BACKGROUND
a major problem in biology is understanding phenotypic plasticity.
phenotypic plasticity, the ability of a single genotype to express alternative forms of morphology, physiology and behaviour in response to environmental conditions  <cit> , provides the opportunity to study the influences of environment on the genome of an individual or group.
within the natural world, phenotypic plasticity is widespread and has been key to speciation and evolution  <cit> .
however, within eusocial species it has resulted in polyphenism, where multiple distinct adult phenotypes result from differential expression of a single genome  <cit> .
revealing how multiple sets of genes are differentially expressed within the distinct phenotypes of eusocial species offers an unprecedented opportunity to understand the molecular mechanisms related to polyphenisms.
the eusocial hymenoptera  present an excellent system in which to study how gene expression relates to numerous types of polyphenism.
firstly, the social hymenoptera undergo complete metamorphosis, or holometabolous development, where four morphologically distinct developmental stages  exist.
holometabolous development is widespread in the superorder endopterygota and the success of the life history trait is reflected in the high rates of diversification of species that undergo complete metamorphosis  <cit> .
secondly, they possess caste differentiation within the female sex, where a clear division of labour is evident between two or more physiologically, behaviourally and, in many cases, morphologically distinct phenotypes  <cit> .
the reproductive duties of the colony are dominated by a queen while the majority of individuals serve as functionally sterile workers that perform altruistic tasks such as larval feeding, resource foraging, nest maintenance and colony defence  <cit> .
third, social hymenoptera display haplodiploid sex determination, with females produced from diploid eggs, while haploid eggs develop into males  <cit> .
transcriptomic studies have the potential to unveil key gene expression differences that govern central biological processes, such as immunity and olfaction, within and across life cycle stages.
a number of studies have been performed in social hymenoptera to determine which genes are differentially expressed across the adult castes  <cit>  and across the sexes  <cit> .
these analyses addressed only subsets of the complete transcriptomes of the target species.
recent advances in sequencing technology, such as roche  <dig> and illumina sequencing platforms, generate vastly higher volumes of data  <cit> .
as a consequence, these technologies have been used in several studies of non-model species, such as the granville butterfly  <cit> , the propertius duskywing and the anise swallowtail butterflies  <cit> , the migratory locust locusta migratoria  <cit>  and the primitively eusocial wasp, polistes metricus  <cit> .
these studies demonstrate the potential of transcriptomics to provide insights into the expression of polyphenisms in insects, including eusocial species.
thus, the utilisation of transcriptomic tools can significantly improve our knowledge of what genes influence the evolution of polyphenism within eusocial insect species.
the buff-tailed bumblebee bombus terrestris, which is common across eurasia, is an important ecological pollinator of a wide variety of crops  <cit> .
the success of b. terrestris as a pollinator is reflected in its increased utilisation in commercial agriculture, a multi-million dollar industry  <cit> .
caste differentiation in the female sex is evident in b. terrestris with a single monandrous queen responsible for the main reproductive duties of the colony while functionally sterile workers perform altruistic tasks.
as in all social hymenoptera, sex determination is haplodiploid.
b. terrestris has an annual life cycle, where queens overwinter for several months, postmating.
post-hibernation, the overwintered queen establishes a colony in early spring.
she constructs the initial nest, and lays eggs.
these hatch after four days into larvae that are completely dependent for feeding from the queen or sister workers for  <dig> to  <dig> days  <cit> .
after spinning a cocoon or pupal case, the larva pupates for approximately fourteen days and metamorphoses into the adult  <cit> .
bombus workers display a range of sizes, with size being correlated to function, such as larger bees functioning as foragers  <cit> .
worker-destined diploid eggs continue to be laid by the queen until a transition point occurs during the colony life cycle, known as the competition phase, where the workers begin to develop functional ovaries and compete with the queen for reproductive output.
workers lay unfertilised male-destined haploid eggs.
the initiation of the competition phase coincides with haploid egg laying by the queen while diploid eggs present in the nest may develop into gynes, or virgin queens.
sexuals leave the colony soon after emergence.
virgin queens are mated and subsequently, locate a suitable site to diapause for the duration of the winter months  <cit> .
two previous transcriptomic studies for b. terrestris have been performed.
sadd et al.
<cit>  generated  <dig>  expressed sequence tags  from the thorax and abdomen of four pooled workers  using sanger sequencing technology.
more recently, woodard et al.
<cit>  generated  <dig> reads from the brains and abdomens of over  <dig> workers, to include in an analysis of genes central to convergent evolution of eusociality within the bees.
while both studies have provided valuable resources for the study of the genomics of b. terrestris, they focused on gene expression within workers and as genes may be differentially regulated throughout development, caste or sex, a study incorporating multiple life cycle stages is required to increase our knowledge of gene expression in this important pollinator.
here we present a deep-sequencing roche  <dig> transcriptome study of the pre-adult stages, adult castes, and sexes of b. terrestris, based on individual specimens.
we identify potentially differentially expressed transcripts related to polyphenism and use differential expression to explore hypotheses for the involvement of two important biological processes, immunity and olfaction, in the life cycle of the bee.
these processes were chosen because they represent generally important aspects of animal biology, they are expected to exhibit specific patterns of differential expression in social insects, and they can act as exemplars for the power of the transcriptomic approach.
immune defence against foreign agents would be expected to be heightened in life cycle stages that are more prone to infection, such as larvae, which are housed in a homeostatic nest environment with a high density of nest-mates and consumable resources  <cit> , compared to those that are more protected, such as pupae, which are enclosed in a sealed cocoon.
in adults, workers, which have increased exposure to the environment through foraging and increased contact with potentially infected individuals in the colony, and gynes, which need to survive mating and hibernation, would require a heightened immune response to increase longevity compared to the non-social short-lived male  <cit> .
olfaction is a key aspect of animal biology, and in bombus is particularly important for nest-mate recognition and communication  <cit> , resource discrimination  <cit> , subordinate control by the queen  <cit>  and also mate selection  <cit> .
we thus predict that specific olfaction-related genes would be upregulated in the adult stages, for purposes of resource discrimination and mate recognition, while distinct repertoires will be active in pre-adult stages, for caste-development pheromone reception.
RESULTS
sequencing, assembly and assembly validation
six cdna preparations were sequenced using the roche  <dig> life sciences gs flx titanium series sequencer, generating a total of  <dig> , <dig> sequences.
after removal of adapters and poly tails, and trimming for base quality, there were  <dig> , <dig> high quality sequences with an average length of  <dig> bases.
using the method outlined by kumar and blaxter  <cit> , these were assembled using two different assemblers, roche 454's gsassembler  and mira, to generate the first-order assemblies that we will refer to as newbler_ <dig> and mira_ <dig>  there were  <dig>  contigs in newbler_ <dig> with a mean length of  <dig> bases, while mira_ <dig> contained  <dig>  with a mean length of  <dig> bases .
using partigene,  <dig>  b. terrestris sanger ests generated by sadd et al.
<cit>  and  <dig> b. terrestris mrna sequences  were clustered into  <dig>  contigs .
the three first order assemblies  were coassembled using cap <dig> to generate the bt_transcriptome_v <dig> contig set.
this contained  <dig>  contigs with contributions from more than one first order contig, of which  <dig>  had contributions from all three first order assemblies, and  <dig>  additional contigs containing single first order contigs .
as expected, the improved data sampling in terms of depth, life cycle stage, caste and sex resulted in there being an excess  of cap <dig> contigs with contribution from only newbler_ <dig> and mira_ <dig>  we regard those contigs with independent evidence from sanger and  <dig> data, and presence in all three primary assemblies as being highly credible.
cap <dig> contigs with contributions from two or only one primary assembly are likely to be of lower credibility.
we noted that there was still some residual redundancy in bt_transcriptome_v <dig> contig set, and thus reclustered using the  <dig>  second order contigs with partigene .
this dataset, bt_transcriptome_v <dig>  contained  <dig>  contigs.
of these,  <dig>  were unchanged from bt_transcriptome_v <dig>  while the remaining  <dig>  bt_transcriptome_v <dig> contigs were clustered into  <dig>  bt_transcriptome_v <dig> contigs.
bt_transcriptome_v <dig> contigs have an average length of  <dig>  bases  with an n <dig> contig size of  <dig>  bases.
the total span of the bt_transcriptome_v <dig> contigs is 40mb.
full details of the assembly are provided in additional file  <dig>
pg_sanger contigs that did not coassemble with the  <dig> primary assemblies were examined for biological credibility.
approximately 75%  of the b. terrestris sanger sequences were included in contigs with  <dig> data while in comparison,  <dig>    <dig> reads mapped to the pg_sanger contigs.
the remaining  <dig>  sanger sequences clustered into  <dig>  contigs, of which  <dig>   were singletons.
thus we conclude that the sanger-sequencing derived contigs that lack  <dig> data support are more likely to be rarely expressed genes or technical artefacts.
however, at content was approximately equal for both sets , suggesting that these isolated contigs do not derive from genomic contamination.
in terms of gene content, 43% of the  <dig>  sanger-only contigs had significant  matches to the nonredundant protein database, again suggesting that many have recognisable coding potential and are less likely to be artefacts.
full information on testing of credibility is provided in additional file  <dig>
the b. terrestris genome project has released a first draft assembly and  <dig> % of the bt_transcriptome_v <dig> contigs  had high significance megablast matches  to these data.
woodard et al.
<cit>  generated an assembly of  <dig>  b. terrestris contigs from roche  <dig> data derived from sampling the brains and abdomens of over  <dig> bees, which were obtained from the tsa database on ncbi.
nearly 75% of these contigs  matched  <dig>  contigs within bt_transcriptome_v <dig>  the contigs unique to the study of woodard et al.
<cit>  may derive from rare transcripts, or from differences in origin of samples as woodard et al.
<cit>  generated transcripts from only brains and abdomens of workers while we used whole bodies of several stages.
annotation of bt_transcriptome_v2
we compared bt_transcriptome_v <dig> to the ncbi nr protein database and found significant matches  for  <dig> %  contigs .
of these contigs,  <dig>  were assembled from more than one first order assembly.
based on the fact that these contigs were independently assembled using two different algorithms, we may have greater confidence in the biological validity of these  <dig>  contigs.
the majority of top-scoring matches  were to hymenoptera, including apis species , the ant species harpegnathos saltator , camponotus floridanus  and solenopsis invicta , the parasitoid wasp nasonia species  and bombus species  .
we compared the transcriptome assembly to the whole-genome derived proteomes of a set of model hymenoptera and other insects.
of the  <dig>  bt_transcriptome_v <dig> contigs,  <dig>   matched a predicted protein from the honeybee apis mellifera genome,  <dig>   matched predicted proteins in the ant c. floridanus, and  <dig>   matched predicted proteins in the ant h. saltator.
there were  <dig>   matches to the drosophila melanogaster transcriptome.
to generate a gene estimate for our bt_transcriptome_v <dig> contig set, we examined the amount of best blast matches  between our transcriptome set and the ogs of the honeybee, a. mellifera.
in total, the bt_transccriptome_v <dig> contig set matched  <dig>   <dig> unique predicted proteins from the latest apis genome ogs suggesting the potential for an equal number of homologous protein-encoding genes within our transcriptome set.
contigs that had no match to a previously predicted protein may derive from non-coding rnas, untranslated regions of mrnas, or from protein coding genes highly diverged in or novel to b. terrestris.
functional annotation classification using the go, ec and kegg ontologies using annot8r resulted in the assignment of  <dig>  go terms,  <dig>  ec terms and  <dig>  kegg terms to bt_transcriptome_v <dig> contigs.
approximately 39%  of contigs were annotated with go terms.
while many other contigs had significant blast matches in protein databases, these were to genes that have no go annotation.
go-slim analyses are provided in additional file  <dig>  interproscan searches were performed generating predicted protein signatures for  <dig>  bt_transcriptome_v <dig> contigs, of which  <dig>  contigs received interproscan  annotations with information regarding protein family, occurrences of functional domains and repeats.
<dig>  contigs had no ipr annotation but were annotated with functional domains.
conservation of major biological processes and pathways across insecta
to assess the completeness of the b. terrestris transcriptome represented in our data, we investigated the presence in bt_transcriptome_v <dig> of genes in conserved developmental and physiological pathways across a set of sequenced insect genomes.
for each pathway or system, a set of canonical genes was collated from the proteomes of four species  and these were compared to bt_transcriptome_v <dig> .
for each species, between  <dig> % and  <dig> % of the surveyed genes had matches in bt_transcriptome_v <dig>  suggesting that deep sequencing with  <dig> technology has indeed yielded comprehensive insight into the core regulatory machinery in b. terrestris.
* pathway components unavailable
biological processes and pathways with the number of pathway proteins with significant matches to bt_transcriptome_v <dig> contigs in the honeybee, a. mellifera, the jewel wasp, n. vitripennis, the fruitfly, d. melanogaster, and the pea aphid, a. pisum.
the number of reference proteins are given in brackets.
life cycle differential expression in b. terrestris
we estimated the expression level of each contig by counting  <dig> reads mapped back to the bt_transcriptome_v <dig> dataset.
the majority  were mapped uniquely to  <dig>  contigs.
<dig>  contigs  received ests from all life cycle stages, and these universally expressed genes represented a majority  of the ests from each life cycle stage.
there were  <dig>   singletons .
for  <dig>  of the  <dig>  contigs expression was detected in only one life cycle stage .
the larval and pupal stages had the highest proportion of stage-restricted contigs .
the adult stages had few stage-restricted contigs .
the two worker samples were very similar, with 95%  or 97%  of reads mapping to  <dig>  shared contigs.
further information regarding the number of unique contigs is provided in additional file  <dig>
r-stat analysis of differential gene expression amongst life cycle stages and castes
although our life cycle stage samples are non-replicated, with the exception of the workers , to provide a preliminary assessment of potential differential expression across the life cycle stages of the bee, we employed the r-stat method devised by stekel et al.
<cit> .
as we did not replicate across life cycle stages, with the exception of the workers, we do not have a full understanding of age or individual variation within the differential expression analysis but operating under the assumption that variation would be minimal between life cycle stages, we examined the following.
in total,  <dig>  bt_transcriptome_v <dig> contigs were identified as having high r-values .
the thirty contigs that had the highest r-values amongst all life cycle stages are detailed in table  <dig>  the ten genes that best distinguish the different life-stages are provided in additional file  <dig>
a = total number of larva ests ; b = total number of pupa ests ; c = total number of worker <dig> ests ; d = total number of worker <dig> ests ; e = total number of male ests ; f = total number of gyne ests ; g = total number of ests .
mapped read contributions from each sequenced life cycle stage were analyzed using r-stat to identify contigs with potential differential expression.
read counts are provided for each life cycle stage with the corresponding r-value.
bt_transcriptome_v <dig> contigs  reference, functional description, best ncbi nonredundant  blast match description and genbank accession are provided for each contig.
gene expression within the workers
the contig with the highest r-value  was identified as alpha-glucosidase, with elevated expression in both workers .
the workers also had elevated expression of genes involved in flight ; defence ; metabolism ; and a protein with domain matches to both haemolymph juvenile hormone binding and mite allergen group- <dig> .
examination of the ten genes that best distinguish the worker caste  identified genes involved in metabolism, chitin binding and defence ).
both workers exhibited high expression of genes involved in metabolism, such as oxidation of organic compounds ), and glucose breakdown,  and glucose oxidase ).
a lipase , involved in the breakdown of lipids essential for periods of high activity, was also highly expressed in the workers.
two contigs  matched chitin-binding, peritrophin-like proteins.
two worker-upregulated contigs matched genes implicated in immune defence: allergen-related g <dig> protein  and bombolitin .
other contigs highly differentially expressed in workers included diapause-related protein  <dig> , and two contigs matching hypothetical proteins from the ant c. floridanus  and the honeybee .
btt05480_ <dig> and btt20743_ <dig> have both transmembrane and signal peptide domains suggesting a cell-surface or membrane-bound location.
gene expression in the gyne
the top ten differentially expressed genes included elevated expression in the gyne of an amino acid storage protein, hexamerin  .
within the ten genes that best distinguish the gyne , five contigs matched storage proteins: four hexamerins  and one arylphorin .
apart from genes involved in storage, one contig  matched acyl-coa delta- <dig> desaturase, a protein involved in fatty acid biosynthesis, another a chitin-binding peritrophin  and a third crooked, a protein involved in maintenance of tracheal tube structure .
two contigs had matches to hypothetical proteins in the ant brachymyrmex patagonicus  and the ant c. floridanus .
btt07422_ <dig> was annotated with a predicted signal peptide domain while btt20597_ <dig> had predicted transmembrane, signal peptide and fibronectin-like  <dig> domains.
gene expression in the male
the male sample had a high expression of genes involved in immunity .
the male contributed a high proportion of ests to a contig similar to the allergen protein g <dig>  and also exhibited high expression of two contigs encoding antimicrobial peptides identified as hymenoptaecins .
within the top ten genes that distinguished the male  a third hymenoptaecin contig was identified .
immunity aside, the male had high expression of genes involved in metabolism  and a serine carboxypeptidase , flight  and cuticle formation .
two contigs of unknown function were also overexpressed in the male.
one had a predicted fibronectin-like domain  while the second contig had transmembrane and signal peptide domains .
gene expression in the larva
the pre-adult stages accounted for  <dig> of the top thirty highest r-value annotated contigs .
the larva  had elevated expression of proteins involved in cuticle formation , and amino acid storage .
a member of the cytochrome p <dig> superfamily  was also highly expressed in the larva.
within the larva we identified elevated expression of additional genes involved in development and cuticle formation .
the enzyme, carbonic anhydrase, which is involved in metabolism, was highly expressed .
two functionally unidentified contigs were also overexpressed, one of which  had predicted transmembrane and signal peptide domains.
gene expression within the pupa
in contrast to the high expression of cuticular and structural proteins in the larva, the pupa had a higher expression of a diversity of amino acid storage proteins .
of the amino acid groups, seven contigs matched hexamerins: four to hexamerin  <dig> , one to hexamerin 70c  and two to an unclassified hexamerin .
there was a high pupal est contribution to a contig matching a peregrin-like protein, which has a role in dorsal/ventral axon guidance, an important biological process during transformation from larva to adult.
of the three contigs putatively matching metabolic enzymes, two contigs  matched beta-ureidopropionase, an enzyme involved in metabolism of pyrimidine and beta-alanine, while one matched a short-chain dehydrogenase-reductase .
vitellogenin, which has a role in lipid transport, was highly expressed in the pupa  in comparison to the larva.
differential expression of immunity-related genes across the b. terrestris life cycle
the development and expression of the immune response in b. terrestris is particularly interesting .
we identified b. terrestris homologues of the four major a. mellifera immune signalling pathways , janus kinase and signal transducer and activator of transcription , and jnk immune signalling) in bt_transcriptome_v <dig> contigs, and analysed these for expression differences among life cycle stages .
of the toll pathway components, expression was uniform across life cycle stages apart from two signalling components, tube and pelle.
neither was expressed in the pupa, while tube was only detected in the female adult stages .
for the imd, jnk, and jak-stat signalling pathways, two components were not present in bt_transcriptome_v2: the transmembrane receptor domeless, and the transcription factor for the jnk pathway, jnk map kinase basket.
components of these three pathways were detectably expressed in all stages, but tepa, dredd, and tgf-β activated kinase  <dig>  were only expressed in workers.
sadd et al.
<cit>  also enumerated pathway component expression in workers, but we identified more complete coverage of expected pathways, including a complete toll signalling pathway.
ten immunity-related effector genes were differentially expressed across the sequenced specimens .
nine of these were expressed in all life cycle stages  abaecin was absent from the larva).
phenoloxidase subunit a <dig> , which functions in the production of melanin and other polyphenic compounds for both cuticle biosynthesis and immune encapsulation, had highest expression in the pupa.
a c-type lectin  and a gram-negative binding protein , which both have roles in immune detection of bacteria, had elevated expression in the larva, while the gram-positive binding peptidoglycan recognition protein  sa was highly expressed in the gyne .
transferrin, an iron chelator that impacts on the survival of bacteria within a host, matched  <dig> contigs with differential expression, of which  <dig> contigs had high est contributions in the male while two contigs had high est contribution from the gyne  and the larva , respectively .
the male had elevated expression of the amps abaecin, apidaecin, hymenoptaecin and defensin  <dig> .
several distinct contigs encoding the bombus-specific amp bombolitin , a constituent of venom, showed elevated expression in the female adults, especially the workers.
a = total number of larva ests ; b = total number of pupa ests ; c = total number of worker <dig> ests ; d = total number of worker <dig> ests ; e = total number of male ests ; f = total number of gyne ests ; g = total number of ests .
immunity-related genes were identified in bt_transcriptome_v <dig> and are shown with read counts for each life cycle stage.
the highest amount of ests per contig is highlighted in bold.
differential expression of olfaction genes in b. terrestris
we expected that olfaction would differ among castes and life cycle stages because of their differing needs for, and responses to, social and other cues.
we examined the presence and differential expression of olfaction-related genes, namely the odorant-binding proteins  and the chemosensory proteins  .
both larva and pupa had low expression of obps, with the exception of high expression of obp <dig> in the pupa .
in addition, obp <dig>  obp <dig>  and obp <dig> , obp <dig>  were expressed in the pupa while obp <dig> and obp <dig> were only expressed in the larva .
four csps, namely csp <dig> , csp <dig> , csp <dig>  and csp <dig> , were expressed in the larva with particularly high expression of csp <dig>  and heightened expression of csp <dig> in comparison to the pupa .
the pupa only expressed three csps .
a = total number of larva ests ; b = total number of pupa ests ; c = total number of worker <dig> ests ; d = total number of worker <dig> ests ; e = total number of male ests ; f = total number of gyne ests ; g = total number of ests .
potential odorant-binding proteins and chemosensory proteins in the bt_transcriptome_v <dig> contigs are shown, with read counts for each of the life cycle stages.
the highest amount of ests per contig are highlighted in bold.
the adult stages had a heightened expression of obp <dig> and obp <dig> in comparison to the larval and pupal stages .
contigs identified as obp <dig>  and obp <dig>  had elevated expression in the adult stages in comparison to the larva, and were not expressed in pupa.
obp13-like proteins were highly expressed in the pupa  and the male .
in addition, obp <dig> and obp <dig> were detected in all adult stages but in neither the larva nor the pupa.
obp <dig> was only expressed in the sexuals  while obp <dig> was only expressed in the male.
four csps expressed in the larva were expressed in the adult stages.
there was an elevated expression of csp <dig> in the female adults in comparison to the male.
discussion
we have used roche  <dig> deep sequencing to define and compare the transcriptomes expressed by life cycle stages and castes of b. terrestris.
together, these data form a comprehensive gene catalogue for this ecologically and economically important species.
the de novo assembly had high contiguity, with a mean contig length of  <dig>  bases.
the g+c content for the contigs was 36%, which is similar to the worker-sequencing results of sadd et al.
<cit> .
the majority of contigs  had significant blast matches to previously described proteins.
the remaining 42% may derive from untranslated regions of unassembled mrnas, noncoding rnas or transcriptional noise , or, more interestingly, from b. terrestris genes that are either novel or have diverged significantly from any sequenced relatives.
comparison of the b. terrestris transcriptome against the emerging b. terrestris genome sequence identified matches for  <dig> % of the contigs, and we expect that our data will be of utility in annotation efforts for this genome .
separate sequencing of individual life stages permitted the identification of potentially differentially expressed genes across the life cycle stages using r-stat, and identified potentially important candidate genes underpinning stage- and caste-specific phenotypes.
while we drew samples from two natal colonies, comparison of expression between the workers from the two colonies demonstrated significant similarity, justifying an overall comparison of pre-adult stages and adult stages.
we used r-stat analyses to identify a list of potentially differentially expressed contigs, in order to provide direction for future studies examining gene expression within or across life cycle stages of b. terrestris.
however, some data already exist in support of this approach.
in a previous study by pereboom et al.
<cit> , northern blot analyses examined expression differences between whole bodied larvae and adults  and identified three genes  that were more highly expressed in larvae than in adults, where expression was absent.
these genes match contigs generated from our transcriptomic r-stat analyses that show the same larva-adult expression pattern, providing independent support for the biological validity of the results discussed below.
differential gene expression linked to developmental processes
we identified a number of key differences in genes implicated in developmental processes across the different life stages.
for example, the larval data showed high expression of genes involved in cuticle biogenesis.
as the larval stage represents a period of feeding and growth, undergoing four developmental moults over a fourteen day period, this upregulation of cuticular proteins and endocuticle structural glycoproteins is expected.
other contigs identified in the transcriptome with similar expression profiles are thus likely candidates for genes with similar stage-specific roles.
for example, a short-chain dehydrogenase/reductase  had elevated expression in the pupa.
sdrs function in hormone and steroid metabolism  <cit> , and in social insects, may be involved in stage and caste differentiation, for example through binding juvenile hormone.
an sdr was demonstrated to be differentially expressed at the mrna level in the ovaries in developing larvae of honeybee workers and queens, and sdr gene expression was higher in the ovaries of worker larvae in comparison to queen larvae resulting in possible inhibition of ovary development  <cit> .
the likely co-orthologues found in our study of the a. mellifera sdr analysed by guidugli et al.
<cit>  were derived solely from pupal reads, while guidugli et al.
<cit>  reported expression being highest in final stage honeybee larvae.
thus, we propose that these sdrs are candidate genes for further investigation into their roles in bumblebee caste determination.
amino-acid storage protein expression across the bumblebee life cycle
hexamerins are amino acid storage proteins, related to tyrosinases  <cit> , used during non-feeding periods of adult development to provide amino acids and energy  <cit> .
hexamerins also have roles in the binding of juvenile hormone during insect development, impacting on growth regulation within the larval stage  <cit> .
a number of studies  <cit>  have demonstrated differential expression of hexamerin proteins  at different stages of development amongst the sexes and castes in the honeybee.
in b. terrestris, the pupal sample had elevated expression of hexamerin 110-like genes and a hexamerin  <dig> homologue, while the larva had elevated expression of distinct hexamerin 70-like genes, and the gyne elevated expression of four additional hexamerin 70-like genes.
thus while only four hexamerin-encoding genes were identified in a. mellifera  <cit> , we have identified potentially ten in b. terrestris that show differential expression between life cycle stages and castes, suggesting that these proteins may play complex roles in bumblebee development.
why would gynes express high levels of amino acid storage proteins?
honeybee virgin queens have a higher expression of hexamerins in comparison to workers, with the hexamerins functioning in gonad development  <cit> , whilst in the wasp, p. metricus, developing gynes have higher quantities of hex  <dig> than workers  <cit> .
studies on hexamerins in other social insects, such as ants and termites, have identified a correlation between depletion of hexamerins within queens and colony formation  <cit> .
therefore, this potentially high expression of hexamerins would be important for a b. terrestris gyne from a colony formation viewpoint.
however, as b. terrestris queens undergo a prolonged hibernation after mating, amino acid storage proteins may be important for maintaining functional operation of crucial biological processes during a period of intense stress.
as hibernation is a key stage in the life cycle of bumblebees, many species of which are in drastic worldwide decline  <cit> , our results provide direction for future work to analyse the mechanisms behind successful hibernation in these insects.
genes involved in adult behaviour and physiology
workers had elevated expression of enzymes, such as alpha-glucosidase and a muscle-specific lipase, that would be important for worker task completion.
alpha-glucosidase is involved in carbohydrate metabolism and utilised by foraging honeybees to metabolise nectar into fructose and glucose  <cit> , while the muscle lipase is important for breaking down lipids during periods of high activity  <cit> .
interestingly, in our study both enzymes were expressed at an early stage in the adult workers' life , in contrast to their temporal pattern of expression in a. mellifera workers.
honeybee workers demonstrate a strict temporal polyethism, where younger workers perform nursing duties while older workers forage  <cit> , and it is these foraging workers that exhibit higher expression of these enzymes.
in comparison, there is no strong age-dependent division of labour within bumblebees  <cit> .
alloethism within bumblebee workers has been correlated with size, with studies identifying larger workers performing foraging tasks while smaller workers perform in-nest functions, although this division of labour can change depending on the requirements of the colony  <cit> .
thus future work might focus on size- and age-related differences in gene expression between b. terrestris workers in relation to their subroles within the colony.
males are underrepresented in genomic studies in social insects as the emphasis has been almost exclusively on females.
in the current study, the male had elevated expression of titin, a muscle protein, expressed in the insect flight muscles  <cit> .
as the male bumblebee requires flight for foraging, patrolling and indeed mating, high expression of flight-specific muscles would be required.
in relation to behaviour, the male had a high expression of a neuroparsin, queen brain selective protein  <dig>  which has been suggested to function in caste determination during honeybee development through manipulation of the insect insulin-like pathway  <cit> .
however, neuroparsins have been suggested to play roles in a wide variety of functions, including reproduction  <cit> .
therefore, a neuroparsin-like protein in the bumblebee may have male-specific expression in relation to its behaviour or physiology.
the male had elevated expression of several genes that matched hypothetical or otherwise unannotated proteins in the genomes of a. mellifera, c. floridanus and s. invicta.
it is particularly interesting that the male received so many fully unannotated protein matches .
this may suggest possible novel expression associates with male-specific behaviour and/or physiology.
thus, these data offer valuable insights into the mechanistic basis of male biology in social insects, which has largely been ignored by previous studies .
the immune response in males
even in the absence of overt infectious challenge, the background level of immune defence is likely to be regulated through the bumblebee life cycle.
we did not explicitly challenge the sampled bees with pathogens, but also did not keep them in germ-free environments , and thus we expect a background level of immune activation.
b. terrestris queens are monandrous, mating only once  <cit> , while males can mate up to eight times  <cit> .
in addition, b. terrestris exhibits highly male-biased sex ratios  <cit> .
together, this suggests high levels of competition among the males to mate with gynes.
consequently, males should invest in reproductive fitness, which has been demonstrated in other insect species to trade-off against immunity .
we identified genes involved in pathogen recognition, the transduction of recognition signals, and immune effectors, and analysed their patterns of expression for data to support this hypothesis.
surprisingly, in contrast to our expectations based on life-history theory, the male had elevated expression  of amps involved in the removal of infectious agents as part of the immune system  <cit> , including hymenoptaecin, defensin, abaecin and apidaecin.
this is the first account of an apidaecin-type protein being expressed in b. terrestris.
wilfert et al.
<cit>  found no trade-offs between either branch of the immune system  and amp) and reproductive investment, but rather a positive correlation between amps and reproduction.
wilfert et al.
<cit>  suggest the basis for the positive correlation may be because males pass on amps with their sperm to mates during copulation.
however, the male in our study was very young and we sampled the whole body rather than just reproductive tissue, making the mating-gift hypothesis less convincing.
unlike the majority of social insect males, bumblebee males do not remain within the colony post-emergence from the pupal case.
bumblebee males forage for themselves and spend the majority of their time patrolling scent-marked trails  <cit> .
bumblebee males can survive outside the colony for up to  <dig> days .
thus males cannot take advantage of proposed colony-level social immunity  <cit>  and a primed immune system might be an adaptation to life outside the colony.
thus, our results suggest that the life-history differences between males  and females  may impose divergent selection on expression of immune system genes in social insects.
olfaction in the bumblebee
olfaction and the ability to discriminate a number of volatiles is of immense importance to insects in general, and social insects in particular.
they must recognise nest-mates, discriminate and control subordinates, select mates, and discriminate between a wide range of plants for food collection.
here we discuss two classes of olfaction genes, the odorant binding proteins  and the chemosensory proteins .
in the honeybee genome,  <dig> obp genes have been identified and examined for patterns of expression  <cit> .
within our transcriptome dataset, we found significant matches for eight a. mellifera obps .
this enables us to compare their expression in bumblebees to that of their homologues in honeybees.
in the honeybee, obp <dig>  obp <dig>  obp <dig> and obp <dig> are expressed exclusively in the antennae of adults  <cit>  and obp <dig> was identified as having gender-specific expression , suggesting a role in female recognition of mates  <cit> .
we found low expression of a b. terrestris orthologue of a. mellifera obp <dig> , which is involved in binding of queen pheromone in honeybees  <cit> .
in honeybees, obp <dig> is expressed in workers' and drones' antennae after approximately  <dig> days post-emergence  <cit> .
as we sampled our bees before this late timepoint, level of expression of the obp <dig> homologue may simply be due to developmental staging.
obp <dig> was expressed in all the adult bumblebee stages, but not the larva and pupa, matching expression patterns in the honeybee.
in contrast, obp <dig> was expressed in only the male and gyne.
consequently, obp <dig> in the bumblebee may have a role in mate recognition within both sexes, as opposed to the putative female-specific role in honeybees.
obp <dig> had elevated expression in all adult stages of b. terrestris.
in honeybees, obp <dig> is expressed specifically in the antenna with weak expression in the legs and head, possibly from chemosensory sensilla in these body parts  <cit> .
in contrast, obp <dig> is ubiquitously expressed in all adult body parts of the honeybee with the exception of the antennae  <cit> .
in b. terrestris, obp <dig> was highly expressed in all the bumblebee adult stages, with higher expression in the gyne than in the worker, but was absent from the larva and pupa, which again matches honeybee expression patterns.
obp <dig> is only expressed in the ovaries and eggs of the queen in honeybees  <cit>  but in b. terrestris the male was the only sample to show expression of obp <dig>  lastly, obp <dig> was highly expressed in the b. terrestris pupa and male, but forêt and maleszka  <cit>  identified expression of a. mellifera obp <dig> in old larvae, with expression continued throughout the pupal stage.
it appears that expression of these odorant binding proteins has been conserved in some cases, whilst diverging in others, presumably in response to taxon-specific selection processes.
we identified four putative csps  in the b. terrestris transcriptome.
b. terrestris csp <dig> and csp <dig> had elevated expression in the larva, matching the results of forêt et al.
<cit>  who found a. mellifera csp <dig> to exhibit highest expression in the larva before pupal and imaginal moults.
while briand et al.
<cit>  proposed that csp <dig> had a role in brood pheromone recognition, forêt et al.
<cit>  proposed that csp <dig> may play a role in cuticle maturation.
in a. mellifera, csp <dig> expression was restricted to olfactory tissues in adult, but not pre-adult stages  <cit> .
in contrast, while we detected csp <dig> expression in adult b. terrestris, the highest expression was in the larva.
in honeybees, csp <dig> was expressed at low levels throughout the life cycle stages while csp <dig> was expressed throughout the larva, pupa and adult stages, with elevated tissue-specific expression in queen ovaries and eggs  <cit> .
the expression of csp <dig> and csp <dig> across the bumblebee life stages is consistent with that of the honeybee.
we were surprised not to have detected a homologue of a. mellifera csp <dig>  which is expressed ubiquitously across honeybee life cycle stages.
finally, a. mellifera csp <dig> is expressed only by mature queen honeybees in eggs and ovaries, stages we did not sample in b. terrestris.
overall, the examination of olfaction-related genes in b. terrestris reveals both similarities and also striking differences from their expression in a. mellifera.
we expect that these divergences reflect the different social structuring of bumblebee compared to honeybee colonies, and in particular the differing roles and strategies adopted by different castes in these two species.
again, our results provide indications of fruitful lines of future research into how patterns of gene expression relate to the evolution of primitive  and advanced  sociality.
CONCLUSIONS
the role of differential gene expression in polyphenism within groups is only beginning to be understood, e.g.
<cit> .
as a step towards understanding genes influential in the expression of specific phenotypes throughout an individual's life cycle stage, we performed a transcriptome-wide analysis of individual specimens of the pre-adult stages, castes and sexes of the bumblebee to improve our knowledge of differential gene expression across the life history of this ecologically and agriculturally important pollinator.
we have identified sets of genes that are candidates for regulators and effectors of different phenotypes, and revealed the differing physiological states of each morph.
these candidate genes will prove important for future genomic and proteomic studies on b. terrestris.
the b. terrestris genome is being sequenced, and a bac library and a number of genetic linkage maps have been constructed to provide greater genomic resources for this important insect  <cit> .
our study contributes to this global effort through  <dig> mb of roche  <dig> transcriptomic data for b. terrestris.
we provide a central, web-based transcriptomics resource for b. terrestris <cit> , that facilitates user querying of sequences and functional annotations.
these data are now a bridgehead into deeper, molecular analyses of the physiological, genetic and evolutionary bases of polyphenism, and the further development of the bumblebee as a model social insect.

 a major problem in biology is understanding phenotypic plasticity. phenotypic plasticity, the ability of a single genotype to express alternative forms of morphology, physiology and behaviour in response to environmental conditions, provides the opportunity to study the influences of environment on the genome of an individual or group. within the natural world, phenotypic plasticity is widespread and has been key to speciation and evolution. however, within eusocial species it has resulted in polyphenism, where multiple distinct adult phenotypes result from differential expression of a single genome. revealing how multiple sets of genes are differentially expressed within the distinct phenotypes of eusocial species offers an unprecedented opportunity to understand the molecular mechanisms related to polyphenisms. the eusocial hymenoptera present an excellent system in which to study how gene expression relates to numerous types of polyphenism. firstly, the social hymenoptera undergo complete metamorphosis, or holometabolous development, where four morphologically distinct developmental stages exist. holometabolous development is widespread in the superorder endopterygota and the success of the life history trait is reflected in the high rates of diversification of species that undergo complete metamorphosis. secondly, they possess caste differentiation within the female sex, where a clear division of labour is evident between two or more physiologically, behaviourally and, in many cases, morphologically distinct phenotypes. the reproductive duties of the colony are dominated by a queen while the majority of individuals serve as functionally sterile workers that perform altruistic tasks such as larval feeding, resource foraging, nest maintenance and colony defence. third, social hymenoptera display haplodiploid sex determination, with females produced from diploid eggs, while haploid eggs develop into males. transcriptomic studies have the potential to unveil key gene expression differences that govern central biological processes, such as immunity and olfaction, within and across life cycle stages. a number of studies have been performed in social hymenoptera to determine which genes are differentially expressed across the adult castes and across the sexes. these analyses addressed only subsets of the complete transcriptomes of the target species. recent advances in sequencing technology, such as roche and illumina sequencing platforms, generate vastly higher volumes of data. as a consequence, these technologies have been used in several studies of non-model species, such as the granville butterfly, the propertius duskywing and the anise swallowtail butterflies, the migratory locust locusta migratoria and the primitively eusocial wasp, polistes metricus. these studies demonstrate the potential of transcriptomics to provide insights into the expression of polyphenisms in insects, including eusocial species. thus, the utilisation of transcriptomic tools can significantly improve our knowledge of what genes influence the evolution of polyphenism within eusocial insect species. the buff-tailed bumblebee bombus terrestris, which is common across eurasia, is an important ecological pollinator of a wide variety of crops. the success of b. terrestris as a pollinator is reflected in its increased utilisation in commercial agriculture, a multi-million dollar industry. caste differentiation in the female sex is evident in b. terrestris with a single monandrous queen responsible for the main reproductive duties of the colony while functionally sterile workers perform altruistic tasks. as in all social hymenoptera, sex determination is haplodiploid. b. terrestris has an annual life cycle, where queens overwinter for several months, postmating. post-hibernation, the overwintered queen establishes a colony in early spring. she constructs the initial nest, and lays eggs. these hatch after four days into larvae that are completely dependent for feeding from the queen or sister workers for to days. after spinning a cocoon or pupal case, the larva pupates for approximately fourteen days and metamorphoses into the adult. bombus workers display a range of sizes, with size being correlated to function, such as larger bees functioning as foragers. worker-destined diploid eggs continue to be laid by the queen until a transition point occurs during the colony life cycle, known as the competition phase, where the workers begin to develop functional ovaries and compete with the queen for reproductive output. workers lay unfertilised male-destined haploid eggs. the initiation of the competition phase coincides with haploid egg laying by the queen while diploid eggs present in the nest may develop into gynes, or virgin queens. sexuals leave the colony soon after emergence. virgin queens are mated and subsequently, locate a suitable site to diapause for the duration of the winter months. two previous transcriptomic studies for b. terrestris have been performed. sadd et al. generated expressed sequence tags from the thorax and abdomen of four pooled workers using sanger sequencing technology. more recently, woodard et al. generated reads from the brains and abdomens of over workers, to include in an analysis of genes central to convergent evolution of eusociality within the bees. while both studies have provided valuable resources for the study of the genomics of b. terrestris, they focused on gene expression within workers and as genes may be differentially regulated throughout development, caste or sex, a study incorporating multiple life cycle stages is required to increase our knowledge of gene expression in this important pollinator. here we present a deep-sequencing roche transcriptome study of the pre-adult stages, adult castes, and sexes of b. terrestris, based on individual specimens. we identify potentially differentially expressed transcripts related to polyphenism and use differential expression to explore hypotheses for the involvement of two important biological processes, immunity and olfaction, in the life cycle of the bee. these processes were chosen because they represent generally important aspects of animal biology, they are expected to exhibit specific patterns of differential expression in social insects, and they can act as exemplars for the power of the transcriptomic approach. immune defence against foreign agents would be expected to be heightened in life cycle stages that are more prone to infection, such as larvae, which are housed in a homeostatic nest environment with a high density of nest-mates and consumable resources, compared to those that are more protected, such as pupae, which are enclosed in a sealed cocoon. in adults, workers, which have increased exposure to the environment through foraging and increased contact with potentially infected individuals in the colony, and gynes, which need to survive mating and hibernation, would require a heightened immune response to increase longevity compared to the non-social short-lived male. olfaction is a key aspect of animal biology, and in bombus is particularly important for nest-mate recognition and communication, resource discrimination, subordinate control by the queen and also mate selection. we thus predict that specific olfaction-related genes would be upregulated in the adult stages, for purposes of resource discrimination and mate recognition, while distinct repertoires will be active in pre-adult stages, for caste-development pheromone reception.  sequencing, assembly and assembly validation six cdna preparations were sequenced using the roche life sciences gs flx titanium series sequencer, generating a total of, sequences. after removal of adapters and poly tails, and trimming for base quality, there were, high quality sequences with an average length of bases. using the method outlined by kumar and blaxter, these were assembled using two different assemblers, roche 454's gsassembler and mira, to generate the first-order assemblies that we will refer to as newbler_ and mira_ there were contigs in newbler_ with a mean length of bases, while mira_ contained with a mean length of bases. using partigene, b. terrestris sanger ests generated by sadd et al. and b. terrestris mrna sequences were clustered into contigs. the three first order assemblies were coassembled using cap to generate the bt_transcriptome_v contig set. this contained contigs with contributions from more than one first order contig, of which had contributions from all three first order assemblies, and additional contigs containing single first order contigs. as expected, the improved data sampling in terms of depth, life cycle stage, caste and sex resulted in there being an excess of cap contigs with contribution from only newbler_ and mira_ we regard those contigs with independent evidence from sanger and data, and presence in all three primary assemblies as being highly credible. cap contigs with contributions from two or only one primary assembly are likely to be of lower credibility. we noted that there was still some residual redundancy in bt_transcriptome_v contig set, and thus reclustered using the second order contigs with partigene. this dataset, bt_transcriptome_v contained contigs. of these, were unchanged from bt_transcriptome_v while the remaining bt_transcriptome_v contigs were clustered into bt_transcriptome_v contigs. bt_transcriptome_v contigs have an average length of bases with an n contig size of bases. the total span of the bt_transcriptome_v contigs is 40mb. full details of the assembly are provided in additional file pg_sanger contigs that did not coassemble with the primary assemblies were examined for biological credibility. approximately 75% of the b. terrestris sanger sequences were included in contigs with data while in comparison, reads mapped to the pg_sanger contigs. the remaining sanger sequences clustered into contigs, of which were singletons. thus we conclude that the sanger-sequencing derived contigs that lack data support are more likely to be rarely expressed genes or technical artefacts. however, at content was approximately equal for both sets, suggesting that these isolated contigs do not derive from genomic contamination. in terms of gene content, 43% of the sanger-only contigs had significant matches to the nonredundant protein database, again suggesting that many have recognisable coding potential and are less likely to be artefacts. full information on testing of credibility is provided in additional file the b. terrestris genome project has released a first draft assembly and % of the bt_transcriptome_v contigs had high significance megablast matches to these data. woodard et al. generated an assembly of b. terrestris contigs from roche data derived from sampling the brains and abdomens of over bees, which were obtained from the tsa database on ncbi. nearly 75% of these contigs matched contigs within bt_transcriptome_v the contigs unique to the study of woodard et al. may derive from rare transcripts, or from differences in origin of samples as woodard et al. generated transcripts from only brains and abdomens of workers while we used whole bodies of several stages. annotation of bt_transcriptome_v2 we compared bt_transcriptome_v to the ncbi nr protein database and found significant matches for % contigs. of these contigs, were assembled from more than one first order assembly. based on the fact that these contigs were independently assembled using two different algorithms, we may have greater confidence in the biological validity of these contigs. the majority of top-scoring matches were to hymenoptera, including apis species, the ant species harpegnathos saltator, camponotus floridanus and solenopsis invicta, the parasitoid wasp nasonia species and bombus species. we compared the transcriptome assembly to the whole-genome derived proteomes of a set of model hymenoptera and other insects. of the bt_transcriptome_v contigs, matched a predicted protein from the honeybee apis mellifera genome, matched predicted proteins in the ant c. floridanus, and matched predicted proteins in the ant h. saltator. there were matches to the drosophila melanogaster transcriptome. to generate a gene estimate for our bt_transcriptome_v contig set, we examined the amount of best blast matches between our transcriptome set and the ogs of the honeybee, a. mellifera. in total, the bt_transccriptome_v contig set matched unique predicted proteins from the latest apis genome ogs suggesting the potential for an equal number of homologous protein-encoding genes within our transcriptome set. contigs that had no match to a previously predicted protein may derive from non-coding rnas, untranslated regions of mrnas, or from protein coding genes highly diverged in or novel to b. terrestris. functional annotation classification using the go, ec and kegg ontologies using annot8r resulted in the assignment of go terms, ec terms and kegg terms to bt_transcriptome_v contigs. approximately 39% of contigs were annotated with go terms. while many other contigs had significant blast matches in protein databases, these were to genes that have no go annotation. go-slim analyses are provided in additional file interproscan searches were performed generating predicted protein signatures for bt_transcriptome_v contigs, of which contigs received interproscan annotations with information regarding protein family, occurrences of functional domains and repeats. contigs had no ipr annotation but were annotated with functional domains. conservation of major biological processes and pathways across insecta to assess the completeness of the b. terrestris transcriptome represented in our data, we investigated the presence in bt_transcriptome_v of genes in conserved developmental and physiological pathways across a set of sequenced insect genomes. for each pathway or system, a set of canonical genes was collated from the proteomes of four species and these were compared to bt_transcriptome_v. for each species, between % and % of the surveyed genes had matches in bt_transcriptome_v suggesting that deep sequencing with technology has indeed yielded comprehensive insight into the core regulatory machinery in b. terrestris. * pathway components unavailable biological processes and pathways with the number of pathway proteins with significant matches to bt_transcriptome_v contigs in the honeybee, a. mellifera, the jewel wasp, n. vitripennis, the fruitfly, d. melanogaster, and the pea aphid, a. pisum. the number of reference proteins are given in brackets. life cycle differential expression in b. terrestris we estimated the expression level of each contig by counting reads mapped back to the bt_transcriptome_v dataset. the majority were mapped uniquely to contigs. contigs received ests from all life cycle stages, and these universally expressed genes represented a majority of the ests from each life cycle stage. there were singletons. for of the contigs expression was detected in only one life cycle stage. the larval and pupal stages had the highest proportion of stage-restricted contigs. the adult stages had few stage-restricted contigs. the two worker samples were very similar, with 95% or 97% of reads mapping to shared contigs. further information regarding the number of unique contigs is provided in additional file r-stat analysis of differential gene expression amongst life cycle stages and castes although our life cycle stage samples are non-replicated, with the exception of the workers, to provide a preliminary assessment of potential differential expression across the life cycle stages of the bee, we employed the r-stat method devised by stekel et al.. as we did not replicate across life cycle stages, with the exception of the workers, we do not have a full understanding of age or individual variation within the differential expression analysis but operating under the assumption that variation would be minimal between life cycle stages, we examined the following. in total, bt_transcriptome_v contigs were identified as having high r-values. the thirty contigs that had the highest r-values amongst all life cycle stages are detailed in table the ten genes that best distinguish the different life-stages are provided in additional file a = total number of larva ests; b = total number of pupa ests; c = total number of worker ests; d = total number of worker ests; e = total number of male ests; f = total number of gyne ests; g = total number of ests. mapped read contributions from each sequenced life cycle stage were analyzed using r-stat to identify contigs with potential differential expression. read counts are provided for each life cycle stage with the corresponding r-value. bt_transcriptome_v contigs reference, functional description, best ncbi nonredundant blast match description and genbank accession are provided for each contig. gene expression within the workers the contig with the highest r-value was identified as alpha-glucosidase, with elevated expression in both workers. the workers also had elevated expression of genes involved in flight; defence; metabolism; and a protein with domain matches to both haemolymph juvenile hormone binding and mite allergen group-. examination of the ten genes that best distinguish the worker caste identified genes involved in metabolism, chitin binding and defence ). both workers exhibited high expression of genes involved in metabolism, such as oxidation of organic compounds ), and glucose breakdown, and glucose oxidase ). a lipase, involved in the breakdown of lipids essential for periods of high activity, was also highly expressed in the workers. two contigs matched chitin-binding, peritrophin-like proteins. two worker-upregulated contigs matched genes implicated in immune defence: allergen-related g protein and bombolitin. other contigs highly differentially expressed in workers included diapause-related protein, and two contigs matching hypothetical proteins from the ant c. floridanus and the honeybee. btt05480_ and btt20743_ have both transmembrane and signal peptide domains suggesting a cell-surface or membrane-bound location. gene expression in the gyne the top ten differentially expressed genes included elevated expression in the gyne of an amino acid storage protein, hexamerin. within the ten genes that best distinguish the gyne, five contigs matched storage proteins: four hexamerins and one arylphorin. apart from genes involved in storage, one contig matched acyl-coa delta- desaturase, a protein involved in fatty acid biosynthesis, another a chitin-binding peritrophin and a third crooked, a protein involved in maintenance of tracheal tube structure. two contigs had matches to hypothetical proteins in the ant brachymyrmex patagonicus and the ant c. floridanus. btt07422_ was annotated with a predicted signal peptide domain while btt20597_ had predicted transmembrane, signal peptide and fibronectin-like domains. gene expression in the male the male sample had a high expression of genes involved in immunity. the male contributed a high proportion of ests to a contig similar to the allergen protein g and also exhibited high expression of two contigs encoding antimicrobial peptides identified as hymenoptaecins. within the top ten genes that distinguished the male a third hymenoptaecin contig was identified. immunity aside, the male had high expression of genes involved in metabolism and a serine carboxypeptidase, flight and cuticle formation. two contigs of unknown function were also overexpressed in the male. one had a predicted fibronectin-like domain while the second contig had transmembrane and signal peptide domains. gene expression in the larva the pre-adult stages accounted for of the top thirty highest r-value annotated contigs. the larva had elevated expression of proteins involved in cuticle formation, and amino acid storage. a member of the cytochrome p superfamily was also highly expressed in the larva. within the larva we identified elevated expression of additional genes involved in development and cuticle formation. the enzyme, carbonic anhydrase, which is involved in metabolism, was highly expressed. two functionally unidentified contigs were also overexpressed, one of which had predicted transmembrane and signal peptide domains. gene expression within the pupa in contrast to the high expression of cuticular and structural proteins in the larva, the pupa had a higher expression of a diversity of amino acid storage proteins. of the amino acid groups, seven contigs matched hexamerins: four to hexamerin, one to hexamerin 70c and two to an unclassified hexamerin. there was a high pupal est contribution to a contig matching a peregrin-like protein, which has a role in dorsal/ventral axon guidance, an important biological process during transformation from larva to adult. of the three contigs putatively matching metabolic enzymes, two contigs matched beta-ureidopropionase, an enzyme involved in metabolism of pyrimidine and beta-alanine, while one matched a short-chain dehydrogenase-reductase. vitellogenin, which has a role in lipid transport, was highly expressed in the pupa in comparison to the larva. differential expression of immunity-related genes across the b. terrestris life cycle the development and expression of the immune response in b. terrestris is particularly interesting. we identified b. terrestris homologues of the four major a. mellifera immune signalling pathways, janus kinase and signal transducer and activator of transcription, and jnk immune signalling) in bt_transcriptome_v contigs, and analysed these for expression differences among life cycle stages. of the toll pathway components, expression was uniform across life cycle stages apart from two signalling components, tube and pelle. neither was expressed in the pupa, while tube was only detected in the female adult stages. for the imd, jnk, and jak-stat signalling pathways, two components were not present in bt_transcriptome_v2: the transmembrane receptor domeless, and the transcription factor for the jnk pathway, jnk map kinase basket. components of these three pathways were detectably expressed in all stages, but tepa, dredd, and tgf-β activated kinase were only expressed in workers. sadd et al. also enumerated pathway component expression in workers, but we identified more complete coverage of expected pathways, including a complete toll signalling pathway. ten immunity-related effector genes were differentially expressed across the sequenced specimens. nine of these were expressed in all life cycle stages abaecin was absent from the larva). phenoloxidase subunit a, which functions in the production of melanin and other polyphenic compounds for both cuticle biosynthesis and immune encapsulation, had highest expression in the pupa. a c-type lectin and a gram-negative binding protein, which both have roles in immune detection of bacteria, had elevated expression in the larva, while the gram-positive binding peptidoglycan recognition protein sa was highly expressed in the gyne. transferrin, an iron chelator that impacts on the survival of bacteria within a host, matched contigs with differential expression, of which contigs had high est contributions in the male while two contigs had high est contribution from the gyne and the larva, respectively. the male had elevated expression of the amps abaecin, apidaecin, hymenoptaecin and defensin. several distinct contigs encoding the bombus-specific amp bombolitin, a constituent of venom, showed elevated expression in the female adults, especially the workers. a = total number of larva ests; b = total number of pupa ests; c = total number of worker ests; d = total number of worker ests; e = total number of male ests; f = total number of gyne ests; g = total number of ests. immunity-related genes were identified in bt_transcriptome_v and are shown with read counts for each life cycle stage. the highest amount of ests per contig is highlighted in bold. differential expression of olfaction genes in b. terrestris we expected that olfaction would differ among castes and life cycle stages because of their differing needs for, and responses to, social and other cues. we examined the presence and differential expression of olfaction-related genes, namely the odorant-binding proteins and the chemosensory proteins. both larva and pupa had low expression of obps, with the exception of high expression of obp in the pupa. in addition, obp obp and obp, obp were expressed in the pupa while obp and obp were only expressed in the larva. four csps, namely csp, csp, csp and csp, were expressed in the larva with particularly high expression of csp and heightened expression of csp in comparison to the pupa. the pupa only expressed three csps. a = total number of larva ests; b = total number of pupa ests; c = total number of worker ests; d = total number of worker ests; e = total number of male ests; f = total number of gyne ests; g = total number of ests. potential odorant-binding proteins and chemosensory proteins in the bt_transcriptome_v contigs are shown, with read counts for each of the life cycle stages. the highest amount of ests per contig are highlighted in bold. the adult stages had a heightened expression of obp and obp in comparison to the larval and pupal stages. contigs identified as obp and obp had elevated expression in the adult stages in comparison to the larva, and were not expressed in pupa. obp13-like proteins were highly expressed in the pupa and the male. in addition, obp and obp were detected in all adult stages but in neither the larva nor the pupa. obp was only expressed in the sexuals while obp was only expressed in the male. four csps expressed in the larva were expressed in the adult stages. there was an elevated expression of csp in the female adults in comparison to the male. discussion we have used roche deep sequencing to define and compare the transcriptomes expressed by life cycle stages and castes of b. terrestris. together, these data form a comprehensive gene catalogue for this ecologically and economically important species. the de novo assembly had high contiguity, with a mean contig length of bases. the g+c content for the contigs was 36%, which is similar to the worker-sequencing results of sadd et al.. the majority of contigs had significant blast matches to previously described proteins. the remaining 42% may derive from untranslated regions of unassembled mrnas, noncoding rnas or transcriptional noise, or, more interestingly, from b. terrestris genes that are either novel or have diverged significantly from any sequenced relatives. comparison of the b. terrestris transcriptome against the emerging b. terrestris genome sequence identified matches for % of the contigs, and we expect that our data will be of utility in annotation efforts for this genome. separate sequencing of individual life stages permitted the identification of potentially differentially expressed genes across the life cycle stages using r-stat, and identified potentially important candidate genes underpinning stage- and caste-specific phenotypes. while we drew samples from two natal colonies, comparison of expression between the workers from the two colonies demonstrated significant similarity, justifying an overall comparison of pre-adult stages and adult stages. we used r-stat analyses to identify a list of potentially differentially expressed contigs, in order to provide direction for future studies examining gene expression within or across life cycle stages of b. terrestris. however, some data already exist in support of this approach. in a previous study by pereboom et al., northern blot analyses examined expression differences between whole bodied larvae and adults and identified three genes that were more highly expressed in larvae than in adults, where expression was absent. these genes match contigs generated from our transcriptomic r-stat analyses that show the same larva-adult expression pattern, providing independent support for the biological validity of the results discussed below. differential gene expression linked to developmental processes we identified a number of key differences in genes implicated in developmental processes across the different life stages. for example, the larval data showed high expression of genes involved in cuticle biogenesis. as the larval stage represents a period of feeding and growth, undergoing four developmental moults over a fourteen day period, this upregulation of cuticular proteins and endocuticle structural glycoproteins is expected. other contigs identified in the transcriptome with similar expression profiles are thus likely candidates for genes with similar stage-specific roles. for example, a short-chain dehydrogenase/reductase had elevated expression in the pupa. sdrs function in hormone and steroid metabolism, and in social insects, may be involved in stage and caste differentiation, for example through binding juvenile hormone. an sdr was demonstrated to be differentially expressed at the mrna level in the ovaries in developing larvae of honeybee workers and queens, and sdr gene expression was higher in the ovaries of worker larvae in comparison to queen larvae resulting in possible inhibition of ovary development. the likely co-orthologues found in our study of the a. mellifera sdr analysed by guidugli et al. were derived solely from pupal reads, while guidugli et al. reported expression being highest in final stage honeybee larvae. thus, we propose that these sdrs are candidate genes for further investigation into their roles in bumblebee caste determination. amino-acid storage protein expression across the bumblebee life cycle hexamerins are amino acid storage proteins, related to tyrosinases, used during non-feeding periods of adult development to provide amino acids and energy. hexamerins also have roles in the binding of juvenile hormone during insect development, impacting on growth regulation within the larval stage. a number of studies have demonstrated differential expression of hexamerin proteins at different stages of development amongst the sexes and castes in the honeybee. in b. terrestris, the pupal sample had elevated expression of hexamerin 110-like genes and a hexamerin homologue, while the larva had elevated expression of distinct hexamerin 70-like genes, and the gyne elevated expression of four additional hexamerin 70-like genes. thus while only four hexamerin-encoding genes were identified in a. mellifera, we have identified potentially ten in b. terrestris that show differential expression between life cycle stages and castes, suggesting that these proteins may play complex roles in bumblebee development. why would gynes express high levels of amino acid storage proteins? honeybee virgin queens have a higher expression of hexamerins in comparison to workers, with the hexamerins functioning in gonad development, whilst in the wasp, p. metricus, developing gynes have higher quantities of hex than workers. studies on hexamerins in other social insects, such as ants and termites, have identified a correlation between depletion of hexamerins within queens and colony formation. therefore, this potentially high expression of hexamerins would be important for a b. terrestris gyne from a colony formation viewpoint. however, as b. terrestris queens undergo a prolonged hibernation after mating, amino acid storage proteins may be important for maintaining functional operation of crucial biological processes during a period of intense stress. as hibernation is a key stage in the life cycle of bumblebees, many species of which are in drastic worldwide decline, our results provide direction for future work to analyse the mechanisms behind successful hibernation in these insects. genes involved in adult behaviour and physiology workers had elevated expression of enzymes, such as alpha-glucosidase and a muscle-specific lipase, that would be important for worker task completion. alpha-glucosidase is involved in carbohydrate metabolism and utilised by foraging honeybees to metabolise nectar into fructose and glucose, while the muscle lipase is important for breaking down lipids during periods of high activity. interestingly, in our study both enzymes were expressed at an early stage in the adult workers' life, in contrast to their temporal pattern of expression in a. mellifera workers. honeybee workers demonstrate a strict temporal polyethism, where younger workers perform nursing duties while older workers forage, and it is these foraging workers that exhibit higher expression of these enzymes. in comparison, there is no strong age-dependent division of labour within bumblebees. alloethism within bumblebee workers has been correlated with size, with studies identifying larger workers performing foraging tasks while smaller workers perform in-nest functions, although this division of labour can change depending on the requirements of the colony. thus future work might focus on size- and age-related differences in gene expression between b. terrestris workers in relation to their subroles within the colony. males are underrepresented in genomic studies in social insects as the emphasis has been almost exclusively on females. in the current study, the male had elevated expression of titin, a muscle protein, expressed in the insect flight muscles. as the male bumblebee requires flight for foraging, patrolling and indeed mating, high expression of flight-specific muscles would be required. in relation to behaviour, the male had a high expression of a neuroparsin, queen brain selective protein which has been suggested to function in caste determination during honeybee development through manipulation of the insect insulin-like pathway. however, neuroparsins have been suggested to play roles in a wide variety of functions, including reproduction. therefore, a neuroparsin-like protein in the bumblebee may have male-specific expression in relation to its behaviour or physiology. the male had elevated expression of several genes that matched hypothetical or otherwise unannotated proteins in the genomes of a. mellifera, c. floridanus and s. invicta. it is particularly interesting that the male received so many fully unannotated protein matches. this may suggest possible novel expression associates with male-specific behaviour and/or physiology. thus, these data offer valuable insights into the mechanistic basis of male biology in social insects, which has largely been ignored by previous studies. the immune response in males even in the absence of overt infectious challenge, the background level of immune defence is likely to be regulated through the bumblebee life cycle. we did not explicitly challenge the sampled bees with pathogens, but also did not keep them in germ-free environments, and thus we expect a background level of immune activation. b. terrestris queens are monandrous, mating only once, while males can mate up to eight times. in addition, b. terrestris exhibits highly male-biased sex ratios. together, this suggests high levels of competition among the males to mate with gynes. consequently, males should invest in reproductive fitness, which has been demonstrated in other insect species to trade-off against immunity. we identified genes involved in pathogen recognition, the transduction of recognition signals, and immune effectors, and analysed their patterns of expression for data to support this hypothesis. surprisingly, in contrast to our expectations based on life-history theory, the male had elevated expression of amps involved in the removal of infectious agents as part of the immune system, including hymenoptaecin, defensin, abaecin and apidaecin. this is the first account of an apidaecin-type protein being expressed in b. terrestris. wilfert et al. found no trade-offs between either branch of the immune system and amp) and reproductive investment, but rather a positive correlation between amps and reproduction. wilfert et al. suggest the basis for the positive correlation may be because males pass on amps with their sperm to mates during copulation. however, the male in our study was very young and we sampled the whole body rather than just reproductive tissue, making the mating-gift hypothesis less convincing. unlike the majority of social insect males, bumblebee males do not remain within the colony post-emergence from the pupal case. bumblebee males forage for themselves and spend the majority of their time patrolling scent-marked trails. bumblebee males can survive outside the colony for up to days. thus males cannot take advantage of proposed colony-level social immunity and a primed immune system might be an adaptation to life outside the colony. thus, our results suggest that the life-history differences between males and females may impose divergent selection on expression of immune system genes in social insects. olfaction in the bumblebee olfaction and the ability to discriminate a number of volatiles is of immense importance to insects in general, and social insects in particular. they must recognise nest-mates, discriminate and control subordinates, select mates, and discriminate between a wide range of plants for food collection. here we discuss two classes of olfaction genes, the odorant binding proteins and the chemosensory proteins. in the honeybee genome, obp genes have been identified and examined for patterns of expression. within our transcriptome dataset, we found significant matches for eight a. mellifera obps. this enables us to compare their expression in bumblebees to that of their homologues in honeybees. in the honeybee, obp obp obp and obp are expressed exclusively in the antennae of adults and obp was identified as having gender-specific expression, suggesting a role in female recognition of mates. we found low expression of a b. terrestris orthologue of a. mellifera obp, which is involved in binding of queen pheromone in honeybees. in honeybees, obp is expressed in workers' and drones' antennae after approximately days post-emergence. as we sampled our bees before this late timepoint, level of expression of the obp homologue may simply be due to developmental staging. obp was expressed in all the adult bumblebee stages, but not the larva and pupa, matching expression patterns in the honeybee. in contrast, obp was expressed in only the male and gyne. consequently, obp in the bumblebee may have a role in mate recognition within both sexes, as opposed to the putative female-specific role in honeybees. obp had elevated expression in all adult stages of b. terrestris. in honeybees, obp is expressed specifically in the antenna with weak expression in the legs and head, possibly from chemosensory sensilla in these body parts. in contrast, obp is ubiquitously expressed in all adult body parts of the honeybee with the exception of the antennae. in b. terrestris, obp was highly expressed in all the bumblebee adult stages, with higher expression in the gyne than in the worker, but was absent from the larva and pupa, which again matches honeybee expression patterns. obp is only expressed in the ovaries and eggs of the queen in honeybees but in b. terrestris the male was the only sample to show expression of obp lastly, obp was highly expressed in the b. terrestris pupa and male, but forêt and maleszka identified expression of a. mellifera obp in old larvae, with expression continued throughout the pupal stage. it appears that expression of these odorant binding proteins has been conserved in some cases, whilst diverging in others, presumably in response to taxon-specific selection processes. we identified four putative csps in the b. terrestris transcriptome. b. terrestris csp and csp had elevated expression in the larva, matching the results of forêt et al. who found a. mellifera csp to exhibit highest expression in the larva before pupal and imaginal moults. while briand et al. proposed that csp had a role in brood pheromone recognition, forêt et al. proposed that csp may play a role in cuticle maturation. in a. mellifera, csp expression was restricted to olfactory tissues in adult, but not pre-adult stages. in contrast, while we detected csp expression in adult b. terrestris, the highest expression was in the larva. in honeybees, csp was expressed at low levels throughout the life cycle stages while csp was expressed throughout the larva, pupa and adult stages, with elevated tissue-specific expression in queen ovaries and eggs. the expression of csp and csp across the bumblebee life stages is consistent with that of the honeybee. we were surprised not to have detected a homologue of a. mellifera csp which is expressed ubiquitously across honeybee life cycle stages. finally, a. mellifera csp is expressed only by mature queen honeybees in eggs and ovaries, stages we did not sample in b. terrestris. overall, the examination of olfaction-related genes in b. terrestris reveals both similarities and also striking differences from their expression in a. mellifera. we expect that these divergences reflect the different social structuring of bumblebee compared to honeybee colonies, and in particular the differing roles and strategies adopted by different castes in these two species. again, our results provide indications of fruitful lines of future research into how patterns of gene expression relate to the evolution of primitive and advanced sociality.  the role of differential gene expression in polyphenism within groups is only beginning to be understood, e.g.. as a step towards understanding genes influential in the expression of specific phenotypes throughout an individual's life cycle stage, we performed a transcriptome-wide analysis of individual specimens of the pre-adult stages, castes and sexes of the bumblebee to improve our knowledge of differential gene expression across the life history of this ecologically and agriculturally important pollinator. we have identified sets of genes that are candidates for regulators and effectors of different phenotypes, and revealed the differing physiological states of each morph. these candidate genes will prove important for future genomic and proteomic studies on b. terrestris. the b. terrestris genome is being sequenced, and a bac library and a number of genetic linkage maps have been constructed to provide greater genomic resources for this important insect. our study contributes to this global effort through mb of roche transcriptomic data for b. terrestris. we provide a central, web-based transcriptomics resource for b. terrestris, that facilitates user querying of sequences and functional annotations. these data are now a bridgehead into deeper, molecular analyses of the physiological, genetic and evolutionary bases of polyphenism, and the further development of the bumblebee as a model social insect. 
for example, examination of immunity-related genes identified high conservation of important immunity pathway components across individual specimens from the life cycle stages while olfactory-related genes exhibited differential expression with a wider repertoire of gene expression within adults, especially sexuals, in comparison to immature stages. a preliminary assessment of differences in gene expression across non-replicated specimens from the pre-adult stages, castes and sexes was performed using r-stat analysis. individual samples from the life cycle stages of the bumblebee differed in the expression of a wide array of genes, including genes involved in amino acid storage, metabolism, immunity and olfaction. cdna was prepared from mrna from five life cycle stages and a total of, expressed sequence tags were generated using roche technology, substantially increasing the sequence data available for this important species. overlapping ests were assembled into b. terrestris putative transcripts, and functionally annotated. detailed analyses of immune and olfaction gene expression across phenotypes demonstrated how transcriptomic analyses can inform our understanding of processes central to the biology of b. terrestris and the social hymenoptera in general. as there is an absence of replication across the samples, the results of this study are preliminary but provide a number of candidate genes which may be related to distinct phenotypic stage expression. polyphenism has been key to the evolution of the hymenoptera, and particularly the social hymenoptera where the genome of a single species regulates distinct larval stages, sexual dimorphism and physical castes within the female sex. transcriptomic analyses of social hymenoptera will therefore provide unique insights into how changes in gene expression underlie such complexity. here we describe gene expression in individual specimens of the pre-adult stages, sexes and castes of the key pollinator, the buff-tailed bumblebee bombus terrestris. understanding polyphenism, the ability of a single genome to express multiple morphologically and behaviourally distinct phenotypes, is an important goal for evolutionary and developmental biology.
 understanding polyphenism, the ability of a single genome to express multiple morphologically and behaviourally distinct phenotypes, is an important goal for evolutionary and developmental biology. polyphenism has been key to the evolution of the hymenoptera, and particularly the social hymenoptera where the genome of a single species regulates distinct larval stages, sexual dimorphism and physical castes within the female sex. transcriptomic analyses of social hymenoptera will therefore provide unique insights into how changes in gene expression underlie such complexity. here we describe gene expression in individual specimens of the pre-adult stages, sexes and castes of the key pollinator, the buff-tailed bumblebee bombus terrestris.  cdna was prepared from mrna from five life cycle stages and a total of, expressed sequence tags were generated using roche technology, substantially increasing the sequence data available for this important species. overlapping ests were assembled into b. terrestris putative transcripts, and functionally annotated. a preliminary assessment of differences in gene expression across non-replicated specimens from the pre-adult stages, castes and sexes was performed using r-stat analysis. individual samples from the life cycle stages of the bumblebee differed in the expression of a wide array of genes, including genes involved in amino acid storage, metabolism, immunity and olfaction.  detailed analyses of immune and olfaction gene expression across phenotypes demonstrated how transcriptomic analyses can inform our understanding of processes central to the biology of b. terrestris and the social hymenoptera in general. for example, examination of immunity-related genes identified high conservation of important immunity pathway components across individual specimens from the life cycle stages while olfactory-related genes exhibited differential expression with a wider repertoire of gene expression within adults, especially sexuals, in comparison to immature stages. as there is an absence of replication across the samples, the results of this study are preliminary but provide a number of candidate genes which may be related to distinct phenotypic stage expression. this comprehensive transcriptome catalogue will provide an important gene discovery resource for directed programmes in ecology, evolution and conservation of a key pollinator. 