Metapenaeus Ensis Research Articles

Differential Gene Expression in Hepatopancreas of the Shrimp Metapenaeus ensis During Ovarian Maturation

Differentially expressed genes were identified in the hepatopancreas of Metapenaeus ensis during ovarian maturation via differential display reverse transcription-polymerase chain reaction (DDRT-PCR). They are G-protein signaling modulator 2 (GPSM2), glutamate carboxypeptide II (GCPII), ligatin, C-type lectin, O-linked N-acetylglucosamine transferase (O-GlcNAc transferase), 1-acylglycerol-3-phosphate O-acyltransferase 4 (AGPAT4), vitellogenin (Vg), and hemocyanin. The hepatopancreas Vg gene identified in this study shows 92% and 49% amino acid sequence homology, respectively, to MeVg1 and MeVg2 previously isolated from this species, suggesting the identification of a new Vg gene in M. ensis. Vg gene expression was highest when the ovary was actively developing. The two metabolic enzymes, O-GlcNAc transferase and AGPAT4, exhibited a similar trend of expression to Vg gene, suggesting their involvement in Vg synthesis. The signal transduction related genes (GPSM2, GCPII, ligatin, and C-type lectin) were highly expressed in the hepatopancreas in the initial phase of maturation. These genes may be important for the signaling in the hepatopancreas for synthesis and mobilization of vitellogenin and nutrients to the developing ovary. The present work provides candidate genes for further investigation on the role of hepatopancreas in shrimp reproduction.

The use of recombinant protein and RNA interference approaches to study the reproductive functions of a gonad‐stimulating hormone from the shrimp Metapenaeus ensis

Although the crustacean crustacean hyperglycemic hormone/molt-inhibiting hormone/gonad-inhibiting hormone neuropeptides have been studied extensively in the last two decades and several neuropeptides from the shrimp Metapenaeus ensis have been cloned, the functions of most of these neuropeptides remained putative. In this article, we describe the use of recombinant protein and an RNA interference approach to study the reproductive function of the previously reported molt-inhibiting hormone (MeMIH-B) in M. ensis. When hepatopancreas and ovary explants were cultured in medium containing recombinant MeMIH-B, the vitellogenin gene (MeVg1) expression level was upregulated in a dose-dependent manner, reaching a maximum in explants treated with 0.3 nm recombinant MeMIH-B. Shrimp injected with recombinant MeMIH-B showed an increase in vitellogenin gene expression in the hepatopancreas. Moreover, a corresponding increase in the vitellogenin-like immunoreactive protein was detected in the hemolymph and ovary of these females. Injection of MeMIH-B dsRNA into the female shrimp caused a decrease in MeMIH-B transcript level in thoracic ganglion and eyestalk. These shrimp also showed reduction of vitellogenin gene expression in the hepatopancreas and ovary. Furthermore, the hemolymph vitellogenin level was also reduced in these animals. In summary, the results from recombinant protein and RNA interference experiments have demonstrated the gonad-stimulatory function of MeMIH-B in shrimp.

Molecular Coordinated Regulation of Gene Expression During Ovarian Development in the Penaeid Shrimp

To understand the molecular events of ovarian development in penaeid shrimp, RNA arbitrarily primed polymerase chain reaction (RAP-PCR) was used to identify differentially expressed genes during ovarian maturation in Metapenaeus ensis. From a screening of 700 clones in a cDNA library of the shrimp ovary by the products of RAP-PCR of different maturation stages, 91 fragments with differentially expressed pattern as revealed by dot-blot hybridization were isolated and sequenced. Forty-two of these fragments show significant sequence similarity to known gene products and the differentially expressed pattern of 10 putative genes were further characterized via Northern hybridization. Putative glyceraldehyde-3-phosphate dehydrogenase and arginine kinase are related to provision of energy for active cellular function in oocyte development. Translationally controlled tumor protein, actin, and keratin are related to the organization of cytoskeleton to accomplish growth and development of oocytes. High mobility group protein DSP1, heat shock protein 70, and nucleoside diphosphate kinase may act as repressors before the onset of ovarian maturation. Peptidyl-prolyl cis-trans isomerase and glutathione peroxidase are related to the stabilization of proteins and oocytes. This study provides new insights on the molecular events in the ovarian development in the shrimp.

Food digestion by cathepsin L and digestion-related rapid cell differentiation in shrimp hepatopancreas

Cathepsin L (CatL) has been readily localized in the large vacuole and in the apical complex of the digestive B-cell of the shrimp hepatopancreas. Immunogold technique revealed the occurrence of CatL in zymogen granule, digestive body and digestive vacuole of the B-cell in the hepatopancreas of Metapenaeus ensis. Coalescences of zymogen granule with sub-apical vacuole, and of two small digestive bodies were observed. This progressive coalescence of CatL vesicles is direct evidence of involvement of CatL in intracellular digestion. Released CatL vesicles and free CatL were found in the lumen of hepatopancreatic tubule. CatL mRNA existed in F-cell, but not in the mature B-cell. This finding supports the previous suggestion that F-cell is the precursor of B-cell. F-cell is a transient form. Transition from F-cell to B-cell is fast. We define F-cell as the transcribing cell, F/B-cell as the enzyme-synthesizing cell and B-cell as the enzyme-secreting cell. For the first time, we suggest that R-cell is the replacing cell for the leaving B-cell. CatL degrades nutrient intracellularly and extracellularly. The most interesting finding is that CatL is transcribed in one type of cell, and the very cell evolves quickly to a morphologically different cell where the enzyme functions.

Acute Toxicity of Ammonia to Penaeus semisulcatus Postlarvae in Relation to Salinity

Stocking densities of cultured penaeid shrimp have been intensified because of the limited availability of land for pond construction. In an intensive culture system, ammonia is the most common toxicant resulting from nitrogen excretion by cultured animals and mineralization of unconsumed feed and feces (Lin and Chen 2003). In decapod crustaceans, nitrogen is excreted mainly as ammonia (60–70% of total ammonia) (Lee and Chen 2003). Concentrations of total ammonia-nitrogen (TAN) increase directly with culture period and might reach as high as 46 mg TAN/L in intensive grow-out ponds (Chen and Lin 1992). Accumulation of ammonia-nitrogen in pond water may deteriorate water quality, reduce growth, increase oxygen consumption, alter concentrations of hemolymph protein and free amino acid levels, and even cause high mortality (Chen and Lin 1992; Chen et al. 1994). TAN is composed of NH3-N (unionized ammonia) andNH4+ (ionized ammonia) (Losordo et al. 1992; Masser et al. 1992). It is the unionized form that is most toxic to aquatic organisms as it can readily diffuse through cell membranes and is highly soluble in lipids (Chin and Chen 1987; Frias-Espericueta et al. 1999). There is an equilibrium between NH4+ and NH3-N in the water. This equilibrium is affected by pH, temperature, and salinity. The higher the pH and temperature, the higher the proportion of NH3-N, while an increase in salinity will lead to lower proportions of the unionized form, hence the toxicity of the TAN to aquatic animals (Losordo et al. 1992;Masser et al. 1992; Sampaio et al. 2002). A number of studies have been conducted on the lethal effects of ammonia at various life stages of penaeid shrimps, such as Penaeus chinensis (Chen and Lin 1992), P. monodon (Chen and Lei 1990), P. paulensis (Ostrensky and Wasielesky 1995), P. penicillatus (Chen and Lin 1991), P. semisulcatus (Wajsbrot et al. 1990), and Metapenaeus ensis (Nan and Chen 1991). However, little information is available on the lethal effect of ammonia at different salinity levels for penaeid shrimps. Chen and Lin (1992) and Lin and Chen (2003) studied ammonia toxicity in P. chinensis and Litopenaeus vannamei at three different salinity levels, but no results have been reported for other penaeid species. Green tiger shrimp (P. semisulcatus) is an Indo-Pacific species, distributed along the coast of the Eastern Mediterranean, and is one of the most important commercial species in this part of the world. This species is less tolerant to low salinity levels (Soyel and Kumlu 2003), and knowledge of the effects of ammonia toxicity at various salinity regimes that may be confronted during the culture and overwintering periods in the subtropical areas is of importance for the farmers. This paper provides information on acute toxicity of ammonia to juveniles of this shrimp species at six different salinity levels (15, 20, 25, 30, 35, and 40 ppt).

Risk assessment of heavy metal contamination in shrimp farming in Mai Po Nature Reserve, Hong Kong

An ecological survey was carried out to determine the sediment concentrations of nutrients and heavy metals and bioaccumulation of heavy metals in fish and shrimp including tilapia (Oreochromis mossambicus x O. nilotica), grey mullet (Mugil cephalus), gei wai shrimp (Metapenaeus ensis) and caridean shrimp (Macrobrachium nipponensis) in the traditional tidal shrimp ponds (gei wais) of Mai Po Nature Reserve, Hong Kong. The sediments collected from the landward sites contained higher nutrient contents, as well as zinc (Zn), chromium (Cr), copper (Cu), nickel (Ni) and cadmium (Cd) than those collected from the seaward sites, but vice versa for lead (Pb) and mercury (Hg). However, the concentrations of all metals were exceptionally high in the two sites located outside the reserve, suggesting that waters from Deep Bay might be the possible source of metal contamination affecting the reserve. All metals studied seemed to accumulate in the viscera of fish. Body size was the determining factor for the accumulation of heavy metals in caridean shrimp and gei wai shrimp but not fish. Concentrations of the metals studied in tissues of grey mullet and gei wai shrimp were found to be safe for human consumption. Concentrations of Cr in tilapia whole body (0.68-1.10 mg kg(-1) wet weight) were close to or over the guideline value of 1 mg kg(-1) set by the Food Adulteration (Metallic Contamination) Regulations of Hong Kong. Tilapia flesh and small caridean shrimp collected from gei wais were contaminated by Cr and Pb but still fit for human consumption. Caution is required if large caridean shrimp is to be consumed in large amounts continuously because the concentration of Pb exceeded the maximum permitted concentration (6 mg kg(-1)). The rather high Cr concentrations in tilapia whole body should not be overlooked as the fish will serve as a food source for migratory birds visiting the site.

Characterization of vitellogenin in the shrimp Metapenaeus ensis: Expression studies and hormonal regulation of MeVg1 transcription in vitro

During gonad maturation, female shrimp accumulate the major egg yolk protein vitellin (Vn) in premolt stage, and the process of molting and reproduction is synchronized. Using a polyclonal anti-Vn antibody, immunopositive signals could be detected in the ovary and among the proteins secreted by the hepatopancreas by Western blot. In the ovary, Vn immunoreactivity was located in the posterior lobe. Hepatopancreas proteins with sizes identical to ovary vitellogenin (Vg) subunits (i.e., 78 and 157 kDa) were immunoreactive to the Vn antibody and these proteins included amino acid sequences identical to parts of the MeVg1 precursor. A major 7.8 kb MeVg1 transcript, was detected in the ovary. In the hepatopancreas, the transcripts were primarily small (<2.3 kb) and while the 7.8 kb transcript which constitutes <50% of the total Vg mRNA. MeVg1 transcript could be detected in the hepatopancreas of juvenile females with a maximum level during late intermolt and early premolt. To study the effect of different hormones on expression of MeVg1, explant cultures of hepatopancreas and ovary were developed. Although several hormones (20-hydroxyecdysone, estradiol (ES), farnesoic acid (FA), juvenile hormone (JH) III, methyl farnesoate, and progesterone (PG)) apparently stimulated MeVg1 gene expression, only FA consistently stimulated MeVg1 expression by the hepatopancreas explants, while both FA and 20-hydroxyecdysone were stimulated ovarian explants. In summary, (i) Vg transcripts can be detected in both reproductive and nonreproductive females; (ii) the presence of large quantities of smaller Vg transcripts and the absence of a large Vg precursor from the hepatopancreas suggests that smaller MeVg1 transcripts provide an important contribution to Vg synthesis in shrimp. Our results suggest that there is differential processing of the MeVg1 precursor in the ovary and hepatopancreas of shrimp.

Ecdysteroid-responsive genes, RXR and E75, in the tropical land crab, Gecarcinus lateralis: Differential tissue expression of multiple RXR isoforms generated at three alternative splicing sites in the hinge and ligand-binding domains

In order to study the potential role of the steroid molting hormone (20-hydroxyecdysone) in regulating molt-induced claw muscle atrophy, full-length cDNAs encoding retinoid-X receptor (Gl-RXR) and E75 early ecdysone inducible gene (Gl-E75) were obtained from land crab ( Gecarcinus lateralis) skeletal muscle mRNA using RT-PCR and 3′ and 5′ RACE. Gl-E75A (3528 bp), which encoded a protein of 828 amino acids, had highest sequence identity to Me-E75A from a shrimp ( Metapenaeus ensis). It was expressed in skeletal muscle and gonads. The deduced amino acid sequence of Gl-RXR was highly similar to that of the fiddler crab RXR (Up-RXR) and insect ultraspiracle (USP). Nine variant sequences occurred in Gl-RXR mRNAs at three alternative splicing sites, one in the “T box” in the linker D domain and two in the ligand-binding domain (LBD). The three T-box variants, termed T(+8), T(+7), and T(+12), contained insertions of 8, 7, or 12 amino acids, respectively. Four variants were generated at the first site in the LBD. Two of the LBD site 1 variants differed in the presence (+33) or absence (−33) of a 33-amino acid sequence; the other two were LBD truncations with or without the 33 amino acid sequence (+33ΔE/F and −33ΔE/F, respectively). Two variants differing in the presence (+35) or absence (−35) of a 35-amino acid sequence were generated at the second site in the LBD. The Gl-RXRa isoform (1516 bp) with the longest open reading frame (+12/+33/+35) encoded a protein of 436 amino acids. Thoracic muscle expressed only isoforms with the T(+12) sequence. In contrast, claw muscle expressed isoforms with T(+7) or T(+12) and fewer isoforms with T(+8). Ovary and testis expressed a greater number of RXR isoforms than skeletal muscle. All tissues expressed full-length and truncated RXR isoforms. These data suggest that differences in response of claw and thoracic muscles to elevated ecdysteroid are due in part to differences in the expression of RXR isoforms.

Organization and expression study of the shrimp ( Metapenaeus ensis) putative 5-HT receptor: Up-regulation in the brain by 5-HT

Abstract To study the mechanism of 5-HT action and the roles of its receptor in the control of reproduction, we have cloned and characterized the gene and the cDNA of a putative 5-HT receptor (5HT1) from the shrimp, Metapenaeus ensis. The 5HT1 gene is intronless in the coding region but consists of two introns in the 5′ untranslated region. 5HT1 transcript is 1.8 kb in size and the cDNA consists of an open reading frame of 1230 bp encoding for a protein of 409 amino acid residues. The deduced 5HT1 consists of the characteristic seven hydrophobic transmembrane (TM1–TM7) domains, which share a high amino acid sequence homology to those of the GPCRs. The results from phylogenetic tree analyses indicate that 5HT1 is more closely related to the octopamine/tyramine of the insect and the 5-HT receptors of the vertebrates than to the other G-protein coupled receptors. Although there is no major difference in the tissues' expression pattern of 5-HT in both sexes, the expression level of 5HT1 is much lower in the females than that in the males. 5HT1 expression in the brain and in the eyestalk is also up-regulated in the 5-HT injected shrimp. By in situ hybridization, no difference in the expression pattern of 5-HT was recorded in the eyestalk of males and females, and the pattern of 5HT1 expression in the eyestalk remained unchanged after 5-HT injection. In the eyestalk, 5HT1 transcripts can be detected in neuronal globuli cells and X-organs. The up-regulation of 5HT1 in the eyestalk and brain may be important for the sustained action in the signal transduction pathway or the regulation of other genes. This may represent an auto-regulation of 5HT1 expression in shrimp by 5-HT. To our knowledge, this is the first demonstration of 5-HT stimulation of a putative 5HT1 gene expression in shrimp.

Vitellogenesis in the red crab Charybdis feriatus: Hepatopancreas‐specific expression and farnesoic acid stimulation of vitellogenin gene expression

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.

Genetic and phenotypic variations of isolates of shrimp Taura syndrome virus found in Penaeus monodon and Metapenaeus ensis in Taiwan.

Distinct Taura syndrome virus (TSV) isolates were found in Metapenaeus ensis (isolate Tw2KMeTSV), Penaeus monodon (isolate Tw2KPmTSV) and Litopenaeus vannamei (isolate Tw02LvTSV). Nucleotide sequence analysis of these three isolates revealed differences in the TSV structural protein (capsid protein precursor) gene orf2. TSV ORF2 amino acid sequence comparison and phylogenetic analysis suggested a comparatively close relationship between these three Taiwanese isolates and the Hawaiian isolate HI94TSV. In P. monodon specimens that were naturally and experimentally infected with the Tw2KPmTSV isolate, the virus was contained and shrimps showed no clinical signs of infection. However, when P. monodon was challenged with the Tw2KMeTSV isolate, the virus replicated freely. The ORF2 amino acid sequence of the Tw2KMeTSV isolate differed from that of isolate Tw2KPmTSV in four positions and these differences may account for their phenotypic differences, at least in terms of their ability to replicate in specific hosts.

Phylogenetic Relationships of 12 Penaeoidea Shrimp Species Deduced from Mitochondrial DNA Sequences

DNA sequences of an 847 bp fragment of mitochondrial cytochrome oxidase subunit I (COI) gene and a 514 bp fragment of 16s rRNA gene were determined to examine the phylogenetic relationships of 12 Penaeoidea shrimp species (Penaeus chinensis, Penaeus japonicus, Penaeus penicillatus, Penaeus vannamei, Penaeus canaliculatus, Trachypenaeus curvirostris, Metapenaeus affinis, Metapenaeus ensis, Metapenaeopsis barbata, Parapenaeus fissuroides, Parapenaeopsis hardiwickii, Solenocera crassicomis). Both fragments of the swimming crab Portunus trituberculaus chosen as the outgroup were also sequenced. Intraspecific sequence divergence of 0.24-1.2% in the COI gene was found in 5 species, while no intraspecific variation was observed in the 16s rRNA gene. Three phylogenetic trees based on the 1361 bp combined sequences of COI and 16s rRNA were concordant in indicating the following suggestions: (1) phylogenetic relationship of the 11 Penaeidae species based on our result support the opinion of Burkenroad (Burkenroad, M.D. (1983). Crustacean Issues 3:279-290) on the basis of morphological features; (2) it seems more reasonable to class Solenocera crassicorni in the family Penaeidae; (3) the fragment of the COI gene chosen here appears to be a good marker for speciation studies and population analysis in Crustaceans, while the 16s rRNA gene fragment here seems suitable for examining phylogenetic relationships at the species or genus levels in Crustaceans. Our time estimates suggest that Penaeus and Metapenaeus might have separated about 6.38 x 10(6)-7.98 x 10(6) years BP in the post-Miocene, and the species separation within Metapenaeus and Penaeus might occur 0.08 x 10(6)-0.4 x 10(6) years BP in the late Pleistocene.

Vitellogenesis in the sand shrimp, metapenaeus ensis: the contribution from the hepatopancreas-specific vitellogenin gene (MeVg2).

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.

Shrimp cathepsin L encoded by an intronless gene has predominant expression in hepatopancreas, and occurs in the nucleus of oocyte

We have cloned the cDNA and genomic DNA of an active intronless cathepsin L from Metapenaeus ensis. The encoded enzyme has the shortest prosequence among cathepsin L subgroup. It was predominantly expressed in hepatopancreas with an expression level of at least 10 times higher than in any other tissues. It also has expression in stomach, intestine, eye, testis, ovary and muscle. Western blots visualized the mature enzyme in hepatopancreas and a procathepsin L in ovary, intestine and stomach. Metapenaeus cathepsin L (MeCatL) is localized in the large digestive vacuole of the digestive B cell of hepatopancreas. MeCatL has a role in food digestion. An interesting finding is that it exists in the nucleus of oocyte. MeCatL might have a specified physiological role in the nucleus of oocyte. MeCatL might also have a house-keeping function as is suggested for mammalian cathepsin L.

Comparative immunohistochemistry and cellular distribution of farnesoic acid O-methyltransferase in the shrimp and the crayfish

Farnesoic acid O-methyltransferase (FAMeT) catalyzes the conversion of farnesoic acid (FA) to methylfarnesoate (MF) by the mandibular organ (MO) of crustaceans. Here we report the cellular localization of FAMeT and radiochemical assay of endogenous FAMeT activity in shrimp ( Metapenaeus ensis) and crayfish ( Procambarus clarkii) tissues. As in the eyestalk (ES), FAMeT is concentrated in specific neurosecretory cells of the ventral nerve cord (VNC) whereas only weak FAMeT immunoreactivity was observed in the MO. FAMeT was also detected in the ventral nerve cord, heart (HET), eyestalk, and muscle of the juvenile shrimp. Although the VNC shows the greatest FAMeT immunoreactivity, the heart extract exhibited the highest FAMeT enzymatic activity. These results suggest that FAMeT in the VNC may be inactive or inactivated at the stages of development tested. Contrary to the previous reports in other crustaceans, MO extract in shrimp shows only low FAMeT activity. The eyestalk, epidermis, ovary and testis show appreciable FAMeT activity. The presence of FAMeT in neurosecretory cells of VNC and eyestalk of shrimp and crayfish implies a possible interaction of FAMeT with the eyestalk CHH-family of neuropeptides. The widespread activity of FAMeT suggests that it has a wide spectrum of action in many tissues that contribute to the function and regulation of MF synthesis in shrimp and crayfish.

Organization of the shrimp vitellogenin gene: evidence of multiple genes and tissue specific expression by the ovary and hepatopancreas

Vitellogenin is the major egg yolk protein synthesized in female shrimp during gonad maturation. Although there are several reports for the cloning of vitellogenin complementary DNA (cDNA) in different crustaceans, little is known of the gene organization of this protein. This study reports the first cloning and characterization of a full-length gene encoding the vitellogenin precursor from the shrimp Metapenaeus ensis. By genomic DNA library screening, six different lambda clones were isolated using shrimp partial gene sequence as probe. Initial DNA sequence determination revealed that these clones are derived from different genes with coding sequence similar to other crustacean vitellogenins. Two of these clones were used for further analysis. One of the lambda clones (λ3.3) carries most of the coding sequence that correspond to the M. ensis vitellogenin gene (MeVg1) and the other clone (λ8.3) carries a smaller portion of the coding sequence of a different vitellogenin gene (MeVg2). The λ3.3 clone was chosen for further characterization. To clone the remaining 5′ end upstream promoter region, 5′ untranslated region and the remaining coding sequence of MeVg1, a polymerase chain reaction (PCR)-based gene walking approach was used. Subsequently, a PCR clone with overlapping sequence identical to the genomic clone was obtained and the organization of MeVg1 gene was constructed. The MeVg1 gene consists of 15 exons and 14 introns spanning approximately 10 kb. Several potential cleavage sites were identified from the deduced vitellogenin precursor. Cleaving of the precursor in these sites would result in the production of several vitellogenin subunits. To clone the cDNA for the vitellogenin, 5′ and 3′ rapid amplification of cDNA ends was performed using ovary cDNA of the shrimp. A 4.4 kb 5′ cDNA clone and a 4 kb 3′ end cDNA clone were isolated. The size of the reconstructed cDNA for M. ensis Vg is 7.97 kb and consists of the longest open reading frame of 7776 bp. Unlike the vitellogenin precursor of most insects and vertebrates, the deduced vitellogenin precursor lacks the polyserine domain important for receptor-mediated endocytosis. Phylogenetic analysis revealed a closer relationship of the MeVg1 with other crustacean vitellogenins but distantly related to other invertebrate and vertebrate vitellogenins. By reverse transcription-PCR, we have demonstrated that the shrimp MeVg1 gene is expressed only in the ovary and hepatopancreas while the MeVg2 gene is expressed exclusively in the hepatopancreas. In conclusion, the shrimp ovary also contribute significantly in the production of vitellogenin at transcription level and the gene organization of the shrimp protein may provide an insight in the evolution of this group of important proteins.

Characterization of an additional molt inhibiting hormone-like neuropeptide from the shrimp Metapenaeus ensis

We have identified a second form of the type-II neuropeptide encoding a molt inhibiting hormone-like (MeeMIH-B) neuropeptide. MeeMIH-B showed only a 70% amino acid identity to the MIH-A (formerly MIH) isolated from the same species, suggesting a possible different function of the deduced neuropeptide. Like other neuropeptide members of the CHH family, the MIH-B gene consists of three exons separated by two introns. The levels of MIH-B mRNA transcript in the eyestalk decrease in the initial phase of gonad maturation and increase towards the end of maturation. The drop in MIH-B level suggests an inhibitory role for this neuropeptide in the initiation of vitellogenesis. MIH-B transcripts can also be detected in the brain, thoracic ganglion and ventral nerve cord. Together with the CHH-B peptide that we have previously described, this is the second peptide of the CHH family that can also be identified in the ventral nerve cord and in the XOSG complex. A recombinant MIH-B was produced and a polyclonal antibody against rMIH-B was subsequently generated. Specific anti-rMIH-B antiserum recognized the presence of MIH-B in the sinus gland, X-organs, as well as a giant neuron of the ventral nerve cord. Injection of rMIH-B delayed the molting cycle of the maturing female. Taken together, the results of this study suggest that a drop in MIH-B level may be required for the delay in the molting of the maturing females.

Genomic instability of prawn white spot bacilliform virus (WSBV) and its association to virus virulence

Prawn White Spot Bacilliform Virus (WSBV) is a major pathogen that causes prawn diseases. In this study, we examined the sequence of WSBV genome DNA in the shrimp Penaeus japonicus, P. vannamei, P. Monodon, P. chinensis and Metapenaeus ensis through successive PCR amplification of the DNA fragments in the whole WSBV genome. We found a sequence deletion hotspot in the WSBV genome that is 305,107 bp in length. The sizes of the deleted fragments were 4.6, 4.8 or 8.1 kbp depending on the species of prawn. Since the mortality of shrimp infected by the intact WSBV was always significantly higher than that of shrimp infected by DNA fragment-deleted WSBV, we suggest that this deletion be somehow linked to the virulence of the virus itself. This result may lead to the discovery of the molecular mechanism of the pathogenicity of WSBV in shrimps.

A change of heart: cardiovascular development in the shrimp Metapenaeus ensis

The larval development of penaeid shrimp is among the most complicated in crustaceans. In Metapenaeus ensis, there are six naupliar, three protozoeal and three mysid larval instars, followed by postlarval development. Irregular heartbeat begins late in naupliar instar 6. Co-ordinated beating at 400–600 beats min −1 commences in the first protozoeal instar and continues throughout larval life. Initially, the contractile region is located more posteriorly in the cephalothorax and has a single pair of ostia, and the arterial distribution is limited to a single anterior vessel. In later mysid instars, a second cardiac pumping site develops posterior to, but connected with, the original site. This extension is more muscular, contains additional ostia and develops additional distribution vessels supplying the cephalothorax and abdominal areas. The original site is gradually merged into the new extension and only small refinements in the circulation occur in postlarval and juvenile life. Changes in physiological responses of the heart also occur throughout development. Responses to intra-pericardial microinjection of 5-hydroxytryptamine change drastically during development, as do cardiac responses to ambient hypoxia. Similarly, heartbeat of later juvenile instars is inhibited by injection of tetrodotoxin, while heartbeat of larval and early juvenile instars is not, suggesting that neurogenic regulation via the cardiac ganglion arises later in development. Our present studies attempt to integrate the anatomical and physiological changes in the development of the crustacean heart.

Function and cellular localization of farnesoic acid O-methyltransferase (FAMeT) in the shrimp, Metapenaeus ensis.

The isoprenoid methyl farnesoate (MF) has been implicated in the regulation of crustacean development and reproduction in conjunction with eyestalk molt inhibiting hormones and ecdysteroids. Farnesoic acid O-methyltransferase (FAMeT) catalyzes the methylation of farnesoic acid (FA) to produce MF in the terminal step of MF synthesis. We have previously cloned and characterized the shrimp FAMeT. In the present study, recombinant FAMeT (rFAMeT) was produced for bioassay and antiserum generation. FAMeT is widely distributed in shrimp tissues with the highest concentration observed in the ventral nerve cord. Interestingly, an additional larger protein in the eyestalk also showed immunoreactivity to anti-FAMeT serum. FAMeT was localized in the neurosecretory cells of the X-organ-sinus gland complex of the eyestalk. As shown by RT-PCR, FAMeT mRNA is constitutively expressed throughout the molt cycle in the eyestalk and the ventral nerve cord. To show that our cloned gene product had FAMeT activity, we demonstrated that expressed rFAMeT gene product catalyzed the conversion of FA to MF in a radiochemical assay. The ubiquitous distribution of FAMeT suggests that this enzyme is involved in physiological processes in addition to gametogenesis, oocyte maturation and development and metamorphosis of the shrimp. We hypothesize that FAMeT directly or indirectly (through MF) modulates the reproduction and growth of crustaceans by interacting with the eyestalk neuropeptides as a consequence of its presence in the neurosecretory cells of the X-organ-sinus gland.