Ovarian primary tissue culture of the kuruma shrimp Marsupenaeus japonicus

Cell growth in ovarian primary culture of the kuruma shrimp, Marsupenaeus japonicus, was examined under various culture conditions. The best growth of ovarian cells was obtained in a culture system consisting of double strength of Leibovitz-15 medium supplemented with 10% fetal bovine serum, glucose (1 g/L), proline (0.1 g/L), TC-Yeastolate (1 g/L), and lactalbumin hydrolysate (1 g/L). The cells survived in this medium at 25 degrees C for 45 d. The epithelial-like cells predominated in 10-d-old cultures, covering >80% of the surface area on the bottom of flask. Cells in mitosis were often observed. Cell proliferation was monitored by incorporation of 5-bromo-2'-deoxyuridine (BrdU), an analog of thymidine. 5-Bromo-2'-deoxyuridine-associated cells accounted for 11.5 and 35.0% of cell populations at 2 and 24 h, respectively, after BrdU treatment. Our results provide an improved culture technique for ovarian tissue of the kuruma shrimp.     

Molecular cloning and expression analysis of alpha 2-macroglobulin in the kuruma shrimp, Marsupenaeus japonicus

The cDNA encoding the kuruma shrimp, Marsupenaeus japonicus alpha(2)-macroglobulin (alpha(2)M) was obtained by screening a haemocyte cDNA library and 5' RACE PCR amplification. The full length cDNA of 4748 bp contains an open reading frame of 4518 nucleotides that translates into a 1505-amino acid putative peptide, with a 5'untranslated region (UTR) of 59 bp and a 3'UTR of 171 bp. The open reading frame encodes an N-terminal signal sequence of 17 residues and a mature protein of 1488 residues. The entire amino acid sequence is similar to the alpha(2)M sequences of arthropods (30-31% identity), mammals (26-27% identity) and fish (25-28% identity). The M. japonicus alpha(2)M sequence contains putative functional domains including a bait region, an internal thiol ester site, and a receptor-binding domain, which are present in mammalian alpha(2)Ms. In a healthy shrimp, the mRNA of alpha(2)M was mainly expressed in haemocytes. In addition, the expression level of alpha(2)M mRNA was dramatically increased by through time upon oral administration of peptidoglycan (PG), which is an immune stimulant. The highest expression of alpha(2)M mRNA was observed 7 days after feeding with PG. These results suggest that the shrimp alpha(2)M is an important molecule in immune system.     

A novel gene of tumor necrosis factor ligand superfamily from kuruma shrimp,Marsupenaeus japonicus

A tumor necrosis factor (TNF) gene has been isolated and characterized in kuruma shrimp, Marsupenaeus japonicus, providing the first conclusive evidence for the existence of the TNF ligand in shrimp. The kuruma shrimp TNF (MjTNF) cDNA was composed of 1868 bp with a 262 bp 5′-untranslated region (UTR) and a 220 bp 3′-UTR, which was translated into a protein of 462 amino acid residues that included a predicted transmembrane domain of 23 amino acid residues (Trp20–Val42) and the TNF family signature (Pro321–Leu448). Homology analysis of MjTNF showed 30.7% and 26.7% identities with fruit fly (Drosophila melanogaster) Eiger and human (Homo sapiens) ectodysplasin A, respectively. The MjTNF gene was constitutively expressed in unstimulated organs of shrimp such as the muscle, stomach, brain and gill. In lymphoid organ cells, an enhanced expression of the MjTNF gene was observed following stimulation with peptidoglycan and polycytidylic acid. A high expression level of MjTNF was observed in vivo 2 h and 4 h after stimulation with lipopolysaccharide and Vibrio penaeicida, respectively. These observations suggest that MjTNF plays a role in the innate immune defense in kuruma shrimp. The discovery of shrimp TNF will allow a more complete and concrete understanding of shrimp inflammatory responses.     

Identification of cDNA encoding Toll receptor, MjToll gene from kuruma shrimp, Marsupenaeus japonicus

Toll receptors are cell-surface receptors acting as pattern recognition receptors (PRRs) that are involved in the signaling pathway for innate immunity activation and are genetically conserved from insects to mammals. Tolls from penaeid shrimp are found in white leg shrimp Litopenaeus vannamei (lToll) and black tiger shrimp Penaeus monodon (PmToll). However, the molecular ligand-recognition patterns and identification of these penaeid Toll classes remain unknown. Here, we report cDNA cloning of a new type of Toll receptor gene (MjToll) from kuruma shrimp, Marsupenaeus japonicus, and the modulation of expression by immunostimulation. The full length cDNA of MjToll gene has 3095 nucleotides coding for a putative protein of 1009 amino acids. The MjToll gene is constitutively expressed in the gill, gut, lymphoid organ, heart, hematopoietic organ, hemocyte, ventral abdominal nerve cord, eyestalk neural ganglia and brain tissues. The MjToll gene expression was significantly increased (76-fold) as compared to a control in lymphoid organ stimulated with peptidoglycan at 12h, in vitro. lToll gene showed high similarity to PmToll gene with 96.9% identity; however, MjToll gene exhibited a percentage identity of 59% with that of penaeid Toll homologues. Therefore, this suggests that the identified MjToll gene belongs to the other class of Toll receptors in shrimp.     

Increase of uricogenesis in the kuruma shrimp Marsupenaeus japonicus reared under hyper-osmotic conditions

Tissues of kuruma shrimp Marsupenaeus japonicus Bate (5.7+/-1.1 g) reared in salinities of 18, 26, 34 and 42 were examined for levels of nucleotide-related compounds, ammonia, urea and uric acid, and activities of xanthine dehydrogenase (XDH), xanthine oxidase (XOD) and uricase. Levels of total nucleotide-related compounds, including xanthine and hypoxanthine, in gill increased directly with salinity, whereas these same levels in hepatopancreas were inversely related with salinity. Hemolymph ammonia, urea and uric acid levels, and epidermal ammonia, urea and uric acid levels increased directly with salinity, whereas hepatopancreas ammonia and uric acid and gill uric acid levels were inversely related to salinity. Activities of XDH and XOD in hepatopancreas increased directly with salinity level, whereas no significant difference of uricase activity in hepatopancreas was observed among the four salinities. It is concluded M. japonicus exhibited uricogenesis and uricolysis, and an increase of uricogenesis occurred for the shrimp under hyper-osmotic conditions (salinity of 42). Uric acid produced in the hepatopancreas was transported and accumulated in the epidermis, and removed along with the spongy connective tissue at the time of molting.     

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.     

Structure-activity relationship of crustacean molt-inhibiting hormone from the kuruma prawn Marsupenaeus japonicus

In crustaceans, molt-inhibiting hormone (MIH) controls molting by suppressing the synthesis and/or secretion of molting hormone. In our previous study, which determined the solution structure of MIH by NMR, we hypothesized that the peptide's functional site spanned the region encompassing the N-terminal alpha-helix and a portion of the C-terminus, both of which are located sterically close to each other [Katayama et al. (2003) J. Biol. Chem. 278, 9620-9623]. To confirm this hypothesis, various mutants of MIH were prepared and their molt-inhibiting activities were assessed. All peptides mutated at the putative functional site exhibited circular dichroism spectra similar to the natural MIH, suggesting that the mutants retained their natural conformation regardless of the mutations. As expected, a majority of the mutants, except for Delta12 (a deletion mutant of Gly(12)) and Delta75-77 (a deletion mutant of the last three residues of the C-terminus), were less active than the natural MIH. In particular, I72G exhibited no molt-inhibiting activity even at 200 nM, while N13A and S71Y exhibited low activity at the same concentration. In contrast, the natural and recombinant MIHs exhibited full inhibitory activity at 20 nM. All these results indicate that the functional site of MIH is located in the region containing the C-terminal ends of the N- and C-terminal alpha-helices, and that Asn(13), Ser(71), and Ile(72) are especially significant for conferring molt-inhibiting activity. Furthermore, these findings agree with the results and the proposed hypothesis presented in previous studies on the structure-activity relationship of MIH and its related peptides.     

Deciphering the DNA repair protein, Rad23 from kuruma shrimp Marsupenaeus japonicus: full-length cDNA cloning and characterization

Aims: Lesions of DNA are removed by nucleotide excision repair (NER) process in the living systems. NER process‐related host factors are believed to aid recovery steps during viral integration. Here, we report identification and characterization of a DNA repair molecule Rad23 from kuruma shrimp Marsupenaeus japonicus. Methods and Results: The full‐length cDNA of M. japonicus Rad23 gene (MjRad23) has 1149 bp coding for a putative protein of 382 amino acids with a 5′ untranslated region (UTR) of 92 bp and 3′ UTR region of 1116 bp. Quantitative expression analysis revealed MjRad23 is constitutively expressed in all the organs of healthy shrimp, whereas with high level in muscle tissue. Although MjRad23 expression is observed in every haemolymph samplings to post‐white spot syndrome virus infection, high expression is recorded at 2 h post infection (h.p.i.). MjRad23 consists of putative functional domains including one ubiquitin domain (UBQ), two ubiquitin‐associated domains (UBA) and one heat‐shock chaperonin‐binding motif (STI1). Multiple alignment of MjRad23 with Rad23 of other species showed highly significant identity ranging from 37 to 53%; however, high homology is observed with Rad23 of Bombyx mori (BmRad23). UBQ domain region alignment revealed maximum of 66% homology with Rad23 of Apis melifera (AmRad23). MjRad23 clustered with invertebrate sector along with insect species in evolution analysis. Three‐dimensional structural analyses demonstrated the highest identity between MjRad23 and human Rad23A (hHR23A). Conclusions: The present work revealed the presence of MjRad23 gene, which is essential in DNA repair process. Further studies are required to clarify the involvement of MjRad23 in NER process. Significance and Impact of the Study: This is the first report on identification and characterization of DNA repair protein in crustaceans, which will lead to further investigation to explore the molecular mechanisms behind the NER process.     

Effect of sulfide on the immune response and susceptibility to Vibrio alginolyticus in the kuruma shrimp Marsupenaeus japonicus

Kuruma shrimp Marsupenaeus japonicus held in 34 per thousand seawater were injected with tryptic soy broth (TSB)-grown Vibrio alginolyticus (2.7x10(6)cfu shrimp(-1)), and then placed in water containing concentrations of sulfide at 0 (control), 51, 106, 528 and 1050microgl(-1), respectively. After 12-144h, mortality of V. alginolyticus-injected shrimp exposed to 528 and 1102microgl(-1) sulfide was significantly higher than that of shrimp exposed to 51microgl(-1) sulfide and the control solution. In another experiment, M. japonicus which had been exposed to 0, 56, 112, 525 and 1076microgl(-1) sulfide for 6, 12, 24 and 48h were examined for immune parameters, and phagocytic activity and clearance efficiency of V. alginolyticus. Sulfide concentrations at 525microgl(-1) or greater for 12h resulted in decreased total haemocyte count (THC) and phenoloxidase activity, phagocytic activity and bacterial clearance efficiency, whereas a sulfide concentration at 1076microgl(-1) for 24h caused a significant increase in respiratory burst and superoxide dismutase activity of M. japonicus. It is concluded that concentrations of sulfide at 528microgl(-1) or greater increased the susceptibility of M. japonicus against V. alginolyticus infection by a depression in immune ability. The increased production of superoxide anion by M. japonicus exposed to 525microgl(-1) sulfide or greater was considered to be cytotoxic to the host.     

Metabolic responses and arginine kinase expression under hypoxic stress of the kuruma prawn Marsupenaeus japonicus

In response to hypoxia at PO(2) 1.3-1.7 mg/L for 6 h, the kuruma prawn Marsupenaeus (Penaeus) japonicus showed a dramatic decrease in phosphoarginine storage in muscle, with normal levels restored during 4-h post-hypoxic recovery. Large stores of muscle glycogen only decreased between 4 and 6 h during hypoxia, but greatly diminished during recovery. Muscle ATP levels and energy charge decreased only slightly under hypoxia. Lactate levels increased slightly during hypoxia and promptly returned to control levels during recovery. These data indicate that phosphoarginine works in muscle as an ATP buffer during hypoxia and glycogen is utilized as an energy source during recovery. Under hypoxia, up- and down-regulated proteins were identified after 2D electrophoresis and partial sequences were obtained after protease digestion. Fructose bisphosphate aldolase was down-regulated during hypoxia, suggesting the suppression of glycolysis under hypoxia. Several partial sequences from three protein spots up-regulated under hypoxia were all assigned to arginine kinase, suggesting the existence of several isoforms of arginine kinase in the muscle of M. japonicus. This arginine kinase up-regulation under hypoxia may indicate a provision for oxygen re-supply after anaerobiosis. This is consistent with the prompt replenishment of phosphoarginine stores during recovery from hypoxia.     

Maturation-related variations in prostaglandin and fatty acid content of ovary in the kuruma prawn (Marsupenaeus japonicus)

Total lipid, fatty acids and prostaglandins (PGF(2 alpha) and PGE(2)) in the ovary of kuruma prawns (Marsupenaeus japonicus) were measured during ovarian development. The level of ovarian total lipid increased with an increase in the gonad-somatic index (GSI). No significant difference was found in fatty acid composition among different stages of ovarian development. However, the content of arachidonic acid (precursor of PG(2)), but not eicosapentanoic acid (precursor of PG(3)), was significantly lower at stages I and II than at stage V (P<0.01). When ovarian PGF(2 alpha) and PGE(2) levels were plotted against GSI, no correlation was found in either PG. However, in terms of ovarian developmental stages, the level of ovarian PGs was high (approx. 20 pg/mg) at stage I, followed by marked decreases at stages IV and V (PGF(2 alpha), P<0.01) and stage IV (PGE(2), P<0.01). These results suggest that ovarian PGs and arachidonic acid are deeply involved in ovarian maturation in kuruma prawns.