Yongxu Cheng

Insights into Hepatopancreatic Functions for Nutrition Metabolism and Ovarian Development in the Crab Portunus trituberculatus: Gene Discovery in the Comparative Transcriptome of Different Hepatopancreas Stages

The crustacean hepatopancreas has different functions including absorption, storage of nutrients and vitellogenesis during growth, and ovarian development. However, genetic information on the biological functions of the crustacean hepatopancreas during such processes is limited. The swimming crab, Portunus trituberculatus, is a commercially important species for both aquaculture and fisheries in the Asia-Pacific region. This study compared the transcriptome in the hepatopancreas of female P. trituberculatus during the growth and ovarian maturation stages by 454 high-throughput pyrosequencing and bioinformatics. The goal was to discover genes in the hepatopancreas involved in food digestion, nutrition metabolism and ovarian development, and to identify patterns of gene expression during growth and ovarian maturation. Our transcriptome produced 303,450 reads with an average length of 351 bp, and the high quality reads were assembled into 21,635 contigs and 31,844 singlets. Based on BLASTP searches of the deduced protein sequences, there were 7,762 contigs and 4,098 singlets with functional annotation. Further analysis revealed 33,427 unigenes with ORFs, including 17,388 contigs and 16,039 singlets in the hepatopancreas, while only 7,954 unigenes (5,691 contigs and 2,263 singlets) with the predicted protein sequences were annotated with biological functions. The deduced protein sequences were assigned to 3,734 GO terms, 25 COG categories and 294 specific pathways. Furthermore, there were 14, 534, and 22 identified unigenes involved in food digestion, nutrition metabolism and ovarian development, respectively. 212 differentially expressed genes (DEGs) were found between the growth and endogenous stage of the hepatopancreas, while there were 382 DEGs between the endogenous and exogenous stage hepatopancreas. Our results not only enhance the understanding of crustacean hepatopancreatic functions during growth and ovarian development, but also represent a basis for further research on new genes and functional genomics of P. trituberculatus or closely related species.

Meat Quality of Chinese Mitten Crabs Fattened with Natural and Formulated Diets

After 42 days feeding on either a Huayi formulated diet (HY) or trash fish (TF), the meat quality of Chinese mitten crab, Eriocheir sinensis, was compared. The meat yield of males (25 vs. 22%) was higher, but the total edible yield was lower than that of the females (p < 0.05). The proximate composition of both genders fed two diets was similar (p > 0.05), and there was no significant difference in the total collagen content of the meat (0.2–0.30%) between both dietary treatments and genders (p > 0.05). The diets had no significant effects on free amino acid content of the meat, while gender significantly affected the contents of some amino acids, such as taurine and glycine. Crabs fed TF had significantly lower contents of 20:5n-3 as well as higher arachidonic acid (ARA)/eicosapentaenoic acid (EPA) value compared with those of same sex crabs fed HY (p < 0.05). There were no significant dietary effects on a range of sensory traits evaluated by panelists, neither in female nor in male crabs. These results suggested no negative effects of formulated diet HY on the meat quality compared to trash fish. Therefore, diet HY could be used to replace the trash fish for fattening mitten crabs.

Cloning and tissue distribution of a fatty acyl Δ6-desaturase-like gene and effects of dietary lipid levels on its expression in the hepatopancreas of Chinese mitten crab (Eriocheir sinensis)

Fatty acyl Δ6-desaturase is the rate-limiting enzyme in the biosynthetic pathway of highly unsaturated fatty acids (HUFAs) in vertebrates. In this report, a fatty acyl Δ6-desaturase-like cDNA was cloned from the hepatopancreas of Eriocheir sinensis (Chinese mitten crab) and characterized by performing rapid-amplification of cDNA ends. The 2278-bp long full-length cDNA encodes a polypeptide with 442 amino acids. Gene expression analysis via real-time quantitative polymerase chain reaction revealed that the fatty acyl Δ6-desaturase-like transcripts are widely distributed in various tissues, with high expression levels in the hepatopancreas and cranial ganglia. This study focuses on the nutritional regulation of genes involved in the HUFA biosynthetic pathway in Chinese mitten crab. A feeding trial was performed whereby crablets were fed for 238 d with four different diets: control diet without oil lipids (added with 3% basic lipid of the fundamental diets); fish oil diet (FO; added with 3% of the fundamental diets); soybean oil diet (SO; added with 3% of the fundamental diets); and FO/SO diet (1:1; added with 3% of the fundamental diets). The hepatopancreas of crabs sampled at 168 d and 238 d to determine the effects on fatty acyl Δ6-desaturase-like mRNA expression. The results show that the expression of fatty acyl Δ6-desaturase-like is higher in the hepatopancreas of crabs fed with SO diet than those fed with FO diet. Furthermore, gene expression increased by 2.45-fold in the hepatopancreas of crabs fed with SO after 238 d than those fed after 168 d but remained steady for those fed with FO after 238 d.

Isolation and characterization of Vibrio metschnikovii causing infection in farmed Portunus trituberculatus in China

In August 2008, a massive epizootic occurred among Portunus trituberculatus reared together with Palaemon carincauda Holthuis and Sinonovacula constricta in the seawater pond of Ningang farm, Rudong district, Jiangsu province, China. The disease occurred in crabs from juveniles to adults, and the mortality rate reached 30–40%. The diseased crabs exhibited lethargy, hepatopancreas turgidity, and elevated levels of turbid hemolymph. A gram-negative, rod-shaped bacterium (designated as strain LGS-1) was isolated from the ascitic fluid of the diseased and moribund crabs and was confirmed as the causative agent by our infectivity study. We conducted morphological and biochemical characterization of LGS-1, and sequenced the 16S rRNA gene, which led us to identify the bacterium as Vibrio metschnikovii. Drug sensitivity tests showed that this pathogenic bacterium is sensitive to florfenicol, orfloxacin, and SXT, but completely resistant to antibacterial drugs like gentamicin, erythrocin, and acheomycin. This is the first report on Vibrio metschnikovii as a virulent pathogen for Portunus trituberculatus.

Effects of glyphosate on immune responses and haemocyte DNA damage of Chinese mitten crab, Eriocheir sinensis

Abstract As a broad‐spectrum organophosphorus herbicide, glyphosate is widely utilized around the world. The toxic effects of glyphosate on Chinese mitten crab, Eriocheir sinensis, were assessed using immunotoxicity and genotoxicity biomarkers in this study. The results showed that 24 h and 96 h LC50 values of glyphosate for E. sinensis were estimated as 461.54 and 97.89 mg/L, respectively, and the safe concentration was 4.4 mg/L. According to the results above, glyphosate was applied at concentrations of 0, 4.4, 9.8, 44 and 98 mg/L, for 96 h in the exposure experiment. Total haemocyte count (THC) and percentage of granulocytes decreased significantly following 6 h exposure to each concentration of glyphosate and tended to gradually stabilize after 12 h except in 4.4 mg/L, which rapidly recovered to a normal level in 12 h. Phagocytic activity in all treatments decreased dramatically at 6 h and maintained stability until the 96‐h mark. Comet tail has been observed early at 24 h in each treatment, and the comet ratio and percentage of DNA (% DNA) in the tail increased as the exposure experiment progressed. Immune‐related enzyme activity varied during the experiment. Acid phosphatase (ACP) and alkaline phosphatase (AKP) activities in 44 and 98 mg/L treatments decreased significantly after 48 h exposure, while AKP activities in all concentrations increased markedly at the beginning of exposure. The superoxide dismutase (SOD) and peroxidase (POD) activities increased significantly after 6 h exposure to 44 and 98 mg/L of glyphosates but decreased at 24 h. In addition, the &bgr;‐glucuronidase (&bgr;‐GD) activities in the 9.8, 44 and 98 mg/L groups, increased after 6‐h exposure and were significantly higher than those in the control at 96 h. These results indicated that glyphosate has evident toxic effect on E. sinensis by immune inhibition that is possibly due to the haemocyte DNA damage and a sharp decline in haemocyte numbers, which subsequently induced changes in activities of immune‐related enzymes and haemocyte phagocytosis. HighlightsAn acute toxic test was conducted in this study to evaluate the effect of glyphosate on immune response and DNA damge from Chinese mitten crab, Erochier Sinensis.Glyphosate could induce different levels of immune inhibition and haemcyte DNA damge by different concentration of exposure.The THC, phagocytic activity, Comet ratio, %DNA in tail, and &bgr;‐GD activity, were considered more sensitive biomarkers.

Immunorecognition and Distribution of Progesterone Receptors in the Chinese Mitten Crab Eriocheir sinensis During Ovarian Development

ABSTRACT Progesterone is an important sex steroid that plays a vital role during ovarian development in crustaceans. In vertebrates, progesterone mediates reproduction via the progesterone receptor (PR). Previous studies have shown that PR is present in the ovary, hepatopancreas, and nerve tissues of some crustacean species. The Chinese mitten crab, Eriocheir sinensis, is an important aquaculture species in China and has become an invasive species in Europe and North America. A better understanding of the relevant reproductive mechanisms could potentially benefit artificial propagation and production of E. sinensis. Our intention was to immunorecognize and immunolocalize PR in the ovary, hepatopancreas, optic ganglion, brain ganglion, and thoracic ganglion of female E. sinensis using Western blot and immunohistochemistry. Changes in the distribution of PR were also investigated in these tissues during ovarian development. With an apparent molecular weight of 70 kDa, PR was identified in the optic, brain, and thoracic ganglion of female E. sinensis. During ovarian development, follicle cells were stained with positive PR at each ovarian stage. In germinal cells, positive PR was found in the cytoplasm only during the early ovarian development stages (I–III), whereas positive PR stained in the nucleus of germinal cells from stage III—stage V. In the hepatopancreas, PR was localized in the nucleus of resorptive cells as well as in the cytoplasm and nucleus of fibrillar cells for all stages of ovarian development. On the contrary, no PR-like substance was found in the other types of hepatopancreatic cells, such as blisterlike cells and embryonic cells, during ovarian development. However, a PR-like substance was detected in the nerve tissues of female E. sinensis. In the optic ganglion, PR was localized in the nucleus only of nerve cells. In the thoracic ganglion, PR was detected in the cytoplasm and nuclei of nerve cells during all ovarian development stages, with stronger detection during late ovarian development (stages III–V) rather than early (stages I and II). In addition, PR was localized in the brain ganglion, which is supported by evidence that the nuclei of nerve cells stained positively for PR antibody during all ovarian development stages. We suggest that progesterone not only regulates vitellogenesis and ovarian development directly by binding PR in the ovary and hepatopancreas, but also modulates indirectly ovarian development through nerve tissue.

Immunolocalization of estrogen receptor α in Neomysis japonica oocytes and follicle cells during ovarian development.

Estrogen induces oocytes development and vitellogenesis in crustacean by interacting with estrogen receptor (ER) subtypes. In the present study, we detect for the first time the ERα in oocytes and follicle cells and hepatopancreas cells of mysis by immunohistochemistry using a specific ERα antibody. ERα was mainly localized in the nuclei of oocytes and follicle cells, while mainly detected in nuclei of oogonia (OG), previtellogenic oocyte (PR) and endogenous vitellogenic oocyte (EN) at previtellogenic and early vitellogenic stage (I-early III). Follicle cells in all stages of ovary (all vitellogenic stages) showed strong ERα positive reaction, and they were able to gradually move to oocytes during the development of oocytes. In addition, ERα was also localized in the nuclei and cytoplasm of four hepatopancreas cells (including E-, R-, F- and B-cell) in all ovary stages. These findings suggest, for the first time to our knowledge, that there could be a close link between oogenesis, follicle cells, hepatopancreas cells and endocrine regulation, and estrogens might be involved in the regulation of oocytes at early ovarian stage in mysis.

Effects of Histamine on Survival and Immune Parameters of the Chinese Mitten Crab, Eriocheir sinensis

ABSTRACT In the current study, we examined the effects of histamine on survival and immune parameters of the Chinese mitten crab, Eriocheir sinensis. The crabs infected with the common bacteria pathogen Aeromonas hydrophila were injected with 0 µg histamine/g/crab, 1 µg histamine/g/crab, 50 µg histamine/g/crab, or 100 µg histamine/g/crab, and their survival after 96 h was recorded. The results showed that histamine alone had no influence on crab survival but can decrease survival of the crabs challenged with A. hydrophila after 36 h. In addition, the survival of the crabs injected with the high level (100 µg/g) of histamine was significantly greater than that of the lower level (50 µg/g) after 60–96 h (P < 0.05), but not different from that of the control. Immune parameters of the hemolymph in crabs, such as the total hemocyte count (THC), phenoloxidase (PO), acid phosphatase (ACP), alkaline phosphatase (AKP), and superoxide dismutase activity (SOD), were measured at 6 h, 12 h, and 24 h after histamine injection. Some immune parameters, such as PO and SOD, showed an increase, whereas THC, ACP, and AKP levels declined at 6 h. Surprisingly, after the injection of histamine, the crabs quickly released their pereiopods and began to twitch, and the number of the crabs with autotomy and spasm increased with the increasing histamine concentration (P < 0.05). This is the first report that indicates that histamine could lead to crab autotomy. The overall results of this study indicate that the effects of histamine on survival and the immune parameters of E. sinensis infected with A. hydrophila depend on its concentration.

De novo transcriptome sequencing and analysis of male and female swimming crab ( Portunus trituberculatus ) reproductive systems during mating embrace (stage II)

BackgroundThe swimming crab Portunus trituberculatus is one of the most commonly farmed crustaceans in China. As one of the most widely known and high-value edible crabs, it crab supports large crab fishery and aquaculture in China. Only large and sexually mature crabs can provide the greatest economic benefits, suggesting the considerable effect of reproductive system development on fishery. Studies are rarely conducted on the molecular regulatory mechanism underlying the development of the reproductive system during the mating embrace stage in this species. In this study, we used high-throughput sequencing to sequence all transcriptomes of the P. trituberculatus reproductive system.ResultsTranscriptome sequencing of the reproductive system produced 81,688,878 raw reads (38,801,152 and 42,887,726 reads from female and male crabs, respectively). Low-quality (quality <20) reads were trimmed and removed, leaving only high-quality reads (37,020,664 and 41,021,030 from female and male crabs, respectively). A total of 126,188 (female) and 164,616 (male) transcripts were then generated by de novo transcriptome assembly using Trinity. Functional annotation of the obtained unigenes revealed that a large number of key genes and some important pathways may participate in cell proliferation and signal transduction. On the basis of our transcriptome analyses and as confirmed by quantitative real-time PCR, a number of genes potentially involved in the regulation of gonadal development and reproduction of P. trituberculatus were identified: ADRA1B, BAP1, ARL3, and TRPA1.ConclusionThis study is the first to report on the whole reproductive system transcriptome information in stage II of P. trituberculatus gonadal development and provides rich resources for further studies to elucidate the molecular basis of the development of reproductive systems and reproduction in crabs. The current study can be used to further investigate functional genomics in this species.

Effects of dietary lipids on the hepatopancreas transcriptome of Chinese mitten crab (Eriocheir sinensis)

Fish oil supplies worldwide have declined sharply over the years. To reduce the use of fish oil in aquaculture, many studies have explored the effects of fish oil substitutions on aquatic animals. To illustrate the effects of dietary lipids on Chinese mitten crab and to improve the use of vegetable oils in the diet of the crabs, 60 male juvenile Chinese mitten crabs were fed one of five diets for 116 days: fish oil (FO), soybean oil (SO), linseed oil (LO), FO + SO (1:1, FSO), and FO + LO (1:1, FLO). Changes in the crab hepatopancreas transcriptome were analyzed using RNA sequencing. There were a total 55,167 unigenes obtained from the transcriptome, of which the expression of 3030 was significantly altered in the FLO vs. FO groups, but the expression of only 412 unigenes was altered in the FSO vs. FO groups. The diets significantly altered the expression of many enzymes involved in lipid metabolism, such as pancreatic lipase, long-chain acyl-CoA synthetases, carnitine palmitoyltransferase I, acetyl-CoA carboxylase, fatty acid synthase, and fatty acyl Δ9-desaturase. The dietary lipids also affected the Toll-like receptor and Janus activated kinase-signal transducers and activators of transcription signaling pathways. Our results indicate that substituting fish oil with vegetable oils in the diet of Chinese mitten crabs might decrease the digestion and absorption of dietary lipids, fatty acids biosynthesis, and immunologic viral defense, and increase β-oxidation by altering the expression of the relevant genes. Our results lay the foundation for further understanding of lipid nutrition in Chinese mitten crab.