Abstract:To explore and effectively identify the laboratory red crucian carp’s mitochondrial DNA barcode, we have identified the species of crucian carp. This research based on the laboratory red crucian carp and the other species of crucian carp (Dongting lake crucian carp, Japanese white crucian carp, Carassius auratus grandoculis, C. auratus langsdorfii, goldfish, goldfish-like fish and red crucian carp-like fish) as the test group, using Sanger sequencing for all individual mitochondrial COⅠ, COⅢ, Cytb, D-loop sequencing, and built the three gene splicing sequences, and analyzed their characteristics and ten kinds of sequence clustering characteristics of crucian carp molecular system evolution. The results showed that the splicing sequence of three genes (COⅠ, COⅢ, Cytb) and the evolutionary tree clustering of COⅠ gene can obviously find that ten different species of crucian carp can form different small branches, while the clustering of COⅢ gene and D-loop sequence can’t distinguish ten species of crucian carp at the same time. The results showed that COⅠ gene and the splicing sequences of three genes can effectively identify laboratory red crucian carp and other species of crucian carp, so this sequence can be used as an effective molecular marker of laboratory red crucian carp. Meanwhile, the evolutionary tree constructed by COⅠ, COⅢ, Cytb and D-loop gene can reflect the evolutionary relationship between ten different species of crucian carp.

Abstract:As the co-transporters, SGLTs (Sodium-glucose co-transporters) play a pivotal role in the absorption of the substances. As the important member of SGLTs, SGLT1 and SGLT2 are involved in keeping blood glucose homeostasis by regulation of glucose uptake. To investigate the role of SGLT1 and SGLT2 in the glucose uptake of grass carp (Ctenopharyngodon idella), a series of experiments were performed. In this study, the C. idella sglt1 and sglt2 were cloned in foregut and kidney by RT-PCR, and the sequences were analyzed by bioinformatics and the mRNA expressions were detected by real-time PCR. The results showed that the ORF of C. idella sglt1 and sglt2 were 1 977 and 1 989 bp, which encode 658 and 662 amino acids. Based on amino acids sequence, the structure of SGLT1 and SGLT2 protein were 14 transmembrane, and the results of sequence alignment and phylogenetic tree showed that the C. idella SGLT1 and SGLT2 proteins were closest to those of Carassius auratus and Sinocyclocheilus grahami. The real-time PCR results indicated that the higher expression of sglt1 was in the intestine and kidney, and the low expression of sglt1 was in other tissues of C. idella. The higher expression of sglt2 was in the kidney, and the low expression of sglt2 was in other tissues of C. idella. In OGTT experiment, the sglt1 mRNA expression was significantly increased in C. idella foregut, and the sglt2 mRNA expression was markedly increased in C. idella kidney by glucose treatment for 1 h. In in vitro experiment, the sglt1 and sglt2 mRNA expressions were markedly increased in the CIK cells by glucose treatment. In conclusion, the C. idella sglt1/2 were cloned and played a role in glucose uptake. The results will provide the data basis for consummating SGLTs functions and theoretical foundation for investigating the regulation of glucose homeostasis in fish.

Abstract:Crassostrea hongkongensis is one of the pillar industries of mariculture in the coastal areas of South China. In recent years, with the continuous expansion of the scale of cultivation, the destruction of the breeding environment and the frequent occurrence of diseases, the large-scale death of C. hongkongensis has occurred, which seriously restricts the healthy and sustainable development of C. hongkongensis. Therefore, it is very important and urgent to carry out immune research for the healthy and sustainable development of C. hongkongensis. In this study, the immune function of T-cell acute lymphocytic leukemia (tal) in C. hongkongensis was studied, RACE technique was used to clone the full-length cDNA sequence of the (T-cell acute lymphocytic leukemia) tal gene of C. hongkongensis, named Chtal. The open reading frame (ORF) is 660 bp, encoding 219 amino acids, with the relative molecular weight of 25.11 ku. Multiple sequence alignment and phylogenetic tree results showed that the Chtal gene had high homology with C. gigas, C. virginica and Aplysia californica, which proves that it is a member of the tal family of mollusks. Fluorescence quantitative PCR results showed that Chtal was the most highly expressed in haemolytic lymphocytes of oyster in C. hongkongensis, followed by heart, gill and digestive gland, suggesting its function in lymphogenesis. After the stimulation of Vibrio alginolyticus and Staphylococcus haemolyticus, the expression levels of Chtal were significantly up-regulated, reaching the highest levels at 24 and 12 h, respectively, and the expression of the gene gradually decreased with time. The results of subcellular localization showed that Chtal was expressed in the nucleus. In vivo injection of recombinant tal can significantly increase the number of hemocytes. Edu proliferation assay further confirmed that Chtal could promote the regeneration of hemocytes in C. hongkongensis. In summary, this study revealed that Chtal was involved in hemocytes regeneration, providing a basis for further research on the function of this gene in immune response of oysters.

Abstract:In aquaculture, fish inevitably suffers from a variety of stressors, leading to stress response, especially oxidative stress. In fish, oxidative stress is related to the occurrence and progress of many diseases, but it is in the absence of direct evidences regarding pathogenic mechanism of oxidative stress. Therefore, the aim of this study was to investigate the antioxidative status and immune responses in common carp (Cyprinus carpio) under oxidative stress. The fish were exposed to four concentrations of H₂O₂ (0, 0.25, 0.50 and 1.00 mmol/L) for 1 h per day for 7 days. After 7 days of exposure, we collected the blood and liver tissues to determine biochemical parameters and genes expression. The data showed that the levels of cortisol and lactic acid (LA) in H₂O₂ treatments were significantly higher than those of the control group. The activities of alkaline phosphatase (AKP) and acid phosphatase (ACP) were also significantly increased in 1.00 mmol/L H₂O₂ group. In serum, 0.50 and 1.00 mmol/L H₂O₂ exposure markedly enhanced the levels of malondialdehyde (MDA) and total antioxidant capacity (T-AOC), but decreased the levels of catalase (CAT) and glutathione (GSH). In liver, 1.00 mmol/L H₂O₂ exposure obviously decreased the level of GSH, but enhanced MDA formation. Meanwhile, the genes expression data showed that 1.00 mmol/L H₂O₂ exposure up-regulated the cytochrome p450 (cyp1a) transcription and down-regulated the cyp1b transcription in the liver. Additionally, the mRNA levels of c3, c-lyz, hep and heat shock proteins (hsp70 and hsp90) in the liver were significantly up-regulated by 0.50 and 1.00 mmol/L H₂O₂ exposure. In conclusion, oxidative stress can induce stress response and lipid peroxidation, impair antioxidant ability, and initiate immune response in C. carpio. These data might contribute to understanding pathogenic mechanism of oxidative stress, and provide theoretical reference for the prevention of diseases associated with oxidative stress in fish.

Abstract:At present, marine aquaculture in China and other countries around the world is mainly carried out in shallow coastal waters, which has caused a series of environmental problems and affected the sustainable development of aquaculture. Chinese researchers have been working on salmon mariculture far offshore in the Yellow Sea, and rainbow trout (Oncorhynchus mykiss) is one of the popular salmonid. Light color, which can affect the entire life cycle of aquatic species, is an important factor in the management of far offshore cultures. The sunlight irradiated on the water surface enters the water body after refraction, and the incident light will be absorbed and scattered strongly by the water and will attenuate rapidly. Blue light has strong penetrating power in clean sea water. Red light and ultraviolet light attenuate rapidly when passing through clean water. It is important to explore the effects of light color on the antioxidant enzyme activities and related hormone concentrations of O. mykiss, which will provide important information for the practical management of far offshore cultures. O. mykiss were injected with different concentrations of melatonin (0 and 3 mg/mL) and were exposed under four light colors (white, red, green, and blue) for 28 hours. Totally eight treatments were set up i. e. white light group of control, red light group of control, green light group of control, blue light group of control, white light group treated with melatonin, red light group treated with melatonin, green light group treated with melatonin, and blue light group treated with melatonin. O. mykiss (42.0±1.5) g were cultured in twenty-four tanks (PVC, length×height×width: 80 cm×60 cm×50 cm) and all tank sides were covered with opaque covers, light sources were placed above the surface of water. Serum and head kidney tissues of O. mykiss were collected at 4, 8, 12, 16, 20, 24, and 28 hours of the experiment to detect superoxide dismutase (SOD), cortisol, and melatonin in serum and catalase (CAT) in head kidney. The results showed that the activities of SOD and CAT, and the concentrations of cortisol were higher at 16 hours and were stable from 24 to 28 hours during the study. The average activities of SOD, CAT, and the average concentrations of cortisol in red light groups were significantly higher than the other light groups (P < 0.05). And the average activities of SOD and the average concentrations of cortisol in green light groups were significantly lower than the other light groups. The average activities of CAT in green light groups were significantly lower than the red and blue light groups. The SOD and CAT activities and cortisol concentrations in O. mykiss injected with melatonin showed a down trend during the experiment. Compared with the non-injection melatonin groups, melatonin injection made the activities of SOD and CAT and the concentrations of cortisol decrease significantly and the concentrations of melatonin increase significantly in O. mykiss. This study indicated that in a 28-hour experiment, O. mykiss were strongly stressed by light at 16 hours, and then the fish adapted to the light environment; red light stressed O. mykiss and green light did not produce stress; injection melatonin could release red light pressure for fish. In sum, green light and intraperitoneal melatonin injection could be used in O. mykiss production to relieve the fish oxidative stress response. Red light stresses fish and is unsuitable for fish cultivation. Long-term experiments should be carried out to study the effects of different light colors on the growth, physiological, and biochemical responses of O. mykiss.

Abstract:Carnosine (β-alanyl-L-histidine) is a natural dipeptide that was first discovered in 1900 by Vladimir Gulevitch as an abundant non-protein, nitrogen-containing compound of meat. Carnosine is an archetype of a family of histidine-containing dipeptides (HCDs), and several members of this family have been identified subsequently, including anserine (β-alanyl-Nπ-methyl-L-histidine), balenine (also called ophidine, β-alanyl-Nτ-methyl-L-histidine), and homocarnosine (γ-aminobutyryl-L-histidine). Recent findings have highlighted the important roles of HCDs in muscular function and homeostasis, including their pH buffering ability, antioxidant capacity, increased Ca²⁺ sensitivity and protein glycation inhibition. The high concentration of HCDs has been observed in skeletal muscle, cardiac muscle, the brain and olfactory bulb, the stomach, and the kidneys of vertebrates. However, the biological role of carnosine and its analogues is not yet entirely known. β-alanine is a key substrate for the synthesis of carnosine, and β-alanine supplementation has been used for elevation of the carnosine content and thus has very important value in the field of medicine, pharmacy and food. Mussel is an important aquaculture shellfish in China. In order to determine the changes of small molecule metabolites and the possible metabolic mode of β-alanine supplementation in the biological body, high performance liquid chromatography-time-of-flight mass spectrometry was used to analyze the metabolite changes of Mytilus coruscus after β-alanine supplementation. Using UPLC-MS/MS technology, a total of 18 023 metabolite peaks were obtained, including 9 555 from POS model and 8 468 from NEG model, and 381 and 309 differential metabolites were therefore generated from POS model and NEG model, respectively. From these data, a set of 60 representative metabolites were screened out with Fold Change >1.3 or <0.73, and P < 0.05, after the injection of β-alanine. These significantly different metabolites were identified as carbohydrate metabolites (maltose, lactose, sucrose, glucose and fructose, etc.); amino acids and their derivatives (serine, cysteine, β-alanine, glutamine, alpha-ketoglutaric acid, etc.); lipid metabolites (photosterol, 2-2-arachidonic glycerol, etc.) and other metabolites (montane trachidine, asimidazole). Differential metabolites were then submitted to KEGG database for metabolic pathway enrichment analysis, and a total of 134 (POS mode) and 113 (NEG mode) metabolic pathways were enriched, among which, 60 representative metabolites were enriched to 25 metabolic pathways, including galactose metabolism, amino acid biosynthesis, β-alanine biosynthesis, arginine biosynthesis, glutathione metabolism, fructose and mannose metabolism, carbohydrate digestion and absorption pathways. After β-alanine injection, the free amino acids in the whole tissue of M. corucus showed that the content of 19 amino acids in the experimental group changed compared with the control group. Compared with the control group, the contents of cysteine, carnosine, and serine were significantly decreased after β-alanine injection, which was consistent with the results of the metabolomics analysis. These results revealed that β-alanine supplementation can effectively increase the energy metabolism of M. coruscus and carnosine content. This study provided a foundation for understanding the regulation of β-alanine in mussel metabolism and its mechanism, and also provided new ideas and means for improving the efficiency and nutritional value of mussel culture through β-alanine supplementation.

Abstract:To reveal the age and growth characteristics of Coreius heterodon, we collected 435 specimens from Wuhan stretch, Yangtze River, between July 2017 and December 2018. The result showed that the body length ranged from 152.1 to 325.2 mm with the mean of (233.2±73.4) mm, and that the body weight ranged from 33.1 to 429.8 g, with the mean of (182.40 ±85.4) g. The age ranged from 1 to 5 years and the dominant population with the mean of 75.6% was of individuals of 2 and 3 years. Although there was no significant difference between the sex ratio (male: female) of 1∶1.07 and 1∶1, there was significant difference in different body length groups. The female dominated in large individuals, while the male dominated in small individuals. The marginal increment ratio (MIR) of scales showed that the formation time of age was from July to September. The relationship between body length and body weight was described by the equation: W=7×10^-6L^3.111(R²=0.963) and significant difference was not found between both sexes in the length-weight relationship. The relationship between body length and scale radius was described by the equation: L=52.983R+72.439 (R²=0.678). The Von Bertalanffy growth function was L_t=482.7 {1-exp [-0.22 (t+0.25)]}; W_t=1 573.7 {1-exp[-0.22 (t+0.25)]}^3.11, respectively. The inflection point age of growth was 5.15 years, and the corresponding body length and weight were 335.6 mm, and 501.7 g, respectively. C. heterodon in Wuhan, Yangtze River had a simple age structure with slow growth rate. It is urgent to take necessary measures to promote the spread of its population.

Abstract:In order to explore the molecular basis of the higher growth rate in FFRC No.2 strain common carp(Cyprinus carpio), we utilized comparative transcriptomics analysis using next-generation sequencing and examined transcript profiles of the muscles from high-growth (HG) (average final body weight FBW= 522.3 g, SE=11.03) and slow-growth (SG) (average FBW= 314.9 g, SE=7.16) individuals. Two poly (A)⁺ RNA-seq libraries were constructed and sequenced using paired-end (PE 2×125) Illumina technique. All sequence data were submitted to NCBI SRA database (SRP078896). A total of 286 118 576 reads were generated, resulting in 392 238 assembled contigs, corresponding to 348 144 unique genes and their length ranged from 200 to 32 499 bp. We firstly performed a functional annotation on the assembly using first aligning by BLASTx with cut-off e-value < 1×10^–10, then all assembled contigs were blasted against the Nr database, UniProt-SwissProt database, KEGG database and COG database, respectively. The contigs hit 56.01%-77.71% of the unique proteins of Danio rerio, Fugu ocellatus, Oryzias latipes, Gasterosteusaculeatus, C. carpio and Oreochromis niloticus. And 93 462 contigs containing 129 047 simple sequence repeats (SSRs) were identified. For the assembled contigs which had hits against with Nr database, 112 001 annotated contigs were found to contain the Open Reading Frame (ORF). The ORF length ranged from 27 to 32 490 bp with an average of 554.68 bp. To reveal the underline mechanism of the muscle growth rate in FFRC No. 2 strain C. carpio, we performed a differential expression analysis between the two muscle transcriptomes. Based on the criteria of |log₂FC| ≥ 1 and FDR ≤ 0.05, we identified 749 differentially expressed genes in HG fishes compared with SG fishes, of which 348 genes were up-regulated and 401 genes were down-regulated genes in HG fishes. Several key genes related to muscle growth, i.e. mb,myl2b, tnni1,fhl1, lamb3,pdk2 and igfbp7 were identified. All these DEGs were subjected to GO term enrichment and KEGG pathway analysis to obtain the potential function annotation. These DEGs were enriched in different gene ontologies and pathways. The KEGG pathway analysis results showed that the cardiac muscle contraction pathway and hypertrophic cardiomyopathy (HCM) pathway were found significantly enriched of DEGs, in which tnni1, tnni2,mybpc1, actn2 and tpm2 serve as key regulatory proteins. These findings will facilitate gene function study and help to understand the molecular mechanisms of muscle growth in C. carpio by breeding and improve the molecular selection method of the specific strain with fast growth.

Abstract:Oysters represent a significant molluscan taxon that is widely distributed in world oceans and is the leading molluscan species by quantity produced. By now, oysters have been cultured on all continents, excluding Antarctica. Among all oysters, Crassostrea gigas, also known as the Pacific oyster, is the most economically important in aquaculture around the world. It is native to China, Japan and Korea, but has been introduced to many countries of the world, because of its potential for rapid growth and tolerance of a wide range of environment conditions. The global aquaculture production of C. gigas continues to expand year by year. In China, C. gigas is also one of the dominant species of marine culture. Like many other oysters, diploid C. gigas have an inferior taste and low meat quality during the reproductive seasons (spring and summer) each year, which can be attributed to the sharp decrease of glycogen content during gametogenesis. At the same time, it will also be accompanied by decreased stress resistance and slower growth rate during spawning. Therefore, reproductive control has been an important research field in genetic breeding of oysters. At present, artificial induction of triploid oysters is the main way to control the fecundity of C. gigas. There are two primary methods to produce triploid C. gigas: by inhibiting polar body formation after fertilization through chemical induction and crossing tetraploid oysters with diploid ones. Chemical induction is not reliable in producing a hundred percent triploids, while crossing tetraploids with diploids can reach very close to pure triploids. Furthermore, to avoid chemical toxicity, triploid C. gigas have been mostly produced by crossing tetraploid males and diploid females in recent years. Triploid oysters have advantage in terms of fast growth, delicious meat, and high survival rate during reproductive seasons, due to the poorly developed gonad, which has improved marketability relative to diploid in the reproductive seasons. Triploid C. gigas has entered into commercial farming in many countries, including China and America. Although triploid C. gigas has generally been considered to be sterile, some triploid C. gigas exhibit the same fecundity as diploids. Two types of gametogenic pattern have been reported in triploid C. gigas: fertile and sterile types. Most triploid oysters cannot produce or produce a very small number of gametes, which are referred to as being sterile (3nβ) and as genetically confined. Nevertheless, about 25% of triploid oysters can produce a significant number of mature gametes, referred to as non-sterile or fertile triploids (3nα). It has been reported that gametes of triploid oysters can reach functional maturity and can produce some viable progeny. The existence of triploid oysters with different fertility provides important materials for researchers to carry out studies on the gonadal development of polyploid molluscs. The development of gonad in molluscs is closely related to the changes in the biochemical composition of the edible part. Under the condition of sufficient food, molluscs can regulate the stored glycogen, protein and fat to provide energy for gametogenesis. The gametogenesis of oysters requires stored glycogen to provide energy. Though the contents of biochemical components of triploid C. gigas have been reported in the previous studies, the authors did not specifically distinguish between fertile and infertile triploid oysters. Whether there are differences in the content of biochemical components during gonadal development of triploid oysters with different fertility has not been reported yet. In addition, glycogen content is an important indicator of the high quality of triploid oysters and the main molecular contributor to flavor quality in oysters, and whether it changes with fertility is also an important issue for researchers. Therefore, in the present study, in order to clarify the relationship between gonadal development and the changes of nutritional components in different fertility triploid C. gigas, we analyzed the main nutritional components (glycogen, total protein and total fat content) in the gonadal development of sterile and fertile triploid C. gigas, and compared them with diploid C. gigas. Accor潤湩敮湧琠獴?椠湴?瑥眠潮?摡楲昭晩敮牦敲湡瑲?瑤礠灲敥獦?潥晣?瑡牮楣灥氠潳楰摥??楲????杰楹朠愨獎??椩???呡桬敹?晩楳渠摭楯湤来獬?椠湆?瑵桲敩?灲爠整獲敡湮瑳?獯瑲畭搠祎?湒漠瑳?潥湣汴祲?慭牥整?楲洠灷潡牳琠慵湳瑥?映潴牯?灡牮潡浬潹瑺楥渠杴?瑥栠敯?慴灩灣污楬挠慤瑥楮潳湩?潹映?瑦爠楴灨汥漠楴摲??楳????来楤朠慯獲??楥??楥湣?瑥桤攠?楩湧摨畴猠瑯牦礠??扥甠瑳?慭汰獬潥?瀠牔潨癥椠摣敯?楴浥灮潴爠瑯慦渠瑤?楦湦晥潲牥浮慴琠楳潵湢?晴潡牮?牥敳瀠牨潡摳甠捡琠楬癩敮?捡潲渠瑲牥潬污?楩湯?瑳桨敩?戠牷敩整摨椠湴杨?漠晡??楯????杯楮朠慰獥??楳??f multiple different wavelength points in the near-infrared region. According to this theory, the glycogen content, total protein content and total fat content of each sample was tested. The results showed that the content of glycogen in gonad-visceral mass, adductor muscle and mantle of triploid C. gigas was significantly higher than that of diploid C. gigas in the same period, and the content of total protein in gonad-visceral mass and adductor muscle of triploid C. gigas was significantly lower than that of diploid C. gigas in the same period. During gonadal development, the glycogen content in gonad-visceral mass of fertile triploid C. gigas decreased by 31.88%, that of diploids decreased by 82.41%, and that of sterile triploid C. gigas decreased by 0.55%, which was closely related to the energy supply of glycogen for gametogenesis. In addition, the content of glycogen, total protein and total fat in gonad-visceral mass of sterile triploid C. gigas did not fluctuate significantly in the reproductive seasons, while the trend of nutrient content of fertile triploid C. gigas was similar to that of diploid because of the development of gonad to a certain extent. The results showed that there were significant differences in nutritional components between sterile and fertile triploid C. gigas during the reproductive seasons, and the glycogen quality of sterile triploids was better than that of fertile triploids. It will be interesting to address the questions why there are two different types of gametogenic pattern in triploid C. gigas and what controls the changes of biochemical components in future. To our knowledge, this is the first demonstration of nutritional comp

Abstract:In order to explore the effect of silymarin on relieving low salinity induced stress in the Pacific white shrimp, Litopenaeus vannamei, four experimental diets with silymarin supplementation of 0 (control group), 0.1, 0.2 and 0.4 g/kg were prepared. The effects of silymarin on shrimp growth, immune-related enzymes, microstructure of hepatopancreas and intestinal microbiota were analyzed after juvenile L. vannamei (0.080 ±0.002) g were cultured at low salinity of 3 for 8 weeks. The results showed that: ① Silymarin significantly increased the weight gain rate and decreased the feed coefficient of shrimp at low salinity, but had no significant effects on survival rate, fatness and body composition. ② Silymarin increased the activities of digestive enzymes of shrimp. The activity of amylase in hepatopancreas of 0.4 g/kg group was significantly higher than that of the control group. Dietary 0.2 and 0.4 g/kg silymarin significantly increased the activities of digestive enzymes in intestinal tract of shrimp. ③ Silymarin alleviated the oxidative damage of shrimp caused by low salinity and improved the antioxidant capacity of shrimp. In 0.4 g/kg group, the activities of total superoxide dismutase (T-SOD), catalase (CAT) and reduced glutathione (GSH) in hepatopancreas were significantly increased, but there was no significant change in intestinal antioxidant capacity. The activities of intestinal acid phosphatase (ACP) and alkaline phosphatase (AKP) in 0.2 g/kg group were significantly higher than those in the control group, but the activities of lysozyme and phenoloxidase had no significant change. ④ With the increase of dietary silymarin content, the structure of hepatopancreas of shrimp bacame more complete, the arrangement of hepatic corpuscles was compact, the crosssection of hepatic tubules displayed the shape of regular polygonal star, and contained a large number of secretory cells (B cells) and embryonic cells (E cells). ⑤ The relative abundance of Bacteroides and Gracilibacteria decreased significantly in L. vannamei fed 0.4 g/kg silymarin. The results suggest that silymarin as a feed additive can effectively alleviate the stress caused by low salinity, improve the growth and antioxidant performance in L. vannamei, and have positive effects on the hepatopancreas health and the composition of intestinal microbiota.

Abstract:Grass carp (Ctenopharyngodon idella) is a unique aquaculture species in China, which is mainly distributed in the Yangtze River, Pearl River and Heilongjiang River, and its aquaculture output has long been the highest in freshwater aquaculture in China. With the increase of culture scale and density, the environment continues to deteriorate, resulting in frequent outbreaks of a variety of diseases. Therefore, it is necessary to study its disease resistance mechanism. B lymphocytes are the key cells of adaptive immune response, and can secrete a variety of immunoglobulins which play an important role in the immune response. Respiratory burst is an oxygen-dependent killing mechanism of immune cells when pathogenic microorganisms invade the body. Previous studies suggested that fish B lymphocytes can participate in non-specific immune process through phagocytosis. However, the study of the phagocytic activity of B lymphocytes in C. idella is still not well known. To elucidate the immune function of IgM, real-time fluorescence quantitative PCR (qRT-PCR) was used to detect the expression of IgM in embryonic development, the distribution of IgM in different tissues and the transcription of IgM stimulated by bacteria. The results showed that during embryonic development, the expression of IgM was not significantly changed from cleavage stage to pre-hatching stage, and it significantly increased after hatching stage; IgM was distributed in all tested tissues, with the highest expression in the head kidney, and its expression was significantly up-regulated after the stimulation of different bacteria. B lymphocytes were isolated and purified from the C. idella blood and verified by Giemsa staining, RT-PCR and indirect immunofluorescence. The results of bacterial phagocytosis test showed that B lymphocytes had phagocytic ability to Aeromonas hydrophila. Furthermore, we also found that the production of reactive oxygen species (ROS) and nitric oxide (NO) in grass carp B cells could be significantly up-regulated after being infected by microbes. Finally, the serum conditioning assay showed that the release of ROS from B lymphocytes could be enhanced through serum conditioning, however, there was no influence on NO production. Taken together, the results of this study showed that B lymphocytes of C. idella have the potential of phagocytic ability to bacteria and can directly participate in anti-bacterial immunity by producing non-specific immunity such as respiratory burst.

Abstract:Researches show that elastase (Ela) is involved in regulation of bacterial ulcer caused by Aerononas hydrophila, and plays an important role in control of immune diseases during fish culture. The full-length cDNA sequence of elastase gene was cloned from Acanthopagrus latus based on the reported sequencing data of other vertebrates by using RT-PCR and RACE, which was further analyzed by bioinformatics analysis. The cloned AlEla gene was 946 bp, containing a 807 bp open reading frame which encoded 269 amino acids. AlEla protein was a hydrophilic stable extracellular protein containing a signal peptide with 16 amino acid residues, which might be located in extracellular matrix. Furthermore, AlEla protein has 3 predicted O-glycosylation sites and 15 predicted phosphorylation sites. The theoretical molecular weight of mature protein was 27 509 u and its isoelectric point was 6.05. The analysis of amino acid sequence homology showed that it has high similarity with Sparus aurata, Spondyliosoma cantharus, Pagrus major, Scophthalmus maximus, Paralichthys olivaceus (84.76%-99.26%). They were also in the same branch in phylogenetic tree, indicating that they are the close relatives. Moreover, AlEla has the conserved structures of Elas, including conserved region Tryp-SPc superfamily, catalytic triad of serine proteinase active site, conserved cysteine residues and the sequence of “GDSGGPL” in C-terminal, which revealed that AlEla might show serine proteinase catalytic activity. In the secondary structure of AlEla protein, the proportion of alpha helix, beta turn, extended strand and random coil was 17.84%, 7.06%, 24.54% and 50.56%, respectively. The result of predicted tertiary structure of AlEla indicated that alpha helix and extended strand might play an important role in its conformation structure. In the present study, AlEla gene was successfully cloned from Acanthopagrus latus and we analyzed its bioinformatics, providing theoretical basis for further elucidation of mechanism of regulation of fish immune diseases by AlEla protein.

Abstract:To further explore the mechanism of Aeromonas hydrophila against oxidative stress, the correlations between the expressions of sodA, sodB and KatG in wild-type strain B11 and the gene silencing strains sodA-RNAi, sodB-RNAi and KatG-RNAi under different oxidative stresses were analyzed. Meanwhile, the correlation between the expressions of sodA, sodB and KatG and bacterial growth and survival was also studied. The results indicated that of the three genes (sodA, sodB and KatG), the silencing of any one gene resulted in significantly suppressed expression of the other two genes when they were under no oxidative stress. This indicated that these antioxidant stress genes were closely correlated in expression. Under H₂O₂ stress, silencing of one gene was always coupled with the significant up-regulation of the other two genes. Under MV-induced ROS stress, the expression of sodA andsodB genes in silencing strains was up-regulated, but the expression of KatG in strains sodA-RNAi and sodB-RNAi was down-regulated, indicating that the expression of two SODs genes was essential in resisting MV induced ROS damage. With the increase of H₂O₂ concentration, the survival rate of wild strain B11 remained at a high level, but the survival rate of gene silencing strains sodA-RNAi, sodB-RNAi and KatG-RNAi decreased significantly, and the expression of sodA, sodB and KatG was correlated with the length of the lag phase of A. hydrophila. Under different concentrations of MV stress, the survival rate of wild strain B11 and silencing strain KatG-RNAi remained at a relatively high level, while the survival rate of silencing strains sodA-RNAi and sodB-RNAi decreased significantly. But all the strains still maintained relatively good growth ability. These results suggested that under different oxidative stress conditions, bacteria can resist ROS damage through the synergism of different antioxidant stress genes. Under H₂O₂ stress, KatG is more important for the survival of A. hydrophila. Under MV-induced endogenous ROS stress, the expression of SODs genes contributes more to the survival of A. hydrophila.

Abstract:How to keep the intestinal health of fishes in the intensive and high-density farming system is an important issue in aquaculture. In 1980s, probiotics were first used in fisheries. Application of probiotics has been regarded as an environment-friendly and effective strategy in aquaculture since then. At present, the commonly used probiotic microorganisms include Bacillus spp., Lactobacillus spp., Lactococcus spp. and Saccharomyces. Probiotics maintain the gut health of fish by inhibiting the proliferation of pathogenic microorganisms, improving the integrity of fish intestinal barriers, modulating the composition of intestinal microbiota and regulating the intestinal immune functions. However, more researches still need to be conducted to reveal the exact function of probiotics. Here, the application of probiotics and their possible mechanisms were summarized, and furthermore, the challenges and the future direction of probiotics in aquaculture were also discussed.