The first identification and functional analysis of two drosophila mothers against decapentaplegic protein genes (SpSmad1 and SpSmad2/3) and their involvement in the innate immune response in Scylla paramamosain.

Smad，a member of the TGF-ß superfamily，controls cell proliferation，growth and guiding cell differentiation, thus playing a crucial role in diseases. However, the presence as well as specific function of Smad in crabs is still unknown. In this study, two Smads (Smad1 and Smad2/3) were identified for the first time from the mud crab Scylla paramamosain. The complete open reading frames of SpSmad1 and SpSmad2/3 were 1,497bp and 1,338bp, encoding deduced proteins of 498 and 445 amino acids respectively. Moreover, under the administration of Vibrio alginolyticus and WSSV, the relative expression levels of SpSmad1 and SpSmad2/3 were significantly increased, indicating their involvement in the innate immune response of mud crabs. Knockdown of SpSmad1 and SpSmad2/3 in vivo not only led to the increasement of the expressions of NF-κB signaling genes and antimicrobial peptides genes, but also significantly affected the bacterial clearance process of mud crabs. Additionally, overexpression of SpSmad1 and SpSmad2/3 in HEK293T cells could markedly activate NF-κB signaling. These results indicated that Smad1 and Smad2/3 participated in the innate immunity of Scylla paramamosain, and might provide a better understanding of the presence and immune regulatory functions of Smad1 and Smad2/3 in crabs and even invertebrates.

Interactive Effect of Dietary Heat-Killed Lactobacillus Plantarum L-137 and Berberine Supplementation on Intestinal Mucosa and Microbiota of Juvenile Black Sea Bream (Acanthopagrus Schlegelii).

To compare the synergistic impact of dietary heat-killed Lactobacillus plantarum and berberine supplementation on intestinal health of juvenile black sea bream, the test fish (5.67 ± 0.05 g) were fed three diets: a basal control diet designated as Con; basal diet supplemented with 400 mg/kg L. plantarum, labelled LP; and basal diet supplemented with 400 mg/kg L. plantarum + 50 mg/k berberine, labelled LPBB. After 56 days of feeding, the control fish had significantly lower intestinal villus height (VH), villus surface area (VSA), and muscularis mucosae (MS) thickness than the rest of the groups (P < 0.05). The LPBB fish had significantly higher VH than the control fish, and wider MS and VSA than the rest of the groups (P < 0.05). Occludin was significantly upregulated in the LPBB fish, and heat shock protein 90 was upregulated in the control fish (P < 0.05). The abundance of Proteobacteria family was significantly higher in the intestinal microbiome of the control and LP fish, the LPBB fish had higher abundance of Cyanobacteria and Spirochaetes, and the LP group had higher Bacteroidetes abundance (P < 0.05). Potentially beneficial Delftia and Brevinema were the significantly abundant genera in the LP and LPBB fish, respectively; potentially pathogenic Elizabethkingia was abundant in the LP fish; and the control fish had higher abundance of potentially pathogenic Burkholderia-Caballeronia-Paraburkholderia (P < 0.05). According to these results, there is possible synergy between L. plantarum and berberine as dietary supplements in fostering healthy intestine for black sea bream than L. plantarum alone.

Comparative study on the effects of crystalline L-methionine and methionine hydroxy analogue calcium supplementations in the diet of juvenile Pacific white shrimp (Litopenaeus vannamei).

An 8-week feeding trial was conducted to evaluate the effects of L-methionine and methionine hydroxy analogue calcium (MHA-Ca) supplements in low-fishmeal diet on growth performance, hepatopancreas morphology, protein metabolism, anti-oxidative capacity, and immunity of Pacific white shrimp (Litopena eus vannamei). Four isonitrogenous and isoenergetic diets were designed: PC (203.3 g/kg fishmeal), NC (100 g/kg fishmeal), MET (100 g/kg fishmeal +3 g/kg L-methionine) and MHA-Ca (100 g/kg fishmeal +3 g/kg MHA-Ca). White shrimp (initial body weight 0.23 ± 0.00 g, 50 shrimp per tank) were allocated to 12 tanks and divided among 4 treatments in triplicates. In response to L-methionine and MHA-Ca supplementations, the shrimp exhibited higher weight gain rate (WGR), specific growth rate (SGR), condition factor (CF), and lower hepatosomatic index (HSI) compared to those fed the NC diet (p < 0.05). The WGR and SGR of shrimp fed L-methionine and MHA-Ca showed no difference with those in the PC diet (p > 0.05). Both of L-methionine and MHA-Ca supplementary diets significantly decreased the malondialdehyde (MDA) levels of shrimp when compared with the NC diet (p < 0.05). L-methionine supplementation improved the lysozyme (LZM) activity and total antioxidant capacity (T-AOC) of shrimp, while the MHA-Ca addition elevated the reduced glutathione (GSH) levels in comparison with those fed the NC diet (p < 0.05). Hypertrophied blister cells in hepatocytes were observed in shrimp fed the NC diet, and alleviated with L-methionine and MHA-Ca supplementations. Shrimp fed the MET and MHA-Ca diets had higher mRNA expression levels of target of rapamycin (tor) than those fed the NC diet (p < 0.05). Compared to the NC group, dietary MHA-Ca supplementation upregulated the expression level of cysteine dioxygenase (cdo) (p < 0.05), while L-methionine supplementation had no significant impact (p > 0.05). The expression levels of superoxide dismutase (sod) and glutathione peroxidase (gpx) were significantly upregulated by L-methionine supplemented diet in comparison with those in the NC group (p < 0.05). Overall, the addition of both L-methionine and MHA-Ca elevated the growth performance, facilitated protein synthesis, and ameliorated hepatopancreatic damage induced by plant-protein enriched diet in L. vannamei. L-methionine and MHA-Ca supplements enhanced anti-oxidants differently.

Effect of dietary supplementation of lauric acid on growth performance, antioxidative capacity, intestinal development and gut microbiota on black sea bream (Acanthopagrus schlegelii).

A feeding trial of eight weeks was conducted to examine the influence of food supplementation with lauric acid (LA) on Acanthopagrus schlegelii (juvenile black sea bream). A 24 percent fish meal baseline diet was created, while the other two diets were generated with dietary supplementation of graded points of LA at 0.1 percent and 0.8 percent, respectively. Each diet was given a triplicate tank with 20 fish weighing 6.22 ± 0.19 g. In comparison with the control group, the weight gain rate, growth rate, as well as feed efficiency of fish fed of 0.1 percent diet of LA were considerably (P < 0.05) greater. The total body and dorsal muscle proximate compositions did not change significantly between groups (P > 0.05). Triglyceride (TG) content was considerably (P < 0.05) greater in the LA-supplemented meals eating group in comparison with the control group. In the group eating LA-supplemented meals, the height of villus and the number of goblet cells/villus were considerably (P < 0.05) larger. The microbial makeup of the gut was also studied. The differences in phyla, class, and family level were not statistically significant (P > 0.05). Firmicutes in the phylum, Betaproteobacteri, Gammaproteobacteria, and Clostridia in the class, and Clostridiaceae in the family were all substantially increased with higher levels of LA supplementation (P < 0.05). According to the findings of this study, an LA-supplemented diet improves fish development, antioxidative capability, gut microbiota and intestinal health.

Experimental Studies on the Impact of the Projected Ocean Acidification on Fish Survival, Health, Growth, and Meat Quality; Black Sea Bream (Acanthopagrus schlegelii), Physiological and Histological Studies.

Acidification (OA), a global threat to the world's oceans, is projected to significantly grow if CO2 continues to be emitted into the atmosphere at high levels. This will result in a slight decrease in pH. Since the latter is a logarithmic scale of acidity, the higher acidic seawater is expected to have a tremendous impact on marine living resources in the long-term. An 8-week laboratory experiment was designed to assess the impact of the projected pH in 2100 and beyond on fish survival, health, growth, and fish meat quality. Two projected scenarios were simulated with the control treatment, in triplicates. The control treatment had a pH of 8.10, corresponding to a pCO2 of 321.37 ± 11.48 µatm. The two projected scenarios, named Predict_A and Predict_B, had pH values of 7.80-pCO2 = 749.12 ± 27.03 and 7.40-pCO2 = 321.37 ± 11.48 µatm, respectively. The experiment was preceded by 2 weeks of acclimation. After the acclimation, 20 juvenile black sea breams (Acanthopagrus schlegelii) of 2.72 ± 0.01 g were used per tank. This species has been selected mainly due to its very high resistance to diseases and environmental changes, assuming that a weaker fish resistance will also be susceptibly affected. In all tanks, the fish were fed with the same commercial diet. The seawater's physicochemical parameters were measured daily. Fish samples were subjected to physiological, histological, and biochemical analyses. Fish growth, feeding efficiency, protein efficiency ratio, and crude protein content were significantly decreased with a lower pH. Scanning electron microscopy revealed multiple atrophies of microvilli throughout the small intestine's brush border in samples from Predict_A and Predict_B. This significantly reduced nutrient absorption, resulting in significantly lower feed efficiency, lower fish growth, and lower meat quality. As a result of an elevated pCO2 in seawater, the fish eat more than normal but grow less than normal. Liver observation showed blood congestion, hemorrhage, necrosis, vacuolation of hepatocytes, and an increased number of Kupffer cells, which characterize liver damage. Transmission electron microscopy revealed an elongated and angular shape of the mitochondrion in the liver cell, with an abundance of peroxisomes, symptomatic of metabolic acidosis.

Knowledge of, and Attitudes towards, Live Fish Transport among Aquaculture Industry Stakeholders in China: A Qualitative Study.

China is the largest food fish producer in the world. Chinese consumers normally purchase fish that are still alive to ensure freshness. Therefore, the live transport of fish is important in China's aquaculture, although it carries potential risks for animal welfare. This study investigated the attitudes and knowledge of stakeholders within Chinese aquaculture towards the live transport and welfare of fish. Semi-structured interviews were conducted with 12 participants who were involved with the aquaculture industry in China. Most participants self-rated their transport-related knowledge as moderate and had some understanding of animal welfare, although this term was generally considered only relevant to terrestrial animals. Participants' responses indicated that the live transport of fish occurs frequently in China, generally using sealed tanks, plastic bags, and foam boxes, in purpose-built vehicles. Seasonal changes, such as changes in ambient and water temperature, are considered to be important contributors to successful live transport, as well as sufficient oxygen supplies and stocking density. The use of anesthetics was not commonly reported, particularly in food fish, and fish capture is predominantly by conventional dipnets. The health status of transported fish is determined mostly by morphology (body injury, body or eye color, and fin condition), as well as vigor and swimming ability. Our results indicate that live transport poses a number of welfare risks to fish but that participants in the process associated welfare concerns more with terrestrial animals, not fish.

Dietary berberine regulates lipid metabolism in muscle and liver of black sea bream (Acanthopagrus schlegelii) fed normal or high-lipid diets.

The present study investigated the influence of berberine (BBR) supplementation in normal and high-lipid (HL) diets on lipid metabolism and accumulation in black sea bream (Acanthopagrus schlegelii). BBR was supplemented at 50 mg/kg to control (Con, 11·1 % crude lipid) and high-lipid (HL, 20·2 % crude lipid) diets and named as ConB and HLB, respectively. After the 8-week feeding trial, fish body length and specific growth rate were significantly reduced by HL diets (P < 0·05). Muscle and whole-body crude lipid contents were significantly influenced by both BBR supplementation and dietary lipid level. Fish fed the HLB diet had significantly lower serum TAG, LDL-cholesterol contents and alanine aminotransferase activity compared with the HL group. The HL group presented vast lipid accumulation in the liver, and hypertrophied hepatocytes along with large lipid droplets, and translocation of nuclear to the cell periphery. These abnormalities in black sea bream were alleviated in the HLB group. BBR supplementation in the HL diet significantly down-regulated the hepatic expression levels of acetyl-CoA carboxylase α, sterol regulatory element-binding protein-1, 6-phosphogluconate dehydrogenase, glucose 6-phosphate dehydrogenase and pparγ, whereas the lipoprotein lipase, hormone-sensitive lipase and carnitine palmitoyltransferase 1a expression levels were significantly up-regulated. However, the expression levels of these genes showed opposite trends in muscle (except for pparγ). In conclusion, dietary BBR supplementation in the HL diet reduced hepatic lipid accumulation by down-regulating lipogenesis gene expression and up-regulating lipolysis gene expression, and it increased muscle lipid contents with opposite trends of the mechanism observed in the liver.

Molecular characterization of a cDNA encoding Na+/K+/2Cl- cotransporter in the gill of mud crab (Scylla paramamosain) during the molt cycle: Implication of its function in osmoregulation.

Although iono-regulatory processes are critical for survival of crustaceans during the molt cycle, the mechanisms involved are still not clear. The Na+/K+/2Cl- cotransporter (NKCC), a SLC12A family protein that transports Na+, K+ and 2Cl- into cells, is essential for cell ionic and osmotic regulation. To better understand the role of NKCC in the molt osmoregulation, we cloned and characterized a NKCC gene from the mud crab, Scylla paramamosain (designated as SpNKCC). The predicted SpNKCC protein is well conserved, and phylogenetic analysis revealed that this protein was clustered with crustacean NKCC. Expression of SpNKCC was detected in all the tissues examined but was highest in the posterior gills. Transmission electron microscopy revealed that posterior gills had a thick type of epithelium for ion regulation while the anterior gills possessed a thin phenotype related to gas exchange. During the molting cycle, hemolymph osmolality and ion concentrations (Na+ and Cl-) increased significantly over the postmolt period, remained stable in the intermolt and premolt stages and then decreased at ecdysis. Meanwhile, the expression of SpNKCC mRNA was significantly elevated (26.7 to 338.8-fold) at the ion re-establishing stages (postmolt) as compared with baseline molt level. This pattern was consistent with the coordinated regulation of Na+/K+-ATPase α-subunit (NKA α), carbonic anhydrase cytoplasmic (CAc) isoform and Na+/H+ exchanger (NHE) genes in the posterior gills. These data suggest that SpNKCC may be important in mediating branchial ion uptake during the molt cycle, especially at the postmolt stages.

A new species of Nilothauma Kieffer from China, with a key to known species of the genus (Diptera: Chironomidae).

A new species of the genus Nilothauma Kieffer, N. pandum sp. n., is described, and its morphological descriptions and illustrations are also given. A key to the males of Nilothauma is given. The adult male of N. pandum sp. n. can be distinguished from known species of the genus by the following combination of characters: anal point very broadly lanceolate with microtrichia in median ridge and apical margin, rounded at apex; superior volsella pad-like, expanded distally; median volsella curved, rounded at apex, with 2 long basal setae and 1 long median seta.