Abstract

Polychaetes (Perinereis sp.) have been used extensively in shrimp broodstock maturation diets because of their nutritional value, being rich in protein, fat and minerals. The aims of this study were to identify the reproduction-related gene in polychaetes, and analyze gene expression levels in polychaetes. Messenger RNA (mRNA) was isolated from mature polychaete samples and used as the template for complementary DNA (cDNA) synthesis. The cDNA was then subjected to cloning and sequencing. However, no cDNA sequence was found to match the reproductive-related gene. As an alternative procedure, a set of cDNA was synthesized and sequenced by the pyrosequencing method. From 454 Pyrosequencing, 40,402 raw sequences were obtained, covering 14,747,824 nucleotides 200-300 bp in length and grouped into 609 isotigs, 712 contigs and 15,975 singletons. In addition, BLASTX analysis showed that 410 sequences (67.3%) matched known genes, whereas, 199 sequences (32.7%) showed no significant homology to known genes. A significant match was found with the vitellogenin structural genes’ (yolk protein genes) family member (vit-l)-like gene, which was grouped as the reproduction-related gene in polychaetes. The gene was then used to analyze the expression levels in polychaetes aged 2, 3,4, 5, 6,7, and 8 months by real-time quantitative RT-PCR. The highest vitellogenin gene expression was found in 6 - month old polychaetes (P<0.05) and dramatically decreased after reaching 6 months. This result suggested that 6 - month old polychaetes will be suitable as the maturation diet for shrimp broodstock; however, the effect on shrimp reproductive development may need to be analyzed further.