| Cathepsins, a superfamily of hydrolytic enzymes produced and enclosed within lysosomes, function in immune response in vertebrates; however, their function within the innate immune system of invertebrates remains largely unknown. Therefore, we investigated the immune functionality of cathepsin L (catL) in Chinese mitten crab (Eriocheir sinensis), a commercially important and disease vulnerable aquaculture species. The full length catL cDNA (1274 bp) was cloned via PCR based upon an initial expressed sequence tag (EST) isolated from a hepatopancreatic cDNA library. The catL cDNA contained a 978 bp open reading frame (ORF) that encoded a putative 325 amino acid (aa) protein. Comparisons with other reported invertebrate and vertebrate sequences revealed conserved gene structure and enzyme active sites common among papain-like cysteine proteases, and high percent identity among other invertebrate cathepsins. CatL mRNA expression in E. sinensis was (a) tissue-specific, with the highest expression observed in hepatotpancreas, gill, stomach, and hemocytes, and (b) responsive in hemocytes to a Vibrio anguillarum challenge, the catL expression level and enzyme activity both with peak exposure observed 8 h post-injection. Collectively, data demonstrate the successful isolation of catL from the Chinese mitten crab, and its involvement in the innate immune system of an invertebrate.The full length catC cDNA (1481 bp) was cloned via PCR based upon an initial expressed sequence tag (EST) isolated from a hepatopancreatic cDNA library. The catC cDNA contained a 1284 bp open reading frame (ORF) that encoded a putative 427 amino acid (aa) protein. Comparisons with other reported invertebrate and vertebrate cathepsins sequences revealed high percent identity. CatC mRNA expression in E. sinensis was responsive in hemocytes to a Vibrio anguillarum challenge, with peak exposure observed 6 h post-injection. Collectively, data demonstrate the successful isolation of catC from the Chinese mitten crab, and its involvement in the innate immune system of an invertebrate.Cathepsins, a superfamily of hydrolytic enzymes produced and enclosed within lysosomes, function in immune response in vertebrates; however, their function within the innate immune system of invertebrates remains largely unknown. Therefore, we investigated the immune functionality of cathepsin A (catA) in Chinese mitten crab(Eriocheir sinensis), a commercially important and disease vulnerable aquaculture species. The full length catA cDNA (2200 bp) was cloned via PCR based upon an initial expressed sequence tag (EST) isolated from a hepatopancreatic cDNA library. The catA cDNA contained a 1398 bp open reading frame (ORF) that encoded a putative 465 amino acid (aa) protein. Comparisons with other reported vertebrate cathepsins sequences revealed percent identity range from 48% to 51%. CatA mRNA expression in E. sinensis was (a) tissue-specific, with the highest expression observed in gill and (b) responsive in hemocytes to a Vibrio anguillarum challenge, with peak exposure observed 12 h post-injection. Collectively, data demonstrate the successful isolation of catA from the Chinese mitten crab, and its involvement in the innate immune system of an invertebrate.Macrophage migration inhibitory factor (MIF) as a multi-functional cytokine mediating both innate and adaptive immune responses, however, their function within the innate immune system of invertebrates remains largely unknown. Therefore, we investigated the immune functionality of MIF in Chinese mitten crab(Eriocheir sinensis), a commercially important and disease vulnerable aquaculture species. The full length MIF cDNA (704 bp) was cloned via PCR based upon an initial expressed sequence tag (EST) isolated from a E. sinensis cDNA library. The MIF cDNA contained a 363 bp open reading frame (ORF) that encoded a putative 120 amino acid (aa) protein. Comparisons with other reported invertebrate and vertebrate MIF sequences revealed conserved enzyme active sites. MIF mRNA expression in E. sinensis was (a) tissue-specific, with the highest expression observed in hepatotpancreas, and (b) responsive in hemocytes, hepantopancreas and gill to a Vibrio anguillarum challenge, with peak exposure observed 8 h,12 h and 12 h post-injection, respectively. Collectively, data demonstrate the successful isolation of MIF from the Chinese mitten crab, and its involvement in the innate immune system of an invertebrate.Fatty acid-binding proteins (FABPs), small cytosolic proteins that function in the uptake and utilization of fatty acids, have been extensively studied in higher vertebrates while invertebrates have received little attention despite similar nutritional requirements during periods of reproductive activity. Therefore, a cDNA encoding Eriocheir sinensis FABP (Es-FABP9) was cloned based upon EST analysis of a testis cDNA library. The full length cDNA was 898 bp and encoded a 136 aa polypeptide that was highly homologous to related genes reported in shrimp. Gene expression analysis, as determined by RT-PCR, revealed the presence of Es-FABP9 transcripts was widely distributed with high and detectable expression levels observed in intestine, ovary, testis and heart, while expression were comparable among hepatopancreas, hemolymphe, gills, muscle, stomach and brain. Real-time quantitative RT-PCR analysis revealed that Es-FABP9 expression in testis, hemolymphe, hepatopancreas and ovarian was dependent on the status of testis development. Evidence provided in the present report supports a role of Es-FABP9 in lipid transport during the period of rapid testis growth in E. sinensis, and indirectly confirms the participation of the hepatopancreas, testis, hemolymphe and ovarian in lipid nutrient absorption and utilization processes.In order to identify the minimal dietary zinc (Zn) requirements of the Chinese mitten crab, Eriocheir sinensis, and assess the influence of dietary zinc levels on fatty acid composition, and mRNA levels of fatty acid binding protein (FABP) and metallothionein-1 (MT-1), an experimental eight weeks feeding regime was employed. Supplemental Zn (0,10,20,40 and 80 mg/kg diet) was orally administered to crabs via their diet. Resultant data indicate that dietary supplementation of 20 mg Zn/kg diet (which corresponded to a quantified level of 27 mg Zn/kg of diet) yielded the largest weight gain. While hepatopancratic concentrations of Zn increased progressively with supplemental dosage, hepatopancreas fatty acid composition was variable and dependent of supplementation. As dietary Zn concentrations increased,18:0 in crab hepatopancreas exhibited a downward trend, while 18:1 n-9,18:3 n-3,20:4 n-6 (ARA) and 22:6 n-3 (DHA) compositions exhibited a bell curve response. FABP and MT-1 mRNA expression levels were highest in crabs receiving 20 or 40 mg Zn/kg diet (which corresponded to quantified levels of 27 or 44 mg Zn/kg diet). Fatty acid composition suggests that dietary zinc deficiency may influence the synthesis and metabolism of fatty acids in the heptaopancreas of the mitten crab, an important aquaculture species.
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