We have isolated a tiger frog virus (TFV) from diseased tiger frogs, Rana tigrina rugulosa. The genome was a linear double-stranded DNA of 105,057 basepairs in length with a base composition of 55.01% G+C. About 105 open reading frames were identified with coding capacities for polypeptides ranging from 40 to 1294 amino acids. Computer-assisted analyses of the deduced amino acid sequences revealed that 39 of 105 putative gene products showed significant homology to functionally characterized proteins of other species in the GenBank/EMBL/DDBJ databases. These proteins included enzymes and structural proteins involved in virus replication, transcription, modification, and virus--host interaction. The deduced amino acid sequences of TFV gene products showed more than 90% identity to FV3, but a low degree of similarity among TFV, ISKNV, and LCDV-1. The results from this study indicated that TFV may belong to the genus Ranavirus of the family Iridoviridae.

Vitellogenesis in the mature female crab Charybdis feriatus occurs all year round during which active synthesis of the vitellogenin (Vg) precursor occurs. Several polypeptides from the ovaries were shown to be immuno-reactive to the shrimp vitellin (Vn) antibody. N-terminal amino acid sequence determination revealed that several ovarian polypeptides and one polypeptide secreted by the hepatopancreas were identical to part of the C. feriatus Vg (CfVg) precursor. The full-length cDNA sequence encoding a protein with high amino acid sequence similarity to the Vg of the shrimp Metapenaeus ensis was cloned. In common with the shrimp M. ensis MeVg2, the crab vitellogenin gene is expressed only in the hepatopancreas. The expression level of CfVg is undetectable in the non-reproductive females, increases to maximum at the middle stages of vitellogenesis and drops to a lower level in late vitellogenesis. Expression of CfVg also extended to females that are undergoing brooding of developing larvae. Although the 8 kb transcript for the full-length cDNA was detected, smaller transcripts specific to CfVg mRNA were also detected, suggesting the occurrence of alternative splicing/expression of the CgVg gene to produce the smaller transcripts. Using a short term in vitro hepatopancreas explant culture assay, we have demonstrated that low concentrations of farnesoic acid (FA) stimulate CfVg gene expression in the hepatopancreas. Although both methyl farnesoate (MF) and juvenile hormone III also caused up-regulation of the CfVg gene, their effects are only significant at much higher concentrations.

An additional vitellogenin gene (MeVg2) that is structurally different from MeVg1 was cloned and characterized from the shrimp Metapenaeus ensis. The MeVg2 gene consists of fewer exons-introns and is most likely evolved from the MeVg1 gene. The cDNA for MeVg2 is 8.0 kilobases (kb) in size, and the deduced MeVg2 precursor shared an overall 54% amino sequence identity to the MeVg1 gene of the same shrimp. As compared to the MeVg1 precursor, MeVg2 precursor consists of more potential subunit cleavage sites, suggesting that the precursor may be processed into many smaller subunits. The MeVg2 is expressed only in the hepatopancreas, and the expression level of MeVg2 in adult female increases from the early vitellogenic stage, reaching a maximum at the middle vitellogenic stage, and remains high toward the end of vitellogenic cycle. In addition to the 8-kb mRNA, smaller transcripts of 1.5-2.5 kb for MeVg2 were identified, and the 8-kb transcript only constitutes less than 10% of the overall MeVg2-derived transcripts. To confirm the presence of the small transcripts, we screened the shrimp hepatopancreas cDNA library and isolated two smaller MeVg2-specific cDNA clones. These clones shared greater than 99% overall identity to the corresponding C-terminal region of the MeVg2 precursor, suggesting that an alternative expression/ splicing of the MeVg2 gene occurred. By immunohistochemical analysis, vitellin-immunopositive signals were localized in the lumen and extracellular fraction of the hepatopancreas. Amino acid sequence determination of the tissue protein and secreted protein from the hepatopancreas revealed that the 76-kDa vitellogenin subunit is most likely processed into smaller-sized subunits. Taken together, these results suggest that the hepatopancreas is an important organ for the synthesis of vitellogenin and may contribute to vitellogenesis by producing a large quantity of smaller MeVg2 subunit for ovarian uptake.