BACKGROUND
many male and female crabs have a number of significant differences in their biology and economic value, such as growth rate and body size.
a study of gonadal differentiation and development in crabs would be helpful for the aquaculture industry.
among the four species of the genus scylla, the green mud crab  is the most prevalent species in the southeast coast of china , and it has become one of the most important marine aquaculture species in china because of its huge economic value.
at present, however, the demand for this crab far exceeds the supply.
a major challenge currently facing the crab aquaculture industry is the need to form a series of viable seed production techniques to help increase supply.
accordingly, it is important to understand the regulatory mechanisms of sex determination, gonadal differentiation and maturation, and gametogenesis in this species.
to understand these mechanisms at the molecular level in more detail, the regulatory pathways associated with gonadal differences need to be studied at the genomic level.
many methods, such as traditional suppression subtractive hybridization , annealing control primer , differential display rt-pcr   <cit> , est sequencing  <cit> , and microarray  <cit> , have been applied in research into the crustacean reproductive system, and many differentially expressed genes involved in sex determination, gametogenesis, and gonadal differentiation and maturation have been identified and characterized.
some of these genes were detected in crabs; for example, cell division cycle  <dig>   <cit> , cyclin b
<cit> , extracellular regulated protein kinase  <dig>   <cit> , and small ubiquitin-related modifier  <dig>   <cit>  in s. paramamosain, and double-sex and mab- <dig> related transcription factor   <cit>  in eriocheir sinensis.
several of the differentially expressed genes were found in shrimps; for example, tra- <dig>  <cit> , mitogen-activated protein kinase  <dig>   <cit> , prostaglandin reductase  <dig>  <cit> , and ubiquitin specific peptidase  <dig>  x-linked  <cit>  in penaeus monodon, and proliferating cell nuclear antigen   <cit> , ubiquitin-conjugating enzyme e2r   <cit> , and heat shock protein  <dig>   <cit>  in marsupenaeus japonicus.
however, because of the limited amount of genomic data that is available for crustaceans, it is difficult to identify genes and construct regulatory networks associated with crab reproduction.
expressed sequence tags  represent effective transcription fragments of whole genomes, avoiding non-coding and repetitive sequences.
ests contain a huge amount of functional information that can greatly accelerate the research and discovery of new genes  <cit>  and molecular markers  <cit> .
next-generation high-throughput sequencing technologies have been used to generate large amounts of large-scale transcript sequences and gene expression data for many species when the genomic sequences are not available.
these technologies include the roche/ <dig> platform  <cit> , the illumina/solexa system  <cit> , and the abi solid technology  <cit> , all of which have high-accuracy, high-speed, and low-cost compared with first-generation sequencing technology .
here a roche/ <dig> gs flx sequencing system was used for deep-coverage rna sequencing of s. paramamosain during different gonadal developing stages to generate a transcriptome database that will enlarge the public est database for this species and help support future studies.
the transcriptomic analysis will help to identify gonad-specific/differential genes by comparing the transcripts in the testis and ovary libraries, which will help build a more complete understanding of the regulatory mechanisms associated with gonadal differentiation and reproductive processes.
RESULTS
<dig> sequencing and assembly of green mud crab cdna libraries
two cdna libraries were constructed from the testes and ovaries of green mud crabs.
the roche/ <dig> sequencing generated  <dig>  reads from the testis and  <dig>  reads from the ovary with average sequence lengths of 290 bp and 281 bp for the testis and ovary reads respectively.
more than half the reads  were between 300 bp and 450 bp in length, the longest read was 735 bp and the shortest read was less than 50 bp, and the overall length of the reads was 104 mb.
the size distribution of the raw reads is shown in figure  <dig>  the raw reads from these two libraries have been deposited in genbank with the following accession numbers:  and .figure  <dig>
length distribution of all reads generated by  <dig> sequencing.
x-axis:size distribution of all raw reads,y-axis:the number of reads in different length range.
of all the reads, more than half  of them were between 300 bp and 450 bp.
the longest read was 735 bp and the shortest read was less than 50 bp.
after filtering out the adaptor primers and low-quality and very short  reads,  <dig>   clean reads with an average sequence length of 274 bp remained.
the clean reads in the two libraries were assembled separately.
altogether,  <dig>  isotigs  were assembled, leaving  <dig>  reads  as singlets.
the isotigs varied from  <dig> to  <dig>  bp in length, and the singlets varied from  <dig> to 500 bp in length.
more than three-fourths of the isotigs  were from  <dig> to 600 bp long.
a statistical analysis of the assembly is presented in table  <dig>  and the length distribution of the isotigs is shown in figure  <dig> table  <dig>
summary statistics of the rna sequencing of the
s.paramamosain
the comparison between two libraries,length distribution of the assembled isotigs.
x-axis:size distribution of assembled isotigs,y-axis:the number of isotigs in different length range.
the length of isotigs varied from  <dig> to 2000 bp, more than three fourths  of the isotigs occupied a range of 200-600 bp long.
annotation of the unique sequences in the two libraries
annotation of the assembled  sequences was carried out using blastx searches against the ncbi non-redundant  protein sequence database.
we identified  <dig>  unique sequences  that shared similarities with known protein sequences;  <dig>   sequences shared significant similarities with the matched sequences , while the other  <dig>  sequences shared a weak similarity .
the e-value distribution for the annotated sequences is shown in figure  <dig> figure  <dig>
distribution of the e-values for annotated sequences.
x-axis:e-values distribution of annotated sequences,y-axis:the number of sequences in different e-values range.
in total,  <dig>   unique sequences share significant similarity with the matched sequences , while  <dig>  of unique sequences left share weak similarity with the matched sequences with e value between 1e- <dig> to 1e- <dig>
the  <dig>  annotated sequences matched known sequences from  <dig> different species.
most numbers of sequence hits were to the water flea daphnia pulex , the red flour beetle tribolium castaneum , the body louse pediculus humanus corporis , and the florida lancelet branchiostoma floridae .
except for the top  <dig> species with sequence hits,  <dig>   of the annotated sequences matched sequences from other species .
the top six crustacean species with the most number of sequence hits were s. paramamosain , p. monodon , litopenaeus vannamei , e. sinensis , procambarus clarkia , and homarus americanus .figure  <dig>
species that match to the annotated sequences of
s. paramamosain.
x-axis:the number of annotated sequences matching each species, y-axis:distribution of top species that match to the annotated sequences.
gene ontology analysis
the blast2go program was used to assign gene ontology  terms to the  <dig>  annotated sequences to predict the functions of the unique sequences and the translated proteins that they encode.
a total of  <dig> ,  <dig>  and  <dig>  sequences were assigned to the biological process, cellular component, and molecular function go categories, respectively.
the distributions of the annotated sequences in the three go categories  are shown in figure  <dig>  in the biological process category, the metabolic process  and cellular process  level  <dig> terms were the most abundant terms.
in the molecular function category, catalytic activity  and binding  were the most abundant, while, in the cellular component category, cell , cell part , organelle part , and macromolecular complex  were the most abundant level  <dig> terms.figure  <dig>
distributions of the annotated sequences in three go categories .
a: biological process, b: molecular function, c: cellular component.
functional classification based on kegg analysis
functional classification and pathway assignments were based on a kyoto encyclopedia of genes and genomes  analysis.
among the  <dig>  annotated sequences,  <dig> that were annotated with an enzyme code ec number were mapped to  <dig> kegg pathways.
the top five kegg pathways were metabolic pathways, biosynthesis of secondary metabolites, spliceosome, microbial metabolism in diverse environments, and oxidative phosphorylation pathway.
some sequences were mapped to several pathways related with reproduction, growth, development, and immunity; for example, cell cycle, mapk signaling pathway, ubiquitin mediated proteolysis, oocyte meiosis, progesterone-mediated oocyte maturation, gnrh signaling pathway, wnt signaling pathway, hedgehog signaling pathway, p <dig> signaling pathway, and toll-like receptor signaling pathway.
the distribution of the mapped kegg pathways is presented in figure  <dig> figure  <dig>
distribution of the mapped kegg pathways.
x-axis: distribution of kegg pathways, y-axis: the number of sequences mapped into each kegg pathway *represents important pathways.
analysis of gonad-differentially and specifically expressed genes
by comparing the sequences in the ovary and testis libraries,  <dig>  gonad-differentially expressed genes ,  <dig>  ovary-specifically expressed genes and  <dig>  testis-specifically expressed genes were identified.
from these,  <dig> differentially,  <dig> testis-specifically, and  <dig> ovary-specifically expressed transcripts were chosen for validation by quantitative real-time pcr  and semi-quantitative pcr.
as shown in figure 7a and b, the qrt-pcr result confirmed that  <dig>  of the  <dig> differentially expressed transcripts were gonad-differentially expressed.
this finding corresponds with the results from the fisher p test.
semi-quantitative pcr confirmed that  <dig> of the  <dig> ovary-specifically expressed transcripts were expressed only in the ovaries , while the other  <dig> transcripts were expressed in both ovaries and testes.
moreover, among these  <dig> transcripts, ten were found to be expressed significantly higher in ovaries than in testes by qrt-pcr validation .
similarly, only  <dig> of the  <dig> testis-specifically expressed transcripts were verified as testis-specifically expressed by semi-quantitative pcr .
the expressions of the other  <dig> transcripts were examined by qrt-pcr, and the result showed that only seven of them  were significantly differentially expressed in ovaries and testes .figure  <dig>
expression analysis of gonad differentially expressed transcripts by qrt-pcr.
x-axis: the serial number of gonad differentially-expressed transcripts, y-axis: relative expression of each transcripts.
18s rrna is as internal control gene.
*indicates significantly differential expression, **indicates most significantly differential expression .
a&b  <dig> differentially expressed transcripts were chosen for quantitative real-time pcr , qrt-pcr result showed that  <dig>  were confirmed as gonad-differentially expressed.
c  <dig> ovary-specifically expressed transcripts were chosen for semi-quantitative pcr validation firstly.
then,  <dig> transcripts that expressed both in ovary and testis were performed for qrt-pcr.
result showed that  <dig> transcripts were expressed significantly higher in ovaries than in testes.
d  <dig> testis-specifically expressed transcripts were chosen for semi-quantitative pcr validation firstly.
then, 41transcripts that expressed both in ovary and testis were performed for qrt-pcr.
result showed that  <dig> transcripts  were expressed in ovaries and testes with significant difference.
expression analysis of testis-specifically expressed transcripts by semi-quantitative pcr.
jc represents the serial number of testis-specifically expressed transcripts, 18s rrna represents endogenous control.
m: marker, t: testis, o: ovary.
expression analysis of ovary-specifically expressed transcripts by semi-quantitative pcr.
lc represents the serial number of ovary-specifically expressed transcripts, 18s rrna represents endogenous control.
m: marker, t: testis, o: ovary.
discovery of molecular markers
isotigs and singlets were both used to predict simple sequence repeats , and isotigs alone were used to predict single nucleotide polymorphisms .
based on the number of reads  that aligned to a ssr or snp, the number of ssrs and snps were calculated when n ≥ <dig>  n ≥ <dig>  and n ≥ <dig> .
we found that when the n value was increased, the numbers of predicted ssrs and snps decreased significantly but the accuracy was greatly improved.
for n ≥ <dig>  we obtained  <dig>  ssrs from the ovary and testis transcriptome libraries .
the largest number of ssr motifs were dinucleotides , which accounted for  <dig> % of all the predicted ssrs, followed by trinucleotides , tetranucleotides , and penta/hexanucleotides .
in addition, of the  <dig>  ssr-containing sequences,  <dig>  sequences were predicted to contain more than two of the ssr types.using the ssahasnp software,  <dig>  indels and  <dig>  putative snps were detected from  <dig>  isotigs .
the predicted snps included  <dig>  transitions and  <dig>  transversions.
indels were the most common snp type, making up  <dig> % of the snp types, while transitions  and transversions  made up the rest.
the most frequent snps types were a/g, c/t, and a/t, while c/g was the least common type .table  <dig>
summary of simple sequence repeat  in the transcriptome of
s.paramamosain
distribution of putative single nucleotide polymorphisms  in the transcriptome of
s. paramamosain.
x-axis: distribution of snp types, y-axis: the number of different snp types.
discussion
large-scale rna sequencing of the green mud crab s. paramamosain  was performed to expand the limited amount of sequence data that are available for crabs in the public est database.
our aim was to identify potentially valuable novel genes that can be used to attain a better understanding of the basic biological mechanisms of reproduction, development, and immunity in crabs.
the rna sequencing produced  <dig>  reads, of which  <dig>  were high-quality reads, which were then assembled into  <dig>  unique sequences.
only  <dig>   of the unique sequences were annotated successfully by blastx searches against the nr database, probably because of the limited number of crustacean sequences in public databases.
besides, some of the unannotated sequences may be 3’ or 5’ untranslated regions, non-coding rnas, or short sequences that do not contain known protein domains.
because sequence similarity is not necessarily indicative of functional homology, in future studies, the functions of these annotated sequences will need to be validated.
further, the model species d. pulex was the first crustacean to have its whole genome sequenced  <cit> , therefore, it is not surprising that the s. paramamosain sequences had the highest number of matches with d. pulex sequences.
even then, only about  <dig> % of all the annotated sequences matched d. pulex, probably because s. paramamosain belongs to the class malacostraca while d. pulex belongs to the class branchiopoda.
go and kegg analysis provide gene function classification labels and gene function background knowledge that can help predict the role of protein interaction networks in cells  <cit> .
in the go analysis, the most abundant level  <dig> go terms in the biological process category, were cellular metabolic process, primary metabolic process, macromolecule metabolic process, nitrogen compound metabolic process, and biosynthetic process.
a similar approach has been applied successfully in the transcriptomic analysis of the housefly musca domestica larva  <cit>  and platy fish xiphophorus maculatus
<cit> .
the focus of our project was to mine gonad-differentially/specifically expressed genes.
we identified  <dig>  common genes in the testis and ovary libraries , of which  <dig>   were considered to have a significant difference , and  <dig>  of them were annotated successfully.
the remaining  <dig> % of the common genes could not be annotated.
besides,  <dig>  ovary-specifically expressed genes  were identified in the ovary library and  <dig>  testis-specifically expressed genes  were identified in the testis library.
three issues arise from these findings.
first, the number of unannotated sequences was much larger than the annotated ones, which made it is almost impossible to exploit the unannotated sequences, specifically the differentially/specifically expressed genes, meaning that potentially useful genetic information was missed.
second, the number of testis-specific transcripts was much more than the number of ovary-specific transcript, indicating that female original development was in a kind of “default” state.
it has been reported that the initial male development occurred by activating a series of testis-specific genes directly or indirectly and/or by repressing genes that were specifically involved in ovarian development  <cit> .
third, the annotation ratio of the testis sequences was less than that of the ovary sequences.
this could be because the study of female animals has been the key focus of aquaculture and researchers have paid much more attention to female crustaceans than to male crustaceans.
correspondingly, genetic information for the female of species is much richer than for the male.
among all the  <dig> tested gonad-differentially expressed transcripts, four testis-predominant transcripts, and  <dig> ovary-predominant transcripts were confirmed by qrt-pcr .
in addition,  <dig> ovary-specifically expressed transcripts and  <dig> testis-specifically expressed transcripts were verified.
in addition, another  <dig> specifically expressed transcripts were proved to be significantly differentially expressed in ovaries and testes .
we speculate that all these transcripts could play important roles in certain parts of the gonadal differentiation physiological process.
we failed to confirm several differentially/specifically expressed transcripts probably because the rna samples came from different sources: the rna used for roche/ <dig> sequencing came from testes/ovaries pools at various developing stages, while the rna for the semi-quantitative pcr and qrt-pcr verification were from the mature stage of the gonads .
perhaps the unconfirmed transcripts were expressed in immature stages rather than in the mature stage of gonad development.
besides, some differences often exist between the bioinformatics analysis of next-generation sequencing technology and the actual experimental analysis.
interestingly, these findings indicate that there might be many more valuable genes to mine and research in this large-scale rna sequencing dataset.
despite the various limitations described above, a large number of key genes and some important pathways that may participate in the gonadal development and reproductive process were detected in the transcriptomic sequencing data .
they include the cyclin-cdk-cki pathway, ubiquitin protease pathway, sumoylation  pathway, mapk signal transduction pathway, and the antioxidant system.
we identified the vasa gene  in our transcriptome database.
vasa is specifically expressed in gonads and plays an important role in germ cell formation and gametogenesis in s. paramamosain
<cit> .
we also discovered some genes such as dmrt and feminization- <dig>  that have not been identified previously in s. paramamosain.
dmrt has been reported to be involved the regulation of sex determination/differentiation pathway in most vertebrates and invertebrates  <cit>  and fem- <dig> was reported to be required for all aspects of testis development  <cit> .
by qrt-pcr validation, we found that dmrt was specifically expressed in the testis, as reported previously in e. sinensis
<cit> , while fem- <dig> was expressed similarly in both ovaries and testes .
whether fem- <dig> participates in the process of sex differentiation or gonad development in the crab has yet to be established.
vitellogenesis, the process of yolk formation and accumulation, is central to oogenesis and ovarian maturation.
in oviparous vertebrates, estrogen hormones, especially the 17β-estradiol e <dig>  increase the synthesis of vitellogenin  through the enhancement of the transcription of the vtg gene.
this process is mediated by an estrogen receptor  and heat shock protein  <dig>   <cit> .
yano and hoshino showed that e <dig> can stimulate synthesis of vitellogenin and oocyte development in m. japonicas
<cit>
.
a study of metapenaeus ensis also indicated that hsp <dig> had some role in the regulation of vitellogenin synthesis  <cit> .
moreover, the discovery of er in mollusks and the estrogen receptor-related receptor  in neomysis japonica suggested that a similar mechanism in vertebrates also exists in crustaceans.
although an er has not been found yet, we identified vtg, vitellogenin receptor , hsp <dig>  and err in our transcriptomic data.
vtg  was previously found to be specifically expressed in the ovaries and hepatopancreas  <cit> .
in the present study, vgr was specifically expressed in ovaries , err were expressed similarly in both ovaries and testes , while hsp <dig> exhibited a higher expression level in ovaries than in testes  .
more work is needed to confirm whether err has the same function of er and whether the e2-er-hsp90-vtg pathway exists in crustaceans.
besides the er-mediated classic nuclear receptor pathway, estrogen can also bind with its corresponding membranous receptors to start a fast non-genetic effect.
the g protein-coupled receptor  <dig>  has been regarded as a new type of estrogen receptor  <cit> .
wang et al.
<cit>  and sirianni et al.
<cit>  found that gpr <dig> mrna was expressed in ovarian theca cells and in the granular cells of hamster and the mouse spermatogonial cell line.
this finding indicated that gpr <dig> played a role in the reproductive system.
the complex of estrogen  and gpr <dig> have been reported to activate the erk pathway and accelerate the process of cell cycle, so as to promote cell growth and proliferation  <cit> .
some researchers believe that there is a relationship between gpr <dig> and the classic nuclear receptor er.
we found the g protein-coupled receptor  <dig>  and g-coupled receptor kinase  <dig> in the transcriptome sequencing data.
the qrt-pcr result showed that sp-gpr <dig>  and g-coupled receptor kinase  <dig> were expressed in both the mature ovaries and testes with no differences .
further, our results  showed that the expression level of sp-gpr <dig> in the ovaries was significantly higher  compared with other tissues .
as the ovary develop, sp-gpr <dig> expression levels rose gradually, and the expression level in the mature ovary  was significantly different than the expression levels in the other four stages .
further investigations are needed to determine whether the function of gpr <dig> in crab is similar to the function of gpr <dig>
oocyte maturation is a complex process that is regulated by multiple factors  <cit>  among which, mature promotion factor  and mitogen-activated protein kinase  play key roles in the process.
mpf, a kind of serine/threonine-protein kinase, consists of cyclin b and cell cycle dependent protein kinase  <dig> .
when the oocyte cell cycle transforms from the g <dig> to the m phase, mpf can be activated and this then induces the rapid maturation of oocytes  <cit> .
the erk1/ <dig> of mapk family participates mainly in the regulation of oocyte meiosis and is not activated in the immature oocytes.
however, erk1/ <dig> was found to be activated in mature oocytes where it mediated oocytes maturation signals from cytoplasm to nuclei  <cit> .
oocyte maturation has been a research focus in reproductive biology, but the molecular regulatory mechanism has not been fully elucidated.
we found some genes that may be related to this regulatory system in our transcriptomic database, namely, anaphase promoting complex subunit  <dig>  protein phosphatase 2a regulatory subunit a, adenylate cyclase type, and serine/threonine-protein phosphatase pp-v. the qrt-pcr validation results for these genes are shown in table s <dig>
the wnt signaling pathway is a conservative signaling network, which takes part in embryonic development, cell differentiation and proliferation, and the process of growth regulation.
wnt is a secreted glycoprotein, and many different wnt gene subtypes have been found in a variety of animals.
wnt <dig> is regarded as a sex determination gene, which plays a key role in the morphological development of female mammals  <cit> .
wnt <dig> can regulate the formation of the mullerian duct and the generation of ovarian steroids  <cit> .
in female mice that have lost the wnt <dig> gene, the mullerian duct did not form, indicating that wnt <dig> is essential to regulate the reproductive cells in the development of female mice.
in male mice, the wnt <dig> signal system was inactive  <cit> .
some genes in the wnt signaling pathway, such as wnt <dig> protein , casein kinase ii alpha  <dig>  and protein phosphatase 2a regulatory subunit a were found in our transcriptomic database.
wnt <dig> and protein phosphatase 2a regulatory subunit a were more highly expressed in ovaries than in testes as indicated by the qrt-pcr validation results .
further research is required to elucidate the roles of these genes in the reproductive process of crab.
vertebrate-type steroids, such as progesterone and 17β-estradiol have been identified in crustaceans.
progesterone stimulates yolk protein synthesis and ovarian maturation in penaeus vannamei
<cit>  and s. paramamosain
<cit> , and stimulates pawning in m. ensis
<cit> .
the important function of progesterone is mediated by the progestin membrane receptor component .
pgrmc <dig> contains a cytochrome b <dig> domain fold and belongs to the membrane-associated progesterone receptor  protein family that is widespread in eukaryotes  <cit> .
in our study, the expression of pgmrc <dig> in ovaries was significantly higher than in testes .
this indicated that pgmrc <dig> may be involved in oogenesis and ovarian development, similar to its involvement in p. monodon
<cit> .
otub  belongs to the otu  superfamily of cysteine proteins  <cit> .
there are two subtypes of otub in mammals, otub <dig> and otub <dig>  which have relative molecular masses of 31 kda and 27 kda respectively.
otub <dig> has been identified as a novel erα-interacting protein with the capability of deubiquitinating erα and then repressing its transcriptional activity  <cit> , which may indicate that otub <dig> plays a major role in the estrogen receptor  signaling pathway in mammals.
our qrt-pcr result showed that the expression levels of sp-otub  in mature ovaries and testes were similar .
however, our results published previously indicated that in the process of ovarian development, sp-otub was expressed at the highest level in the proliferation stage and exhibited significant differences with its expression in other development stages.
in the secondary spermatocytes stage of testis development, the expression of sp-otub reached its highest level and was significantly higher than in the primary spermatocytes and spermatids stages  <cit> .
these preliminary results showed that sp-otub may play an important role in reproductive regulation.
its physiological function, however, needs further investigation.
wds  was first identified and characterized in drosophila melanogaster
<cit> .
it is an essential gene that codes for a wd-repeat protein with seven repeats.
northern blots and in situ hybridization showed that the expression level of wds increased significantly during oogenesis; however, in male germ cells, wds transcript levels were low  <cit> .
loss of wds function during oogenesis was found to be lethal in d. melanogaster
<cit> .
similarly, the wds gene isolated from the chinese oak silkworm antheraea pernyi, was more abundant in the ovaries than in spermaries  <cit> .
in our study, the qrt-pcr results demonstrated that the expression level of wds  was significantly higher in ovaries than in testes .
ssrs and snps are important molecular markers that have been used as powerful tools for genetic mapping, gender identification, population genetic analysis, and breeding.
for instance, li et al.
<cit>  found  <dig> est clusters that were involved in immune and defense functions in meretrix meretrix.
an atp-dependent dna helicase gene, ruvb-like  <dig>  with different snps in the untranslated region was reported to influence the body weight of p. monodon during ovarian development  <cit> .
in addition, two snps that were detected within the coding region of the cyp17-i gene, were found to be significantly associated with the reproductive traits of paralichthys olivaceus
<cit> .
in this study, a large number ssrs and snps were detected from the roche/ <dig> sequencing data.
it is expected that these ssrs and snps will play a tremendous role in the exploration and utilization of new genes s. paramamosain, and will be developed as a tool for genomics and functional genomics in the future.
CONCLUSIONS
this is the first large-scale rna sequencing of the green mud crab s. paramamosain to be reported.
more than  <dig>  reads were obtained, and a large number of novel genes were found.
some gonad-differentially and gonad-specifically expressed genes were identified.
many potential ssrs and snps were detected that could be used for further genetic breeding, genomic mapping, and gene localization studies.
we constructed a deep-coverage est database from the sequencing data.
the read sequences have been deposited in genbank, thereby enriching the genomic information of crustaceans that is available.
work is still needed to improve the molecular database for understanding the mechanisms of sex determination/differentiation and gonadal differentiation/development in crustaceans.
the est information gathered will contribute greatly to our further studies.

 many male and female crabs have a number of significant differences in their biology and economic value, such as growth rate and body size. a study of gonadal differentiation and development in crabs would be helpful for the aquaculture industry. among the four species of the genus scylla, the green mud crab is the most prevalent species in the southeast coast of china, and it has become one of the most important marine aquaculture species in china because of its huge economic value. at present, however, the demand for this crab far exceeds the supply. a major challenge currently facing the crab aquaculture industry is the need to form a series of viable seed production techniques to help increase supply. accordingly, it is important to understand the regulatory mechanisms of sex determination, gonadal differentiation and maturation, and gametogenesis in this species. to understand these mechanisms at the molecular level in more detail, the regulatory pathways associated with gonadal differences need to be studied at the genomic level. many methods, such as traditional suppression subtractive hybridization, annealing control primer, differential display rt-pcr, est sequencing, and microarray, have been applied in research into the crustacean reproductive system, and many differentially expressed genes involved in sex determination, gametogenesis, and gonadal differentiation and maturation have been identified and characterized. some of these genes were detected in crabs; for example, cell division cycle, cyclin b, extracellular regulated protein kinase, and small ubiquitin-related modifier in s. paramamosain, and double-sex and mab- related transcription factor in eriocheir sinensis. several of the differentially expressed genes were found in shrimps; for example, tra-, mitogen-activated protein kinase, prostaglandin reductase, and ubiquitin specific peptidase x-linked in penaeus monodon, and proliferating cell nuclear antigen, ubiquitin-conjugating enzyme e2r, and heat shock protein in marsupenaeus japonicus. however, because of the limited amount of genomic data that is available for crustaceans, it is difficult to identify genes and construct regulatory networks associated with crab reproduction. expressed sequence tags represent effective transcription fragments of whole genomes, avoiding non-coding and repetitive sequences. ests contain a huge amount of functional information that can greatly accelerate the research and discovery of new genes and molecular markers. next-generation high-throughput sequencing technologies have been used to generate large amounts of large-scale transcript sequences and gene expression data for many species when the genomic sequences are not available. these technologies include the roche/ platform, the illumina/solexa system, and the abi solid technology, all of which have high-accuracy, high-speed, and low-cost compared with first-generation sequencing technology. here a roche/ gs flx sequencing system was used for deep-coverage rna sequencing of s. paramamosain during different gonadal developing stages to generate a transcriptome database that will enlarge the public est database for this species and help support future studies. the transcriptomic analysis will help to identify gonad-specific/differential genes by comparing the transcripts in the testis and ovary libraries, which will help build a more complete understanding of the regulatory mechanisms associated with gonadal differentiation and reproductive processes.  sequencing and assembly of green mud crab cdna libraries two cdna libraries were constructed from the testes and ovaries of green mud crabs. the roche/ sequencing generated reads from the testis and reads from the ovary with average sequence lengths of 290 bp and 281 bp for the testis and ovary reads respectively. more than half the reads were between 300 bp and 450 bp in length, the longest read was 735 bp and the shortest read was less than 50 bp, and the overall length of the reads was 104 mb. the size distribution of the raw reads is shown in figure  the raw reads from these two libraries have been deposited in genbank with the following accession numbers: and.figure length distribution of all reads generated by sequencing. x-axis:size distribution of all raw reads,y-axis:the number of reads in different length range. of all the reads, more than half of them were between 300 bp and 450 bp. the longest read was 735 bp and the shortest read was less than 50 bp. after filtering out the adaptor primers and low-quality and very short reads, clean reads with an average sequence length of 274 bp remained. the clean reads in the two libraries were assembled separately. altogether, isotigs were assembled, leaving reads as singlets. the isotigs varied from to  bp in length, and the singlets varied from to 500 bp in length. more than three-fourths of the isotigs were from to 600 bp long. a statistical analysis of the assembly is presented in table  and the length distribution of the isotigs is shown in figure  table summary statistics of the rna sequencing of the s.paramamosain the comparison between two libraries,length distribution of the assembled isotigs. x-axis:size distribution of assembled isotigs,y-axis:the number of isotigs in different length range. the length of isotigs varied from to 2000 bp, more than three fourths of the isotigs occupied a range of 200-600 bp long. annotation of the unique sequences in the two libraries annotation of the assembled sequences was carried out using blastx searches against the ncbi non-redundant protein sequence database. we identified unique sequences that shared similarities with known protein sequences; sequences shared significant similarities with the matched sequences, while the other sequences shared a weak similarity. the e-value distribution for the annotated sequences is shown in figure  figure distribution of the e-values for annotated sequences. x-axis:e-values distribution of annotated sequences,y-axis:the number of sequences in different e-values range. in total, unique sequences share significant similarity with the matched sequences, while of unique sequences left share weak similarity with the matched sequences with e value between 1e- to 1e- the annotated sequences matched known sequences from different species. most numbers of sequence hits were to the water flea daphnia pulex, the red flour beetle tribolium castaneum, the body louse pediculus humanus corporis, and the florida lancelet branchiostoma floridae. except for the top species with sequence hits, of the annotated sequences matched sequences from other species. the top six crustacean species with the most number of sequence hits were s. paramamosain, p. monodon, litopenaeus vannamei, e. sinensis, procambarus clarkia, and homarus americanus.figure species that match to the annotated sequences of s. paramamosain. x-axis:the number of annotated sequences matching each species, y-axis:distribution of top species that match to the annotated sequences. gene ontology analysis the blast2go program was used to assign gene ontology terms to the annotated sequences to predict the functions of the unique sequences and the translated proteins that they encode. a total of, and sequences were assigned to the biological process, cellular component, and molecular function go categories, respectively. the distributions of the annotated sequences in the three go categories are shown in figure  in the biological process category, the metabolic process and cellular process level terms were the most abundant terms. in the molecular function category, catalytic activity and binding were the most abundant, while, in the cellular component category, cell, cell part, organelle part, and macromolecular complex were the most abundant level terms.figure distributions of the annotated sequences in three go categories. a: biological process, b: molecular function, c: cellular component. functional classification based on kegg analysis functional classification and pathway assignments were based on a kyoto encyclopedia of genes and genomes analysis. among the annotated sequences, that were annotated with an enzyme code ec number were mapped to kegg pathways. the top five kegg pathways were metabolic pathways, biosynthesis of secondary metabolites, spliceosome, microbial metabolism in diverse environments, and oxidative phosphorylation pathway. some sequences were mapped to several pathways related with reproduction, growth, development, and immunity; for example, cell cycle, mapk signaling pathway, ubiquitin mediated proteolysis, oocyte meiosis, progesterone-mediated oocyte maturation, gnrh signaling pathway, wnt signaling pathway, hedgehog signaling pathway, p signaling pathway, and toll-like receptor signaling pathway. the distribution of the mapped kegg pathways is presented in figure  figure distribution of the mapped kegg pathways. x-axis: distribution of kegg pathways, y-axis: the number of sequences mapped into each kegg pathway *represents important pathways. analysis of gonad-differentially and specifically expressed genes by comparing the sequences in the ovary and testis libraries, gonad-differentially expressed genes, ovary-specifically expressed genes and testis-specifically expressed genes were identified. from these, differentially, testis-specifically, and ovary-specifically expressed transcripts were chosen for validation by quantitative real-time pcr and semi-quantitative pcr. as shown in figure 7a and b, the qrt-pcr result confirmed that of the differentially expressed transcripts were gonad-differentially expressed. this finding corresponds with the results from the fisher p test. semi-quantitative pcr confirmed that of the ovary-specifically expressed transcripts were expressed only in the ovaries, while the other transcripts were expressed in both ovaries and testes. moreover, among these transcripts, ten were found to be expressed significantly higher in ovaries than in testes by qrt-pcr validation. similarly, only of the testis-specifically expressed transcripts were verified as testis-specifically expressed by semi-quantitative pcr. the expressions of the other transcripts were examined by qrt-pcr, and the result showed that only seven of them were significantly differentially expressed in ovaries and testes.figure expression analysis of gonad differentially expressed transcripts by qrt-pcr. x-axis: the serial number of gonad differentially-expressed transcripts, y-axis: relative expression of each transcripts. 18s rrna is as internal control gene. *indicates significantly differential expression, **indicates most significantly differential expression. a&b differentially expressed transcripts were chosen for quantitative real-time pcr, qrt-pcr result showed that were confirmed as gonad-differentially expressed. c ovary-specifically expressed transcripts were chosen for semi-quantitative pcr validation firstly. then, transcripts that expressed both in ovary and testis were performed for qrt-pcr. result showed that transcripts were expressed significantly higher in ovaries than in testes. d testis-specifically expressed transcripts were chosen for semi-quantitative pcr validation firstly. then, 41transcripts that expressed both in ovary and testis were performed for qrt-pcr. result showed that transcripts were expressed in ovaries and testes with significant difference. expression analysis of testis-specifically expressed transcripts by semi-quantitative pcr. jc represents the serial number of testis-specifically expressed transcripts, 18s rrna represents endogenous control. m: marker, t: testis, o: ovary. expression analysis of ovary-specifically expressed transcripts by semi-quantitative pcr. lc represents the serial number of ovary-specifically expressed transcripts, 18s rrna represents endogenous control. m: marker, t: testis, o: ovary. discovery of molecular markers isotigs and singlets were both used to predict simple sequence repeats, and isotigs alone were used to predict single nucleotide polymorphisms. based on the number of reads that aligned to a ssr or snp, the number of ssrs and snps were calculated when n ≥ n ≥ and n ≥. we found that when the n value was increased, the numbers of predicted ssrs and snps decreased significantly but the accuracy was greatly improved. for n ≥ we obtained ssrs from the ovary and testis transcriptome libraries. the largest number of ssr motifs were dinucleotides, which accounted for % of all the predicted ssrs, followed by trinucleotides, tetranucleotides, and penta/hexanucleotides. in addition, of the ssr-containing sequences, sequences were predicted to contain more than two of the ssr types.using the ssahasnp software, indels and putative snps were detected from isotigs. the predicted snps included transitions and transversions. indels were the most common snp type, making up % of the snp types, while transitions and transversions made up the rest. the most frequent snps types were a/g, c/t, and a/t, while c/g was the least common type.table summary of simple sequence repeat in the transcriptome of s.paramamosain distribution of putative single nucleotide polymorphisms in the transcriptome of s. paramamosain. x-axis: distribution of snp types, y-axis: the number of different snp types. discussion large-scale rna sequencing of the green mud crab s. paramamosain was performed to expand the limited amount of sequence data that are available for crabs in the public est database. our aim was to identify potentially valuable novel genes that can be used to attain a better understanding of the basic biological mechanisms of reproduction, development, and immunity in crabs. the rna sequencing produced reads, of which were high-quality reads, which were then assembled into unique sequences. only of the unique sequences were annotated successfully by blastx searches against the nr database, probably because of the limited number of crustacean sequences in public databases. besides, some of the unannotated sequences may be 3’ or 5’ untranslated regions, non-coding rnas, or short sequences that do not contain known protein domains. because sequence similarity is not necessarily indicative of functional homology, in future studies, the functions of these annotated sequences will need to be validated. further, the model species d. pulex was the first crustacean to have its whole genome sequenced, therefore, it is not surprising that the s. paramamosain sequences had the highest number of matches with d. pulex sequences. even then, only about % of all the annotated sequences matched d. pulex, probably because s. paramamosain belongs to the class malacostraca while d. pulex belongs to the class branchiopoda. go and kegg analysis provide gene function classification labels and gene function background knowledge that can help predict the role of protein interaction networks in cells. in the go analysis, the most abundant level go terms in the biological process category, were cellular metabolic process, primary metabolic process, macromolecule metabolic process, nitrogen compound metabolic process, and biosynthetic process. a similar approach has been applied successfully in the transcriptomic analysis of the housefly musca domestica larva and platy fish xiphophorus maculatus. the focus of our project was to mine gonad-differentially/specifically expressed genes. we identified common genes in the testis and ovary libraries, of which were considered to have a significant difference, and of them were annotated successfully. the remaining % of the common genes could not be annotated. besides, ovary-specifically expressed genes were identified in the ovary library and testis-specifically expressed genes were identified in the testis library. three issues arise from these findings. first, the number of unannotated sequences was much larger than the annotated ones, which made it is almost impossible to exploit the unannotated sequences, specifically the differentially/specifically expressed genes, meaning that potentially useful genetic information was missed. second, the number of testis-specific transcripts was much more than the number of ovary-specific transcript, indicating that female original development was in a kind of “default” state. it has been reported that the initial male development occurred by activating a series of testis-specific genes directly or indirectly and/or by repressing genes that were specifically involved in ovarian development. third, the annotation ratio of the testis sequences was less than that of the ovary sequences. this could be because the study of female animals has been the key focus of aquaculture and researchers have paid much more attention to female crustaceans than to male crustaceans. correspondingly, genetic information for the female of species is much richer than for the male. among all the tested gonad-differentially expressed transcripts, four testis-predominant transcripts, and ovary-predominant transcripts were confirmed by qrt-pcr. in addition, ovary-specifically expressed transcripts and testis-specifically expressed transcripts were verified. in addition, another specifically expressed transcripts were proved to be significantly differentially expressed in ovaries and testes. we speculate that all these transcripts could play important roles in certain parts of the gonadal differentiation physiological process. we failed to confirm several differentially/specifically expressed transcripts probably because the rna samples came from different sources: the rna used for roche/ sequencing came from testes/ovaries pools at various developing stages, while the rna for the semi-quantitative pcr and qrt-pcr verification were from the mature stage of the gonads. perhaps the unconfirmed transcripts were expressed in immature stages rather than in the mature stage of gonad development. besides, some differences often exist between the bioinformatics analysis of next-generation sequencing technology and the actual experimental analysis. interestingly, these findings indicate that there might be many more valuable genes to mine and research in this large-scale rna sequencing dataset. despite the various limitations described above, a large number of key genes and some important pathways that may participate in the gonadal development and reproductive process were detected in the transcriptomic sequencing data. they include the cyclin-cdk-cki pathway, ubiquitin protease pathway, sumoylation pathway, mapk signal transduction pathway, and the antioxidant system. we identified the vasa gene in our transcriptome database. vasa is specifically expressed in gonads and plays an important role in germ cell formation and gametogenesis in s. paramamosain. we also discovered some genes such as dmrt and feminization- that have not been identified previously in s. paramamosain. dmrt has been reported to be involved the regulation of sex determination/differentiation pathway in most vertebrates and invertebrates and fem- was reported to be required for all aspects of testis development. by qrt-pcr validation, we found that dmrt was specifically expressed in the testis, as reported previously in e. sinensis, while fem- was expressed similarly in both ovaries and testes. whether fem- participates in the process of sex differentiation or gonad development in the crab has yet to be established. vitellogenesis, the process of yolk formation and accumulation, is central to oogenesis and ovarian maturation. in oviparous vertebrates, estrogen hormones, especially the 17β-estradiol e increase the synthesis of vitellogenin through the enhancement of the transcription of the vtg gene. this process is mediated by an estrogen receptor and heat shock protein. yano and hoshino showed that e can stimulate synthesis of vitellogenin and oocyte development in m. japonicas. a study of metapenaeus ensis also indicated that hsp had some role in the regulation of vitellogenin synthesis. moreover, the discovery of er in mollusks and the estrogen receptor-related receptor in neomysis japonica suggested that a similar mechanism in vertebrates also exists in crustaceans. although an er has not been found yet, we identified vtg, vitellogenin receptor, hsp and err in our transcriptomic data. vtg was previously found to be specifically expressed in the ovaries and hepatopancreas. in the present study, vgr was specifically expressed in ovaries, err were expressed similarly in both ovaries and testes, while hsp exhibited a higher expression level in ovaries than in testes. more work is needed to confirm whether err has the same function of er and whether the e2-er-hsp90-vtg pathway exists in crustaceans. besides the er-mediated classic nuclear receptor pathway, estrogen can also bind with its corresponding membranous receptors to start a fast non-genetic effect. the g protein-coupled receptor has been regarded as a new type of estrogen receptor. wang et al. and sirianni et al. found that gpr mrna was expressed in ovarian theca cells and in the granular cells of hamster and the mouse spermatogonial cell line. this finding indicated that gpr played a role in the reproductive system. the complex of estrogen and gpr have been reported to activate the erk pathway and accelerate the process of cell cycle, so as to promote cell growth and proliferation. some researchers believe that there is a relationship between gpr and the classic nuclear receptor er. we found the g protein-coupled receptor and g-coupled receptor kinase in the transcriptome sequencing data. the qrt-pcr result showed that sp-gpr and g-coupled receptor kinase were expressed in both the mature ovaries and testes with no differences. further, our results showed that the expression level of sp-gpr in the ovaries was significantly higher compared with other tissues. as the ovary develop, sp-gpr expression levels rose gradually, and the expression level in the mature ovary was significantly different than the expression levels in the other four stages. further investigations are needed to determine whether the function of gpr in crab is similar to the function of gpr oocyte maturation is a complex process that is regulated by multiple factors among which, mature promotion factor and mitogen-activated protein kinase play key roles in the process. mpf, a kind of serine/threonine-protein kinase, consists of cyclin b and cell cycle dependent protein kinase. when the oocyte cell cycle transforms from the g to the m phase, mpf can be activated and this then induces the rapid maturation of oocytes. the erk1/ of mapk family participates mainly in the regulation of oocyte meiosis and is not activated in the immature oocytes. however, erk1/ was found to be activated in mature oocytes where it mediated oocytes maturation signals from cytoplasm to nuclei. oocyte maturation has been a research focus in reproductive biology, but the molecular regulatory mechanism has not been fully elucidated. we found some genes that may be related to this regulatory system in our transcriptomic database, namely, anaphase promoting complex subunit protein phosphatase 2a regulatory subunit a, adenylate cyclase type, and serine/threonine-protein phosphatase pp-v. the qrt-pcr validation results for these genes are shown in table s the wnt signaling pathway is a conservative signaling network, which takes part in embryonic development, cell differentiation and proliferation, and the process of growth regulation. wnt is a secreted glycoprotein, and many different wnt gene subtypes have been found in a variety of animals. wnt is regarded as a sex determination gene, which plays a key role in the morphological development of female mammals. wnt can regulate the formation of the mullerian duct and the generation of ovarian steroids. in female mice that have lost the wnt gene, the mullerian duct did not form, indicating that wnt is essential to regulate the reproductive cells in the development of female mice. in male mice, the wnt signal system was inactive. some genes in the wnt signaling pathway, such as wnt protein, casein kinase ii alpha and protein phosphatase 2a regulatory subunit a were found in our transcriptomic database. wnt and protein phosphatase 2a regulatory subunit a were more highly expressed in ovaries than in testes as indicated by the qrt-pcr validation results. further research is required to elucidate the roles of these genes in the reproductive process of crab. vertebrate-type steroids, such as progesterone and 17β-estradiol have been identified in crustaceans. progesterone stimulates yolk protein synthesis and ovarian maturation in penaeus vannamei and s. paramamosain, and stimulates pawning in m. ensis. the important function of progesterone is mediated by the progestin membrane receptor component. pgrmc contains a cytochrome b domain fold and belongs to the membrane-associated progesterone receptor protein family that is widespread in eukaryotes. in our study, the expression of pgmrc in ovaries was significantly higher than in testes. this indicated that pgmrc may be involved in oogenesis and ovarian development, similar to its involvement in p. monodon. otub belongs to the otu superfamily of cysteine proteins. there are two subtypes of otub in mammals, otub and otub which have relative molecular masses of 31 kda and 27 kda respectively. otub has been identified as a novel erα-interacting protein with the capability of deubiquitinating erα and then repressing its transcriptional activity, which may indicate that otub plays a major role in the estrogen receptor signaling pathway in mammals. our qrt-pcr result showed that the expression levels of sp-otub in mature ovaries and testes were similar. however, our results published previously indicated that in the process of ovarian development, sp-otub was expressed at the highest level in the proliferation stage and exhibited significant differences with its expression in other development stages. in the secondary spermatocytes stage of testis development, the expression of sp-otub reached its highest level and was significantly higher than in the primary spermatocytes and spermatids stages. these preliminary results showed that sp-otub may play an important role in reproductive regulation. its physiological function, however, needs further investigation. wds was first identified and characterized in drosophila melanogaster. it is an essential gene that codes for a wd-repeat protein with seven repeats. northern blots and in situ hybridization showed that the expression level of wds increased significantly during oogenesis; however, in male germ cells, wds transcript levels were low. loss of wds function during oogenesis was found to be lethal in d. melanogaster. similarly, the wds gene isolated from the chinese oak silkworm antheraea pernyi, was more abundant in the ovaries than in spermaries. in our study, the qrt-pcr results demonstrated that the expression level of wds was significantly higher in ovaries than in testes. ssrs and snps are important molecular markers that have been used as powerful tools for genetic mapping, gender identification, population genetic analysis, and breeding. for instance, li et al. found est clusters that were involved in immune and defense functions in meretrix meretrix. an atp-dependent dna helicase gene, ruvb-like with different snps in the untranslated region was reported to influence the body weight of p. monodon during ovarian development. in addition, two snps that were detected within the coding region of the cyp17-i gene, were found to be significantly associated with the reproductive traits of paralichthys olivaceus. in this study, a large number ssrs and snps were detected from the roche/ sequencing data. it is expected that these ssrs and snps will play a tremendous role in the exploration and utilization of new genes s. paramamosain, and will be developed as a tool for genomics and functional genomics in the future.  this is the first large-scale rna sequencing of the green mud crab s. paramamosain to be reported. more than reads were obtained, and a large number of novel genes were found. some gonad-differentially and gonad-specifically expressed genes were identified. many potential ssrs and snps were detected that could be used for further genetic breeding, genomic mapping, and gene localization studies. we constructed a deep-coverage est database from the sequencing data. the read sequences have been deposited in genbank, thereby enriching the genomic information of crustaceans that is available. work is still needed to improve the molecular database for understanding the mechanisms of sex determination/differentiation and gonadal differentiation/development in crustaceans. the est information gathered will contribute greatly to our further studies. 
moreover, ovary-specific, testis-specific, and gonad-differential transcripts were confirmed by semi-quantitative pcr and quantitative real-time pcr. a large number of candidate ssrs and snps were detected, which could be used as genetic markers for population genetics and functional genomics in this species. we have identified many important functional genes and made a preliminary attempt to construct the regulatory network involved in the gonadal development of crustaceans. the molecular regulatory mechanism of sex determination and gonadal differentiation in this species has received considerable attention in recent years because of the huge differences—both biological and economic—between male and female crabs. in this study, next-generation sequencing technology was used to develop deep-coverage transcriptomic sequencing data for the testis and ovary of s. paramamosain. this is the first large-scale rna sequencing of s. paramamosain to be reported. the annotated transcriptome data will provide fundamental support for future research into the reproduction biology of s. paramamosain. using the blastx program, unique sequences were annotated with known protein sequences from the ncbi non-redundant protein sequence database. a total of reads with an average sequence length of 285 bp were produced from testis and ovary cdna libraries. in addition, putative simple sequence repeats and potential single nucleotide polymorphisms were identified. after comparing the ovary and testis libraries, gonad-differentially, ovary-specifically, and testis-specifically expressed genes were identified. the green mud crab is the most prevalent crustacean on the southeast coast of china. the gene ontology and kegg analyses allowed the unique sequences that were annotated with enzyme code numbers to be mapped into kegg pathways.
 the green mud crab is the most prevalent crustacean on the southeast coast of china. the molecular regulatory mechanism of sex determination and gonadal differentiation in this species has received considerable attention in recent years because of the huge differences—both biological and economic—between male and female crabs. in this study, next-generation sequencing technology was used to develop deep-coverage transcriptomic sequencing data for the testis and ovary of s. paramamosain.  a total of reads with an average sequence length of 285 bp were produced from testis and ovary cdna libraries. after filtering out contaminating reads, the clean reads were assembled, producing a total of isotigs and leaving reads as singlets. using the blastx program, unique sequences were annotated with known protein sequences from the ncbi non-redundant protein sequence database. the gene ontology and kegg analyses allowed the unique sequences that were annotated with enzyme code numbers to be mapped into kegg pathways. after comparing the ovary and testis libraries, gonad-differentially, ovary-specifically, and testis-specifically expressed genes were identified. moreover, ovary-specific, testis-specific, and gonad-differential transcripts were confirmed by semi-quantitative pcr and quantitative real-time pcr. in addition, putative simple sequence repeats and potential single nucleotide polymorphisms were identified.  this is the first large-scale rna sequencing of s. paramamosain to be reported. we have identified many important functional genes and made a preliminary attempt to construct the regulatory network involved in the gonadal development of crustaceans. the annotated transcriptome data will provide fundamental support for future research into the reproduction biology of s. paramamosain. a large number of candidate ssrs and snps were detected, which could be used as genetic markers for population genetics and functional genomics in this species. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords testisovaryspecifically/differentially expressed genestranscriptomescylla paramamosainissue-copyright-statement© the author 2014 