Transcriptomic bases of reproduction in two portuguese populations of the european clam ruditapes decussatus

The European clam, Ruditapes decussatus (Linnaeus, 1758) is a bivalve mollusc of the family Veneridae native to the European Atlantic and Mediterranean coastal waters. The “carpet-shell clam” is one of the most important bivalve species in Southern European countries, like Italy, Spain and Portugal. This clam is commercially more important and more appreciated by consumers than the Manila clam, Ruditapes philippinarum (Matias et al., 2009). Despite a relatively low European production (8 200 tons/year; FAO, 2006), R. decussatus is a high value seafood product, economically important. The culture of R. decussatus is clearly limited by the availability of seed. Its production is exclusively based on natural recruitment, which is subject to high annual fluctuations due to pollution and other environmental factors. To address this situation, artificial spawning and larval rearing programs were developed to provide an alternative source of seed (Matias et al., 2009). To establish successful hatchery based production, the knowledge of the natural reproductive cycle of bivalves has become fundamental, which may benefit from a better knowledge of the genes underlying reproduction, as they will help to elucidate how and to what extent environmental factors affect relevant gene expression and thus the physiological function. In the present work, we designed a custom oligonucleotide microarray containing 51,709 ESTs, with 86% of annotated transcripts, to characterize the transcriptomic bases of reproduction of R. decussatus. The microarray analysis was performed in four gonadal maturation stages of R. decussatus, sampled in two different Portuguese wild populations from different latitudes, Ria Formosa (37°1’ N) in South Portugal and Ria Aveiro (40°40’ N) in North Portugal. These are the two locations in Portugal with the highest production of this species and their populations have different responses to spawning induction. Indeed, it was previously demonstrated that, living in different areas, this species can strongly differ in terms of their fecundity levels (Shafee and Daoudi, 1991; Trigui-El Menif et al., 1995). In order to compare the gene expression between the two sampled populations of R. decussatus, considering all stages and sexes, a two-unpaired class Significance Analysis of Microarray (SAM) test was carried out. A list of 2900 significant probes was obtained. The environment and the metabolic reactions seemed to have a main influence, since “ABC transporters” (dre02010) and “N-Glycan biosynthesis” (dre00510) were the only represented in the enriched KEGG pathway terms. Hierarchical clustering, using Pearson’s correlation, was also performed considering the set of differentially expressed genes. With some exceptions, three main clusters were identified: Ria Formosa, Ria de Aveiro and Females Stage III and IV from Ria de Aveiro (Figure 1). These results suggest that changes in gene expression profiles between the two populations were higher between females than males, and may reflect the differences observed in terms of success in the spawning induction, between the mature females of the two populations. A comparison between the females stage IV was then performed and a list of 6765 significant probes was obtained, with “Cell cycle” (dre04110) and “Oocyte meiosis” (dre04114) represented in the enriched KEGG pathway terms, with 45 and 28 genes, respectively. Additionally, in order to compare gene expression between the gametogenic stages, a total of 6 comparisons were performed between stages I/II, stages II/III and stages III/IV in females and males of the two populations, separately. For females, the data revealed a list of 7087 significant probes between stage I and stage II and a list of 10633 significant probes between stage II and stage III. “Cell cycle” (dre04110), “Progesterone-mediated oocyte maturation” (dre04914), “Purine metabolism” (dre00230) and “Calcium signaling pathway” (dre04020) were the most affected pathways over the course of oogenesis, suggesting their role in the oocyte maturation of bivalves (Tosti, 2006; Corporeau et al., 2012). In relation to males, the data revealed a list of 14126 significant probes between stage I and stage II and a list of 10633 significant probes between stage II and stage III. “Cell cycle” (dre04110) and “Calcium signaling pathway” (dre04020) were also strongly represented over the course of spermatogenesis. No differentially expressed genes were found, for both sexes, between stages III/IV. This study provides new highly valuable genomic information for the understanding of reproduction of this species and emphasizes some candidate genes as possible starting points for further functional studies.