Isolation and functional identification of secretin family G-protein coupled receptor from Y-organ of the mud crab, Scylla olivacea.

Recently, genes in the superfamily of GPCR are gaining more interest in crustaceans as more evidence shows that they are involved in molting. This study identified four forms of the secretin family of G-protein coupled receptor (GPCR) from the Y-organ of mud crab, Scylla olivacea (ScoGPCR). A full-length sequence of ScoGPCR-B2 was isolated and identified as a lipoprotein receptor while three forms of GPCR in Methuselah-like (Mthl) or B3 subfamilies were reported as ScoGPCR-B3a, -B3b, and -B3c. These four forms exhibit common features of the 7-trans membrane (7TM) domain and distinct aspects in the extracellular region (ECR) at the N-terminus. At the ECR, disulfide bridges are predicted to generate structural stability in all four forms while the putative ScoGPCR-B3 proteins retain conserved Tyr, Trp, Pro, and Phe residues, possibly to form the aromatic-proline interactions and function as key residues for receptor recognition. Expression levels of ScoGPCR-B2 and -B3 in eyestalk, thoracic ganglion, and hindgut between intermolt and premolt stages are similar. Only ScoGPCR-B2 and ScoGPCR-B3a in Y-organ (YO) seem to be premolt-specific responses. An upregulation of ScoGPCR-B2 in YO at the premolt stage is correlated with the demand for cholesterol used in ecdysteroid synthesis, resulting in increased ecdysteroid titers. The effects of ecdysone on YO were pursued by in vitro incubation and revealed that ScoGPCR-B3a and -B3b expressions were induced in a different time frame: early in ScoGPCR-B3b and late in ScoGPCR-B3a. The early response of ScoGPCR-B3b was followed through immunohistology and showed that the newly synthesized protein was located primarily in the cytosol.

Mendelian inheritance of endogenous viral elements (EVE) of white spot syndrome virus (WSSV) in shrimp.

Previous work has shown that non-retroviral endogenous viral elements (EVE) are common in crustaceans, including penaeid shrimp. So far, they have been reported for infectious hypodermal and hematopoietic necrosis virus (IHHNV) and white spot syndrome virus (WSSV). For the latter, it was shown that shrimp sperm were positive for an EVE of WSSV called EVE366, suggesting that it was heritable, since shrimp sperm (non-motile) do not contain mitochondria. However, to prove this hypothesis that EVE366 was heritable and located in chromosomal DNA, it was necessary to carry out mating tests to show that EVE366 could be detected in parental shrimp and distributed in their offspring in a Mendelian fashion. To do this, we analyzed two shrimp crosses using polyacrylamide gels with a multiple-allele, microsatellite marker Pmo11 as a quality control for single allele detection. In both crosses, all of the shrimp (parents and siblings) were positive for 2 Pmo11 alleles as expected. In Cross 1, the female was PCR-positive for EVE366 while the male was negative, and in Cross 2, both the female and male were PCR-positive for EVE366. Individual analysis of the offspring of Cross 1 revealed a distribution of 1:1 for EVE366, indicating that the EVE366-positive female parent was heterozygous for EVE366. In the second cross, the distribution of EVE366 in the offspring was 3:1, indicating that both PCR-positive parents were heterozygous for EVE366. These results supported the hypothesis that EVE366 was present in shrimp chromosomal DNA and was heritable in a Mendelian fashion. This work provides a model to screen for heritable EVE in shrimp and shows that selection of one parent heterozygous for an EVE and the other negative for it can result in approximately half of the siblings positive and half negative for that EVE as expected. Dividing the siblings of such a cross into an EVE positive group and an EVE negative group followed by challenge with the originating lethal virus should reveal whether or not possession of that specific EVE results in any significant protection against disease caused by the homologous virus.