Liqiao Chen

Functional Annotation and Analysis of Expressed Sequence Tags from the Hepatopancreas of Mitten Crab (Eriocheir sinensis)

Due to its popularity as a traditional food, intensive harvesting of the mitten crab (Eriocheir sinensis) is common and has lead to an increase in disease incidence, resulting in catastrophic losses to crab aquaculture. The hepatopancreas of E. sinensis is not only an important digestive organ but also an indispensable immune organ. We constructed a nonnormalized cDNA library from the hepatopancreas of E. sinensis and acquired 3,297 high-quality expressed sequence tags representing 1,178 unigenes. More than half of these unigenes were novel genes for this species; the remaining had homologs in public databases, which is of great importance for future functional research. We also investigated the association of these genes with immune processes for insight into one of the main functions of the hepatopancreas besides metabolism. Despite the relatively low sampling scalar of our cDNA library, we were able to demonstrate several important properties of the hepatopancreatic transcriptome and identified numerous genes that were closely associated with immune responses. These results might serve as the basis for an in-depth genomics study of E. sinensis, including transcriptome analysis, physical mapping, and whole genome sequencing.

Gene discovery from an ovary cDNA library of oriental river prawn Macrobrachium nipponense by ESTs annotation

The oriental river prawn, Macrobrachium nipponense, is an important crustacean species in aquaculture. However, early gonad maturity is a ubiquitous problem which devalues the product quality. While husbandry and nutritional management have achieved little success in tackling this issue, a molecular approach may discover the genes involved in reproduction and development, which will provide the basic knowledge on reproductive control. In this study, a high-quality cDNA library of prawn was constructed from the ovary tissue. A total of 3294 successful sequencing reactions yielded 3256 expressed sequence tags (ESTs) longer than 100 bp. The cluster and assembly analyses yielded 1514 unique sequences including 414 contigs and 1168 singletons. About 719 (47.49%) unique sequences were identified as orthologs of genes from other organisms. By sequence comparability analysis, 28 important genes including cathepsin B, chromobox protein, Cdc2, cyclin B, DEAD box protein and ADF/cofilin protein were expressed. These genes may be involved in reproductive and developmental functions in prawn. Peritrophin consisting of cortical rods was also found in this species. The identification of these EST sequences in M. nipponense would improve our understanding on the genes that regulate reproduction and development in prawn species. This study also lays the groundwork for development of molecular markers related to ovary development in other prawn species.

The bioaccumulation of fluoride ion (F(-)) in Siberian sturgeon (Acipenser baerii) under laboratory conditions.

A 90-d growth trial was conducted to determine the accumulation of fluoride ions (F(-)) in juvenile Siberian sturgeon (Acipenser baerii). Fish were exposed to nominal F(-) concentrations of 4, 10, 25, 62.5 mg L(-1) (added as NaF), along with a control group. Fish growth was determined and F(-) concentrations in bone, cartilage, skin, gill, muscle, liver, gut and pylorus were analyzed by fluoride specific electrode method. Fish growth was inhibited for groups exposed to 10, 25, and 62.5 mg F(-) L(-1) compared to the control group. Significant increases in F(-) concentrations were observed in bone, cartilage, skin and gill of Siberian sturgeon exposed to 4 mg F(-) L(-1). The highest F(-) concentration was in bone (3204.4 mg kg(-1), dry weight), followed by cartilage (1401.2 mg kg(-1)), gill (389.4 mg kg(-1)), skin (100.1 mg kg(-1)). Muscle, liver, gut and pylorus did not show increasing accumulation with increasing F(-) levels in the water, but liver, gut, pylorus accumulated higher F(-) concentration compared to muscle. It is summarized that F(-) accumulates mainly in bone and cartilage in Siberian sturgeon, with 10 mg F(-) L(-1) in water harmful to growth. Even 4 mg F(-) L(-1) may be harmful to Siberian sturgeon growth during a long-term waterborne exposure.

Characterization and Expression of Glutamate Dehydrogenase in Response to Acute Salinity Stress in the Chinese Mitten Crab, Eriocheir sinensis

Background Glutamate dehydrogenase (GDH) is a key enzyme for the synthesis and catabolism of glutamic acid, proline and alanine, which are important osmolytes in aquatic animals. However, the response of GDH gene expression to salinity alterations has not yet been determined in macro-crustacean species. Methodology/Principal Findings GDH cDNA was isolated from Eriocheir sinensis. Then, GDH gene expression was analyzed in different tissues from normal crabs and the muscle of crabs following transfer from freshwater (control) directly to water with salinities of 16‰ and 30‰, respectively. Full-length GDH cDNA is 2,349 bp, consisting of a 76 bp 5′- untranslated region, a 1,695 bp open reading frame encoding 564 amino acids and a 578 bp 3′- untranslated region. E. sinensis GDH showed 64–90% identity with protein sequences of mammalian and crustacean species. Muscle was the dominant expression source among all tissues tested. Compared with the control, GDH expression significantly increased at 6 h in crabs transferred to 16‰ and 30‰ salinity, and GDH expression peaked at 48 h and 12 h, respectively, with levels approximately 7.9 and 8.5 fold higher than the control. The free amino acid (FAA) changes in muscle, under acute salinity stress (16‰ and 30‰ salinities), correlated with GDH expression levels. Total FAA content in the muscle, which was based on specific changes in arginine, proline, glycine, alanine, taurine, serine and glutamic acid, tended to increase in crabs following transfer to salt water. Among these, arginine, proline and alanine increased significantly during salinity acclimation and accounted for the highest proportion of total FAA. Conclusions E. sinensis GDH is a conserved protein that serves important functions in controlling osmoregulation. We observed that higher GDH expression after ambient salinity increase led to higher FAA metabolism, especially the synthesis of glutamic acid, which increased the synthesis of proline and alanine to meet the demand of osmoregulation at hyperosmotic conditions.

Molecular cloning, characterization and mRNA expression of copper-binding protein hemocyanin subunit in Chinese mitten crab, Eriocheir sinensis.

Hemocyanin is a copper-binding protein and plays a crucial role in the physiological processes in crustacean. In this study, the cDNA encoding hemocyanin subunit from Chinese mitten crab Eriocheir sinensis (EsHc) was cloned by using EST analysis and rapid amplification of cDNA ends (RACE) approach. The full-length cDNA of EsHc was 2573 bp, consisting of a 5 untranslated region of 51 bp, a 3 untranslated region of 458 bp, and an open reading frame of 2064 bp. The deduced protein had 688 amino acid residues with molecular mass of 77,997.31 Da. Quantitative real-time RT-PCR analysis showed that the EsHc gene was expressed in haemocytes, hepatopancreas, muscles, gills, and intestines with the highest level of expression in the hepatopancreas and the lowest in the muscles. After Aeromonas hydrophila challenge, the relative expression level of EsHc in hemolymph was up-regulated at 3 h post-injection of bacteria followed by a gradual recovery from 12 to 24 h. In the second set of transcriptional studies, the mRNA expression patterns of EsHc in haemocytes and hepatopancreas were measured by quantitative real-time RT-PCR after the Chinese mitten crab were fed six diets containing different levels of copper (0, 10, 20, 40, 80 and 400 mg kg(-1)) for 8 weeks, respectively. The feeding trial showed that the expression levels of EsHc mRNA significantly increased at the copper levels of 20-40 mg kg(-1). This study implies that the expression levels of EsHc could be affected by dietary copper in the hepatopancreas and haemocytes, and hemocyanin may be potentially involved in the immune responses of the Chinese mitten crab.

Effect of dietary copper on the growth performance, non-specific immunity and resistance to Aeromonas hydrophila of juvenile Chinese mitten crab, Eriocheir sinensis.

An 8-week feeding trial was conducted to determine the dietary copper (Cu) on growth performance and immune responses of juvenile Chinese mitten crab Eriocheir sinensis. Six semi-purified diets with six copper levels (1.88, 11.85, 20.78, 40.34, 79.56 and 381.2xa0mgxa0kg(-1) diet) of CuSO4·5H2O were fed to E.xa0sinensis (0.45xa0±xa00.01xa0g). Each diet was fed to the crab in five replicates. The crab fed diets with 20.78 and 40.34xa0mgxa0Cuxa0kg(-1) diet had significantly greater weight gain and hemolymph oxyhemocyanin content than those fed diets with 1.88 and 381.2xa0mgxa0Cuxa0kg(-1) diet. Survival rates of crab were not significantly different between all treatment groups. The activities of copper-zinc superoxide dismutase (Cu-Zn SOD), phenoloxidase (PO), and total hemocyte count (THC) significantly increased when the supplementation of dietary copper reached 20.78-40.34xa0mgxa0Cuxa0kg(-1) diets. In the bacteria challenge experiment with Aeromonas hydrophila, survival rates significantly increased and reached a plateau when the dietary copper increased from 1.88 to 40.34xa0mgxa0kg(-1), whereas significantly decreased when the dietary copper increased from 40.34 to 381.2xa0mgxa0kg(-1). This study indicates that the level of dietary copper is important in regulating growth and immune response in crab.

MnHSP90 cDNA characterization and its expression during the ovary development in oriental river prawn, Macrobrachium nipponense

Heat shock protein 90 (HSP90) is not only involved in environmental stress but also plays roles in the ovary development in some vertebrates. To understand its role in crustacean, we examined the HSP90 cDNA for the first time in the ovary and hepatopancreas of the oriental river prawn, Macrobrachium nipponense and designated this protein as MnHSP90 in this study. The MnHSP90 was cloned by the methods of degenerated oligonucleotide primers and rapid amplification of the cDNA ends (RACE). Bioinformatics analysis showed that the MnHSP90 cDNA was 2,684xa0bp in length, containing a 126xa0bp 5′ untranslated region (UTR), a 359xa0bp 3′ UTR, and an open reading frame (ORF) of 2,199xa0bp encoding a 732-amino acid polypeptide with predicted molecular mass of 84.3 KDa. Sequence alignment showed that the MnHSP90 shared 72–79% identity with other animals. Real-time quantitative PCR (qPCR) analysis demonstrated that the MnHSP90 mRNA was ubiquitously detected in all tested tissues, with the highest expression in the thoracic ganglia, the mediate in heart, muscle and intestine, and the lowest in haemocytes and gills. The MnHSP90 mRNA levels in the hepatopancreas and ovary of M. nipponense reached a maximum at the stage III (early vitellogenic stage) and stage IV (later vitellogenic stage) ovaries, respectively, and then decreased significantly in both tissues as the ovarian development proceeded. The level of MnHSP90 expression in the hepatopancreas was higher than that in the ovary when compared with in the same ovarian developmental stage. Our results indicate that MnHSP90 is involved in ovarian development in oriental river prawn and may play a regulatory role in ovary maturation.

cDNA Cloning and Expression Analysis of Gustavus Gene in the Oriental River Prawn Macrobrachium nipponense

The gustavus gene is required for localizing pole plasm and specifying germ cells. Research on gustavus gene expression will advance our understanding of the biological function of gustavus in animals. A cDNA encoding gustavus protein was identified and termed MnGus in the oriental river prawn Macrobrachium nipponense. Bioinformatic analyses showed that this gene encoded a protein of 262 amino acids and the protein belongs to the Spsb1 family. Real-time quantitative PCR analyses revealed that the expression level of MnGus in prawn embryos was slightly higher at the cleavage stage than at the blastula stage, and reached the maximum level during the zoea stage of embryos. The minimum level of MnGus expression occurred during the perinucleolus stage in the ovary, while the maximum was at the oil globule stage, and then the level of MnGus expression gradually decreased with the advancement of ovarian development. The expression level of MnGus in muscle was much higher than that in other tissues in mature prawn. The gustavus cDNA sequence was firstly cloned from the oriental river prawn and the pattern of gene expression was described during oocyte maturation, embryonic development, and in other tissues. The differential expression patterns of MnGus in the embryo, ovary and other somatic tissues suggest that the gustavus gene performs multiple physiological functions in the oriental river prawn.

Molecular cloning, characterization and expression of a C-type lectin cDNA in Chinese mitten crab, Eriocheir sinensis.

C-type lectins are pattern-recognition proteins which are functionally important for pathogen recognition and immune regulation in vertebrates and invertebrates. In this study, a lectin cDNA named as Es-Lectin was cloned and characterized from the Chinese mitten crab, Eriocheir sinensis. The full-length sequence of this Es-Lectin cDNA was 651 bp, including an open reading frame of 483 bp encoding 160 amino acids. The predicted molecular weight of the Es-Lectin was 11.8 kDa. A typical signal peptide of 21 amino acids was deduced at the N-terminus of the predicted protein. This Es-Lectin belongs to a C-type lectin and contains six cysteines, a conserved EPN motif (Glu-Pro-Asn) and an imperfect WND (Trp-Asn-Asp) motif (FND, Phe-Asn-Asp). This Es-Lectin had 55% and 32% identity with other two C-type lectins in E. sinensis, and 29-36% homology with decapods. Although the Es-Lectin was also expressed in gill, hepatopancreas, intestine, muscle and stomach, its expression in haemocytes was the greatest. The expression of Es-Lectins in haemocytes increased at 1.5 h after the Aeromonas hydrophila challenge. After a slight decrease, the Es-Lectin expression in haemocytes significantly increased at 48 h post-challenge. The diverse distribution of Es-Lectin and its enhancement by bacterial challenge indicate that C-type lectins are important in the innate immune response to bacterial infection, and can be activated for innate immune response in crab at the initial stage after pathogen infection.

Partial or complete substitution of fish meal with soybean meal and cottonseed meal in Chinese mitten crab Eriocheir sinensis diets

The practical level of fish meal replacement by plant proteins in aquaculture feed varies greatly among species. This study investigated partial or complete replacement of fish meal (FM) by cottonseed and soybean meal (CS) in Chinese mitten crab Eriocheir sinensis. Cottonseed and soybean meals were equally mixed to form five isonitrogenous and isocaloric diets to replace 0 (CS0, control), 21xa0% (CS21), 43xa0% (CS43), 64xa0% (CS64), and 100xa0% (CS100) of FM. The highest crab growth and feed utilization were observed in the CS21 diet, followed by the CS43 diet. Crab fed CS64 had similar weight gain, specific growth rate, feed conversion ratio, protein efficiency ratio, protein retention and energy retention to those fed the control diet. Dry matter digestibility decreased with increasing dietary CS and was significantly lower in the CS64 and CS100 treatments than in the control. Apparent protein and energy digestibilities of the crab fed CS0, CS21, or CS43 were similar but significantly higher than the crab fed CS64 or CS100. Crab fed CS100 had poorer digestibility of nutrients than those fed other diets. The dry matter of the crab fed CS21 was significantly higher than the crab fed CS100. The protein contents in crabs fed CS21 or CS43 were similar but were higher than those fed other diets. Crab fed CS21 contained higher lipid and gross energy than in other treatments. This study indicates that 64xa0% of FM can be replaced by CS in crab diet without compromising growth performance and body composition.