Mitten Crab Eriocheir Japonica Sinensis Research Articles

Comparative transcriptome analysis reveals the expression and characterization of digestive enzyme genes in the hepatopancreas of the Chinese mitten crab

The Chinese mitten crab Eriocheir japonica sinensis is one of the most common aquaculture species cultivated in China. The crab is an omnivore, and its hepatopancreas absorbs and stores nutrients. The aim of this study was to elucidate the expressions of the digestive enzyme genes and determine their respective roles in regulating digestive capacity in E. j. sinensis. We sequenced the hepatopancreatic transcriptomes of crabs fed a meat diet (MD), a vegetarian diet (VD), or a mixed diet (MV) and compared the gene expression patterns of these three groups of crabs. A total of 305,887 unigenes were obtained, of which 8747, 10,963, and 8877 were significantly differentially expressed in the comparisons between the MD and MV, VD and MV, and MD and VD diets, respectively. Kyoto Encyclopedia of Genes and Genomes (KEGG) database-based enrichment analysis revealed that the differentially expressed gene (DEG) responses in the hepatopancreases to the MD mainly involved the “pancreatic secretion,” “glutathione metabolism,” “sphingolipid metabolism,” “fatty acid metabolism,” and “glycerolipid metabolism pathways.” DEG responses to the VD based on KEGG analysis mainly involved the “galactose metabolism,” “starch and sucrose metabolism,” and “fructose and mannose metabolism” pathways. The key digestive enzymes, including trypsin, β-glucosidase, chitinase, and triacylglycerol lipase, were identified. Our results further our understanding of crustacean hepatopancreatic functions during food digestion and provide resources for further studies regarding the molecular basis of omnivorous diets in crustaceans.

Reproductive potential of individual male Chinese mitten crabsEriocheir japonica sinensisin a local pond-reared broodstock: Implications for parent crab selection and sex ratio optimization

Culture of the Chinese mitten crab Eriocheir japonica sinensis has become a large industry in China because of improved hatchery techniques in the past two decades. In this study, we investigated the reproductive potential of individual male crabs in a local pond-reared breeding stock [with a 2.5:1 female-biased sex ratio] to assess the current broodstock management. Relative to virgin (unmated) males, vasa deferentia of the removed pond males markedly changed and some even showed extremely sperm-depleted conditions, such as wizened tube and accessory gland mass and no spermatophores and/or a small quantity of seminal fluid in the lumen. According to calculating individual VSIs, these males could be divided into two groups as small (≤139 g/ind., mean VSI = 2.82%) and large males (>139 g/ind., 1.70%). Combined with the baseline value (2.08%), approximately 50% male (more than 90% from large males) could be graded as “depleted.” These findings strongly indicated that the current broodstock management had a risk of sperm limitation. However, given that “depleted” large males had a relatively high seminal replenishing capacity, and posthatch females always retained a certain amount of sperm load, we could further postulate that other ways should be adopted to offset party of individuals excessive consumption of sperm load at a broodstock level. Thus, we might optimize such an artificial breeding stock and adjust a female-biased sex ratio to 4:1–5:1 by selecting a large male broodstock (>150 g/ind.) to reduce the number of male and increase female to change the entire broodstock structure while maintaining its density.

Identification of Differentially Expressed Genes During the Brachyurization of the Chinese Mitten Crab Eriocheir japonica sinensis

Expressed sequence tags (ESTs) obtained from two-directional suppression subtractive hybridization (SSH) of cDNA libraries derived from the pleon of the Chinese mitten crab were analyzed using mRNA subtractive hybridization. After cDNA cloning and sequencing, gene expression profiles were examined, focusing on brachyurization. We assembled 211 non-redundant ESTs from SSH library 1 (yielding 130 unique transcripts: 25 consensi, 105 singletons) and 669 from SSH library 2 (195: 51 consensi, 144 singletons). Functional analysis shows that these genes are putatively involved in various cellular processes (33%), ribosomal RNA/proteins (30%), molting (12%), signal transduction (6%), immune factor and stress proteins (6%), development (4%), energy metabolism (3%), and mitochondrial membrane protein, hormone metabolizing, and chaperone (1% each) of other organisms. Some 3% are of unknown function in SSH library 1. The results facilitate the functional study of candidate genes involved in early developmental processes, especially the regulation of brachyurization in this crab.

Identification, mRNA expression and characterization of proliferating cell nuclear antigen gene from Chinese mitten crab Eriocheir japonica sinensis

The sliding clamp proliferating cell nuclear antigen (PCNA) plays important roles in nucleic acid metabolism. In this work, we isolated a PCNA gene (designated as EjsPCNA, accession: FJ483830) by rapid amplification of cDNA ends approach from the Chinese mitten crab Eriocheir japonica sinensis. The full-length cDNA of EjsPCNA consists of 1123 nucleotides with an open-reading frame of 780 bp encoding 259 amino acids (28.62 kDa) and containing an interdomain connecting loop, C-terminal tail, and center loop. Sequence alignment, phylogenetic analyses, and structure comparison revealed that EjsPCNA is a member of the PCNA family. Real-time RT-PCR results indicate that EjsPCNA is expressed throughout three developmental stages. EjsPCNA mRNA expression levels at the first crab stage are significantly higher than that of the other two stages. Present data showed that the expression levels of EjsPCNA in E. j. sinensis are likely related to proliferation activity of tissues, and suggested that EjsPCNA gene is probably involved in the crabs' early developmental regulation.

Identification, mRNA expression and characterization of a novel ANK-like gene from Chinese mitten crab Eriocheir japonica sinensis

Ankyrins are a family of adapter molecules mediating linkages between integral membrane and cytoskeletal proteins. Ankyrin repeat is one of the most frequently observed amino acid motifs in protein databases. A novel ANK-like gene of Chinese mitten crab Eriocheir japonica sinensis (denoted as EjsANK) was identified and cloned by expressed sequence tag and rapid amplification of cDNA end approaches. The full-length cDNA of EjsANK is 4375 bp and contains an open reading frame of 1095 bp which encodes a 364 amino acids polypeptide (40.23 kD) bearing seven ankyrin repeats. EjsANK cDNA has a 3073 bp uniquely long 3' untranslated region with three K-box elements, one GY-like box domain and one Brd-like box domain. Sequence alignment and three-dimensional structural analyses revealed that EjsANK should be a novel cytosolic member of the ankyrin family. Fluorescent real-time quantitative RT-PCR approach was performed to examine the expression profiles of EjsANK mRNA by testing its relative level in three types of tissues at three different developmental stages, respectively. We found that the relative level of EjsANK mRNA expression was significantly higher in the abdomen at the first crab stage. Functional bioinformatics prediction analyses indicated that EjsANK has an analogical effect like IkappaB which is a key component of IkappaB/NF-kappaB complex in mammalian cells playing very important roles in the development process. Results suggest that EjsANK gene is involved in the early developmental regulation of Chinese mitten crab, especially brachyurization regulation.

Studies on the interaction between benzidine and hemocyanin from Chinese mitten crab Eriocheir japonica sinensis (Decapoda, Grapsidae)

The interaction of benzidine (BZ) with hemocyanin (Hc) from Chinese mitten crab ( Eriocheir japonica sinensis) was studied by fluorescence spectrum. BZ can quench the fluorescence of Hc by forming a new complex. The process of binding BZ on Hc was a spontaneous molecular interaction procedure. The thermodynamic parameters, the enthalpy change and entropy change were estimated to be 47.36 kJ mol −1, 248.51 J mol −1 K −1 according to the van’ Hoff equation. This indicates that hydrophobic interaction played a major role in stabilizing the complex. The results of synchronous fluorescence spectroscopy and three-dimensional fluorescence spectra indicated that the structure of these tyrosine residues environments was altered by BZ, which could enter into the hydrophobic pocked of Hc.

Molecular cloning, mRNA expression, and characterization of HSP90 gene from Chinese mitten crab Eriocheir japonica sinensis

HSP90 is a highly conserved molecular chaperone important in the maturation of a broad spectrum of proteins. Using expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE) techniques, an HSP90 gene designated as EjsHSP90 was cloned and characterized from the Chinese mitten crab Eriocheir japonica sinensis. The full-length cDNA of EjsHSP90 is 2,517 bp and contains an open reading frame of 2157 bp which encodes a 718 amino acid polypeptide (82.8 kDa) bearing characteristics of the HSP90 family and an ATP binding domain. Sequence alignment shows that EjsHSP90 shared 79%-96% identity with HSP90 sequences reported in other animals, and it shares identical structural features. Fluorescent real-time quantitative RT-PCR approach was performed to examine the expression profiles of EjsHSP90 mRNA by testing its relative level in three types of tissues at three different developmental stages, respectively. We found that EjsHSP90 is expressed throughout the three developmental stages but expression levels varied among different body parts of crabs. EjsHSP90 mRNA expression in the abdomen of the first crab stage is consistently higher than that of the other two stages, suggesting that EjsHSP90 gene is involved in the crabs' early developmental process, especially in the crab brachyurization process. Results from quantitative RT-PCR excluded the possibility that the expression of EjsHSP90 mRNA is induced primarily by osmotic stress. Phylogenetic analyses reveal that HSP90 gene is informative and complementary for reconstruction of arthropod phylogenetic relationships.