Identification and characterization of a laccase from Litopenaeus vannamei involved in anti-bacterial host defense.

Phenoloxidases (POs) are a family of enzymes including tyrosinases, catecholases and laccases, which play an important role in immune defences of various invertebrates. Whether or not laccase exists in shrimp and its function is still poorly understood. In this study, a laccase (LvLac) was cloned and identified from Litopenaeus vannamei for the first time. The full length of LvLac is 3406 bp, including a 2034 bp open reading frame (ORF) coding for a putative protein of 677 amino acids with a signal peptide of 33 aa. LvLac contains three Cu-oxidase domains with copper binding centers formed by 10 histidines, one cysteine and one methionine, respectively. Phylogenetic analysis revealed that LvLac was close to insects laccase 1 family. LvLac expression was most abundant in heart and the crude LvLac protein could catalyze the oxidation of hydroquinone. Real-time PCR showed that LvLac expression was responsive to Vibrio parahaemolyticus, Micrococcus lysodeikticus and white spot syndrome virus (WSSV) infection. Knockdown of LvLac enhanced the sensitivity of shrimps to V. parahaemolyticus and M. lysodeikticus challenge, suggesting that LvLac may play a positive role against bacterial pathogens.

cDNA cloning and mRNA expression of retinoid-X-receptor in the ovary of the shrimp Metapenaeus ensis.

Retinoid-X-receptor (RXR) plays an essential role in the molting process of decapod crustaceans, by forming a heterodimeric complex with the ecdysteroid receptor. However, its role during female reproduction, especially in the process of ovarian maturation, has not been characterized. To get an insight into the molecular events governing the process of ovarian maturation in shrimps, the full-length cDNA of RXR from Metapenaeus ensis was cloned by extension of truncated cDNA by using the RACE technique. The open reading frame of MeRXR encodes a 410 amino acid protein with a deduced molecular weight of 44.8 kDa, and putative pI of 6.64, which roughly matched our observation from 2DE gel. Phylogenetic analysis showed that MeRXR has high similarity to RXR of Penaeus chinensis and P. japonicus. RT-PCR revealed that MeRXR was universally expressed in all tissues investigated. The variation in MeRXR mRNA expression pattern during ovarian maturation was further analyzed by real-time PCR. In contrast to the decrease in MeRXR at protein level with ovarian maturation, MeRXR mRNA level was low in pre-vitellogenic and mid-vitellogenic ovaries, and increased significantly from mid-vitellogenic to late-vitellogenic stages. This result suggests that MeRXR transcripts in the mature ovary probably act as maternal messages for regulating early molting events during embryonic development.

The effects of farnesoic acid and 20-hydroxyecdysone on vitellogenin gene expression in the lobster, Homarus americanus, and possible roles in the reproductive process.

Reproduction in female lobster (Homarus americanus) is characterized by the maturation of the ovary, with a gradual increase in its size as a result of uptake of yolk protein precursor, vitellogenin (Vg) to the final product vitellin (Vn). Vn is formed by aggregation of several Vg subunits. In most decapods, the hepatopancreas is the major site of vitellogenin biosynthesis. The production of vitellogenin is controlled by endocrine factors. In this study, the effect of farnesoic acid (FA) and 20-hydroxyecdysone (20E) on production of vitellogenin by hepatopancreas (HaVg1) was investigated by in vitro organ explant HaVg1 gene expression was stimulated by FA or 20E in a dose-dependent manner. A 2-fold and 2.2-fold increase in HaVg1 gene expression was observed with 4.2 microM FA and 0.7 microM 20E, respectively. The stimulatory effect by either FA or 20E was observed principally during the first 90 min. Stimulation of HaVg1 gene expression by FA and 20E together is greater (3.3-fold increase) than that of either hormone alone. This stimulation was also observed within the first 90 min. To study the synergistic effect of these two hormones, FA and 20E were tested separately and together at low concentration (42.3 nM and 6.7 nM, respectively). Combined use of FA and 20E increased HaVg1 gene expression synergistically, but not additively. These findings should contribute to our understanding of lobster reproduction and provide insights into manipulation of lobster reproduction in aquaculture or under captive conditions.

Cloning and expression study of the lobster (Homarus americanus) vitellogenin: Conservation in gene structure among decapods.

This study reports the molecular characterization of the vitellogenin (Vg) of the lobster, Homarus americanus. Based on the annual collection of female lobsters, vitellogenesis commences in early March and continues through to September of each year. Using an antibody to vitellin of the lobster, H. americanus, several immunoreactive ovarian proteins were initially identified by Western blot analysis. The 80kDa protein contained the amino acid sequence APWGGNTPRC, identified subsequently by cDNA cloning to be identical to the lobster Vg. In common with the shrimp Metapenaeus ensis and crab Charybdis feriatus, the lobster HaVg1 gene comprises 14 introns and 15 exons. The deduced HaVg1 precursor is most similar to the Vg of the crayfish Cherax quadricarinatus (57%), followed by M. ensis (40-43% identity) and C. feriatus (38%). The results from genomic and RT-PCR cloning also confirmed the presence of multiple Vg genes in lobster. At early reproductive stages, the hepatopancreas HaVg1 transcript levels are low but increased to a maximum in animals with mature oocytes. The ovary, however, also expressed low levels of HaVg1. Using in vitro explant culture, treatment of hepatopancreas fragments with farnesoic acid or 20-hydroxyecdysone resulted in a significant stimulation in HaVg1 expression. From this study, it appears that Vg gene organization and expression pattern in decapods is highly conserved. Similar endocrine mechanisms may govern the process of vitellogenesis across the decapods.

Characterization of a novel cellular retinoic acid/retinol binding protein from shrimp: expression of the recombinant protein for immunohistochemical detection and binding assay.

Members of the cellular retinoic acid (CRABP) and retinol binding (CRBP) proteins family are involved in the metabolic pathways of retinoic acid (RA) and retinal respectively. The objective of this study is to determine whether such proteins are present in crustaceans. We report here the cloning and isolation of a novel complementary DNA (cDNA) that showed characteristics of the CRABP/CRBP from the ovary and eyestalk of the shrimp. The cDNA is 0.9 Kb in size and the deduced shrimp protein is encoded for a protein of 14 kDa. Although it shows high amino acids sequence similarity to both the vertebrate and invertebrate CRABP, some conserved amino acids identified in other CRABPs were not found in MeCRABP. MeCRABP is expressed in the ovary, eyestalk, testis, epidermis and early larvae. The presence of MeCRABP in early larval stages suggests that the protein may be involved in the early larval development. Recombinant MeCRABP was produced and used to generate a polyclonal antibody. In the immunohistochemical detection study, anti-rCRABP antibody recognized the presence of CRABP in several cell types of the eyestalk as well as the smaller oocytes of the ovary. Although MeCRABP messenger RNA transcripts can be detected in the ovary throughout the ovarian maturation period, CRABP was detected only in the primary oocytes of the ovary. The results suggest that CRABP transcripts in the mature ovary are not translated and may be supplied to the oocyte as maternal messages. The binding property of the recombinant MeCRABP was also tested by a fluorometeric method. The result indicates that rMeCRABP binds to both RA and retinal with similar affinity. This study represents the first cloning and characterization of a cDNA that belongs to a member of retinoid/fatty acid binding protein family in crustaceans.