Identification and characteristics of a novel gene, <Emphasis Type="Italic">EJO1</Emphasis>, in the Chinese mitten crab (<Emphasis Type="Italic">Eriocheir japonica sinensis</Emphasis>) ovary

EJO1, a novel gene presumably involved in the ovary development of the Chinese mitten crab (Eriocheir japonica sinensis), was identified and characterized by suppression subtractive hybridization and cDNA macroarray analysis. EJO1 expression was 2.6-fold higher at stage III than at stage II during the ovary development of the mitten crab. EJO1 is 876 bp in length containing a 759 bp open reading frame which encodes a 252-amino-acid protein. Homology analysis showed that no sequence significantly matching EJO1 was found in SwissProt, so it was deduced as a novel gene (GenBank accession number: AY185917). The EJO1 protein is probably a secretion protein with a signal peptide of 17 amino acids. The pI/Mw deduced from the amino acid sequence was 6.18/28.18 kDa. Expression profile showed that EJO1 mRNA is highly expressed in the heart, intestine, and ovary of the crab, while there is little or no expression in muscle and hepatopancreas. The differential expression of EJO1 at the different developmental stages of the ovary was further confirmed by Northern blot analysis. In conclusion, EJO1 is a novel gene differentially expressed in the ovary of the Chinese mitten crab, which may play an important role in the ovary development.     

Effects of the complete replacement of fish oil with linseed oil on growth,fatty acid composition,and protein expression in the Chinese mitten crab (<Emphasis Type="Italic">Eriocheir sinensis</Emphasis>)

Background

The finite marine resources make it difficult for us to obtain enough fish oil (FO) used in aquatic feeds. Another sustainable ingredients should be found to substitute FO. The effects of replacing FO with vegetable oil have been studied in a variety of crustaceans, but most studies have focused on the phenotypic effects. Little is known about the mechanisms of the effects.

Methods

To understand the molecular responses during the replacement of FO in Eriocheir sinensis, we investigated the effects of feeding FO or linseed oil (LO) on growth performance, digestive enzyme activity, fatty acid composition and protein expression in E. sinensis. Twenty-four juvenile crabs were fed diets containing FO or LO for 112 days. Weight, carapace length and width were recorded. Fatty acid composition of the diets and the hepatopancreas and protein expression in the hepatopancreas were analyzed.

Results

Growth performance and molting interval were unchanged by diet. Crabs fed FO and LO had same activity of lipase and amylase, but comparing with crabs fed LO, crabs fed FO had higher trypsin activity and lower pepsin activity. Hepatopancreas fatty acid composition changed to reflect the fatty acid composition of the diets. In total, 194 proteins were differentially expressed in the hepatopancreas between the diets. Expression of heat shock proteins was higher in crabs fed LO. Expression of fatty acid synthase, long-chain fatty acid transport protein 4, acyl-CoA delta-9 desaturase, and fatty acid-binding protein 1, was higher in crabs fed FO.

Conclusions

The substitution of FO with LO didn’t have any effects on the growth and molting of mitten crab, but could significantly decrease the ability of mitten crab to cope with stress. The high content of HUFAs in the hepatopancreas of mitten crab fed FO is due to the high abundance of the proteins relative to the transport of the HUFAs. These findings provide a reason of the high content of EPA and DHA in crabs fed with FO, and provide new information for the replacement of FO in diets of mitten crab.

     

Characteristic of PGDS potential regulation role on spermatogenesis in the Chinese mitten crab Eriocheir sinensis

Prostaglandin D synthase (PGDS) catalyzes the isomerization of PGH2 to produce PGD2 in the presence of sulfhydryl compounds. In this study, a full length PGDS gene comprising 1250 nucleotides from the Chinese mitten crab Eriocheir sinensis (Es-PGDS) was characterized, with a 615 bp open reading frame encoding 204 amino acid residues. Its deduced peptide has high homology with other species' PGDS protein. The Es-PGDS mRNA expression was tissue-related, with the highest expression observed in the hepatopancreas, accessory sex gland, testis and ovaries. We also detected the different stages of tissue expression and the enzyme activity for Es-PGDS in the testis and male crab hepatopancreas. The different expression patterns and its corresponding enzyme activity level indicated that PGDS is involving in the regulation of reproductive action during the period of rapid development in E. sinensis. Furthermore our research could arouse a heat debate on the PGDS reproductive function in invertebrate and further study will be needed to determine the molecular mechanism(s) linking PGDS functions to spermatogenesis and ontogenesis if this gene is to be exploited as a molecular biomarker in further studies of development.     

Metabolic effects of eyestalk removal in the crab Varuna litterata (Fabricius)

Metabolic effects of eyestalk removal in the crab V. litterata is studied. Bilateral eyestalk ablation results in hypoglycaemia and a fall and rise in the glycogen content of hepatopancreas and muscle respectively. Injection of unboiled eyestalk extract evokes hyperglycaemia, whereas boiled eyestalk extract does not produce any significant change in the glycaemia level. The other effect of eyestalk removal is a fall in the fat content of the hepatopancreas and these results are discussed.     

Reproductive function of Selenoprotein M in Chinese mitten crabs (Eriocheir sinesis)

Selenoproteins are present in all major forms of life, including eukaryotes, bacteria and archaea. In eukaryotic animals, selenoproteins often function as antioxidants, but rare or absent in other phyla, such as plants and fungi (except for the green alga Chlamydomonas). Selenoprotein M (SelM) is a selenocysteine containing protein with redox activity, which is involved in the antioxidant response. However, information remains limited about SelM physiology and function in marine invertebrates, particularly in crustaceans. Hence, we investigated the reproductive functionality of SelM in the Chinese mitten crab (Eriocheir sinensis), which is a commercially important yet disease vulnerable aquaculture species. The full-length SelM cDNA (928bp) strand was cloned by using PCR, based on an initial expressed sequence tag (EST) that was isolated from a hepatopancreatic cDNA library. The SelM cDNA contained a 390bp open reading frame (ORF) that encoded a putative 129 amino acid (aa) protein. SelM mRNA expression in E. sinensis was (a) tissue-specific, with the highest expression observed in the hepatopancreas, testis, ovaries and intestines. Based on this information, we then detected the different stages of tissue expression for SelM in the testis, ovary, and male crab hepatopancreas and hemolymph, and the enzyme activity of SelM in the testis. Overall, SelM was isolated successfully from the Chinese mitten crab, and its involvement in the regulation of reproduction during the period of rapid development in E. sinensis was confirmed.     

Molecular Cloning and Gene Expression Analysis of the Leptin Receptor in the Chinese Mitten Crab Eriocheir sinensis

Background

Leptin is an adipocyte-derived hormone with multiple functions that regulates energy homeostasis and reproductive functions. Increased knowledge of leptin receptor function will enhance our understanding of the physiological roles of leptin in animals.

Methodology/Principal Findings

In the present study, a full-length leptin receptor (lepr) cDNA, consisting of 1,353 nucleotides, was cloned from Chinese mitten crab (Eriocheir sinensis) using rapid amplification of cDNA ends (RACE) following the identification of a single expressed sequence tag (EST) clone in a cDNA library. The lepr cDNA consisted of a 22-nucleotide 5′-untranslated region (5′ UTR), a 402-nucleotide open reading frame (ORF) and a 929-nucleotide 3′ UTR. Multiple sequence alignments revealed that Chinese mitten crab lepr shared a conserved vacuolar protein sorting 55 (Vps55) domain with other species. Chinese mitten crab lepr expression was determined in various tissues and at three different reproductive stages using quantitative real-time RT-PCR. Lepr expression was highest in the intestine, thoracic ganglia, gonad, and accessory gonad, moderate in hepatopancreas and cranial ganglia, and low in muscle, gill, heart, haemocytes, and stomach. Furthermore, lepr expression was significantly higher in the intestine, gonad and thoracic ganglia in immature crabs relative to precocious and mature crabs. In contrast, lepr expression was significantly lower in the hepatopancreas of immature crabs relative to mature crabs.

Conclusions/Significance

We are the first to identify the lepr gene and to determine its gene expression patterns in various tissues and at three different reproductive stages in Chinese mitten crab. Taken together, our results suggest that lepr may be involved in the nutritional regulation of metabolism and reproduction in Chinese mitten crabs.     

Bilateral eyestalk ablation of the blue swimmer crab, Portunus pelagicus, produces hypertrophy of the androgenic gland and an increase of cells producing insulin-like androgenic gland hormone

The androgenic glands (AG) of male decapod crustaceans produce insulin-like androgenic gland (IAG) hormone that controls male sex differentiation, growth and behavior. Functions of the AG are inhibited by gonad-inhibiting hormone originating from X-organ-sinus gland complex in the eyestalk. The AG, and its interaction with the eyestalk, had not been studied in the blue swimmer crab, Portunus pelagicus, so we investigated the AG structure, and then changes of the AG and IAG-producing cells following eyestalk ablation. The AG of P. pelagicus is a small endrocrine organ ensheathed in a connective tissue and attached to the distal part of spermatic duct and ejaculatory bulb. The gland is composed of several lobules, each containing two major cell types. Type I cells are located near the periphery of each lobule, and distinguished as small globular cells of 5-7 μm in diameter, with nuclei containing mostly heterochromatin. Type II cells are 13-15 μm in diameter, with nuclei containing mostly euchromatin and prominent nucleoli. Both cell types were immunoreactive with anti-IAG. Following bilateral eyestalk ablation, the AG underwent hypertrophy, and at day 8 had increased approximately 3-fold in size. The percentage of type I cells had increased more than twice compared with controls, while type II cells showed a corresponding decrease.     

Calcium–calmodulin dependent protein kinase I from Macrobrachium nipponense: cDNA cloning and involvement in molting

Calcium–calmodulin dependent protein kinase I is a component of a calmodulin-dependent protein kinase cascade and involved in many physiological processes. The full-length cDNA of calcium–calmodulin dependent protein kinase I (MnCaMKI) was cloned from the freshwater prawn Macrobrachium nipponense and its expression pattern during the molt cycle and after eyestalk ablation is described. The full-length cDNA of MnCaMKI is 3262 bp in length and has an open reading frame (ORF) of 1038 bp, encoding a 345 amino acid protein. The expression of MnCaMKI in three examined tissues was upregulated in the premolt stage of the molt cycle. Its expression was induced after eyestalk ablation (ESA): the highest expression level was reached 1 day after ESA in hepatopancreas, and 3 days after ESA in muscle. By dsRNA-mediated RNA interference assay, expression of MnCaMKI and ecydone receptor gene (MnEcR) was significantly decreased in prawns treated by injection of dsMnCaMKI, while expression of these two genes was also significantly decreased in prawns treated by injection of dsMnEcR, demonstrating a close correlation between the expression of these two genes. These results suggest that CaMKI in M. nipponense is involved in molting.     

Characterization and expression of calcium-activated potassium channels (Slo) in ovary of the mud crab,Scylla paramamosain

Large conductance calcium-activated potassium channels (Slo) play important roles in controlling neuronal excitability. At present, very little is known about the function of Slo channels on ovarian development. We cloned the SPSlo gene from the mud crab, Scylla paramamosain. This gene shows 91 and 93 % sequence identity to PISlo from the spiny lobster, Panulirus interruptus and CBSlo from the jonah crab, Cancer borealis, respectively. We isolated six variants of the SPSlo cDNA within S. paramamosain ovary tissue. Sequence analysis indicated that there were at least seven alternative sites in SPSlo, each with multiple alternative segments. Real-time PCR showed that the SPSlo gene was expressed in various tissues, and highly expressed in brain and ovary. In addition, the expression of SPSlo changed throughout ovarian development, highest at the early-developing stage (Stage II) followed by a slow decrease in subsequent stages. These results suggested that SPSlo channels may be implicated in the ovarian development of the mud crab.     

Molecular cloning, characterization and expression analysis of two apoptosis genes, caspase and nm23, involved in the antibacterial response in Chinese mitten crab, Eriocheir sinensis

Apoptosis is a central regulatory feature of the immune system, and the most common form of death among immunological cells. However, the function of apoptosis, within the innate immune system of invertebrates, remains largely unknown. For this reason, we investigated the immune functionality of two apoptosis genes, caspase and nm23, in the Chinese mitten crab (Eriocheir sinensis), which is a commercially important and disease vulnerable aquaculture species. The entire length caspase and nm23 cDNA genes were cloned using PCR, based on an initial expressed sequence tag (EST) isolated from a hepatopancreatic cDNA library. The caspase cDNA contained an 1119 bp open reading frame that encoded a putative 372 amino acid protein, while nm23 cDNA contained a 456 bp open reading frame that encoded a putative 151 amino acid protein. Comparison, with other reported invertebrate and vertebrate sequences, revealed the presence of conserved enzyme active sites that were common among caspase and nm23 superfamilies. In brief, caspase and nm23 mRNA expression in E. sinensis were (a) both detected in all tissues, including the hemocytes, heart, hepatopancreas, gill, stomach, muscle, intestine, brain and eyestalk, and (b) responsive in hemocytes, gill and hepatopancreas to a Vibrio anguillarum immuno-challenge all appeared sharp increase. Collectively, the data presented here demonstrate the successful isolation of caspase and nm23 apoptosis genes from the Chinese mitten crab, and their role in the innate immune system of an invertebrate.     

Characterization and expression profile of Vitellogenin gene from Scylla paramamosain

The full-length (7816 bp) cDNA of Vitellogenin (Vtg) encoding 2560 aa with an estimated molecular mass of 287.743 kDa was cloned from the green mud crab Scylla paramamosain. Semi-quantitative PCR (sq-PCR) revealed a specific expression pattern of Sp-vtg gene in ovaries and hepatopancreas. With the development of ovaries, the expression level of Sp-vtg gene showed an increasing trend both in ovaries and hepatopancreas, and the expression level of Sp-vtg gene in hepatopancreas and ovary was stable after stage IV. By in situ hybridization, the positive signals of Sp-vtg gene were detected in the cytoplasm of oocytes in stage I, in the follicle cell and the surrounding of the nucleus in stage III, and in the nucleus in stage V. Furthermore, the signals become stronger with the later development stages of ovary. Moreover, in situ hybridization analysis revealed that positive signals of Sp-vtg gene are present in the hepatopancreatic tubule, and the signals increase during the development, becoming the strongest in stage V. Our results indicate that both ovaries and hepatopancreas are sites of Vitellogenin gene synthesis in S. paramamosain.