BACKGROUND
the annual arrival of winter in temperate habitats represents a fundamental challenge to the survival and reproduction of a wide variety of insects.
many temperate insects surmount the harsh conditions of winter by undergoing photoperiodic diapause, a process in which day length  provides a token cue that initiates a pre-programmed and hormonally controlled developmental arrest in advance of the onset of unfavorable conditions .
photoperiodic diapause is thus a crucial ecological adaptation enabling temperate insects to coordinate growth, development, reproduction and dormancy in a seasonal environment.
processes related to regulation of development, metabolic depression, stress tolerance and nutrient storage appear to be particularly important physiological components of the diapause response  <cit> .
while many aspects of the physiological and ecological controls of diapause are known, research on the molecular bases of diapause has been hampered by the lack of genetic information from a suitable model organism  <cit> .
drosophila melanogaster has a weak diapause response that is highly temperature-dependent  <cit> , and thus can give only limited insight into the mechanistic basis of photoperiodic diapause  <cit> .
bombyx mori provides a rich source of information on gene expression during diapause.
however, diapause entry in b. mori is controlled by diapause hormone, which is poorly conserved throughout insects  <cit> , and is thus of limited use for comparative analyses.
recently, the advent of high-throughput sequencing methods has facilitated genetic and genomic analyses of life-history traits in non-model systems  <cit> .
these new technologies allow for de novo characterization of genome-wide expression in non-model organisms, and have already led to exciting recent progress on the transcriptional bases of diapause in several insect taxa  <cit> .
an additional factor that has limited progress on understanding the molecular bases of diapause is the wide diversity of diapause syndromes among different insect species.
the diapause program is characterized by three eco-physiological phases: pre-diapause, diapause, and post-diapause  <cit> .
during the pre-diapause phase the individual is sensitive to token environmental cue and in response to appropriate stimuli will initiate preparation for entry into diapause.
during the diapause phase metabolism is reduced and direct development is arrested.
finally, during the post-diapause phase, the individual emerges from diapause and post-diapause direct development is resumed.
all three eco-physiological phases can occur in every stage of the insect life cycle, but an individual species is usually constrained to the diapause phase  during a single stage of the life-cycle  <cit> .
the diversity of life-cycle timing of eco-physiological phases among insects implies a corresponding diversity of molecular and physiological pathways underlying diapause regulation in different insect species.
most studies on the transcriptional bases of diapause have focused on gene expression during the phase of actual developmental arrest .
however, the pre-diapause phase can reveal important insights into the regulation and physiological trajectory of diapausing animals  <cit> .
the asian tiger mosquito, aedes albopictus, is an outstanding emerging model organism for the study of diapause within a well-defined ecological and evolutionary context.
currently considered the most invasive mosquito species in the world  <cit> , in the last  <dig> years, ae.
albopictus has rapidly spread from its native asian range across the world and is currently found in at least  <dig> countries on every continent except antarctica  <cit> .
temperate populations of ae.
albopictus undergo a maternally controlled egg diapause in which exposure of the maternal pupa and adult to short day lengths initiates diapause of the offspring as a pharate larvae inside the chorion of the egg  <cit> .
increased egg desiccation resistance during diapause  <cit>  and rapid evolution of both diapause incidence  and diapause timing  during the range expansion of ae.
albopictus in the us imply that the diapause response has facilitated the rapid global spread of this invasive mosquito.
furthermore, because ae.
albopictus is a vector of dengue and chikungunya viruses, identifying the genetic basis of diapause could potentially provide a platform for developing novel vector control methods  <cit> .
finally, the complete genome sequence has been determined for aedes aegypti  <cit> , a closely related mosquito in the same subgenus  as ae.
albopictus.
the ae.
aegypti genome sequence thus provides a valuable "reference genome" that can be used to annotate ae.
albopictus transcriptome sequences.
here, we use a gs-flx  <dig> platform to sequence and assemble the transcriptome of ae.
albopictus oocytes from females reared under diapause-inducing  and non-diapause-inducing  photoperiods.
our goals were to 1) generate a transcriptome database for the study of ae.
albopictus functional genetics because limited genetic information is currently available for this species; and 2) to identify and verify candidate transcripts involved in the transcriptional bases of diapause preparation.
we leverage the close evolutionary relationship of ae.
albopictus to ae.
aegypti for the annotation of the assembly.
we use differential expression, based on normalized read counts from the di and ndi transcriptomes, to identify a series of candidate genes for diapause preparation, and verify these candidates using quantitative pcr .
we then discuss the putative functional significance of verified differentially expressed genes relative to the molecular physiology of pre-diapause and diapause in other insects.
our transcriptome data enrich the limited sequence information for ae.
albopictus, contribute to our knowledge of gene expression during the pre-diapause phase, and set the stage for comparative analyses both amongst other taxa and relative to other diapause phases of ae.
albopictus.
RESULTS
sequencing, read cleaning and de novo assembly
<dig> gs-flx sequencing was performed on cdna libraries from mature  oocytes of ae.
albopictus females reared under diapause-inducing  or non-diapause-inducing  photoperiods .
we merged reads from both libraries for assembly and annotation.
our quality filtering procedure  removed ~14% of the raw sequenced reads, leaving  <dig> , <dig> reads remaining for further analysis.
the average read length was  <dig> bp, with an n <dig> of  <dig> bp .
these lengths are broadly comparable with other  <dig> transcriptome sequencing studies  <cit> .
raw reads and quality scores are archived at ncbi sequence read archive  under accession srp <dig>
summary statistics of reads from two cdna libraries derived from oocytes from di  and ndi  photoperiod treatments are shown.
our de novo assembly using mira  <dig> .
<dig>  <cit>  yielded  <dig>  contigs, with an n <dig> of  <dig>  bp .
these contigs are accessible at ncbi''s transcript shotgun archive  under accession numbers jo845359-jo <dig>  contig length is one of the many benchmarks that can be used to assess assembly quality, and our assembly shows a comparable mode of contig length relative to similar studies  <cit> .
annotated transcripts from the ae.
aegypti genome, the most suitable reference genome for ae.
albopictus, have an n <dig> of  <dig>  bp .
this result suggests that many of the contigs in our assembly are shorter than the actual transcripts from which they are derived.
this disparity in contig length distributions between de novo transcriptome assemblies and annotated transcripts from genome assemblies is expected, because transcript predictions from the genome assembly are based on both extensive computational  and empirical  evidence  <cit> , whereas the ae.
albopictus transcriptome is currently limited to data from a single source.
de novo assemblies can seldom merge all reads into contigs  <cit> , especially when the abundance of some transcripts is too low for representative reads to be assembled.
however, the "singleton" reads still represent useful sequence information on low-coverage transcripts, and can be included in subsequent analyses of the transcriptome.
in our assembly, 8% of the quality-filtered reads were not assembled , and these singletons were merged with the contig set into a preliminary est set.
functional annotation
to functionally annotate the preliminary est set, we performed a series of blast searches  <cit>  to several reference organisms .
our functional annotations drew heavily from two culicid genomes, ae.
aegypti and culex quinquefasciatus, due to their close phylogenetic relationship with ae.
albopictus.
most ests  matched to ae.
aegypti transcripts and peptides .
percent identities are frequently used as a measure of blast quality  <cit> .
here, percent identities between the ests and their putative homologs declined with phylogenetic relatedness, with the highest percent identities found to putative homologs in aedes spp., and the lowest to c. elegans .
28% of ests  had no significant similarity to any of the databases that we searched, likely due to the conservative e-value cutoffs we used when assigning homology .
the minimum e-value used for the initial est set, and the minimum % identity used for the final est set, varied based on phylogenetic distance of the organism/database and ae.
albopictus.
both the preliminary est set and final, trimmed est set are listed .
homology-based est filtering and trimming
the mira assembler tends to assemble contigs with high redundancy  <cit> , and our dataset is no exception: on average, more than eight ests matched the same gene .
there are both biological and technical reasons for this high redundancy.
for example, alternatively spliced isoforms, or alleles of the same gene, could assemble into separate contigs.
additionally, sequencing errors, incomplete transcript coverage during sequencing, chimeric reads or contigs, or paralogous genes from recently diverged gene families could also contribute to the redundancy.
biologically valid transcript variants are difficult to distinguish from sequencing and assembly errors, particularly in the absence of a reference genome.
to systematically reduce our est set to a non-redundant transcriptome, we took advantage of the homology of the ae.
albopictus ests to the closely related ae.
aegypti genome, as well as homology to other, more distantly related genomes.
for each group of ests that matched a putative homolog, we identified one ''representative'' est, and trimmed this est to its overlap with the putative homolog .
this resulted in  <dig>  non-redundant, ''conservative'' ests with annotations to known genes.
in the subsequent text, we refer to this non-redundant, trimmed est set as the ''final'' est set.
the n <dig> of the final est set improved over that of the redundant ests .
while this conservative approach certainly removed perfectly legitimate sequence data, it systematically avoided chimeric contigs that could occur due to contig mis-assembly, thereby yielding a higher-confidence est set.
the advantage of this approach is that it eliminated redundant ests, as well as annotations with little support from other organisms.
the disadvantages are that genes novel to ae.
albopictus are eliminated, and that some valid sequence information from ests with homology information was discarded.
a similar approach was used by crawford et al.
<cit>  for a non-model mosquito species .
here, the authors utilized significant homology of their ests to other reference genomes to validate contigs and eliminate redundancy, reflecting the fact that est redundancy and uncertainty are common problems in transcriptome assembly of organisms without a genome sequence, and that unique, taxon-specific approaches will often be necessary.
trimmed ests covered an average of 43% of their putative homologs , which indicates that the majority of the final ests do not represent full transcripts.
this result is expected, given the incomplete nature of de novo transcriptome assembly of non-model organisms, as well as our rigorous trimming procedure.
other de novo transcriptome assemblies of mosquito species have also documented incomplete transcript coverage based on comparisons to a closely related reference genome  <cit> .
of the ests that remained in the final contig set, 93% were annotated to aedes spp.
, with high percent identities .
not surprisingly, percent identities of blast matches to other, less closely related organisms were much lower .
we provide fasta files of the trimmed ests, as well as an excel spreadsheet with relevant annotation information at http://albopictusexpression.org.
to evaluate whether our sequencing efforts maximized the number of transcripts captured, we generated a bootstrapped gene accumulation curve  <cit> , based on blastn matches of individual reads to the ae.
aegypti transcriptome.
after an initial steep increase in the number of ae.
aegypti transcripts discovered with increasing read number, the slope of the curve rapidly asymptotes, which implies that our sequencing depth captured most of the ae.
aegypti transcript homologs found in our cdna libraries .
paired with other results indicating that our coverage of putative homologs is incomplete , we conclude that our assembly maximized the number of putative homologs found in our libraries, but that the ests representing these homologs are incomplete.
because of this, our measures of expression under di and ndi conditions will underestimate actual expression, although we do not expect a bias in this estimate between di and ndi libraries.
however, differentially expressed genes with inherently low expression are more likely to go undetected than genes with high expression, in particular when sequencing coverage is low  <cit> .
differential expression  analysis-  <dig> sequence data
we estimated expression levels for each est under di and ndi treatments by mapping reads from each treatment to the final est set.
62% and 61% of reads from the ndi and di libraries, respectively, uniquely mapped to the final est set .
<dig> % of all reads were discarded because they did not map uniquely.
almost all remaining unmapped reads corresponded to the untrimmed est set ; those that did not had low complexity regions over the majority of the read length, and thus were not assembled.
some viral and bacterial genes  were discovered during the annotation process.
these genes were retained for read mapping, but the genes were removed before measuring differential expression, as measuring viral loads was outside the scope of this study.
using tmm-normalized read counts  to initially determine differential expression of ests between photoperiod treatments ,  <dig> ests had significantly higher expression under ndi conditions, and  <dig> ests were over-expressed under di conditions .
additionally, the range of log fold-change tended to be higher under ndi relative to di conditions .
verification of de ests by qpcr
we evaluated differential expression of  <dig> candidate genes using qpcr to validate expression patterns in the  <dig> dataset.
of these,  <dig> were predicted over-expressed under ndi conditions,  <dig> under di conditions, and  <dig> were of a priori interest.
we first asked whether expression levels from the  <dig> dataset and qpcr experiments were correlated, and whether the normalization method of the  <dig> dataset affected these correlations.
in addition to the tmm method that we used to identify candidate genes  <cit> , we calculated rpkm , and asked whether these two measures correlated with mean mrna abundance from the qpcr experiments for ndi and di treatments.
we removed one gene with much higher qpcr than  <dig> expression because it is extremely likely that paralogs were co-amplified by qpcr but were undetected by the qpcr melt-curve analysis.
this gene, a histone 2a homolog , has  <dig> near-identical paralogs in ae.
aegypti, of which eight are 100% identical at the peptide level .
subsequently, correlation coefficients of differential expression for the remaining  <dig> candidate genes assessed by  <dig> sequencing and qpcr were high and resembled those found in previous comparisons of qpcr and rna-seq data .
correspondence between  <dig> and qpcr expression levels was notably higher when  <dig> expression was measured as rpkm, suggesting that rpkm is a more accurate measure of gene expression for our dataset.
correlation coefficients are derived from linear regressions between qpcr mrna abundance and both tmm and rpkm gene expression measures from  <dig> read counts for each gene verified by qpcr.
while correlation coefficients between  <dig> expression and mrna abundance calculated within each photoperiod treatment were very high for expression measures in the di treatment, correlation coefficients were lower for expression measures from the ndi treatment for all analyses .
this raises the question whether this difference is biological  or technical .
to address this question, we calculated coefficients of variation  of mrna abundance from the five replicates of the qpcr data for each gene and photoperiod treatment.
if gene expression were more variable in animals reared under ndi conditions, we would expect higher cvs of mrna abundance for ndi vs. di treatments.
while the median cv was marginally higher among ndi replicates , cvs were not significantly different between photoperiod treatments .
this result suggests that the lower correlation between qpcr and  <dig> data in the ndi dataset is more likely due to technical artifacts, rather than inherently higher variation in gene expression under ndi conditions.
verification of de ests - significance comparisons
perfect congruence between the levels of significance detected using qpcr and  <dig> sequencing is not to be expected, because 1) the  <dig> results were based only on one biological replicate, thus increasing the possibility of both type i and type ii error, and 2) the datasets were acquired using fundamentally different approaches, necessitating alternative analyses to determine significance of de.
while qpcr has some methodological caveats  <cit> , it is generally considered a benchmark for gene expression analyses.
to determine whether significant differential expression in the  <dig> dataset corresponds to significant de in the qpcr data, we calculated sensitivity  and specificity  statistics of the  <dig> dataset.
because our correlation analyses between  <dig> and qpcr expression levels suggest that rpkm performs better at predicting mrna abundance than the tmm normalization, we compared both expression measures'' performance for predicting qpcr significance.
despite the better predictive ability of rpkm on mrna abundance, both expression measures of the  <dig> data were poor predictors of qpcr significance .
this is perhaps not surprising, given the dramatically different methods used to determine significance between  <dig> and qpcr noted above.
despite lack of congruence between  <dig> and qpcr results regarding genes predicted as significantly de,  <dig> of  <dig> genes from the di category had higher average expression under di conditions based on qpcr results, and as such were directionally consistent between both methods .
as expected based on the lower correlations in the ndi data noted above, directional consistency was lower for ndi genes .
these results indicate that  <dig> sequencing as applied in this study can be useful guide to de novo identification of transcriptional differences underlying complex phenotypes, but that replication, either by verification with qpcr, or by sequencing replicated libraries, is essential for verification.
below, we restrict our discussion of transcriptional elements of diapause response in ae.
albopictus to ests verified as de by qpcr.
values for both rpkm and tmm expression measures from the  <dig> data are shown.
sensitivity is calculated as the number of true positives over the number of all positives; specificity is the number of true negatives divided by all negatives.
verification of de ests - functional categories of verified genes
several themes in the insect diapause program have emerged from comparative analyses of diapause-mediated developmental arrest, including dna replication and transcription, endocrine signaling, metabolism, and response to stress  <cit> .
these processes can be important during diapause preparation, but other pathways leading to preparatory functions, such as growth regulation in advance of developmental arrest, accumulation of metabolic reserves, and behavioral or morphological changes to protect against the physiological stresses of the harsh environment  <cit> , may dominate the transcriptional profile during the preparatory phase.
in ae.
albopictus, maternally provisioned transcripts from females reared under short day lengths dictate the development of diapause-destined oocytes; it is not known in ae.
albopictus, nor in most other pre-diapause insects, which of these processes should be transcriptionally dominant.
from qpcr experiments, we found differential expression of ests belonging to the main categories of developmental arrest during diapause, and identified additional ests from pathways that have not yet received attention in previous studies on the molecular bases of diapause .
information on each gene verified as differentially expressed is shown.
putative homologs were derived from blast searches . "
<dig> category" refers to the differential expression status assigned to the est based on read counts: di or ndi, significantly over-expressed under di or ndi conditions, respectively; and a priori, not de based on  <dig> read counts, but of a priori interest based on previous diapause research.
fold-change calculations are based on qpcr results, and were calculated as log <dig>  where di and ndi are the average mrna abundance for di and ndi treatments, respectively .
the corrected p-value is derived from student''s t-test of mrna abundances derived from qpcr reactions.
developmental arrest is the definitive diapause phenotype.
studies of pre-diapause gene expression in other insects suggest that precursors of developmental arrest are initiated during the preparatory phase  <cit> .
in our study, two ests with homologs involved in dna replication and transcription were more abundant in di oocytes: inhibitor of growth protein  and bhlhzip transcription factor bigmax  .
in various systems, ing <dig> has shown involvement in oncogenesis, apoptosis, dna repair and negative cell cycle regulation .
in d. melanogaster, ing <dig> is thought to interact with p <dig>  which is a transcription factor that can respond to stress by affecting cell-cycle arrest, dna repair, apoptosis, or senescence  <cit> .
cell-cycle arrest is one of the unifying themes of diapause  <cit> , thus genes that may affect this arrest are good candidates as developmental regulators.
among its various functions, the transcription factor bhlhzip bigmax appears to be a target of foxo and is involved in regulating metabolism and energy sensing  <cit> .
the importance of foxo in the diapause of culex pipiens, another mosquito  <cit> , and the critical role of energy sensing during diapause  <cit>  suggests possible roles for these two ests in the preparatory phase of diapause in ae.
albopictus.
the endocrine system plays a central role in regulating diapause  <cit> .
although our understanding of the endocrine regulation of embryonic diapause is restricted to just a few species, high levels of ecdysteroids are critical for the induction and maintenance of embryonic diapause in the gypsy moth, lymantria dispar  <cit> .
in pre-diapause ae.
albopictus oocytes, two homologs of genes potentially involved in ecdysone signaling were differentially expressed.
the first, rack <dig>  encodes a receptor for activated protein kinase c, and was more abundant under di conditions.
rack <dig> is known to bind to several different signaling molecules  <cit> , including a molt-associated transcription factor that is linked to the action of 20-hydroxyecdysone  <cit> .
it is highly expressed in ovary tissue and is thought to be required for oogenesis in d. melanogaster; female d. melanogaster homozygous for rack <dig> null alleles show reduced ovary size  <cit> .
rack <dig> has also been implicated in diapause in other insects.
for example, in a study of gene expression in pre-diapause and diapause cricket embryos , rack <dig> expression was higher in pre-diapause embryos, but subsequently decreased during diapause  <cit> .
intriguingly, it has recently been shown that rack <dig> affects circadian rhythm function in mouse  <cit> .
a classic hypothesis proposes that circadian rhythm underlies the expression of photoperiodic diapause  <cit> , although evidence supporting this hypothesis is limited  <cit>  and highly debated .
however, these findings suggest a possibly complex role for rack <dig> in diapause preparation.
the second differentially expressed transcript potentially related to endocrine signaling is ecdysone inducible protein l <dig> , which was less abundant in di oocytes.
the d. melanogaster homolog of eip, imp-l <dig>  has been implicated in ectoderm and neural development  <cit> , is essential for starvation resistance  <cit> , and may have a role in the regulation of growth  <cit> .
loss-of-function alleles of imp-l <dig> in d. melanogaster result in female size increase, which appears to be primarily driven by enlarged ovaries  <cit> .
interestingly, both rack <dig> and eip have phenotypes related to ovary size in d. melanogaster  <cit> .
eggs from ae.
albopictus reared under di conditions are larger and contain more lipids than eggs from females reared under ndi conditions ; we speculate that decreased pre-diapause expression of the ae.
albopictus imp-l <dig> homolog, and/or increased expression of the rack <dig> homolog, could affect ovary and subsequent egg size.
ongoing studies of the expression levels of these genes during embryological development and developmental arrest in pharate ae.
albopictus larvae will provide further insight into the diapause-related function of these transcripts, as well as important comparative results regarding the ecdysone signaling pathway.
diapause is often accompanied by a decrease in metabolism.
one gene involved in metabolism was more abundant in di oocytes: phosphoenolpyruvate carboxykinase .
pepck is part of the gluconeogenesis pathway, and has higher expression levels during diapause in sarcophaga crassipalpis  <cit> .
diapausing insects appear to enhance gluconeogenic pathways, in part due to a shift towards anaerobic metabolism  <cit> .
we hypothesize that the over-expression of this transcript during the pre-diapause phase in ae.
albopictus oocytes may either represent a maternally provisioned regulatory cue, or initiation of the gluconeogenic pathway in advance of the onset of developmental arrest.
the transcription factor bhlhzip bigmax, discussed above, could also contribute to the down-regulation of metabolism  <cit>  associated with diapause.
increased longevity and stress resistance are also important features of the diapause program.
potentially relevant to these phenotypes, a gene encoding a g-protein coupled receptor  from the methuselah family was more abundant in pre-diapause oocytes.
in d. melanogaster, the methuselah  gene is associated with longevity and stress resistance  <cit> .
mth haplotype distributions vary latitudinally in d. melanogaster  <cit> , and studies of allelic variation in mth suggest that the gene contributes to differences in lifespan among populations  <cit> .
while down-regulation of mth, rather than over-expression, should result in greater stress resistance based on results from d. melanogaster, other gpcrs are potentially up-regulated in response to diapause-related stimuli in other organisms.
some gpcrs in c. elegans show strong responses to dauer formation  <cit> , which is analogous to insect diapause.
in bombyx, a gpcr shows high affinity to diapause hormone  <cit> , suggesting it could be important in mediating developmental arrest.
three other genes that did not fall into established functional categories related to diapause had higher expression in pre-diapause oocytes.
the putative ae.
aegypti homologs of all three genes are annotated as "conserved hypothetical proteins".
these genes may be particularly intriguing, as they could relate specifically to diapause preparation in ae.
albopictus or other insects.
however, because their annotations are uncertain, the genes'' functions are highly speculative.
we provide brief descriptions of the genes'' domains and gene ontology categories, if available, recognizing that much more work is necessary to understand the function of these genes in the context of ae.
albopictus diapause.
aael008645''s gene ontology association is ''protein binding''.
it contains evidence for a b <dig> /spry domain, a lish motif, and a cra domain.
aael004873''s gene ontology associations are protein binding and cell adhesion.
it contains a gilt motif , which is thought to be associated with disulphide bond reduction.
while it was predicted to be over-expressed in non-diapause destined oocytes based on rpkm, qpcr experiments show that it is actually more abundant in pre-diapause oocytes.
aael <dig> has several motifs; a galactose-binding-domain-like motif ; and a lish dimerization motif.
previously, we investigated the expression patterns of a putative phosphatidylethanolamine-binding protein  in diapause and non-diapause oocytes in multiple populations of ae.
albopictus  <cit> .
while our present data show significant up-regulation of pebp in a single temperate population under di conditions, the previous study suggested a regional effect on pebp expression in these populations, rather than a simple over-expression of pebp under di conditions.
these results are not necessarily contradictory, as the studies differ in their experimental design and replication: while the previous study utilized more populations, our analysis includes more replicates per photoperiod treatment, and thus may have more statistical power to capture subtle differences in expression levels.
this could indicate that the differential expression of pebp under different photoperiods is population-specific.
there is little information about the role of pebp in insects, but studies from plants have documented differential expression of a pebp gene family member ft  in response to photoperiod  <cit> , which shows differential expression to photoperiod treatments in latitudinally disparate populations in picea abies  <cit> .
these results raise the intriguing possibility that genes from the pebp family may have similar roles or be involved in photoperiodically mediated life history transitions across both plant and animal kingdoms.
three additional genes  implicated in the diapause response of ae.
albopictus based on differential abundance in oocyte tissue  <cit>  exhibited non-significant up-regulation under di conditions in the current study.
because the experimental design of the previous studies differed from the current experiments, exact comparison of results is not possible.
nevertheless, the direction of differential expression is the same, indicating that our current results are qualitatively consistent with these previous studies.
CONCLUSIONS
our goals for this study were: 1) to generate a comprehensive oocyte transcriptome for ae.
albopictus, an emerging model system for studying the evolutionary and ecological genomics of diapause, and 2) to identify candidate genes for diapause preparation in this species.
our assembly of a high-quality, conservative transcriptome for ae.
albopictus oocytes highlights useful approaches to utilizing a closely related reference genome to generating high-confidence ests in a non-model organism.
additionally, the assembly enabled us to identify several candidate genes for diapause preparation that are consistent with established themes of the insect diapause program as well as additional candidates that are potentially unique to ae.
albopictus.
in addition to the potential relevance to diapause, many of these ests are related to fundamental biological processes such as metabolism, stress tolerance, and the endocrine control of development and thus may ultimately provide useful targets for developing novel forms of vector control in this medically significant vector of human disease.
finally, the comprehensive oocyte est database should provide a useful resource for comparative genomics and vector molecular physiology.

 the annual arrival of winter in temperate habitats represents a fundamental challenge to the survival and reproduction of a wide variety of insects. many temperate insects surmount the harsh conditions of winter by undergoing photoperiodic diapause, a process in which day length provides a token cue that initiates a pre-programmed and hormonally controlled developmental arrest in advance of the onset of unfavorable conditions. photoperiodic diapause is thus a crucial ecological adaptation enabling temperate insects to coordinate growth, development, reproduction and dormancy in a seasonal environment. processes related to regulation of development, metabolic depression, stress tolerance and nutrient storage appear to be particularly important physiological components of the diapause response. while many aspects of the physiological and ecological controls of diapause are known, research on the molecular bases of diapause has been hampered by the lack of genetic information from a suitable model organism. drosophila melanogaster has a weak diapause response that is highly temperature-dependent, and thus can give only limited insight into the mechanistic basis of photoperiodic diapause. bombyx mori provides a rich source of information on gene expression during diapause. however, diapause entry in b. mori is controlled by diapause hormone, which is poorly conserved throughout insects, and is thus of limited use for comparative analyses. recently, the advent of high-throughput sequencing methods has facilitated genetic and genomic analyses of life-history traits in non-model systems. these new technologies allow for de novo characterization of genome-wide expression in non-model organisms, and have already led to exciting recent progress on the transcriptional bases of diapause in several insect taxa. an additional factor that has limited progress on understanding the molecular bases of diapause is the wide diversity of diapause syndromes among different insect species. the diapause program is characterized by three eco-physiological phases: pre-diapause, diapause, and post-diapause. during the pre-diapause phase the individual is sensitive to token environmental cue and in response to appropriate stimuli will initiate preparation for entry into diapause. during the diapause phase metabolism is reduced and direct development is arrested. finally, during the post-diapause phase, the individual emerges from diapause and post-diapause direct development is resumed. all three eco-physiological phases can occur in every stage of the insect life cycle, but an individual species is usually constrained to the diapause phase during a single stage of the life-cycle. the diversity of life-cycle timing of eco-physiological phases among insects implies a corresponding diversity of molecular and physiological pathways underlying diapause regulation in different insect species. most studies on the transcriptional bases of diapause have focused on gene expression during the phase of actual developmental arrest. however, the pre-diapause phase can reveal important insights into the regulation and physiological trajectory of diapausing animals. the asian tiger mosquito, aedes albopictus, is an outstanding emerging model organism for the study of diapause within a well-defined ecological and evolutionary context. currently considered the most invasive mosquito species in the world, in the last years, ae. albopictus has rapidly spread from its native asian range across the world and is currently found in at least countries on every continent except antarctica. temperate populations of ae. albopictus undergo a maternally controlled egg diapause in which exposure of the maternal pupa and adult to short day lengths initiates diapause of the offspring as a pharate larvae inside the chorion of the egg. increased egg desiccation resistance during diapause and rapid evolution of both diapause incidence and diapause timing during the range expansion of ae. albopictus in the us imply that the diapause response has facilitated the rapid global spread of this invasive mosquito. furthermore, because ae. albopictus is a vector of dengue and chikungunya viruses, identifying the genetic basis of diapause could potentially provide a platform for developing novel vector control methods. finally, the complete genome sequence has been determined for aedes aegypti, a closely related mosquito in the same subgenus as ae. albopictus. the ae. aegypti genome sequence thus provides a valuable "reference genome" that can be used to annotate ae. albopictus transcriptome sequences. here, we use a gs-flx platform to sequence and assemble the transcriptome of ae. albopictus oocytes from females reared under diapause-inducing and non-diapause-inducing photoperiods. our goals were to 1) generate a transcriptome database for the study of ae. albopictus functional genetics because limited genetic information is currently available for this species; and 2) to identify and verify candidate transcripts involved in the transcriptional bases of diapause preparation. we leverage the close evolutionary relationship of ae. albopictus to ae. aegypti for the annotation of the assembly. we use differential expression, based on normalized read counts from the di and ndi transcriptomes, to identify a series of candidate genes for diapause preparation, and verify these candidates using quantitative pcr. we then discuss the putative functional significance of verified differentially expressed genes relative to the molecular physiology of pre-diapause and diapause in other insects. our transcriptome data enrich the limited sequence information for ae. albopictus, contribute to our knowledge of gene expression during the pre-diapause phase, and set the stage for comparative analyses both amongst other taxa and relative to other diapause phases of ae. albopictus.  sequencing, read cleaning and de novo assembly gs-flx sequencing was performed on cdna libraries from mature oocytes of ae. albopictus females reared under diapause-inducing or non-diapause-inducing photoperiods. we merged reads from both libraries for assembly and annotation. our quality filtering procedure removed ~14% of the raw sequenced reads, leaving, reads remaining for further analysis. the average read length was bp, with an n of bp. these lengths are broadly comparable with other transcriptome sequencing studies. raw reads and quality scores are archived at ncbi sequence read archive under accession srp summary statistics of reads from two cdna libraries derived from oocytes from di and ndi photoperiod treatments are shown. our de novo assembly using mira. yielded contigs, with an n of bp. these contigs are accessible at ncbi''s transcript shotgun archive under accession numbers jo845359-jo contig length is one of the many benchmarks that can be used to assess assembly quality, and our assembly shows a comparable mode of contig length relative to similar studies. annotated transcripts from the ae. aegypti genome, the most suitable reference genome for ae. albopictus, have an n of bp. this result suggests that many of the contigs in our assembly are shorter than the actual transcripts from which they are derived. this disparity in contig length distributions between de novo transcriptome assemblies and annotated transcripts from genome assemblies is expected, because transcript predictions from the genome assembly are based on both extensive computational and empirical evidence, whereas the ae. albopictus transcriptome is currently limited to data from a single source. de novo assemblies can seldom merge all reads into contigs, especially when the abundance of some transcripts is too low for representative reads to be assembled. however, the "singleton" reads still represent useful sequence information on low-coverage transcripts, and can be included in subsequent analyses of the transcriptome. in our assembly, 8% of the quality-filtered reads were not assembled, and these singletons were merged with the contig set into a preliminary est set. functional annotation to functionally annotate the preliminary est set, we performed a series of blast searches to several reference organisms. our functional annotations drew heavily from two culicid genomes, ae. aegypti and culex quinquefasciatus, due to their close phylogenetic relationship with ae. albopictus. most ests matched to ae. aegypti transcripts and peptides. percent identities are frequently used as a measure of blast quality. here, percent identities between the ests and their putative homologs declined with phylogenetic relatedness, with the highest percent identities found to putative homologs in aedes spp., and the lowest to c. elegans. 28% of ests had no significant similarity to any of the databases that we searched, likely due to the conservative e-value cutoffs we used when assigning homology. the minimum e-value used for the initial est set, and the minimum % identity used for the final est set, varied based on phylogenetic distance of the organism/database and ae. albopictus. both the preliminary est set and final, trimmed est set are listed. homology-based est filtering and trimming the mira assembler tends to assemble contigs with high redundancy, and our dataset is no exception: on average, more than eight ests matched the same gene. there are both biological and technical reasons for this high redundancy. for example, alternatively spliced isoforms, or alleles of the same gene, could assemble into separate contigs. additionally, sequencing errors, incomplete transcript coverage during sequencing, chimeric reads or contigs, or paralogous genes from recently diverged gene families could also contribute to the redundancy. biologically valid transcript variants are difficult to distinguish from sequencing and assembly errors, particularly in the absence of a reference genome. to systematically reduce our est set to a non-redundant transcriptome, we took advantage of the homology of the ae. albopictus ests to the closely related ae. aegypti genome, as well as homology to other, more distantly related genomes. for each group of ests that matched a putative homolog, we identified one''representative'' est, and trimmed this est to its overlap with the putative homolog. this resulted in non-redundant,''conservative'' ests with annotations to known genes. in the subsequent text, we refer to this non-redundant, trimmed est set as the''final'' est set. the n of the final est set improved over that of the redundant ests. while this conservative approach certainly removed perfectly legitimate sequence data, it systematically avoided chimeric contigs that could occur due to contig mis-assembly, thereby yielding a higher-confidence est set. the advantage of this approach is that it eliminated redundant ests, as well as annotations with little support from other organisms. the disadvantages are that genes novel to ae. albopictus are eliminated, and that some valid sequence information from ests with homology information was discarded. a similar approach was used by crawford et al. for a non-model mosquito species. here, the authors utilized significant homology of their ests to other reference genomes to validate contigs and eliminate redundancy, reflecting the fact that est redundancy and uncertainty are common problems in transcriptome assembly of organisms without a genome sequence, and that unique, taxon-specific approaches will often be necessary. trimmed ests covered an average of 43% of their putative homologs, which indicates that the majority of the final ests do not represent full transcripts. this result is expected, given the incomplete nature of de novo transcriptome assembly of non-model organisms, as well as our rigorous trimming procedure. other de novo transcriptome assemblies of mosquito species have also documented incomplete transcript coverage based on comparisons to a closely related reference genome. of the ests that remained in the final contig set, 93% were annotated to aedes spp., with high percent identities. not surprisingly, percent identities of blast matches to other, less closely related organisms were much lower. we provide fasta files of the trimmed ests, as well as an excel spreadsheet with relevant annotation information at http://albopictusexpression.org. to evaluate whether our sequencing efforts maximized the number of transcripts captured, we generated a bootstrapped gene accumulation curve, based on blastn matches of individual reads to the ae. aegypti transcriptome. after an initial steep increase in the number of ae. aegypti transcripts discovered with increasing read number, the slope of the curve rapidly asymptotes, which implies that our sequencing depth captured most of the ae. aegypti transcript homologs found in our cdna libraries. paired with other results indicating that our coverage of putative homologs is incomplete, we conclude that our assembly maximized the number of putative homologs found in our libraries, but that the ests representing these homologs are incomplete. because of this, our measures of expression under di and ndi conditions will underestimate actual expression, although we do not expect a bias in this estimate between di and ndi libraries. however, differentially expressed genes with inherently low expression are more likely to go undetected than genes with high expression, in particular when sequencing coverage is low. differential expression analysis- sequence data we estimated expression levels for each est under di and ndi treatments by mapping reads from each treatment to the final est set. 62% and 61% of reads from the ndi and di libraries, respectively, uniquely mapped to the final est set. % of all reads were discarded because they did not map uniquely. almost all remaining unmapped reads corresponded to the untrimmed est set; those that did not had low complexity regions over the majority of the read length, and thus were not assembled. some viral and bacterial genes were discovered during the annotation process. these genes were retained for read mapping, but the genes were removed before measuring differential expression, as measuring viral loads was outside the scope of this study. using tmm-normalized read counts to initially determine differential expression of ests between photoperiod treatments, ests had significantly higher expression under ndi conditions, and ests were over-expressed under di conditions. additionally, the range of log fold-change tended to be higher under ndi relative to di conditions. verification of de ests by qpcr we evaluated differential expression of candidate genes using qpcr to validate expression patterns in the dataset. of these, were predicted over-expressed under ndi conditions, under di conditions, and were of a priori interest. we first asked whether expression levels from the dataset and qpcr experiments were correlated, and whether the normalization method of the dataset affected these correlations. in addition to the tmm method that we used to identify candidate genes, we calculated rpkm, and asked whether these two measures correlated with mean mrna abundance from the qpcr experiments for ndi and di treatments. we removed one gene with much higher qpcr than expression because it is extremely likely that paralogs were co-amplified by qpcr but were undetected by the qpcr melt-curve analysis. this gene, a histone 2a homolog, has near-identical paralogs in ae. aegypti, of which eight are 100% identical at the peptide level. subsequently, correlation coefficients of differential expression for the remaining candidate genes assessed by sequencing and qpcr were high and resembled those found in previous comparisons of qpcr and rna-seq data. correspondence between and qpcr expression levels was notably higher when expression was measured as rpkm, suggesting that rpkm is a more accurate measure of gene expression for our dataset. correlation coefficients are derived from linear regressions between qpcr mrna abundance and both tmm and rpkm gene expression measures from read counts for each gene verified by qpcr. while correlation coefficients between expression and mrna abundance calculated within each photoperiod treatment were very high for expression measures in the di treatment, correlation coefficients were lower for expression measures from the ndi treatment for all analyses. this raises the question whether this difference is biological or technical. to address this question, we calculated coefficients of variation of mrna abundance from the five replicates of the qpcr data for each gene and photoperiod treatment. if gene expression were more variable in animals reared under ndi conditions, we would expect higher cvs of mrna abundance for ndi vs. di treatments. while the median cv was marginally higher among ndi replicates, cvs were not significantly different between photoperiod treatments. this result suggests that the lower correlation between qpcr and data in the ndi dataset is more likely due to technical artifacts, rather than inherently higher variation in gene expression under ndi conditions. verification of de ests - significance comparisons perfect congruence between the levels of significance detected using qpcr and sequencing is not to be expected, because 1) the results were based only on one biological replicate, thus increasing the possibility of both type i and type ii error, and 2) the datasets were acquired using fundamentally different approaches, necessitating alternative analyses to determine significance of de. while qpcr has some methodological caveats, it is generally considered a benchmark for gene expression analyses. to determine whether significant differential expression in the dataset corresponds to significant de in the qpcr data, we calculated sensitivity and specificity statistics of the dataset. because our correlation analyses between and qpcr expression levels suggest that rpkm performs better at predicting mrna abundance than the tmm normalization, we compared both expression measures'' performance for predicting qpcr significance. despite the better predictive ability of rpkm on mrna abundance, both expression measures of the data were poor predictors of qpcr significance. this is perhaps not surprising, given the dramatically different methods used to determine significance between and qpcr noted above. despite lack of congruence between and qpcr results regarding genes predicted as significantly de, of genes from the di category had higher average expression under di conditions based on qpcr results, and as such were directionally consistent between both methods. as expected based on the lower correlations in the ndi data noted above, directional consistency was lower for ndi genes. these results indicate that sequencing as applied in this study can be useful guide to de novo identification of transcriptional differences underlying complex phenotypes, but that replication, either by verification with qpcr, or by sequencing replicated libraries, is essential for verification. below, we restrict our discussion of transcriptional elements of diapause response in ae. albopictus to ests verified as de by qpcr. values for both rpkm and tmm expression measures from the data are shown. sensitivity is calculated as the number of true positives over the number of all positives; specificity is the number of true negatives divided by all negatives. verification of de ests - functional categories of verified genes several themes in the insect diapause program have emerged from comparative analyses of diapause-mediated developmental arrest, including dna replication and transcription, endocrine signaling, metabolism, and response to stress. these processes can be important during diapause preparation, but other pathways leading to preparatory functions, such as growth regulation in advance of developmental arrest, accumulation of metabolic reserves, and behavioral or morphological changes to protect against the physiological stresses of the harsh environment, may dominate the transcriptional profile during the preparatory phase. in ae. albopictus, maternally provisioned transcripts from females reared under short day lengths dictate the development of diapause-destined oocytes; it is not known in ae. albopictus, nor in most other pre-diapause insects, which of these processes should be transcriptionally dominant. from qpcr experiments, we found differential expression of ests belonging to the main categories of developmental arrest during diapause, and identified additional ests from pathways that have not yet received attention in previous studies on the molecular bases of diapause. information on each gene verified as differentially expressed is shown. putative homologs were derived from blast searches. " category" refers to the differential expression status assigned to the est based on read counts: di or ndi, significantly over-expressed under di or ndi conditions, respectively; and a priori, not de based on read counts, but of a priori interest based on previous diapause research. fold-change calculations are based on qpcr results, and were calculated as log where di and ndi are the average mrna abundance for di and ndi treatments, respectively. the corrected p-value is derived from student''s t-test of mrna abundances derived from qpcr reactions. developmental arrest is the definitive diapause phenotype. studies of pre-diapause gene expression in other insects suggest that precursors of developmental arrest are initiated during the preparatory phase. in our study, two ests with homologs involved in dna replication and transcription were more abundant in di oocytes: inhibitor of growth protein and bhlhzip transcription factor bigmax. in various systems, ing has shown involvement in oncogenesis, apoptosis, dna repair and negative cell cycle regulation. in d. melanogaster, ing is thought to interact with p which is a transcription factor that can respond to stress by affecting cell-cycle arrest, dna repair, apoptosis, or senescence. cell-cycle arrest is one of the unifying themes of diapause, thus genes that may affect this arrest are good candidates as developmental regulators. among its various functions, the transcription factor bhlhzip bigmax appears to be a target of foxo and is involved in regulating metabolism and energy sensing. the importance of foxo in the diapause of culex pipiens, another mosquito, and the critical role of energy sensing during diapause suggests possible roles for these two ests in the preparatory phase of diapause in ae. albopictus. the endocrine system plays a central role in regulating diapause. although our understanding of the endocrine regulation of embryonic diapause is restricted to just a few species, high levels of ecdysteroids are critical for the induction and maintenance of embryonic diapause in the gypsy moth, lymantria dispar. in pre-diapause ae. albopictus oocytes, two homologs of genes potentially involved in ecdysone signaling were differentially expressed. the first, rack encodes a receptor for activated protein kinase c, and was more abundant under di conditions. rack is known to bind to several different signaling molecules, including a molt-associated transcription factor that is linked to the action of 20-hydroxyecdysone. it is highly expressed in ovary tissue and is thought to be required for oogenesis in d. melanogaster; female d. melanogaster homozygous for rack null alleles show reduced ovary size. rack has also been implicated in diapause in other insects. for example, in a study of gene expression in pre-diapause and diapause cricket embryos, rack expression was higher in pre-diapause embryos, but subsequently decreased during diapause. intriguingly, it has recently been shown that rack affects circadian rhythm function in mouse. a classic hypothesis proposes that circadian rhythm underlies the expression of photoperiodic diapause, although evidence supporting this hypothesis is limited and highly debated. however, these findings suggest a possibly complex role for rack in diapause preparation. the second differentially expressed transcript potentially related to endocrine signaling is ecdysone inducible protein l, which was less abundant in di oocytes. the d. melanogaster homolog of eip, imp-l has been implicated in ectoderm and neural development, is essential for starvation resistance, and may have a role in the regulation of growth. loss-of-function alleles of imp-l in d. melanogaster result in female size increase, which appears to be primarily driven by enlarged ovaries. interestingly, both rack and eip have phenotypes related to ovary size in d. melanogaster. eggs from ae. albopictus reared under di conditions are larger and contain more lipids than eggs from females reared under ndi conditions; we speculate that decreased pre-diapause expression of the ae. albopictus imp-l homolog, and/or increased expression of the rack homolog, could affect ovary and subsequent egg size. ongoing studies of the expression levels of these genes during embryological development and developmental arrest in pharate ae. albopictus larvae will provide further insight into the diapause-related function of these transcripts, as well as important comparative results regarding the ecdysone signaling pathway. diapause is often accompanied by a decrease in metabolism. one gene involved in metabolism was more abundant in di oocytes: phosphoenolpyruvate carboxykinase. pepck is part of the gluconeogenesis pathway, and has higher expression levels during diapause in sarcophaga crassipalpis. diapausing insects appear to enhance gluconeogenic pathways, in part due to a shift towards anaerobic metabolism. we hypothesize that the over-expression of this transcript during the pre-diapause phase in ae. albopictus oocytes may either represent a maternally provisioned regulatory cue, or initiation of the gluconeogenic pathway in advance of the onset of developmental arrest. the transcription factor bhlhzip bigmax, discussed above, could also contribute to the down-regulation of metabolism associated with diapause. increased longevity and stress resistance are also important features of the diapause program. potentially relevant to these phenotypes, a gene encoding a g-protein coupled receptor from the methuselah family was more abundant in pre-diapause oocytes. in d. melanogaster, the methuselah gene is associated with longevity and stress resistance. mth haplotype distributions vary latitudinally in d. melanogaster, and studies of allelic variation in mth suggest that the gene contributes to differences in lifespan among populations. while down-regulation of mth, rather than over-expression, should result in greater stress resistance based on results from d. melanogaster, other gpcrs are potentially up-regulated in response to diapause-related stimuli in other organisms. some gpcrs in c. elegans show strong responses to dauer formation, which is analogous to insect diapause. in bombyx, a gpcr shows high affinity to diapause hormone, suggesting it could be important in mediating developmental arrest. three other genes that did not fall into established functional categories related to diapause had higher expression in pre-diapause oocytes. the putative ae. aegypti homologs of all three genes are annotated as "conserved hypothetical proteins". these genes may be particularly intriguing, as they could relate specifically to diapause preparation in ae. albopictus or other insects. however, because their annotations are uncertain, the genes'' functions are highly speculative. we provide brief descriptions of the genes'' domains and gene ontology categories, if available, recognizing that much more work is necessary to understand the function of these genes in the context of ae. albopictus diapause. aael008645''s gene ontology association is''protein binding''. it contains evidence for a b /spry domain, a lish motif, and a cra domain. aael004873''s gene ontology associations are protein binding and cell adhesion. it contains a gilt motif, which is thought to be associated with disulphide bond reduction. while it was predicted to be over-expressed in non-diapause destined oocytes based on rpkm, qpcr experiments show that it is actually more abundant in pre-diapause oocytes. aael has several motifs; a galactose-binding-domain-like motif; and a lish dimerization motif. previously, we investigated the expression patterns of a putative phosphatidylethanolamine-binding protein in diapause and non-diapause oocytes in multiple populations of ae. albopictus. while our present data show significant up-regulation of pebp in a single temperate population under di conditions, the previous study suggested a regional effect on pebp expression in these populations, rather than a simple over-expression of pebp under di conditions. these results are not necessarily contradictory, as the studies differ in their experimental design and replication: while the previous study utilized more populations, our analysis includes more replicates per photoperiod treatment, and thus may have more statistical power to capture subtle differences in expression levels. this could indicate that the differential expression of pebp under different photoperiods is population-specific. there is little information about the role of pebp in insects, but studies from plants have documented differential expression of a pebp gene family member ft in response to photoperiod, which shows differential expression to photoperiod treatments in latitudinally disparate populations in picea abies. these results raise the intriguing possibility that genes from the pebp family may have similar roles or be involved in photoperiodically mediated life history transitions across both plant and animal kingdoms. three additional genes implicated in the diapause response of ae. albopictus based on differential abundance in oocyte tissue exhibited non-significant up-regulation under di conditions in the current study. because the experimental design of the previous studies differed from the current experiments, exact comparison of results is not possible. nevertheless, the direction of differential expression is the same, indicating that our current results are qualitatively consistent with these previous studies.  our goals for this study were: 1) to generate a comprehensive oocyte transcriptome for ae. albopictus, an emerging model system for studying the evolutionary and ecological genomics of diapause, and 2) to identify candidate genes for diapause preparation in this species. our assembly of a high-quality, conservative transcriptome for ae. albopictus oocytes highlights useful approaches to utilizing a closely related reference genome to generating high-confidence ests in a non-model organism. additionally, the assembly enabled us to identify several candidate genes for diapause preparation that are consistent with established themes of the insect diapause program as well as additional candidates that are potentially unique to ae. albopictus. in addition to the potential relevance to diapause, many of these ests are related to fundamental biological processes such as metabolism, stress tolerance, and the endocrine control of development and thus may ultimately provide useful targets for developing novel forms of vector control in this medically significant vector of human disease. finally, the comprehensive oocyte est database should provide a useful resource for comparative genomics and vector molecular physiology. 
albopictus is a medically significant vector and is currently considered the most invasive mosquito in the world. our transcriptome database provides a rich resource for the comparative genomics and functional genetics of ae. our contig filtering approach, where we took advantage of strong sequence similarity to the fully sequenced genome of aedes aegypti, as well as other reference organisms, resulted in high-quality, conservative ests. analysis of expression by qpcr for of these genes indicated a high correlation of expression levels between sequence data and qpcr, but congruence of statistically significant differential expression was low. the asian tiger mosquito, aedes albopictus, represents an outstanding emerging model to investigate the molecular basis of photoperiodic diapause in a well-defined ecological and evolutionary context. albopictus oocytes destined to become diapausing or non-diapausing pharate larvae. finally, our analysis illustrates a useful approach that draws from a closely related reference genome to generate high-confidence ests in a non-model organism. many temperate insects survive the harsh conditions of winter by undergoing photoperiodic diapause, a pre-programmed developmental arrest initiated by short day lengths. to generate novel sequence information for this species, as well as to discover transcripts involved in diapause preparation, we sequenced the transcriptome of ae. seven genes identified as differentially expressed based on qpcr have putative functions that are consistent with the insect diapause syndrome; three genes have unknown function and represent novel candidates for the transcriptional basis of diapause. additionally, the identification of differentially expressed transcripts related to diapause enriches the limited knowledge base for the molecular basis of insect diapause, in particular for the preparatory stage. despite the well-established ecological significance of photoperiodic diapause, the molecular basis of this crucial adaptation remains largely unresolved.
 many temperate insects survive the harsh conditions of winter by undergoing photoperiodic diapause, a pre-programmed developmental arrest initiated by short day lengths. despite the well-established ecological significance of photoperiodic diapause, the molecular basis of this crucial adaptation remains largely unresolved. the asian tiger mosquito, aedes albopictus, represents an outstanding emerging model to investigate the molecular basis of photoperiodic diapause in a well-defined ecological and evolutionary context. ae. albopictus is a medically significant vector and is currently considered the most invasive mosquito in the world. traits related to diapause appear to be important factors contributing to the rapid spread of this mosquito. to generate novel sequence information for this species, as well as to discover transcripts involved in diapause preparation, we sequenced the transcriptome of ae. albopictus oocytes destined to become diapausing or non-diapausing pharate larvae.  gs-flx transcriptome sequencing yielded > million quality-filtered reads, which we assembled into contigs. our contig filtering approach, where we took advantage of strong sequence similarity to the fully sequenced genome of aedes aegypti, as well as other reference organisms, resulted in high-quality, conservative ests. differential expression estimates based on normalized read counts revealed genes with higher expression, and with lower expression under diapause-inducing conditions. analysis of expression by qpcr for of these genes indicated a high correlation of expression levels between sequence data and qpcr, but congruence of statistically significant differential expression was low. seven genes identified as differentially expressed based on qpcr have putative functions that are consistent with the insect diapause syndrome; three genes have unknown function and represent novel candidates for the transcriptional basis of diapause.  our transcriptome database provides a rich resource for the comparative genomics and functional genetics of ae. albopictus, an invasive and medically important mosquito. additionally, the identification of differentially expressed transcripts related to diapause enriches the limited knowledge base for the molecular basis of insect diapause, in particular for the preparatory stage. finally, our analysis illustrates a useful approach that draws from a closely related reference genome to generate high-confidence ests in a non-model organism. 