Tissue expression profiles unveil the gene interaction of hepatopancreas, eyestalk, and ovary in the precocious female Chinese mitten crab, Eriocheir sinensis.

BACKGROUND: Sexual precocity is a common biological phenomenon in animal species. A large number of precocity individuals were identified in Chinese mitten crab Eriocheir sinensis, which caused huge economic loss annually. However, the underlying genetic basis of precocity in E. sinensis remains unclear to date. RESULTS: In this study, morphological and histological observation and comparative transcriptome analysis were conducted among different stages of precocious one-year-old and normal two-year-old sexually mature E. sinensis. The expression profiles of the ovary, hepatopancreas, and eyestalk tissues were presented and compared. Genes associated with lipid metabolic process, lipid transport, vitelline membrane formation, vitelline synthesis, and neuropeptide hormone-related genes were upregulated in the ovary, hepatopancreas, and eyestalk of precocious E. sinensis. Our results indicated that the eyestalk was involved in the neuroendocrine system providing neuropeptide hormones that may induce vitellogenesis in the hepatopancreas and further stimulate ovary development. The hepatopancreas is a site for energy storage and vitellogenin synthesis, and it may assist oogenesis through lipid transport in precocious E. sinensis. CONCLUSION: We provided not only an effective and convenient phenotype measurement method for the identification of potential precocious E. sinensis detection but also valuable genetic resources and novel insights into the molecular mechanism of precocity in E. sinensis. The genetic basis of precocity in E. sinensis is an integrated gene regulatory network of eyestalk, hepatopancreas, and ovary tissues.

Comparative skin transcriptome of two Oujiang color common carp (Cyprinus carpio var. color) varieties.

Body color variation has long been a hot research topic in evolutionary and functional biology. Oujiang color common carp (Cyprinus carpio var. color) is a well-known economical and ornamental fish. Three main types of pigments and four distinct color patterns are typical characters of Oujiang color common carp, which makes it an excellent fish model to study body coloration. In this study, skin transcriptome assembly and comparisons were conducted in two Oujiang color common carp varieties: whole red and whole white. Transcriptome comparison revealed that more differentially expressed energy metabolism genes were upregulated in whole white compared to whole red. The results indicated that energy metabolism genes might be strongly associated with environmental adaption and growth performance and likely affect the red and white color formation in Oujiang color common carp. Our study provided direct guidance for the aquaculture industrials of Oujiang color common carp and presented valuable genetic resources for body color research in fish.

Hepatopancreas transcriptome analysis of Chinese mitten crab (Eriocheir sinensis) with white hepatopancreas syndrome.

White hepatopancreas is a syndrome that has recently emerged in aquaculture of Chinese mitten crab (Eriocheir sinensis). High lethality of the disease caused large economic loss, which drew considerable attention of fish farmers and scientific researchers. In this study, hepatopancreas reference transcriptome was de novo assembled and differential expression analysis was conducted between white hepatopancreas and normal (yellow) hepatopancreas of E. sinensis. A total of 90,687 transcripts were assembled, and 27,387 were annotated. Transcriptomic comparison revealed 69 differentially expressed genes between individuals featuring white hepatopancreas and yellow hepatopancreas. Genes associated with immune response and cell death, include thioredoxin-related transmembrane protein 1, hemocytin, methuselah-like 1, and E3 ubiquitin-protein ligase, and they were up-regulated, whereas titin and 5-formyltetrahydrofolate cyclo-ligase, which are genes related to cell proliferation, were down-regulated in E. sinensis with white hepatopancreas syndrome. Our study provides novel insights into genetic causes of formation and novel gene markers for detection of white hepatopancreas syndrome in aquaculture of E. sinensis.

"Omics" data unveil early molecular response underlying limb regeneration in the Chinese mitten crab, Eriocheir sinensis.

Limb regeneration is a fascinating and medically interesting trait that has been well preserved in arthropod lineages, particularly in crustaceans. However, the molecular mechanisms underlying arthropod limb regeneration remain largely elusive. The Chinese mitten crab Eriocheir sinensis shows strong regenerative capacity, a trait that has likely allowed it to become a worldwide invasive species. Here, we report a chromosome-level genome of E. sinensis as well as large-scale transcriptome data during the limb regeneration process. Our results reveal that arthropod-specific genes involved in signal transduction, immune response, histone methylation, and cuticle development all play fundamental roles during the regeneration process. Particularly, Innexin2-mediated signal transduction likely facilitates the early stage of the regeneration process, while an effective crustacean-specific prophenoloxidase system (ProPo-AS) plays crucial roles in the initial immune response. Collectively, our findings uncover novel genetic pathways pertaining to arthropod limb regeneration and provide valuable resources for studies on regeneration from a comparative perspective.

Plant and Animal-Type Feedstuff Shape the Gut Microbiota and Metabolic Processes of the Chinese Mitten Crab Eriocheir sinensis.

In animals, growth and development are strongly correlated with the gut microbiota and metabolic profiles. In this study, gut microbiome communities, metabolic profiles, and growth performance of Eriocheir sinensis under three dietary feed types based on waterweed plants only, freshwater snails only, and waterweed plants combined with freshwater snails were studied by using 16S rRNA gene sequencing and liquid chromatography-mass spectrometry. Results indicated that different feed types dramatically affected the growth performances of E. sinensis by altering the gut microbiota and metabolic profiles. Aquatic plants, such as waterweeds, played essential roles in shaping gut microbiome communities, and the optimal Bacteroides-to-Firmicutes ratio might strongly promote growth performance. Waterweed plants also helped decrease maleficent Proteobacteria caused by excess animal-type feedstuff, such as freshwater snails, and might have positive roles in antibacterial functions in gut. A diet based on waterweeds only resulted in lipid metabolism disorders, which significantly retarded the growth of E. sinensis. In summary, E. sinensis cultured with a diet of waterweeds and freshwater snails showed superior growth performance due to their healthy gut microbiota and metabolic homeostasis. Our findings unveiled the roles of aquatic plants and animal-type food such as freshwater snail in shaping the gut microbiota and metabolic processes and provided guidance for the aquaculture of E. sinensis in future.

V-ATPase subunit B plays essential roles in the molting process of the Chinese mitten crab, Eriocheir sinensis.

Vacuolar ATPase (V-ATPase) is a proton pump driven by ATP hydrolysis, and it plays an important role in numerous biological processes, such as protein degradation and synthesis, cell growth, and cell autophagy. The V-ATPase subunit B (VATB) is a conservative and regulatory subunit required for ATP hydrolysis during proton pumping. The VATB of Eriocheirsinensis (EsVATB), which includes an open reading frame (ORF) length of 1467 bp encoding 489 amino acids, was cloned to unveil the biological function of VATB during the molting process of crustaceans. Spatial and temporal expression profiles showed that EsVATB was highly expressed in the posterior gill accompanied with the highest osmotic pressure in the premolt (PrM) stage. Meanwhile, the highest expression level of EsVATB was identified in the hepatopancreas and heart during the postmolt stage and epidermis in the intermolt stage, indicating that EsVATB may perform diverse biological functions in different tissues during the molting process. The individual crabs in the interference group showed a high mortality rate (74%) and a low molting rate (26%) and failed to form a new epicuticle in the PrM stage. Meanwhile, a significant difference in osmotic pressure was identified between the interference and control groups. Our results indicate that EsVATB is an indispensable functional gene that may participate in osmoregulation and help with the new epicuticle formation during the molting process of E. sinensis.

Molecular characteristic of activin receptor IIB and its functions in growth and nutrient regulation in Eriocheir sinensis.

Activin receptor IIB (ActRIIB) is a serine/threonine-kinase receptor binding with transforming growth factor-ß (TGF-ß) superfamily ligands to participate in the regulation of muscle mass in vertebrates. However, its structure and function in crustaceans remain unknown. In this study, the ActRIIB gene in Eriocheir sinensis (Es-ActRIIB) was cloned and obtained with a 1,683 bp open reading frame, which contains the characteristic domains of TGF-ß type II receptor superfamily, encoding 560 amino acids. The mRNA expression of Es-ActRIIB was the highest in hepatopancreas and the lowest in muscle at each molting stage. After injection of Es-ActRIIB double-stranded RNA during one molting cycle, the RNA interference (RNAi) group showed higher weight gain rate, higher specific growth rate, and lower hepatopancreas index compared with the control group. Meanwhile, the RNAi group displayed a significantly increased content of hydrolytic amino acid in both hepatopancreas and muscle. The RNAi group also displayed slightly higher contents of saturated fatty acid and monounsaturated fatty acid but significantly decreased levels of polyunsaturated fatty acid compared with the control group. After RNAi on Es-ActRIIB, the mRNA expressions of five ActRIIB signaling pathway genes showed that ActRI and forkhead box O (FoxO) were downregulated in hepatopancreas and muscle, but no significant expression differences were found in small mother against decapentaplegic (SMAD) 3, SMAD4 and mammalian target of rapamycin. The mRNA expression s of three lipid metabolism-related genes (carnitine palmitoyltransferase 1ß (CPT1ß), fatty acid synthase, and fatty acid elongation) were significantly downregulated in both hepatopancreas and muscle with the exception of CPT1ß in muscles. These results indicate that ActRIIB is a functionally conservative negative regulator in growth mass, and protein and lipid metabolism could be affected by inhibiting ActRIIB signaling in crustacean.

Global Analysis of Gene Expression Profiles Provides Novel Insights into the Development and Evolution of the Large Crustacean Eriocheir sinensis.

Chinese mitten crab (Eriocheir sinensis) is an important aquaculture species in Crustacea. Functional analysis, although essential, has been hindered due to the lack of sufficient genomic or transcriptomic resources. In this study, transcriptome sequencing was conducted on 59 samples representing diverse developmental stages (fertilized eggs, zoea, megalopa, three sub-stages of larvae, juvenile crabs, and adult crabs) and different tissues (eyestalk, hepatopancreas, and muscle from juvenile crabs, and eyestalk, hepatopancreas, muscle, heart, stomach, gill, thoracic ganglia, intestine, ovary, and testis from adult crabs) of E. sinensis. A comprehensive reference transcriptome was assembled, including 19,023 protein-coding genes. Hierarchical clustering based on 128 differentially expressed cuticle-related genes revealed two distinct expression patterns during the early larval developmental stages, demonstrating the distinct roles of these genes in "crab-like" cuticle formation during metamorphosis and cuticle calcification after molting. Phylogenetic analysis of 1406 one-to-one orthologous gene families identified from seven arthropod species and Caenorhabditis elegans strongly supported the hypothesis that Malacostraca and Branchiopoda do not form a monophyletic group. Furthermore, Branchiopoda is more phylogenetically closely related to Hexapoda, and the clade of Hexapoda and Branchiopoda and the clade of Malacostraca belong to the Pancrustacea. This study offers a high-quality transcriptome resource for E. sinensis and demonstrates the evolutionary relationships of major arthropod groups. The differentially expressed genes identified in this study facilitate further investigation of the cuticle-related gene expression networks which are likely associated with "crab-like" cuticle formation during metamorphosis and cuticle calcification after molting.

Structure and function of the alternatively spliced isoforms of the ecdysone receptor gene in the Chinese mitten crab, Eriocheir sinensis.

Alternative splicing is an essential molecular mechanism that increase the protein diversity of a species to regulate important biological processes. Ecdysone receptor (EcR), an essential nuclear receptor, is essential in the molting, growth, development, reproduction, and regeneration of crustaceans. In this study, the whole sequence of EcR gene from Eriocheir sinensis was obtained. The sequence was 45,481 bp in length with 9 exons. Moreover, four alternatively spliced EcR isoforms (Es-EcR-1, Es-EcR-2, Es-EcR-3 and Es-EcR-4) were identified. The four isoforms harbored a common A/B domain and a DNA-binding region but different D domains and ligand-binding regions. Three alternative splicing patterns (alternative 5' splice site, exon skipping, and intron retention) were identified in the four isoforms. Functional studies indicated that the four isoforms have specific functions. Es-EcR-3 may play essential roles in regulating periodic molting. Es-EcR-2 may participate in the regulation of ovarian development. Our results indicated that Es-EcR has broad regulatory functions in molting and development and established the molecular basis for the investigation of ecdysteroid signaling related pathways in E. sinensis.

Transcriptomic variation of hepatopancreas reveals the energy metabolism and biological processes associated with molting in Chinese mitten crab, Eriocheir sinensis.

Molting is a critical developmental process for crustaceans, yet the underlying molecular mechanism is unknown. In this study, we used RNA-Seq to investigate transcriptomic profiles of the hepatopancreas and identified differentially expressed genes at four molting stages of Chinese mitten crab (Eriocheir sinensis). A total of 97,398 transcripts were assembled, with 31,900 transcripts annotated. Transcriptomic comparison revealed 1,189 genes differentially expressed amongst different molting stages. We observed a pattern associated with energy metabolism and physiological responses during a molting cycle. In specific, differentially expressed genes enriched in postmolt were linked to energy consumption whereas genes enriched in intermolt were related to carbohydrates, lipids metabolic and biosynthetic processes. In premolt, a preparation stage for upcoming molting and energy consumption, highly expressed genes were enriched in response to steroid hormone stimulus and immune system development. The expression profiles of twelve functional genes detected via RNA-Seq were corroborated through real-time RT-PCR assay. Together, our results, including assembled transcriptomes, annotated functional elements and enriched differentially expressed genes amongst different molting stages, provide novel insights into the functions of the hepatopancreas in energy metabolism and biological processes pertaining to molting in crustaceans.