviewers for their helpful comments on the manuscript, and suzanne peyer for reviewing the english text.
we would like to express our sincere thanks to christine oger  for her help in using the microlabstar hamilton.
a. tabida sequences were obtained within the framework of the “functional genomics and immune signaling in invertebrate endosymbiosis” program, conducted in collaboration with the centre national de séquençage, genoscope .
this work was supported by funding from umr cnrs  <dig>  ifr  <dig> and gdr  <dig>  a grant from the agence nationale de la recherche , and a grant from the fondation innovations en infectiologie .
this article has been published as part of bmc microbiology volume  <dig> supplement  <dig>  2012: arthropod symbioses: from fundamental studies to pest and disease mangement.
the full contents of the supplement are available online at http://www.biomedcentral.com/1471-2180/12?issue=s <dig>

 symbiotic communities of eukaryotic organisms are known to influence host developmental programs and also to shape immune response against pathogens. interestingly, some genes/pathways have a pleiotropic role in immunity and development, and could play a major role in the maintenance of a specific bacterial community. for instance, the homeobox gene caudal is involved in the formation of the antero-posterior body axis of drosophila, but also in the regulation of the commensal gut microbiota. in the squid-vibrio association, it has recently been shown that the regulation of a peptidoglycan recognition protein, classically involved in innate immunity, plays a role in the activation of the apoptotic process initiating the morphogenetic changes of the symbiont-harboring organ. the generality of the interplay between immunity and development during symbiosis is currently unknown. wolbachia is among the most abundant intracellular bacteria. it infects both arthropods and nematodes, and is known to be a master manipulator of host biology. wolbachia is generally a facultative reproductive parasite in arthropods, and invades the host population by inducing cytoplasmic incompatibility, male-killing, feminization or thelytokous parthenogenesis. another extended phenotype due to the presence of wolbachia is observed in the parasitoid wasp asobara tabida, in which aposymbiotic females exhibit a strong developmental defect. surprisingly, wolbachia has become necessary for egg production in this wasp, since aposymbiotic females are unable to produce viable offspring. interestingly, a. tabida is the only member of the genus asobara to be dependent on wolbachia for oogenesis, which suggests that the dependence has evolved recently, and makes it possible to study the molecular mechanisms underlying this transition. in addition, polymorphism of the ovarian phenotype is observed in natural populations after the elimination of wolbachia: some aposymbiotic females do not produce eggs, whereas others produce a few eggs that die prematurely. this polymorphism constitutes a tool to better understand the influence of these molecular mechanisms on the severity of the ovarian phenotype and on the evolution of dependence. at a mechanistic level, cytological analysis of the ovarian phenotype has begun to shed light on the mechanisms underlying dependence in a. tabida. indeed, eliminating wolbachia triggers programmed cell death in the egg chambers within the ovaries of a. tabida females. as egg production is tightly controlled by two main apoptotic checkpoints during oogenesis, the deregulation of pcd in aposymbiotic wasps must result in female inability to complete oogenesis. because pcd is frequently involved in infection processes by bacterial pathogens, it has been hypothesised that a mechanism underlying the maintenance of wolbachia at the individual level may have given rise to the evolution of dependence through its pleiotropic role in immunity and development. this hypothesis is supported by recent findings showing that consequences of wolbachia infection in insects may extend far beyond the classical effect on reproduction, by impacting host physiology and immunity. wolbachia could play a role as a nutritional mutualist, by influencing iron utilization by its drosophila hosts. wolbachia infection has also been shown to generate oxidative stress in one aedes aegypti cell line, which reacts by the over-expression of host antioxidant genes. interestingly, reactive oxygen species are known to play a major role in immunity as a first line of defence but also as a mechanism insuring microbe homeostasis. finally, wolbachia is known to confer resistance against rna viral infection in d. melanogaster and d. simulans, and against various pathogens in the mosquito a. aegypti, notably by priming the innate immune system. to summarize, increasing evidence is emerging on the phenotypic effects of wolbachia infection on host physiology and immunity. however, few studies have attempted to describe the molecular mechanisms underlying these phenotypic effects in natural systems. the objective of this paper is to clarify the effect of wolbachia on gene expression in a particular symbiotic association in which wolbachia affects developmental processes, through its effect on wasp oogenesis. for that purpose, we used both global and dedicated transcriptomic approaches. even though a. tabida is a model system in host/parasitoid and host/wolbachia interactions, no genetic data were available for this parasitoid wasp. thus, the first aim of this study was to build a reference transcriptome based on several tissues and physiological conditions. by sequencing cdna libraries, we provide here the first large-scale, genetic information on this wasp. the second aim of the study was to better understand how dependence arose in this particular species by deciphering the molecular mechanisms underlying this evolutionary transition. an overview of functions that could be differentially expressed in response to symbiosis was outlined through in silico analyses on ovaries est libraries and in vitro subtractions. then, we focused on candidate genes involved in immunity, programmed cell death and oogenesis; functions which could play a major role in the control of ovarian phenotype through pleiotropy. using quantitative real-time pcr, we thus characterized the effect of symbiosis on host gene expression in both males and females, in two populations exhibiting extreme ovarian phenotypes. methods biological system ecology asobara tabida is a solitary endoparasitoid laying its eggs into the first or second instar larvae of drosophila species. after drosophila pupation, the parasitoid becomes an ectoparasite, and consumes its host before it itself pupates prior to emerging. a. tabida is naturally infected by three strains of the intracellular bacterium wolbachia: watab and watab induce cytoplasmic incompatibility, and only watab is required for oogenesis completion. polymorphism of ovarian phenotype in populations after wolbachia removal, the ovarian phenotype displays a high level of intra-species variation: whereas uninfected females of the pi strain produce no eggs, uninfected females of the na strain produce a small number of aborting eggs. in this study, we used the na strain and a pi-derived strain. pi was obtained by moderate antibiotic treatment, and contains only the obligatory wolbachia strain watab. the lines are stable, and have been maintained by regular sib-matings without antibiotic treatment for about generations. when comparing na and pi aposymbiotic individuals, three things must be kept in mind. any differences observed may be explained by the host genotype, whether they are directly linked to the ovarian phenotype or not. because na is triply infected whereas pi is singly infected, differences could also be due to the presence or absence of watab and watab na and pi symbiotic individuals have differing bacterial community compositions due to the moderate antibiotic treatment of pi. general procedures rearing wasps were allowed to parasite wolbachia-free d. melanogaster. insects were reared on axenic medium and maintained under controlled conditions. young adults were collected and anesthetized on ice before being dissected in a drop of pbs and/or stored until use at -80°c. antibiotic treatment because we were interested in determining the effect of symbiosis, we performed antibiotic treatments to produce wolbachia-free wasps. even though antibiotics could also affect host gene expression directly or indirectly, antibiotic treatment is the only efficient method to eliminate wolbachia from a. tabida. aposymbiotic females are sterile, and so it is impossible to establish and maintain aposymbiotic lines. hence, antibiotic treatments had to be administered just before the experiment to obtain aposymbiotic wasps, as described in. briefly, rifampicin 2% was added to the axenic nutritive medium to reach a final concentration of mg/g of standard diet. seventy d. melanogaster eggs were deposited in this medium, and allowed to be parasitized by three female wasps. the developing drosophila thus transferred the antibiotic to each of the endoparasitoid wasp larvae, rendering them aposymbiotic. as a control, the same procedure was performed without the antibiotic treatment. bacterial challenge because we were interested in identifying immunity-related genes, we performed a challenge by the intracellular bacteria salmonella typhimurium to enhance the immune response of a. tabida. bacteria were prepared from a h-culture initially started with a 1/ dilution of an overnight culture. bacteria were rinsed twice and concentrated in ml of fresh lb medium. immune challenge was performed by injecting nl of the mother solution in the thorax of young females. as a control, nl of fresh lb medium was injected as described above. individuals were collected 3h, 6h and 12h after challenge, and stored until use at -80°c. constitution of a reference transcriptome and comparison of gene expression profiles between est libraries preparation of a normalized library for cdna sequences acquisition in order to build a transcriptome of reference for a.tabida, we constructed a normalized library based on both whole females and ovaries, in various physiological conditions. to limit host genetic variability, only the pi strain was used for the library preparation. the normalized library was constructed by evrogen from an equimolar proportion of total rna prepared from aposymbiotic ovaries, symbiotic ovaries, and 3h-, 6h-, 12h-challenged symbiotic females. total rna samples were used for ds cdna synthesis using the smart approach. smart-prepared, amplified cdna was then normalized using the dsn normalization method. normalization included cdna denaturing/re-association, treatment by duplex-specific nuclease and amplification of normalized fraction by pcr. normalized cdna was purified using qiaquick pcr purification kit, digested with restriction enzyme sfi purified, and ligated into pal vector for escherichia coli transformation. preparation of est libraries for in silico comparisons between symbiotic and aposymbiotic ovaries in order to increase the number of transcripts from the ovaries and to determine the influence of symbiosis on host gene expression, we constructed est libraries on aposymbiotic and symbiotic ovaries. total rna was extracted from a large number of ovaries as described in, and treated with dnase, following the manufacturer’s instructions. tissue libraries were prepared using creator smart cdna library construction kit, following the manufacturer’s instructions. cdna was digested by sfi purified, and ligated into pdnrlib vector for e. coli transformation. preparation of suppression subtractive hybridizations libraries for in vitro comparisons because in silico comparisons of est libraries can be limited by the depth coverage, we also used a complementary technique to compare gene expression by directly screening differentially-expressed transcripts through ssh. in order to better understand the influence of ovarian phenotype, we performed sshs between aposymbiotic and symbiotic ovaries in two populations exhibiting extreme phenotypes. total rna was extracted from a large number of ovaries and treated with dnase, following the manufacturer’s instructions. amplified ds cdna was prepared using a smart approach. smart oligo ii oligonucleotide and cds primer were used for first-strand cdna synthesis. smart-amplified cdna samples were further digested by rsai endonuclease. subtractive hybridizations were performed using the ssh method in both directions as described in using the pcr-select cdna subtraction kit. in order to reduce the number of false-positive clones in the ssh-generated libraries, the mos procedure was performed by evrogen for ssh2s a-s, as described in. purified subtracted cdnas from ssh1s a-s were cloned into the pcr topo vector and used for e.coli transformation. and clones, respectively, were selected for further confirmation. purified cdna from ssh2s a-s were cloned into the pal vector and used for e. coli transformation. clones for each subtraction were selected for further confirmation. pcr-amplified inserts from clones representing differentially-expressed gene products were confirmed by differential hybridization using either dig-labeled or p-32-labeled, subtracted cdna probes. finally, in order to characterize genes responding to bacterial challenge, we performed sshs between extracts from whole females, challenged or not challenged by s. typhimurium, see above for bacterial challenge procedure. the preparation of these sshs has been performed by evrogen with the same procedure as for ssh2s a-s. est sequencing, data processing and analysis all clones from the libraries were sequenced using the sanger method, and have been deposited in the genbank database. a general overview of the expressed sequence tags data processing is given in figure raw sequences and traces files were processed with phred software in order to eliminate any low quality bases in sequences. sequence trimming, which includes polya tails/vector/adapter removal, was performed by cross_match. chimeric sequences were computationally digested into independent ests. clustering and assembly of the ests were performed with tgicl to obtain putative unique transcripts composed of contiguous ests and unique ests. to do this, a pairwise comparison was first performed using a modified version of megablast. clustering was done with tclust, which proceeds by a transitive approach. assembly was done with cap. to detect unigene similarities with other species, several blasts were performed against the following databases: ncbi nr; e-value < hsp length > 33aa), refseq genomic database, unigene division arthropods, nasonia vitripennis nvit ogs_v ) and wolbachia sequences from genbank; e-value < e-20). gene ontology annotation was carried out using blast2go software. during the first step, a pool of candidate go terms was obtained for each unigene by retrieving go terms associated with the hits obtained after a blastx search against ncbi nr. during the second step, reliable go terms were selected from the pool of candidate go terms by applying the score function of blast2go with permissive annotation parameters. in the third step of the annotation procedure, the pool of go terms selected during the annotation step was merged with go terms associated with interpro domain. finally, the annex augmentation step was run to modulate the annotation by adding go terms derived from implicit relationships between go terms. in order to extract the biological processes and molecular functions statistically over-represented in aposymbiotic libraries, we performed a hyper-geometrical test between go terms from the aposymbiotic libraries and those from the os library, which corresponds to natural physiological conditions. the p-values were then adjusted using bonferroni’s correction. to perform a functional enrichment analysis of the unigenes extracted from the ssh, we used the fatigo web tool on the os library. with respect to the go analysis, levels and were chosen to describe biological processes, and level was chosen to describe molecular functions. gene expression measurement by quantitative rt-pcr we sought to determine the effect of symbiosis on the expression of a set of candidate genes involved in immunity, programmed cell death and oogenesis. for that purpose, we first compared gene expression between symbiotic and aposymbiotic samples, in ovaries and then in males. in order to limit the influence of the presence of eggs in symbiotic vs. aposymbiotic ovaries of the pi strain, only the distal part that does not contain eggs was dissected in that strain. because the dependence phenotype is determined by the host genotype, we compared gene expression between two populations exhibiting extreme ovarian phenotypes. total rna was extracted from replicates of males or full /partial ovaries, as described in. total rna was purified from potential dna contamination by dnase treatment. first-strand cdna synthesis was performed from ng of total rna using the superscript iii enzyme and oligodt primers, according to the manufacturer’s instructions. for each biological sample, ng of cdna was spotted in duplicate in a 96-well plate. quantitative pcr was performed using lightcycler lc system as follows: min at 95°c, times, s at 70°c. a melting curve was recorded at the end of the pcr amplification to confirm that a unique transcript product had been amplified. the reaction mixture consisted of µm of each primer, µl of fast sybr-green master mix, and µl of diluted cdna. primers used for quantitative pcr are summarized in additional file in order to calculate pcr efficiencies, standard curves were plotted using seven dilutions of a previously amplified pcr product purified using nucleospin extract ii kit. expression data were estimated by calculating e−cp, where e corresponds to the efficiency of the pcr reaction, and cp to the crossing point. candidate gene expression was normalized by the geometric mean of the expression level of three housekeeping genes, and analyzed by wilcoxon’s test. the p-values were then adjusted using false discovery rate’s correction.  more than unigenes sequenced in cdna libraries to construct a major dataset on the transcriptome of a. tabida, ests were generated from several strains and tissues of wasps with different wolbachia-infection and immune-challenge status. the different combinations represent a total of cdna libraries, including subtractive suppression hybridization libraries, non-normalized libraries, and one normalized library. characteristics of these cdna libraries are summarized in figure 2a. in brief, a total of ests were generated using the sanger sequencing approach. the average length of these sequences after cleaning was ± bp. est assembly was done by tgicl on all est sequences, leading to unique transcripts composed of contiguous ests or unique ests. the average length of these unigenes was ± bp, for an average depth of. the average gc content was %. sequences covered around mb vs. mb of predicted transcripts in nasonia vitripenis, and mb in drosophila. consequently, this first sequencing data set gives reliable information about the transcriptome of a. tabida. however, most unigenes were obtained from the normalized library and the ovary libraries. in addition, the overlap between libraries was low, suggesting that the sampling effort should be increased to perform a transcriptomic analysis at the gene level. indeed, 60% of the unigenes were defined by a single est. furthermore, the two aposymbiotic libraries only partially overlapped, sharing unigenes, corresponding to 16% of oa and 26% of oa respectively. functional annotation was performed on the unigenes using blast against various databases and using the gene ontology procedure. gene ontology is a structured vocabulary describing gene products with terms taken from different ontologies, such as the molecular function or biological process resulting from the coordinate action of molecular functions. we chose high e-value cut-offs because of the ancient divergence between a. tabida and the closest sequenced genomes. in addition, divergence can be very high for fast-evolving genes like immune effectors. the principal database sources for the go annotation were uniprotkb, flybase and mouse genome informatics. around 70% of the unigenes had blast similarities, mainly against n. vitripennis, apis mellifera, harpegnathos saltator, camponotus floridanus, solenopsis invicta and tribolium castaneum, with an e-value lower than e- for more than 55% of the unigenes. undetectable similarity could correspond to the utr part of the cdna, or to species-specific genes. around 40% of unigenes were annotated after the blast2go annotation procedure for high scoring pair over a hit length coverage cut-off of 0%. we used permissive annotation parameters since our goal was to keep the maximum functional annotation even if it involves only a very short portion of the unigene. adding interproscan prediction and running the annex augmentation procedure increased the number of unigenes annotated. while we kept the unigenes/go datatset corresponding to the minimum hsp coverage percentage, the mean number of go terms assigned per unigene was go. functional analysis of the symbiotic interaction to determine the effect of wolbachia on host gene expression, we first compared the libraries from aposymbiotic ovaries to the reference library based on symbiotic ovaries, which represents the natural physiological condition of the wasp. this analysis was performed in the pi strain, which exhibits a strong ovarian phenotype. in total, unigenes were present in these three libraries, of which occurred only once. the low sequencing depth made it difficult to detect significant differences at the gene level. hence, to get a better idea of the biological functions that respond to symbiosis, we extracted all the functional annotations from the unigenes, and performed a function-based analysis. autophagic and apoptotic processes were over-represented in aposymbiotic ovaries. developmental processes including female gonad development ) and interspecies interactions between organisms were also over-represented in the aposymbiotic ovaries library. interestingly, numerous molecular functions over-represented in the aposymbiotic ovaries library were linked to stress regulation or immune recognition. go terms represented differently in libraries from aposymbiotic and symbiotic ovaries. the proportion of ests related to each go function is indicated in the oa libraries and in the reference library. functions are sorted relative to their a/s ratio, representing the enrichment percentage in the oa library compared to the os library. an asterisk indicates a function over-represented in both oa and oa libraries. another way of detecting biological functions responding to symbiosis is to directly screen for genes that are differentially expressed after in vitro subtractions between cdna libraries. we therefore performed two different suppressive subtraction hybridizations in populations exhibiting extreme ovarian phenotypes after the removal of wolbachia, in order to determine the influence of the ovarian phenotype on gene expression. the first ssh was carried out on the pi strain, in which aposymbiotic females do not produce eggs; and the second was carried out on the na strain, in which aposymbiotic females produce a few ‘abnormal’ eggs. functions over-represented in aposymbiotic ovaries relative to symbiotic ovaries were analyzed by the fatigo web tool. in the pi strain, genes involved in ferric iron binding were over-represented in aposymbiotic ovaries, whereas those involved in cell cycle regulation and ribosomal machinery were over-represented in the na strain. interestingly, both in silico and in vitro subtractions between symbiotic and aposymbiotic ovaries highlighted the role of host homeostasis, and the ferritin gene was over-expressed in aposymbiotic individuals in all these comparisons. go terms represented differently in ssh experiments comparing aposymbiotic ovaries and symbiotic ovaries in two different populations. ssh was performed in the pi strain in which females do not produce eggs. ssh2+mos was performed in the na strain in which females produce a small number of ‘abnormal’ eggs. suppressive subtraction hybridizations were performed between wasps challenged with s. typhimurium and unchallenged wasps in order to detect immune genes. however, the ssh-c was saturated with the antimicrobial peptide hymenoptaecin, and so was not informative. expression of genes related to immunity, programmed cell death, and oogenesis previous cytological analyses had shown that the oogenetic defects due to the elimination of wolbachia are associated with an increase in programmed cell death in the ovaries. in addition to these findings, the global transcriptomics analysis highlighted the fact that removing wolbachia might interfere with signaling pathways related to immunity in its broad sense, including stress regulation. we used our reference transcriptome to choose unigenes related to these pathways, and studied their expression by qrt-pcr. unfortunately, it was not possible to study all the genes in these signaling pathways. hence, we chose those that were the most characteristic of a given pathway and the best annotated using blast. we first studied their expression in response to wolbachia removal, by comparing symbiotic and aposymbiotic samples, in both ovaries and males. indeed, the comparison of the two tissue types can provide additional information about the specificity of the process: gene expression can be observed throughout the male, in which case there is no evidence of apoptotic phenotype or expression can be specific to the ovaries, in which case an apoptotic phenotype and an oogenetic defect are detected. in the latter case however, the response could also reflect female specificity or any degree of tissue specificity. as the ovarian phenotype is controlled by the host genotype, we finally compared gene expression in response to symbiosis between two different populations with contrasting ovarian phenotypes. overall, the expression patterns observed in males and ovaries differed considerably in terms of expression level and response to wolbachia removal, highlighting either tissue-specific or sex-specific expression and response. while most genes displayed a differential response to bacterial infection under at least one condition, the difference in expression was greater than 2-fold in only one in six of the comparisons, showing that the impact of wolbachia removal on expression was qualitatively important, but quantitatively limited. as expected, expression was more affected in the ovaries than in the males for both strains. the fact that expression was affected to a greater extent in pi than in na ovaries was also expected. more surprisingly, the same pattern was observed in males, although no clear phenotype has ever been identified in males. this indicates that the difference in gene expression between pi and na ovaries was not solely attributable to the ovarian phenotype. differentially-expressed genes are those of which the expression, estimated by qrt-pcr, was statistically different under aposymbiotic and symbiotic conditions. de> corresponds to the number of de genes with an aposymbiotic/symbiotic expression ratio that is greater than or smaller than. the pi strain exhibits a strong ovarian phenotype after wolbachia removal, while the na strain produces a few eggs that fail to develop normally. if we focus on genes involved in immunity, expression patterns were relatively clear in males. indeed, na males showed a very limited response to wolbachia removal, whereas pi males mainly showed an under-expression of these immune genes in symbiotic individuals. on the opposite, immune genes were mainly over-expressed in symbiotic ovaries of both strains, with however a higher differential expression in pi ovaries. this difference could be attributable to the ovarian phenotype, but also to other phenotypic traits controlled by the female genotype. furthermore, numerous genes involved in immune functions may also play an important role during the development. since their transcripts may accumulate during oogenesis, expression results associated with these genes have to be interpreted with caution in aposymbiotic females whose oogenetic process is markedly affected. curiously, in most of these immune pathways, but particularly the toll and jak-stat pathways, expression profiles depended on the gene being investigated. indeed, genes upstream in the pathways were mainly over-expressed in symbiotic individuals, whereas downstream effectors, such as anti-microbial peptides and teps, were mainly down-regulated in response to symbiosis. it is also interesting to note that gene expression was generally much lower in ovaries than in males, suggesting that this tissue may display limited immuno-competency. in order to study immunity in its broad sense, we also took into account processes involved in the stress response and programmed cell death, as they can also be involved in limiting bacterial infection. unfortunately, very few genes involved in canonical pathways of apoptosis and autophagy were detected among the libraries, which limited the scope of our investigation. expression patterns were once again very different in na males and pi males. in pi males, genes involved in stress and programmed cell death were mainly under-expressed in response to symbiosis. it is difficult to interpret the response of na males to symbiosis, since the very few genes that were differentially regulated were either up or down-regulated within a given pathway. in the ovaries, where cytological analyses have highlighted apoptotic and autophagic processes in aposymbiotic ovaries, processes associated with pcd were either unchanged in response to symbiosis or, surprisingly, over-expressed in symbiotic ovaries. in pi and na ovaries, genes involved in the stress response were mainly under-expressed in response to symbiosis, which confirms the trend highlighted by the analyses of est libraries. wolbachia is known to play a role in oogenesis completion in a. tabida, and to restore fertility to the sxlf d. melanogaster mutant. therefore, we studied the expression of genes known to be involved in sex determination in drosophila and also in oogenesis and embryogenesis. expression of sxl and ix was not limited to one sex, as shown by, and varied in response to symbiosis in all the populations investigated. however, the function of sxl in sex determination seems to be restricted to drosophila and could have another role in other insect species. genes involved in oogenesis and embryogenesis were all over-expressed in symbiotic ovaries, and more significantly so in the pi ovaries. these findings are thus congruent with the ovarian phenotype of aposymbiotic females. patterns in gene expression could be explained by the ovarian phenotype’s being related either to a direct role in oogenesis or to mrna storage in the eggs for subsequent embryo development. discussion phenotypic effects of wolbachia on host biology are being increasingly reported in arthropod species. furthermore, growing numbers of wolbachia genomes have now been sequenced from strains inducing various phenotypic effects, which provides essential information about the biology and evolution of the symbiont. however, very few studies have focused on the overall response of the host to the presence of wolbachia in natural associations. most studies have focused on host response after stable or transient infection by wolbachia, or in cell cultures. the first goal of this work was to generate a first reference transcriptome of a. tabida, a model system both for host/wolbachia and host/parasitoid interactions. the unigenes we isolated from the wasp a. tabida constitute a valuable resource for further genetic studies of these interactions. for example, the host transcriptional response to parasitoid attack has been studied in d. melanogaster using microarrays, but large-scale analyses in wasps are currently lacking. the genetic information provided here may help to fill this gap. the second objective was to detect differentially-represented functions in response to symbiosis. direct analysis of the libraries was limited by the sequencing depth at the gene level, and thus required an analysis based on the go term level. several genes associated with candidate functions were extracted from the current ests dataset, and were thoroughly studied through qrt-pcr. the current transcriptomic map can now be used as a backbone for high-throughput sequencing to provide an accurate global analysis of genes that are differentially expressed in response to symbiosis. through different approaches, we identified various biological processes that were transcriptionally affected by wolbachia removal. indeed, almost all the genes we studied using qrt-pcr were differently regulated in male and/or females at least in one population. the difference in gene expression was generally less than 2-fold, and could not have been detected by microarray analyses. the influence of wolbachia removal on gene expression was expected in the ovaries, where the absence of wolbachia dramatically alters the ovarian structure. in males however, the current absence of extended phenotype in response to wolbachia removal would not have suggested changes in gene expression. as has already been pointed out, these results must be treated with caution. in aposymbiotic individuals, antibiotic treatment could indeed have directly influenced mitochondrial metabolism and gene expression because of its general cytotoxic effect. antibiotics could also have indirectly influenced gene expression through the elimination of other bacteria. we are confident that the variations observed must have been due to wolbachia infection. indeed, we would expect the direct effects of antibiotics to affect both strains similarly. however, we found that direct effects of the antibiotic treatment may be very limited, as very few genes were differentially regulated in na males, no gene was differentially expressed in all comparisons, and as expected, the pi strain was more sensitive to wolbachia removal than the na strain. these results suggest either that changes in gene expression are due to the host genotype in response to wolbachia removal, or that the potential antibiotic effect impacts the expression of genes also involved in the ovarian phenotype. as variation in dependence phenotype is determined by the host nuclear genotype, we studied transcriptional response to symbiosis in two populations with extreme ovarian phenotypes. however, the comparison between pi and na populations could have been obscured by their different evolutionary histories and symbiotic status regarding wolbachia strains and other bacteria. to discard this hypothesis, we subsequently measured the expression of some genes in two strains originating from a same population, but exhibiting different ovarian phenotypes. these strains were genetically related and both triply-infected, and similar patterns were observed as in the comparison between pi and na ovaries. hence, variation in gene expression in response to symbiosis must be driven by the genetic background associated with the dependence phenotype. growing evidence shows that the presence of a symbiont can dramatically affect host immunity. for instance, wigglesworthia reduces susceptibility of the tsetse fly to infection by trypanosoma by modulating pgrp-lb, and the male-killer spiroplasma weakens antimicrobial expression in d. melanogaster. immuno-modulation by a symbiont could thus be a way of circumventing the host’s immune system and/or to increase host fitness and ability to cope with common pathogens, thus ensuring that the symbiont is maintained within the host. although wolbachia is hidden in a host-derived vacuole, the transcriptomic analyses presented here suggest that the host organism detects its presence, and that wolbachia may not only adopt an ‘immune-escape’ strategy. indeed, wolbachia seems to influence host immune system in its broad sense, including both canonical pathways and the stress response to external stimuli. these observations led us to wonder how wolbachia is detected within the cell, how wolbachia evades the host immune system, and what are the consequences of these manipulations on host cell physiology. in the present study, most of the canonical immune pgrp receptors were differentially-regulated in the presence of wolbachia, probably through lipoprotein or polysaccharide binding, and the outcome of the interaction tended towards under-expression of immune effectors of the toll, imd and jak-stat pathways. even when the regulation cascade was too complex to analyze, the expression patterns of most immune genes were modified in response to symbiosis, suggesting that wolbachia may adopt an active strategy of immune evasion in a. tabida. however, as few immune genes from the toll signaling pathway are also known to play a role in development, expression data have to be interpreted with caution with respect to the important development defect of ovaries in aposymbiotic females. the regulation appeared to be tissue or sex-specific, immune genes being expressed to a greater extent in males than in ovarian tissues. wolbachia is mainly concentrated in the ovaries of females, whereas they are spread more widely throughout the male body. hence, modulation of immune pathways could be both gene- and tissue-specific, as shown in the differential immune regulation of bacteriocytes vs. whole body in sitophilus zeamais. the immune response to wolbachia also seems to be host strain-specific, with the pi strain generally exhibiting a more pronounced pattern than the na strain. finally, the immune response to wolbachia seems to be host-specific, as drosophila simulans did not repress or induce antimicrobial peptides production, whereas the d. melanogaster cell line over-expressed antimicrobial peptides in response to wolbachia infection. similarly, the presence of wolbachia tends to increase immune gene expression in the mosquito hosts when stably introduced. by comparing aposymbiotic and symbiotic tissues of a. tabida, we also highlighted the influence of wolbachia on host immunity in its broad sense, and especially on the regulation of cell homeostasis and the oxidative environment, which are known to play a key role in physiological responses to invasion by pathogens. indeed, processes involved in the control of the oxidative environment were highlighted both in in silico and in vitro subtractions, and confirmed by qrt-pcr. given these observations, we further demonstrated the influence of wolbachia on iron homeostasis and oxidative stress regulation in a. tabida. we confirmed the differential expression of ferritin, a protein involved in iron storage and transport, in males, females and ovaries from the pi strain. since the control of iron homeostasis pleiotropically affects apoptotic and oogenetic processes, its perturbation may have played a role in the evolution of dependence. interestingly, ros also interfere with oogenesis in mosquitoes and drosophila, probably by controlling apoptotic checkpoints. the influence of wolbachia on iron homeostasis was not restricted to a. tabida, since we demonstrated a similar effect in d. simulans and in an a. aegypti cell line. hence, processes highlighted in an association in which wolbachia induces an extreme phenotype also shed light on more general processes in host/wolbachia interactions. in the present study, the stress response was not restricted to iron regulation, as other chaperones and enzymes involved in detoxification were also differentially expressed in response to wolbachia symbiosis, in both males and females. these results suggest a general regulation of the oxidative environment, not solely restricted to the ovaries where the phenotype is observed. genes involved in the stress response were generally over-expressed in aposymbiotic individuals, suggesting either that wolbachia has a protective effect on host physiology/immunity or that host compensatory mechanisms have been developed to reduce the harmful impact of the presence of wolbachia. interestingly, we observed a differential response in pi vs. na strains through quantitative rt-pcr, which was confirmed in another population with similar phenotypes. these results suggest that host gene expression has evolved to tolerate the presence of wolbachia, and that the pi genotype is more sensitive to its presence. finally, some striking similarities emerge when these results are compared with two other models that have been used in similar studies, but which have radically different extended phenotypes and types of relationships. functions such as oxidative stress regulation and classical immune pathways have already been highlighted, and appear again as being shared between symbiotic associations. apoptosis has previously been highlighted in a. tabida, owing to the strong cellular phenotype induced by the removal of wolbachia, but also appears to be shared by the other associations. finally, new functions, such as autophagy, have been detected in all three associations, raising the possibility that this pathway also plays a central role in symbiotic interactions. all these functions are also shared in host-pathogen interactions, suggesting the existence of a common language between bacteria and their hosts, whatever the form their interaction takes. however, a detailed analysis of these pathways revealed that they may be under- or over-regulated, depending on the symbiotic association. these differences in gene regulation may reflect different co-evolutionary dynamics, and/or different selective pressures due to symbiont location. when symbionts are not restricted to specific tissues, deleterious side effects on other traits, e.g. the response to pathogens or developmental processes modulated by the pleiotropic action of genes, may indeed limit or shape the expression of these pathways.  in this study, we identified unigenes from the parasitoid wasp a. tabida, which can now facilitate future genetic studies on host/wolbachia and host/parasitoid interactions. we also highlighted that wolbachia might interfere with the expression of genes involved in development, pcd and immunity, especially through the regulation of oxidative stress. these results confirm that wolbachia does not only impact its host reproduction, but may also influence more globally the biology and physiology of its hosts with potential unprecedented effects on the evolution of their life history. authors' contributions nk was involved in designing the experiments, prepared the libraries, carried out the quantitative pcr analysis, participated in the sequence analysis and drafted the manuscript. dc was involved in designing the experiments, carried out the est data processing and analysis, and helped with the statistical analysis of expression data. hh was involved in the design and supervision of the molecular studies. fg and pw sequenced the libraries. pm was involved in designing the experiments. fv conceived and coordinated the study, was involved in its design, and helped to draft the manuscript. all the authors have read and approved the final manuscript. competing interests the authors declare that they have no competing interests. supplementary material additional file 1 primers used for quantitative rt-pcr. click here for file additional file 2 functions under-represented in wasp ovaries in response to wolbachia infection, biological process level go terms differentially-represented in libraries from aposymbiotic and symbiotic ovaries. the proportion of ests related to each go function is indicated in the oa library and in the reference library. biological processes are sorted relative to their a/s ratio, representing the enrichment percentage in the oa library compared to the os library. an asterisk indicates functions shared by oa and oa click here for file additional file 3 expression profiles of genes studied in quantitative rt-pcr quantitative rt-pcr was performed from symbiotic or aposymbiotic extracts. the pi strain exhibits a strong ovarian phenotype after wolbachia removal, while the na strain produces a few eggs that do not develop normally. rna was extracted either from males or from ovaries. expression of each candidate gene was normalized by the geometric mean of three housekeeping genes. the mean of biological replicates is shown on the graph. *: conditions that are significantly different. click here for file acknowledgements we would like to thank two anonymous reviewers for their helpful comments on the manuscript, and suzanne peyer for reviewing the english text. we would like to express our sincere thanks to christine oger for her help in using the microlabstar hamilton. a. tabida sequences were obtained within the framework of the “functional genomics and immune signaling in invertebrate endosymbiosis” program, conducted in collaboration with the centre national de séquençage, genoscope. this work was supported by funding from umr cnrs ifr and gdr a grant from the agence nationale de la recherche, and a grant from the fondation innovations en infectiologie. this article has been published as part of bmc microbiology volume supplement 2012: arthropod symbioses: from fundamental studies to pest and disease mangement. the full contents of the supplement are available online at http://www.biomedcentral.com/1471-2180/12?issue=s 
we also showed that wolbachia may interact with immune gene expression to ensure its persistence within the host. this study allowed us to constitute the first major dataset of the transcriptome of a. tabida, a species that is a model system for both host/wolbachia and host/parasitoid interactions. more specifically, our results highlighted that symbiont infection may interfere with numerous pivotal processes at the individual level, suggesting that the impact of wolbachia should also be investigated beyond reproductive manipulations. as pleiotropic functions involved in immunity and development could play a major role in the establishment of dependence, the expression of genes involved in oogenesis, programmed cell death and immunity was analyzed by quantitative rt-pcr. in the parasitoid wasp, asobara tabida, wolbachia is necessary for oogenesis completion, and aposymbiotic females are unable to produce viable offspring. as no genetic data were available on a. tabida, we constructed several expressed sequence tags libraries, and obtained unigenes from this species. gene expression was compared between symbiotic and aposymbiotic ovaries through in silico analysis and in vitro subtraction. in order to decipher the general mechanisms underlying host-wolbachia interactions and the origin of the dependence, we developed transcriptomic approaches to compare gene expression in symbiotic and aposymbiotic individuals. the absence of egg production is also associated with an increase in programmed cell death in the ovaries of aposymbiotic females, suggesting that a mechanism that ensures the maintenance of wolbachia in the wasp could also be responsible for this dependence. apart from these reproductive manipulations, recent findings indicate that wolbachia may also modify the host’s physiology, notably its immune function. we showed that wolbachia might interfere with these numerous biological processes, in particular some related to oxidative stress regulation.
 wolbachia are intracellular bacteria known to be facultative reproductive parasites of numerous arthropod hosts. apart from these reproductive manipulations, recent findings indicate that wolbachia may also modify the host’s physiology, notably its immune function. in the parasitoid wasp, asobara tabida, wolbachia is necessary for oogenesis completion, and aposymbiotic females are unable to produce viable offspring. the absence of egg production is also associated with an increase in programmed cell death in the ovaries of aposymbiotic females, suggesting that a mechanism that ensures the maintenance of wolbachia in the wasp could also be responsible for this dependence. in order to decipher the general mechanisms underlying host-wolbachia interactions and the origin of the dependence, we developed transcriptomic approaches to compare gene expression in symbiotic and aposymbiotic individuals.  as no genetic data were available on a. tabida, we constructed several expressed sequence tags libraries, and obtained unigenes from this species. gene expression was compared between symbiotic and aposymbiotic ovaries through in silico analysis and in vitro subtraction. as pleiotropic functions involved in immunity and development could play a major role in the establishment of dependence, the expression of genes involved in oogenesis, programmed cell death and immunity was analyzed by quantitative rt-pcr. we showed that wolbachia might interfere with these numerous biological processes, in particular some related to oxidative stress regulation. we also showed that wolbachia may interact with immune gene expression to ensure its persistence within the host.  this study allowed us to constitute the first major dataset of the transcriptome of a. tabida, a species that is a model system for both host/wolbachia and host/parasitoid interactions. more specifically, our results highlighted that symbiont infection may interfere with numerous pivotal processes at the individual level, suggesting that the impact of wolbachia should also be investigated beyond reproductive manipulations. 