Cellular localization and potential ligands of a novel scavenger receptor class B/CD36 protein homolog (Pt-SRB2) identified in the marine crab, Portunustrituberculatus.

The scavenger receptor class B family proteins (SRB) are multiligand membrane receptor proteins. Herein, a novel SRB homolog (Pt-SRB2) was identified in Portunus trituberculatus. The open reading frame of Pt-SRB2 was predicted to encode 520 amino acid residues comprising a typical CD36 domain. Phylogenetic analysis showed that Pt-SRB2 distinctly clustered with the SRB homologs of most crustaceans and Drosophila but was separate from all vertebrate CD36/SRB. Semi-quantitative and Real-time quantitative PCR revealed that the abundance of Pt-SRB2 transcripts was the highest in hepatopancreas than in other tested tissues. Overexpressed Pt-SRB2 was distributed primarily in the cell membrane and cytoplasm of HEK293T or Drosophila Schneider 2 cells. In crab hemocytes, Pt-SRB2 was distributed primarily in the cell membrane by immunofluorescence staining. In addition, the immunofluorescence staining showed that green fluorescence signals were mainly located in the inner lumen membrane of the hepatopancreatic tubules. Moreover, solid-phase enzyme-linked immunosorbent assay revealed that rPt-SRB2-L exhibited relative high affinity with lipopolysaccharides, and relative moderate binding affinity with lipoteichoic acid or peptidoglycan. Of note, rPt-SRB2-L showed high binding affinity with eicosapentaenoic acid among a series of long-chain polyunsaturated fatty acids. Taken together, this study provided valuable data for understanding the functions of the crab CD36/SRB.

High-fat-diet induced inflammation and apoptosis via activation of Ire1α in liver and hepatocytes of black seabream (Acanthopagrus schlegelii).

The present study aimed to reveal the role of inositol-requiring enzyme 1α (Ire1α) in mediating high-fat-diet (HFD) induced inflammation and apoptosis in fish and elucidate underling mechanisms of action. In experiment 1, black seabream juveniles were fed a control diet (Control, 12 % dietary lipid) or a high fat diet (HFD, 19 % dietary lipid) for eight weeks. In experiment 2, primary hepatocytes were isolated from black seabream juveniles and treated with oleic acid (OA, 200 µmol/L), OA + transfection with non-silencing control siRNA (negative control) (OA + NC), and OA + transfection with ire1α-small interfering RNA (OA + siire1α) for 48 h versus untreated (Control). Results indicated that fish fed HFD increased lipid deposition in the liver and caused hepatic steatosis. HFD group had significantly higher ire1α/Ire1α mRNA and phosphorylated protein expression and endoplasmic reticulum stress (ERS) related genes expression compared to the Control group, indicating that ERS was triggered. Meanwhile, feeding HFD induced inflammation and apoptosis by evaluated nuclear factor kappa B (nf-κb) mRNA and phosphorylated Nf-κb p65 protein expression, and c-Jun N-terminal kinase (jnk) mRNA and protein expression. However, knock down of ire1α (OA + siire1α) in primary hepatocytes alleviated OA-induced increased expression of ire1α/Ire1α mRNA and protein expression, nf-κb/Nf-κb p65 mRNA and phosphorylated protein expression, and jnk/Jnk mRNA and phosphorylated protein expression. These findings revealed the underling mechanism of action of HFD in fish, confirming that HFD increased ESR stress and Ire1α that, in turn, activated Nf-κb and Jnk pathways in hepatocytes and liver mediating HFD-induced inflammation and apoptosis.

Molecular cloning and characterization of endoplasmic reticulum stress related genes grp78 and atf6α from black seabream (Acanthopagrus schlegelii) and their expressions in response to nutritional regulation.

Glucose-regulated protein 78 (grp78) and activating transcription factor 6α (atf6α) are considered vital endoplasmic reticulum (ER) molecular chaperones and ER stress (ERS) sensors, respectively. In the present study, the full cDNA sequences of these two ERS-related genes were first cloned and characterized from black seabream (Acanthopagrus schlegelii). The grp78 cDNA sequence is 2606 base pair (bp) encoding a protein of 654 amino acids (aa). The atf6α cDNA sequence is 2168 base pair (bp) encoding a protein of 645 aa. The predicted aa sequences of A. schlegelii grp78 and atf6α indicated that the proteins contain all the structural features, which were characteristic of the two genes in other species. Tissues transcript abundance analysis revealed that the mRNAs of grp78 and atf6α were expressed in all measured tissues, but the highest expression of these two genes was all recorded in the gill followed by liver/ brain. Moreover, in vivo experiment found that fish intake of a high lipid diet (HLD) can trigger ERS by activating grp78/Grp78 and atf6α/Atf6α. However, it can be alleviated by dietary betaine supplementation, similar results were also obtained by in vitro experiment using primary hepatocytes of A. schlegelii. These findings will be beneficial for us to evaluate the regulator effects of HLD supplemented with betaine on ERS at the molecular level, and thus provide some novel insights into the functions of betaine in marine fish fed with an HLD.

Dietary choline activates the Ampk/Srebp signaling pathway and decreases lipid levels in Pacific white shrimp (Litopenaeus vannamei).

An 8-week feeding trial was conducted in Pacific white shrimp (Litopenaeus vannamei) to evaluate the effects of dietary choline supplementation on choline transport and metabolism, hepatopancreas histological structure and fatty acid profile, and regulation of lipid metabolism. Six isonitrogenous and isolipidic diets were formulated to contain different choline levels of 2.91 (basal diet), 3.85, 4.67, 6.55, 10.70 and 18.90 g/kg, respectively. A total of 960 shrimp (initial weight, 1.38 ± 0.01 g) were distributed randomly into twenty-four 250-L cylindrical fiber-glass tanks, with each diet assigned randomly to 4 replicate tanks. The results indicated that dietary choline significantly promoted the deposition of choline, betaine and carnitine (P < 0.05). The diameters and areas of R cells, total lipid and triglyceride contents in hepatopancreas, and triglyceride and non-esterified fatty acid contents in hemolymph were negatively correlated with dietary choline level. The contents of functional fatty acids in hepatopancreas, the activity of acetyl-CoA carboxylase (Acc), and the mRNA expression of fas, srebp and acc were highest in shrimp fed the diet containing 4.67 g/kg choline, and significantly higher than those fed the diet containing 2.91 g/kg, the lowest level of choline (P < 0.05). The number of R cells, content of very low-density lipoprotein (VLDL), activities of carnitine palmitoyl-transferase (Cpt1), lipoprotein lipase and hepatic lipase, and the mRNA expression levels of cpt1, fabp, fatp, ldlr, and ampk in hepatopancreas increased significantly as dietary choline increased (P < 0.05). In addition, hepatopancreas mRNA expression levels of ctl1, ctl2, oct1, badh, bhmt, ck, cept, and cct were generally up-regulated as dietary choline level increased (P < 0.01). In conclusion, dietary choline promoted the deposition of choline and its metabolites by up-regulating genes related to choline transport and metabolism. Moreover, appropriate dietary choline level promoted the development of hepatopancreas R cells and maintained the normal accumulation of lipids required for development, while high dietary choline not only promoted hepatopancreas lipid export by enhancing VLDL synthesis, but also promoted fatty acid ß-oxidation and inhibited de novo fatty acid synthesis by activating the Ampk/Srebp signaling pathway. These findings provided further insight and understanding of the mechanisms by which dietary choline regulated lipid metabolism in L. vannamei.

High dietary lipid level promotes low salinity adaptation in the marine euryhaline crab (Scylla paramamosain).

The physiological processes involved in adaptation to osmotic pressure in euryhaline crustaceans are highly energy demanding, but the effects of dietary lipids (fat) on low salinity adaptations have not been well evaluated. In the present study, a total of 120 mud crabs (Scylla paramamosain, BW = 17.87 ± 1.49 g) were fed control and high-fat (HF) diets, at both medium salinity (23‰) and low salinity (4‰) for 6 wk, and each treatment had 3 replicates with each replicate containing 10 crabs. The results indicated that a HF diet significantly mitigated the reduction in survival rate, percent weight gain and feed efficiency induced by low salinity (P < 0.05). Low salinity lowered lipogenesis and activated lipolysis resulting in lipid depletion in the hepatopancreas of mud crabs (P < 0.05). Thus, HF diets enhanced the process of lipolysis to supply more energy. In the gills, low salinity and the HF diet increased the levels of mitochondrial biogenesis markers, the activity of mitochondrial complexes, and the expression levels of genes related to energy metabolism (P < 0.05). Consequently, the positive effects of the HF diet on energy metabolism in mud crabs at low salinity promoted osmotic pressure regulation. Specifically, significantly higher haemolymph osmotic pressure and inorganic ion content, as well as higher osmotic pressure regulatory enzyme activity in gills, and gene and protein expression levels of NaK-ATPase were observed in crabs fed the HF diet at low salinity (P < 0.05). In summary, high dietary lipid levels improved energy provision to facilitate mitochondrial biogenesis, which increased ATP provision for osmotic pressure regulation of mud crabs. This study also illustrates the importance of dietary lipid nutrition supplementation for low salinity adaptations in mud crabs.

Effects of dietary chitosan on the growth, health status and disease resistance of golden pompano (Trachinotus ovatus).

The effects of dietary chitosan (0, 2, 4, 6, 8 and 10 g/kg) on the growth, health condition and disease resistance of golden pompano Trachinotus ovatus were evaluated. Dietary chitosan significantly enhanced weight gain, with the highest observed in fish fed the 6 g/kg chitosan diet. This chitosan level significantly promoted gut health by increasing villus length, lipase and protease activities and intestinal barrier-related genes expression. Meanwhile, dietary 6 g/kg chitosan improved the inflammatory response and anti-oxidative capacity of fish by regulating the expression of genes involved in NF-κB pathway and Nrf2 pathway, respectively. Furthermore, after challenge with Vibrio harveyi for 2 weeks, the survival rate increased significantly when dietary chitosan level was 6 g/kg. Overall, our results indicate that 6 g/kg chitosan is the optimal dose for enhancing growth, health and disease resistance of fish, but excessive chitosan (10 g/kg) weakens its beneficial effects.

Dietary choline promotes growth, antioxidant capacity and immune response by modulating p38MAPK/p53 signaling pathways of juvenile Pacific white shrimp (Litopenaeus vannamei).

The objective of the present study was to evaluate the effects of dietary choline levels on growth performance, antioxidant capacity, innate immunity and hemocyte apoptosis of Litopenaeus vannamei. Six isonitrogenous and isolipidic diets were formulated to contain different choline levels: 2.91 (basal diet), 3.85, 4.67, 6.55, 10.70 and 18.90 g kg-1choline, respectively. The results indicated that shrimp fed diet with 4.67 g kg-1 choline had the highest final body weight (FBW), percent weight gain (PWG), specific growth rate (SGR), feed efficiency (FE), and activities of alkaline phosphatase (AKP) and phenoloxidase (PO) in hemolymph among all treatments. Shrimp fed diet with 18.90 g kg-1 choline exhibited significantly lower crude lipid in hepatopancreas than those fed diets with 2.91, 3.85, 4.67 and 6.55 g kg-1 choline (P < 0.05). The concentration of reactive oxygen species (ROS) and apoptosis rate in hemocytes significantly decreased with the increase of dietary choline levels (P < 0.05). Shrimp fed diets with 6.55, 10.70 and 18.90 g kg-1 choline had significantly higher scavenging ability of hydroxyl radical (SAHR) and total antioxidant capacity (T-AOC) in hemolymph than those fed diet with 2.91 g kg-1 choline (P < 0.05). Dietary choline supplementation down-regulated the expression of genes related to apoptosis such as caspase-1, caspase-3, caspase-8, p53, and p38MAPK in hemocytes (P < 0.05), while up-regulated the expression of anti-apoptosis gene bcl2 in hemocytes (P < 0.05). Overall, the results of the present study demonstrated that appropriate dietary choline could improve growth performance and feed utilization, enhance antioxidant capacity and innate immunity, and mitigate apoptosis in Litopenaeus vannamei. Moreover, the inhibition of hemocyte apoptosis by dietary choline may be regulated by the p38MAPK-p53 signaling pathway.

Dietary cholesterol promotes growth and ecdysone signalling pathway by modulating cholesterol transport in swimming crabs (Portunus trituberculatus).

Cholesterol, as an indispensable nutrient, regulates molting and growth in crustacean. As crustaceans are unable to biosynthesize cholesterol de novo, it is central to understand how dietary cholesterol affects molting in crustaceans. An 8-week feeding trial was conducted to evaluate the effects of dietary cholesterol level (0.12%, 0.43%, 0.79%, 1.00%, 1.30% and 2.50%) on growth, cholesterol metabolism and expression of genes related to lipid and ecdysone metabolism in female swimming crabs (Portunus trituberculatus). A total of 192 crabs (1.41 ± 0.05 g) were randomly distributed into 192 aquaria. Each treatment had 4 replicates with each replicate containing 8 crabs. Crabs fed the 1.00% cholesterol diet showed best growth performance, and thus based on percent weight gain, the optimal dietary cholesterol requirement was calculated at 1.01%. Tissue cholesterol concentrations were positively correlated with dietary cholesterol level. The contents of functional fatty acids in hepatopancreas significantly increased as dietary cholesterol increased from 0.12% to 2.50% (P < 0.05). The expression levels of genes related to lipogenesis pathway, lipid catabolism and fatty acid oxidation were significantly down-regulated with increased dietary cholesterol level (P < 0.05). The highest expression levels of cholesterol transport genes, low-density lipoprotein receptor (ldlr) and low-density lipoprotein receptor-related protein 2 (lrp2) occurred in crabs fed the 1.30% cholesterol diet. Moreover, hormones related to molting such as crustacean hyperglycemic hormone (CHH), methyl farnesoate (MF), molt-inhibiting hormone (MIH), and ecdysone in hemolymph were significantly influenced by dietary cholesterol level (P < 0.05). The highest expression levels of ecdysone receptor (ecr) and chitinase 1 (chi1) in eyestalk and hepatopancreas were found in crabs fed the diet containing 1.00% cholesterol (P < 0.05). In conclusion, the optimal dietary level was beneficial to functional fatty acid accumulation, regulated lipid metabolism, promoted the ecdysone signalling pathway by improving the cholesterol transport, and improved the molting rate and growth of swimming crabs.

Lipid metabolic disorders and physiological stress caused by a high-fat diet have lipid source-dependent effects in juvenile black seabream Acanthopagrus schlegelii.

This study was conducted to evaluate the effects of different dietary lipid sources on growth performance, lipid metabolism, and physiological stress responses including oxidative stress (OS) and endoplasmic reticulum stress (ERS) of juvenile Acanthopagrus schlegelii (initial weight 0.88 ± 0.01 g) fed a high-fat diet (HFD). Four isonitrogenous and isolipidic experimental diets containing different lipid sources were formulated: fish oil (FO), palm oil (PO), linseed oil (LO), and soybean oil (SO), respectively. Results indicated that fish fed HFD supplemented with FO significantly improved growth than SO treatment. The high concentrations of aspartate aminotransferase and alanine transaminase were found in HFD supplemented with SO. Fish fed dietary LO supplementation showed significantly lower serum cholesterol, triglyceride, low-density lipoprotein, and high-density lipoprotein contents than those in SO group. Likewise, hepatic paraffin section analysis indicated that HFD with PO or SO supplementation increased fat drop. The expression levels of peroxisome proliferators-activated receptor alpha (pparα) and silent regulator 1 (sirt1) were significantly elevated by HFD with FO or LO supplementation. Additionally, the key marker of OS malonaldehyde was significantly increased in FO and SO groups. ERS-related genes were activated in dietary PO or SO supplementation and, hence, triggering inflammation and apoptosis by promoting the expression levels of nuclear factor kappa B (nf-κb) and c-Jun N-terminal kinase (jnk). Overall, the present study reveals that lipid metabolic disorders and physiological stress caused by a HFD have significant lipid source-dependent effects, which have important guiding significance for the use of HFD in marine fish.

A New Insight Into the Underlying Adaptive Strategies of Euryhaline Marine Fish to Low Salinity Environment: Through Cholesterol Nutrition to Regulate Physiological Responses.

Salinity is an important environmental factor that can affect the metabolism of aquatic organisms, while cholesterol can influence cellular membrane fluidity which are vital in adaption to salinity changes. Hence, a 4-week feeding trial was conducted to evaluate the effects of water salinity (normal 23 psu and low 5 psu) and three dietary cholesterol levels (CH0.16, 0.16%, CH1.0, 1.0% and CH1.6, 1.6%) on osmoregulation, cholesterol metabolism, fatty acid composition, long-chain polyunsaturated fatty acid (LC-PUFA) biosynthesis, oxidative stress (OS), and endoplasmic reticulum stress (ERS) of the euryhaline fish black seabream (Acanthopagrus schlegelii). The results indicated that in low salinity, fish fed with the CH1.0 diet improved ion reabsorption and osmoregulation by increased Na+ concentration in serum as well as expression levels of osmoregulation-related gene expression levels in gills. Both dietary cholesterol level and water salinity significantly affected most cholesterol metabolic parameters in the serum and tissues, and the results showed that low salinity promoted cholesterol synthesis but inhibited cholesterol catabolism. Besides, in low salinity, hepatic expression levels of LC-PUFA biosynthesis genes were upregulated by fed dietary cholesterol supplementation with contents of LC-PUFAs, including EPA and DHA being increased. Malondialdehyde (MDA) was significantly increased in low-salinity environment, whereas MDA content was decreased in fish fed with dietary CH1.0 by activating related antioxidant enzyme activity and gene expression levels. A similar pattern was recorded for ERS, which stimulated the expression of nuclear factor kappa B (nf-κb), triggering inflammation. Nevertheless, fish reared in low salinity and fed with dietary CH1.0 had markedly alleviated ERS and downregulated gene expression levels of pro-inflammatory cytokines. Overall, these findings demonstrate that cholesterol, as an important nutrient, plays vital roles in the process of adaptation to low salinity of A. schlegelii, and provides a new insight into underlying adaptive strategies of euryhaline marine fish reared in low salinity.

Dietary vitamin K3 activates mitophagy, improves antioxidant capacity, immunity and affects glucose metabolism in Litopenaeus vannamei.

An 8-week feeding experiment was conducted to appraise the influence of dietary vitamin K3 on the growth performance, antioxidant capacities, immune responses, mitophagy and glucose metabolism in Litopenaeus vannamei. Six diets containing graded dietary vitamin K3 (0.40(control), 9.97, 20.29, 39.06, 79.81 and 156.02 mg kg-1 of vitamin K3, respectively) levels were formulated. A total of 900 shrimp with 0.90 g initial weight were randomly assigned to six diets with three replications. Our results revealed that diets supplemented with 9.97-156.02 mg kg-1 vitamin K3 didn't affect the growth performance in L. vannamei. In general, compared with the control group, 39.06 mg kg-1 vitamin K3 group significantly increased (P < 0.05) the total antioxidative capacity, and the activities of catalase, glutathione, nitric oxide synthase, alkaline phosphatase and acid phosphatase in serum and hepatopancreas. 39.06 mg kg-1 vitamin K3 group significantly decreased (P < 0.05) the malondialdehyde in serum and hepatopancreas. The mRNA levels of antioxidant and immune related genes were increased synchronously (P < 0.05). In addition, 39.06 mg kg-1 vitamin K3 group increased glycogen content and levels of mitophagy (pink1, ampkα, parkin, lc3, atg13, atg12) genes. Expression levels of glucose transport related gene (glut1), glycolysis related genes (hk, pfk), glycogen synthesis related genes (gsk-3ß, gys), insulin-like peptides (ILPs)/AKT/PI3K pathway related genes (insr, irsl, akt, pi3k, pdpk1) were increased in the hepatopancreas of 39.06 mg kg-1 vitamin K3 group. In conclusion, the present results indicated that although dietary supplementing vitamin K3 had no influence on the growth performance, 39.06 mg kg-1 vitamin K3 could activate ampkα/pink1/parkin mediated mitophagy, improve antioxidant capacity and immune response. Moreover, vitamin K3 could trigger ILPs/AKT/PI3K signaling pathways and influence glucose metabolism in L. vannamei. This finding would help to advance the field of vitamin K3 nutrition and guide the development of future crustacean feeds.

Effects of faba bean (Vicia faba L.) on fillet quality of Yellow River carp (Cyprinus carpio) via the oxidative stress response.

In order to further explain the fillet texture improvement of Yellow River carp (Cyprinus carpio) fed with faba bean (Vicia faba L.), a three-month rearing trial was conducted to investigate fatty acid composition, antioxidant capacity, myofiber development, collagen deposition and transcriptome in white muscle of two farmed carp groups (One was fed only faba bean, the other was fed commercial diet). As a strong oxidant, faba bean changed fatty acids composition in white muscle, especially DHA and EPA, up-regulated the levels of reactive oxygen species (ROS) and down-regulated major antioxidant enzyme activities in the hepatopancreas and white muscle. Through the analysis of transcriptome and subsequent verification analysis, we speculated that the increase of ROS led to the decrease of myofiber diameter and collagen metabolism. This study provides a theoretical basis for further understanding the regulation of faba bean on fillet texture characteristic of Yellow River carp.

Effects of dietary Astragalus polysaccharides on growth, health and resistance to Vibrio harveyi of Lates calcarifer.

It is generally accepted that Astragalus polysaccharides (APS) supplementation can makes beneficial effects to fish. However, the adverse effects of APS to fish remains poorly understood. In the present study, Asian seabass Lates calcarifer were studied to assess the influence of different doses of APS on growth, health and resistance to Vibrio harveyi. Results showed that supplemental APS with 0.10 to 0.20% significantly boosted the growth performance, the protease and lipase activities of L. calcarifer. Compared with control diet, the villus length of L. calcarifer fed with APS supplemented diets was significantly higher. L. calcarifer fed with APS supplementation diets also significantly facilitated the antioxidant capacity and immune function. Meanwhile, supplemental APS with 0.10 to 0.15% significantly promoted liver health by up-regulating the expression of anti-inflammatory cytokines and down-regulating the expression of pro-inflammatory cytokines. Furthermore, survival rate of L. calcarifer challenged with V. harveyi was higher in diets supplemented with APS compared to the control. However, 0.20% APS significantly hindered the growth performance and caused immunostimulatory fatigue in L. calcarifer compared to 0.10% APS. Taken together, the present study demonstrates that supplementation APS with 0.10% is the optimal level for promoting the growth performance, health and resistance to V. harveyi of L. calcarifer, while 0.20% APS exerts adverse effects on L. calcarifer. Our findings provide novel recommendations for the application of APS supplementation in farmed fish.

Excess iron supplementation induced hepatopancreas lipolysis, destroyed intestinal function in Pacific white shrimp Litopenaeus vannamei.

So far, the adverse effects of excess Fe in shrimp have been ignored for years as it was thought that extra Fe supplementation was not needed in the practical diets. Nowadays, Fe concentration in commercial shrimp feed from feed enterprises could be around 301.34-545.5 mg/kg, which is mainly due to the fish meal containing up to 1500 mg/kg Fe. Therefore, the purpose of this experiment was to investigate the effects of Fe supplementation on the growth performance, tissue Fe deposition, hepatopancreas lipid metabolism, intestinal function in L. vannamei. The results showed that although growth performance was not influenced by the dietary Fe supplementation, excess Fe supplementation (955.00 mg/kg) significantly increased hepatopancreas Fe deposition and induced lipolysis. Moreover, excess Fe supplementation impaired intestinal immune function and disrupted microbiota homeostasis. These findings might provide partial theoretical evidence for the effect of dietary Fe supplementation on physiological metabolism in L. vannamei.

Identification and function analysis of an immune deficiency homolog in swimming crab, Portunus trituberculatus.

The immune deficiency (IMD) pathway is involved in both antiviral and antibacterial immune responses in Drosophila. IMD protein is the key adaptor to link the extracellular signal and the intracellular reaction to initiate the signal transduction in IMD pathway. In present study, the cDNA of the IMD (Pt-IMD) was identified from a marine crab, Portunus trituberculatus. The Pt-IMD is predicted to encode 170 amino acids with a death domain. Real-Time quantitative PCR analysis showed that Pt-IMD was constitutively expressed in hemocytes, intestine, gill, heart, muscle and hepatopancreas in normal crab. Moreover, the transcript of Pt-IMD in large-granule hemocytes is approximately 6-fold higher than semi-granular cells and agranular cells. Intracellular localization showed Pt-IMD was distributed mainly in the cytoplasm when it was over-expressed in Drosophila Schneider 2 (S2) cell. Functionally, over-expression of Pt-IMD could activate the promoters of Drosophila antimicrobial peptide genes (AMPs) in S2 cell. Furthermore, Pt-IMD expression was also knock-down by RNAi to determine the function of Pt-IMD on regulation of the expression of different antimicrobial peptides (AMPs) in crab. In the primary cultured hemocytes challenged with or without Vibrio alginolyticus, after Pt-IMD was knocked-down by specific long double strand RNA, the expression of anti-lipopolysaccharide factor1 (ALF1), ALF3, crustin1, crustin3, arasin2, hyastatin1and hyastatin3 have been significantly inhibited in normal cell or bacterial infected cell, while the expression of lysozyme was normal in non-infected cells and was significantly induced in bacterial infected cells, which compared to the non-specific dsRNA treated cells.

Alteration of Growth Performance, Antioxidant Capacity, Tissue Fatty Acid Profiles, and Lipid Metabolism of Mud Crab (Scylla paramamosain) Juvenile in Response to Different Dietary Arachidonic Acid Levels.

An eight-week feeding trail was carried out to investigate the impacts of different dietary arachidonic acid (ARA) supplementations on growth performance, antioxidant capacity, tissue fatty acid profiles, and lipid metabolism of mud crab (Scylla paramamosain) juvenile. Six isonitrogenous (480 g kg-1 crude protein) and isolipidic (80 g kg-1 crude lipid) diets were formulated to contain 0.40, 2.50, 4.60, 8.90, 12.50, and 15.70 g ARA kg-1 (dry matter), respectively. Each experimental treatment included 24 mud crab juveniles (initial weight 11.29 ± 0.09 g) and was assigned to triplicate groups (n = 3). Crabs fed diets with 2.50, 4.60, and 8.90 g kg-1 ARA presented significantly higher percent weight gain (PWG) and specific growth rate (SGR) than those fed the other diets. Based on two-slope broken-line and quadratic curve regression analysis of PWG against dietary ARA levels, optimal dietary ARA levels were determined to be 5.20 g kg-1 and 6.20 g kg-1, respectively. Crabs fed with 4.60 g kg-1 ARA diet showed the lowest activities of alanine aminotransferase (ALT) as well as aspartate aminotransferase (AST) in hemolymph among all treatments. In hemolymph and hepatopancreas, total antioxidant capacity (T-AOC), the activities of total superoxide dismutase (T-SOD), and glutathione peroxidase (GSH-Px) as well as the contents of reduced glutathione (GSH) rose first and then dropped with the increase of dietary ARA levels, while the concentration of malondialdehyde (MDA) showed an opposite trend. Tissue fatty acid profiles reflected diets fatty acid compositions. The ARA contents in hepatopancreas and muscle significantly increased with the increase of dietary ARA levels. Furthermore, the areas of blasenzellen (B) cells and restzellen (R) cells were significantly downregulated with the increase of dietary ARA levels. Crabs fed with 0.40 g kg-1 ARA diet showed significantly higher gene expression levels of fatty acid synthase (fas) as well as acetyl-CoA carboxylase (acc) among all treatments. Relative gene expression levels of 6-phosphogluconate dehydrogenase (6pgd) as well as glucose-6-phosphate dehydrogenase (g6pd) have been significantly upregulated in 0.40 and 2.50 g kg-1 ARA groups. Relative gene expression level of fatty acid binding protein 1 (fabp1) significantly increased in 4.60, 8.90, 12.50, and 15.70 g kg-1 ARA groups. However, the gene expression levels of fatty acid binding protein 4 (fabp4) as well as scavenger receptor class 2 (srb2) have not been influenced by dietary ARA levels. What is more, crabs fed diets with 4.60, 8.90, 12.50, and 15.70 g kg-1 ARA had a significantly higher expression level of carnitine palmitoyltransferase 1 (cpt1) than those fed diets with 0.40 and 2.50 g kg-1 ARA. In summary, optimum dietary ARA can promote growth, enhance antioxidant capacity, and improve health of mud crab juveniles. It also demonstrated that lipogenesis has been restrained with the increasing dietary ARA levels. These findings could provide theoretical guidance and reference for the lipid nutrition research as well as the development of the commercial diet in mud crab.

Dietary Corn Starch Levels Regulated Insulin-Mediated Glycemic Responses and Glucose Homeostasis in Swimming Crab (Portunus trituberculatus).

Carbohydrate is the cheapest source of energy among the three major nutrient groups, an appropriate amount of carbohydrates can reduce feed cost and improve growth performance, but carnivorous aquatic animals cannot effectively utilize carbohydrates. The objectives of the present study are aimed at exploring the effects of dietary corn starch levels on glucose loading capacity, insulin-mediated glycemic responses, and glucose homeostasis for Portunus trituberculatus. After two weeks of feeding trial, swimming crabs were starved and sampled at 0, 1, 2, 3, 4, 5, 6, 12, and 24 hours, respectively. The results indicated that crabs fed diet with 0% corn starch exhibited lower glucose concentration in hemolymph than those fed with the other diets, and glucose concentration in hemolymph remained low with the extension of sampling time. The glucose concentration in hemolymph of crabs fed with 6% and 12% corn starch diets reached the peak after 2 hours of feeding; however, the glucose concentration in hemolymph of crabs fed with 24% corn starch attained the highest value after 3 hours of feeding, and the hyperglycemia lasted for 3 hours and decreased rapidly after 6 hours of feeding. Enzyme activities in hemolymph related to glucose metabolism such as pyruvate kinase (PK), glucokinase (GK), and phosphoenolpyruvate carboxykinase (PEPCK) were significantly influenced by dietary corn starch levels and sampling time. Glycogen content in hepatopancreas of crabs fed with 6% and 12% corn starch first increased and then decreased; however, the glycogen content in hepatopancreas of crabs fed with 24% corn starch significantly increased with the prolongation of feeding time. In the 24% corn starch diet, insulin-like peptide (ILP) in hemolymph reached a peak after 1 hour of feeding and then significantly decreased, whereas crustacean hyperglycemia hormone (CHH) was not significantly influenced by dietary corn starch levels and sampling time. ATP content in hepatopancreas peaked at 1 h after feeding and then decreased significantly in different corn starch feeding groups, while the opposite trend was observed in NADH. The activities of mitochondrial respiratory chain complexes I, II, III, and V of crabs fed with different corn starch diets significantly increased first and then decreased. In addition, relative expressions of genes related to glycolysis, gluconeogenesis, glucose transport, glycogen synthesis, insulin signaling pathway, and energy metabolism were significantly affected by dietary corn starch levels and sampling time. In conclusion, the results of the present study reveal glucose metabolic responses were regulated by different corn starch levels at different time points and play an important role in clearing glucose through increased activity of insulin, glycolysis, and glycogenesis, along with gluconeogenesis suppression.

The Benefit of Optimal Dietary Lipid Level for Black Seabream Acanthopagrus schlegelii Juveniles under Low-Salinity Environment.

The present study was aimed at evaluating the regulatory effects of dietary lipid levels on growth performance, osmoregulation, fatty acid composition, lipid metabolism, and physiological response in Acanthopagrus schlegelii under low salinity (5 psu). An 8-week feeding trial was conducted in juvenile A. schlegelii with an initial weight of 2.27 ± 0.05 g, and six isonitrogenous experimental diets were formulated with graded levels of lipid: 68.7 g/kg (D1), 111.7 g/kg (D2), 143.5 g/kg (D3), 188.9 g/kg (D4), 239.3 g/kg (D5), and 269.4 g/kg (D6), respectively. Results indicated that fish fed with diet containing 188.9 g/kg lipid significantly improved growth performance. Dietary D4 improved ion reabsorption and osmoregulation by increasing the concentrations of Na+, K+, and cortisol in serum and activities of Na+/K+-ATPase as well as expression levels of osmoregulation related to gene expression levels in the gill and intestine. The expression levels of long chain polyunsaturated fatty acid biosynthesis-related genes were dramatically upregulated when dietary lipid levels increased from 68.7 g/kg to 189.9 g/kg with levels of docosahexaenoic (DHA), eicosapentaenoic (EPA), and DHA/EPA ratio being highest in the D4 group. When fish fed dietary lipid levels from 68.7 g/kg to 188.9 g/kg, lipid homeostasis could be maintained by upregulating sirt1 and pparα expression levels, whereas lipid accumulation was observed in dietary lipid levels of 239.3 g/kg and over. Fish fed with high dietary lipid levels resulted in physiological stress related to oxidative stress and endoplasmic reticulum stress. In conclusion, based on weight gain, the optimal dietary lipid requirement of juvenile A. schlegelii reared at low-salinity water is 196.0 g/kg. These findings indicate that the optimal dietary lipid level can improve growth performance, n-3 LC-PUFA accumulation, and osmoregulatory ability and maintain lipid homeostasis and normal physiological functions of juvenile A. schlegelii.

Evaluation of Krill Meal in Commercial Diets for Juvenile Swimming Crab (Portunus trituberculatus).

An 8-week feeding trial was carried out to assess the effect of dietary krill meal on growth performance and expression of genes related to TOR pathway and antioxidation of swimming crab (Portunus trituberculatus). Four experimental diets (45% crude protein and 9% crude lipid) were formulated to obtain different replacements of fish meal (FM) with krill meal (KM); FM was replaced with KM at 0% (KM0), 10% (KM10), 20% (KM20), and 30% (KM30); fluorine concentration in diets were analyzed to be 27.16, 94.06, 153.81, and 265.30 mg kg-1, respectively. Each diet was randomly divided into 3 replicates; ten swimming crabs were stocked in each replicate (initial weight, 5.62 ± 0.19 g). The results indicated that crabs fed with the KM10 diet had the highest final weight, percent weight gain (PWG), and specific growth rate (SGR) among all treatments (P < 0.05). Crabs fed with the KM0 diet had the lowest activities of total antioxidant capacity (T-AOC), total superoxide dismutase (SOD), glutathione (GSH), and hydroxyl radical scavenging activity and had the highest concentration of malondialdehyde (MDA) in the hemolymph and the hepatopancreas (P < 0.05). In the hepatopancreas, the highest content of 20:5n-3 (EPA) and the lowest content of 22:6n-3 (DHA) were shown in crabs fed with the KM30 diet among all treatments (P < 0.05). With the substitution level of FM with KM gradually increasing from 0% to 30%, the color of the hepatopancreas changed from pale white to red. Expression of tor, akt, s6k1, and s6 in the hepatopancreas was significantly upregulated, while 4e-bp1, eif4e1a, eif4e2, and eif4e3 were downregulated with dietary replacement of FM with KM increasing from 0% to 30% (P < 0.05). Crabs fed with the KM20 diet had notably higher expression of cat, gpx, cMnsod, and prx than those fed with the KM0 diet (P < 0.05). Results demonstrated that 10% replacement of FM with KM can promote growth performance and antioxidant capacity and notably upregulate the mRNA levels of genes related to TOR pathway and antioxidant of swimming crab.