Molecular cloning, tissue distribution and nutritional regulation of four acyl-coenzyme A oxidase (acox) isoforms in Scylla paramamosain

As rate-limiting enzymes of peroxisomal β-oxidation, acyl-coenzyme A oxidase (ACOXs) play vital roles in maintaining energy homeostasis and regulating reactive oxygen species (ROS) metabolism. However, there are no studies on the functions of ACOXs in crustaceans. In the present study, four full-length cDNA sequences of acoxs, namely the acox-1a (2403 bp), acox-1b (2733 bp), acox-3a (2878 bp) and acox-3b (3445 bp), were successfully isolated from mud crab Scylla paramamosain, which encoded 666, 673, 701 and 658 amino acids, respectively. Sequence analysis showed that the ACOX-1a, ACOX-1b and ACOX-3a possessed conserved structural domains like FAD-binding motif, fatty acyl CoA oxidase domain and peroxisomal targeting signal, while the ACOX-3b lacked peroxisomal targeting signal. Results of phylogenetic tree indicated that the four ACOXs of mud crab grouped gathered with their corresponding orthologues from crustaceans. The acox-1a, acox-3a and acox-3b were highly expressed in hepatopancreas, and the acox-1b was mainly distributed in muscle and hepatopancreas. Compared with feeding groups, the expression levels of acox-1a, acox-3a and acox-3b in hepatopancreas and acox-3a in muscle were markedly up-regulated in fasting groups, suggesting that the acoxs had significant effects in modulating energy balance during fasting. In addition, fasting significantly increased the transcriptional levels of nuclear factor erythroid 2-related factor (nrf2) and its downstream antioxidant genes (catalase (cat), glutathione peroxidase (gpx) and glutathione S-transferase (gst)) to improve antioxidant capacity for removing excessive ROS produced by ACOX-mediated peroxisomal β-oxidation. These results would be conducive to providing new insights into evolutionary characteristics and functions of acoxs in crustaceans

Cloning and expression characterization of elongation of very long-chain fatty acids protein 6 (elovl6) with dietary fatty acids, ambient salinity and starvation stress in Scylla paramamosain

Introduction: Elongation of very long-chain fatty acids protein 6 (ELOVL6) played crucial roles in regulating energy expenditure and fatty acid metabolism. Many studies have performed to investigate the physiological roles and regulatory mechanisms of elovl6 in fish and animals, while few studies were reported in crustaceans.Methods: Here we reported on the molecular cloning, tissue distribution and expression profiles in response to dietary fatty acids, ambient salinity and starvation stress in Scylla paramamosain by using rapid amplification of cDNA ends (RACE) and quantitative real-time PCR.Results: Three elovl6 isoforms (named elovl6a, elovl6b and elovl6c) were isolated from S. paramamosain in the present study. The complete sequence of elovl6a was 1345 bp, the full-length sequence of elovl6b was 1419 bp, and the obtained elovl6c sequence was 1375 bp in full length. The elovl6a, elovl6b and elovl6c encoded 287, 329 and 301 amino acids respectively, and exhibited the typical structural features of ELOVL protein family members. Phylogenetic analysis showed that the ELOVL6a from S. paramamosain clustered most closely to ELOVL6 from Portunus trituberculatus and Eriocheir sinensis, while the ELOVL6b and ELOVL6c from S. paramamosain gathered alone into a single branch. Quantitative real-time PCR exhibited that the relatively abundant expression of elovl6b was observed in intestine and stomach, and the elovl6a and elovl6c were highly expressed in hepatopancreas. In addition, studies found that replacing fish oil with soybean oil could significantly increase the transcriptional levels of three elovl6 in hepatopancreas of S. paramamosain, and the expression of elovl6a and elovl6c in hepatopancreas were more sensitive to dietary fatty acids than the elovl6b. Compared with the normal sea water group (27‰), the expression of sterol-regulatory element binding protein1c (srebp-1), elovl6a, elovl6b and elovl6c were upregulated in the low salinity groups, particularly in 7‰. On the contrary, the starvation stress suppressed the expression of srebp-1, elovl6a, elovl6b and elovl6c.Discussion: These results may contribute to understand the functions of elovl6 in fatty acid synthesis and regulatory mechanisms in crustaceans

Molecular cloning and characterization of three carnitine palmitoyltransferase (cpt) isoforms from mud crab (Scylla paramamosain) and their roles in respond to fasting and ambient salinity stress

As rate-limiting enzymes of β-oxidation of fatty acids in mitochondria, the carnitine palmitoyltransferase (CPT) played an important role in regulating energy homeostasis of aquatic animals. However, there was very little research on β-oxidation of fatty acids in crustaceans. In the present study, the full-length cDNA sequences of cpt-1a, cpt-1b and cpt-2 were isolated from the hepatopancreas of Scylla paramamosain, and contained 4206, 5303 and 3486 bp respectively. Sequence analysis showed that the CPT-1A, CPT-1B and CPT-2 encoded proteins with 777, 775 and 672 amino acids respectively, and only the CPT-1A possessed a transmembrane region. In addition, both the CPT-1B and CPT-2 contained conservative functional domains like N-terminal domain and acyltransferases choActase 2, while the CPT-1A lacked. The results of phylogenetic tree indicated that the CPT-1A, CPT-1B and CPT-2 of S. paramamosain gathered together with their corresponding orthologues from crustaceans. The tissue distribution exhibited that the cpt-1a was highly expressed in hepatopancreas, followed by muscle, eyestalk and cranial ganglia, and the muscle, eyestalk and heart were main expressed tissues of cpt-1b. Furthermore, the high expression levels of cpt-2 were mainly detected in hepatopancreas, muscle and heart. The transcriptional levels of cpt-1a, cpt-1b and cpt-2 were significantly up-regulated under chronic low salinity stress. Besides, at the acute low salinity stress condition, the expression levels of cpt-1a, cpt-1b and cpt-2 in hepatopancreas were dramatically increased in 14‰ and 4‰ salinity groups at the 6h and 48h, while the transcriptional levels of cpt-1a, cpt-1b and cpt-2 in muscle were signally up-regulated in 14‰ and 4‰ salinity groups at the 12h and 24h, showing an alternate response pattern. Similarly, the present study found that fasting could markedly increase the expression levels of cpt-1a, cpt-1b and cpt-2 in hepatopancreas and muscle, especially cpt-1a in hepatopancreas as well as cpt-1a and cpt-1b in muscle. The results above indicated that the cpt-1a, cpt-1b and cpt-2 played a crucial part in providing energy for coping with fasting and salinity stress. These results would contribute to enhancing the knowledge of cpt phylogenetic evolution and their roles in energy metabolism of crustaceans

Beneficial effects of dietary organic acids on growth and health of juvenile Chinese mitten crab (Eriocheir sinensis)

The present study determined the effects of five dietary organic acids on the growth and health status of juvenile Eriocheir sinensis. A control diet was without organic acids, and five experimental diets were supplemented with 0.2 % citric acid (CA), malic acid (MA), α-ketoglutaric acid (KG), fumaric acid (FA), and lactic acid (LA), respectively. The initial body weight of E. sinensis was 3.09 ± 0.01 g, and the experiment lasted eight weeks. The present study indicates that dietary CA, KG and FA can markedly improve growth performance and feed utilization for E. sinensis. In addition, all organic acids used in the present study, especially CA, KG and FA, promoted calcium (Ca) and phosphorus (P) deposition, possibly through increasing available P levels in the diet and up-regulating the gene expression of Ca and P absorption, leading to higher ash content of the whole body. Dietary CA, KG and FA significantly reduced whole-body total lipid content, suggesting that these organic acids may facilitate lipid utilization. Besides, all organic acids increased the activities of digestive enzymes, antioxidant enzymes and phosphatases. These results indicate that dietary organic acids benefit digestive ability, antioxidant state, and nonspecific immunity for juvenile E. sinensis. Considering the response of various indicators, we conclude that among the five tested organic acids, CA, KG, and FA have better potential as feed additives in the diets of E. sinensis