Coccidian Parasite in Sea Cucumber (Apostichopus japonicus) Ovaries.

We investigated an unknown ellipsoidal body that is sometimes found in the ovaries of the sea cucumber Apostichopus japonicus. Its external morphology, comprising an ellipsoidal dark central body (about 150 µm in length) and a surrounding transparent layer (about 50 µm in thickness), resembled that of a protozoan cyst, particularly an oocyst. Histological observations of the developing A. japonicus ovaries clarified that a small mass of organisms appeared in the cytoplasm of young oocytes, proliferated in these cells through budding, became rod shaped and arranged radially, and, finally, formed an outer layer. These processes were considered to be the formation of a cyst by a protozoan parasite. The small subunit ribosomal RNA (18S rRNA) gene was amplified from the DNA extracted from unknown ellipsoidal bodies by using polymerase chain reaction with universal primers for eukaryote 18S rRNA. The determined sequence was not identical to any of the known sequences in DNA databases, but it clustered in a clade of coccidian species belonging to Eucoccidiorida in phylogenetic analyses. From these results, we concluded that the unknown ellipsoidal body is a cyst (possibly an oocyst) of a coccidian parasite (order Eucoccidiorida) that is formed in the A. japonicus oocyte, though its lower taxonomic position is uncertain. In a survey of the gonads of wild A. japonicus at Esashi, Hokkaido, during the reproductive season, these cysts were detected in more than 50% of females but were never found in males. We consider that the cysts of this parasite can only be formed in A. japonicus ovaries.

Molecular cloning and expression analysis of the Mitogen-activating protein kinase 1 (MAPK1) gene and protein during ovarian development of the giant tiger shrimp Penaeus monodon.

Isolation and characterization of genes and/or proteins differentially expressed in ovaries are necessary for understanding ovarian development in the giant tiger shrimp (Penaeus monodon). In this study, the full-length cDNA of P. monodon mitogen-activating protein kinase 1 (PmMAPK1) was characterized. PmMAPK1 was 1,398 bp in length containing an open reading frame of 1,098 bp that corresponded to a polypeptide of 365 amino acids. PmMAPK1 was more abundantly expressed in ovaries than in testes of P. monodon. Quantitative real-time PCR revealed differential expression levels of PmMAPK1 mRNA during ovarian development of intact broodstock, where it peaked in early cortical rod (stage III) ovaries (P < 0.05) and slightly decreased afterwards (P > 0.05). Likewise, the expression level of PmMAPK1 in early cortical rod and mature (IV) ovaries was significantly greater than that in previtellogenic (I) and vitellogenic (II) ovaries of eyestalk-ablated broodstock (P < 0.05). The PmMAPK1 transcript was localized in ooplasm of previtellogenic oocytes. In intact broodstock, the expression of the PmMAPK1 protein was clearly increased from previtellogenic ovaries in subsequent stages of ovarian development (P < 0.05). In contrast, the level of ovarian PmMAPK1 protein was comparable during oogenesis in eyestalk-ablated broodstock (P > 0.05). The PmMAPK1 protein was localized in ooplasm of previtellogenic and vitellogenic oocytes. It was also detected around the nuclear membrane of early cortical rod oocytes in both intact and eyestalk-ablated broodstock. Results indicated that PmMAPK1 gene products seem to play functional roles in the development and maturation of oocytes/ovaries in P. monodon.

Molecular cloning and expression of progestin membrane receptor component 1 (Pgmrc1) of the giant tiger shrimp Penaeus monodon.

Knowledge on molecular mechanisms of steroid hormonal induction on oocyte development may lead to the possible ways to effectively induce ovarian maturation in shrimp. In this study, progestin membrane receptor component 1 (Pgmrc1) of the giant tiger shrimp (Penaeus monodon) initially identified by EST analysis was further characterized. The full-length cDNA of Pgmrc1 was 2015bp in length containing an ORF of 573bp corresponding to a polypeptide of 190 amino acids. Northern blot analysis revealed a single form of Pgmrc1 in ovaries of P. monodon. Quantitative real-time PCR indicated that the expression level of Pgmrc1 mRNA in ovaries of both intact and eyestalk-ablated broodstock was greater than that of juveniles (P<0.05). Pgmrc1 was up-regulated in mature (stage IV) ovaries of intact broodstock (P<0.05). Unilateral eyestalk ablation resulted in an earlier up-regulation of Pgmrc1 since the vitellogenic (II) ovarian stage. Moreover, the expression level of Pgmrc1 in vitellogenic, early cortical rod and mature (II-IV) ovaries of eyestalk-ablated broodstock was greater than that of the same ovarian stages in intact broodstock (P<0.05). Pgmrc1 mRNA was clearly localized in the cytoplasm of follicular cells, previtellogenic and early vitellogenic oocytes. Immunohistochemistry revealed the positive signals of the Pgmrc1 protein in the follicular layers and cell membrane of follicular cells and various stages of oocytes. Taken the information together, Pgmrc1 gene products seem to play the important role on ovarian development and may be used as the bioindicator for monitoring progression of oocyte maturation of P. monodon.

Expression and purification of active recombinant cathepsin C (dipeptidyl aminopeptidase I) of kuruma prawn Marsupenaeus japonicus in insect cells.

Cathepsin C (CTSC) is a lysosomal cysteine protease belonging to the papain superfamily. Our previous study showed that CTSC precursor (zymogen) is localized exclusively in cortical rods (CRs) of mature oocyte in the kuruma prawn Marsupenaeus japonicus, suggesting that CTSC might have roles on regulating release and/or formation of a jelly layer. In this study, enzymically active CTSC of the kuruma prawn was prepared by recombinant expression in the High Five insect cell line. The recombinant enzyme with a polyhistidine tag at its C-terminus was considered to be initially secreted into the culture medium as an inactive form of zymogen, because Western blot with anti-CTSC antibody detected a 51 kDa protein corresponding to CTSC precursor. After purification by affinity chromatography on nickel-iminodiacetic acid resin, the enzyme displayed three forms of 51, 31, and 30 kDa polypeptides. All of the forms can be recognized by antiserum raised against C-terminal polyhistidine tag, indicating that the 31 and 30 kDa forms were generated from 51 kDa polypeptide by removal of a portion of the N-terminus of propeptide. Following activation at pH 5.5 and 37 degrees C for 40 hours under native conditions, the recombinant CTSC (rCTSC) exhibited increased activity against the synthetic substrate Gly-Phe-beta-naphthylamide and optimal pH at around 5. The purified rCTSC will be useful for further characterization of its exact physiological role on CRs release and/or formation of a jelly layer in kuruma prawn.

Expressed sequence tags from eyestalk of kuruma prawn, Marsupenaeus japonicus.

We analyzed the expressed sequence tags (ESTs) obtained from a cDNA library of the eyestalk of the kuruma prawn, Marsupenaeus japonicus, to examine gene expression profile with special focus on female reproduction. The assembly of 1988 ESTs created 136 contigs from 738 ESTs; however 1250 ESTs remained singletons. Significant similarities (blast score > or = 50 bits) to the DNA sequences in the databank were found for only 16.7% of the 1386 sequences (136 contigs plus 1250 singletons), suggesting that the eyestalk library contains many unknown genes. Ribosomal RNA and mitochondrial respiration enzymes with significant similarities were found abundantly in the ESTs, whereas genes related to maturation or endocrine systems were scarce. Three ESTs were assumed to encode novel eyestalk hormones with marked similarities to pigment-dispersing hormone, molt-inhibiting hormone and crustacean hyperglycemic hormone. Sequences encoding a product highly homologous to farnesoic acid O-methyltransferase, an enzyme that produces methyl farnesoate, were also found.