Abstract:The hard clam (Meretrix meretrix) is one of the most commercially important cultured shellfish in China. The red shell clam is richer in carotenoids, which makes its shell color red and has higher nutritional value compared with the white shell clam. In order to understand the mutation of scavenger receptor class B type I (SRBI) and its association with the formation of red shell color in M. meretrix, we analyzed the SNPs of Mm-SRBI gene CDS by using direct sequencing among 181 red shell clams and 207 white shell clams. Immunofluorescence (IF) and Western blot (WB) were used to analyze the expression characteristics of Mm-SRBI protein in the mantle of two shell color strains. The results showed that a total of 15 single nucleotide polymorphisms (SNPs) were detected, and there were only two types of mutation in Mm-SRBI: transition and transversion, and the ratio of the two was about 3∶1. Among the 15 loci, 4 loci were significantly associated with shell color. The statistical results indicated that in red shell strain the range of H_o was 0.309 4−0.442 0, H_e 0.468 8−0.494 6, and N_e 1.863 4−1.973 4, while in the white shell strain H_o was 0.231 9−0.328 5, H_e 0.324 4−0.500 0, and N_e 1.639 9−1.995 3. The PIC value evidenced that these loci were all moderately polymorphic (0.5>PIC>0.25). Additionally, all of them were in conserved sequences, in which c.723 A/G site was non-synonymous mutation that led to amino acid changes Ile723Val, and 4 loci were genetically strongly linked (D′>0.75). The results of IF and WB revealed that Mm-SRBI protein was higher expressed in the mantle of red-shelled clams compared to that of the white-shelled clams and was mainly expressed at the outer epithelium of the mantle. Moreover, the quantitative analysis of gray values demonstrated that Mm-SRBI protein expression in red-shell clams was approximately 4.5 times higher than that in white-shell clams. In summary, our results suggested that the mutations of Mm-SRBI and the high expression of its protein may lead to differences in the metabolism of carotenoids in M. meretrix, which may further lead to the formation of red shell color. It also provided a helpful basis to explore the molecular mechanisms of carotenoids metabolism underlying shell coloration and could be potentially applied to marker-assisted selection breeding programs for M. meretrix.

Abstract:Infectious hematopoietic necrosis disease (IHN) is an acute infectious viral disease that can cause sudden death of salmon. Vaccine immunization is the most effective way to prevent and control the disease. At present, there is no commercial vaccine to prevent the disease in China. The objective of the present study was to prepare an inactivated vaccine against IHN and evaluate its protective immunity in Oncorhynchus mykiss. In this study, infectious hematopoietic necrosis viruses (IHNV) were successively cultured on Epithelioma papulosum cyprinid (EPC) cells with different multiplicity of infection (MOI). The optimal proliferation pattern of IHNV on EPC cells was determined by measuring the titer of IHNV in each passage combined with the virus harvest time. IHNV was inactivated by β-propanolactone (BPL) at different final concentrations at 24 °C, and the inactivity was then verified in vitro and in vivo to determine the optimal inactivation condition. Inactivated IHNV prepared with the optimal inactivation protocol was intraperitoneally injected into O. mykiss [(10±2) g)] with different doses, and the protective effect of the inactivated vaccine was analyzed by detecting relative percent survival (RPS) after challenge, expression levels of immune-related factors and serum neutralizing antibody titers at different time post vaccination. It was shown that different proliferation patterns had some effects on the proliferation of IHNV on EPC. We chose MOI of 0.0001 as the best inoculation dose on EPC cells, and the virus was harvested on the 3rd day post inoculation at 15 °C. The in vivo and in vitro safety tests showed that the best inactivation condition was to inactivate IHNV at 24 °C for 24 h with the final concentration of 3.0 mmol/L BPL. 10 μL per fish was chosen as the optimal immunization dose, and more O. mykiss were immunized. The RPS was 91.37%, 84.28%, 84.15% and 47.5% at 7, 21, 45 and 60 d post immunization (d.p.i), respectively, and significant difference was observed on RPS between 60 d.p.i and other time points. Compared with the negative group, the expression levels of Mx-1 and IFN-γ were significantly up-regulated in spleen and head-kidney at 7, 15 and 30 d.p.i, and reached the maximum at 7 d.p.i (5 folds). The expressions of CD4 and IgM genes were significantly up-regulated in spleen and head-kidney at 15 d.p.i. In the detection of neutralizing antibody titer, the average neutralizing antibody titer in O. mykiss serum was 67.25, 43.40 and 29.78 at 30, 45 and 60 d.p.i respectively, with a decreasing trend and significant differences among different groups. The results indicated that the IHN-BPL inactivated vaccine developed in this study could induce specific and non-specific immune response in O. mykiss, and could provide significant immunoprotection, which will provide references for the development of inactivated vaccines against IHN.

Abstract:Cobia(Rachycentron canadum) is a highly prized recreational species worldwide as well as a promising candidate for aquaculture because of its rapid growth rate, strong disease resistance and high quality flesh. Studying the morphometrics and physiology of embryos, larvae and juveniles to obtain information about their early life history is the first step and key to the successful aquaculture of a fish species. In order to find out the characteristics and rules of the early development stages of cobia, the morphological characteristics and developmental characteristics of embryo, larvae and juvenile were studied by microscopic observation. The fertilized eggs obtained by artificial spawning were spherically shaped and buoyant, there was an oil globule in the centre, with an egg diameter of (1.245±0.065) mm and an oil globule diameter of (0.325±0.027) mm. The fertilized eggs hatched 26 h 30 min after fertilization in the sea water at (27.0±0.5)℃, salinity of 29 and pH of 8.3. The embryonic development process was divided into 7 stages, including fertilized egg stage, the cleavage stage, the blastocyst stage, the gastrula stage, the neurula stage, the organogenesis stage and the hatching stage (totally 24 developing periods). The total length (TL) of newly hatched larvae was (3.254±0.096) mm. The larvae opened their mouths and exhibited blackened eyes at 3 days post hatching (dph) with a TL of (4.453±0.267) mm, meanwhile, the yolk-sac decreased in volume by approximately 80%. The yolk-sac and oil globule were completely depleted at 5 dph (TL of 6.007 mm±0.171 mm) and 7 dph (TL of 8.173 mm±0.317 mm) respectively, and the larvae completely entered the exogenous nutritional stage. The intestine of the newly hatched larvae was thin, short, and straight, and the intestinal physiological curve formed at 9 dph when the larvae were (10.053±0.594) mm in TL. The larvae started to develop into juveniles at 14 dph (TL of 19.933 mm±1.118 mm) when the development of the dorsal fin, pectoral fin, anal fin and caudal fin was completed. At 22 dph, the juvenile had a TL of (41.140±3.779) mm, some scales on the caudal peduncle formed. The 46 dph juvenile reached (116.667±5.916) mm in TL, the whole body surface was covered with cycloid scales and their general appearance was similar to that of the adults, except for the shape of caudal fin. These results indicated that the fertilized egg diameter and newly hatched larvae size of cobia were both larger, and its early developmental characteristics had certain adaptability to the ecological behaviors. The present study could provide basic knowledge for investigating biology and artifical propagation of cobia.

Abstract:Pathogenic bacteria in the water environment are mainly monitored in the public health, food safety, aquaculture and other industries due to their major threats to the health of humans and aquatic animals, and the biosafety of aquatic products. However, pathogenic database involved in water environment pathogen is mainly constructed according to independent disciplines, and scattered in the fields of clinical medicine and aquatic animal diseases, which can no longer meet the high-throughput identification and biosafety evaluation of pathogenic bacteria involved in the water environment in the regional scale or ecological perspective. In this study, a database of pathogenic bacteria involved in water environment (DPiWE) was constructed by collecting the taxonomic information of pathogenic bacteria from humans, aquatic animals, mammals, plants, and cross-host comorbidities. A multi-threaded schedulable communication model and a multi-task mode global sequence matching algorithm were developed to construct DPiWE. The database collected 9 070 pathogenic bacteria strains, which belong to 14 phyla, 27 classes, 54 orders, 116 families, 221 genera and 1 097 species. The corresponding 16S rRNA gene sequences, host information and infection types of these strains were also collected in DPiWE. This database was deployed at a website (http://dayuz.com/) with the functions including web user management, pathogenic information retrieval, sequence upload, storage and alignment, and visualization of annotation result. Two examples were used to test the functions of DPiWE. The first example showed that, DPiWE can accurately construct a phylogenetic network of an unidentified bacterium (strain DS10?D19) isolated from cultural seawaters, according to its 16S rRNA gene sequence, and identified it as Photobacterium leiognathid. The result of network also showed that the network structure of strain DS10?D19 was similar to P. leiognathid and P. angustum. The second example showed that the compositions of pathogens in the intestines of three mariculture animals were significantly different through annotating the high-throughput sequencing data using DPiWE, and the rearing water in diseased groups had potential risk of spreading the comorbid pathogenic bacteria of human and fish. The DPiWE and its supporting data analysis process can provide new ideas and data foundations for high-throughput detection of the biosafety of water environment, protecting health of fishery ecology, and controlling diseases of aquatic animals, in the future.

Abstract:Litopenaeus vannamei is one of the most important breeding economic varieties in the world because of its fast growth, strong environmental adaptability, and short growth cycle. Along with the rapid promotion of the intensive culture model of L. vannamei, large quantities of harmful substances accumulate in the culture environment which seriously affect the growth and health of L. vannamei. Nitrite enters through gills and accumulates in tissues, which has severely toxic effects on cultured L. vannamei. In the present study, to explore the accumulation of nitrite and changes in enzymes activities related to energy metabolism exposed to nitrite in L. vannamei [body length (6.8 ± 0.3) cm, weight (4.0 ± 0.6 g)], shrimps were exposed to four nitrite concentrations of 0 (control), 0.8 (11.2 mg/L NO₂-N), 4.0 (56 mg/L NO₂-N) and 8.0 mmol/L (112 mg/L NO₂-N) for 96 h and then recovered for 12 h. Each experimental treatment included six replicates. Three replicates were used for sample collection, and the other three replicates were used to calculate the cumulative mortality of L. vannamei. At 0, 6, 12, 24, 48 and 96 h of the nitrite stress, the hemolymph, hepatopancreas, muscle, gill and intestine of 9 L. vannamei in each group were randomly selected for the determination of the index. Additionally, death number were recorded every 12 h. The results indicated that the shrimp mortality rate increased with nitrite concentrations. At the end of nitrite stress, shrimp mortality in control group, 0.8 mmol/L group, 4.0 mmol/L group and 8.0 mmol/L group were 10.7%, 32.0%, 42.7% and 52.0%, respectively. Within 6 hours of exposure, nitrite accumulated significantly in the gill, hemolymph, intestine, hepatopancreas, and muscle tissues of L. vannamei, and was positively correlated with stress concentrations. The maximum accumulation of nitrite in gills, hemolymph, hepatopancreas, intestine and muscle were 50.1 mg/kg, 43.2 mg/L, 20.7 mg/kg, 33.5 mg/kg and 14.9 mg/kg. In the same stress concentration group, nitrite accumulated the most in gill and the least in muscle, and the accumulation in gill was about 3 times greater than that in muscle. The activity of Na⁺-K⁺-ATPase in hepatopancreas and muscle of L. vannamei were significantly increased at 0.8 and 4.0 mmol/L, but significantly decreased in muscle of 8.0 mmol/L during the exposure period. The activity of AMPK in the hepatopancreas in the stress groups increased compared to the control group, and showed a positive correlation with the stress concentration. During the recovery period, except for hemolymph (8.0 mmol/L group), 1-hour recovery rate of nitrite in L. vannamei tissues in the stress groups were above 50%, and the hepatopancreas and gill showed the highest recovery efficiency exceeding 74%. The recovery time of hemolymph, gill and intestine were the shortest within 6 hours. In addition, the content of nitrite in water increased significantly. This study indicates that nitrite can be accumulated to shrimp tissues in a short time and can accelerate the process of energy metabolism. Nitrite would excrete from the body rapidly during recovery in order to reduce the toxic effects. The results of this study will provide reference for alleviating the toxic effects of nitrite on cultured shrimp.

Abstract:The primary source of ALDFG is from the coastal and oceanic fishery and the maximum risk to produce ALDFG is from the coastal fishing industry. Gillnetters along the coast of China accounted for about 56% of the total number of fishing boats along the coast of China in 2018, and were one of the biggest potential source to produce ALDFG in China. Lost netting of gill net can keep the high rate of ghost fishing for more than 2 years. ALDFG produced by the gill net fishery along the coast of China will cause great influence to the coastal marine ecosystems in China. In order to understand the factors and their impact degree on the netting loss rate of gillnet in waters near China, a questionnaire survey was conducted among the Chinese coastal fishermen from June to December, 2019 and the areas covered Liaoning, Shandong, Jiangsu, Zhejiang and Guangdong provinces. The data from 41 gillnetters were collected including 13 variables, i.e. gross tonnage, power, vessel length, vessel age, captain age, annual fishing days, average voyage days, voyage gear carrying weight, net average weight, annual procurement number, annual gear procurement weight, fishing depth and the lost numbers of gillnet netting. Based on the questionnaire survey data, software SPSS 23 was used to analyze the multicollinearity among 12 independent variables, and eliminate gradually 3 variables that had multicollinearity with other independent variables. The remaining 9 independent variables that had no multicollinearity among them were used to analyze the relationship with the netting loss rate of gillnet in waters near China by GAM and to determine the best GAM based on the AIC value. The results indicate that, (1) The factors (explanation deviation) that effect the netting loss rate of gillnet (R_G) in waters near China are as follows: average voyage days (86.8%), annual fishing days (4.2%), voyage carrying netting weight (2.8%), fishing depth (2.4%), gross tonnage (0.9%) and annual gear procurement number (0.1%); (2) The GAM can be used to study the relationship between the netting loss rate of gillnetters and the fishing boat parameters, annual fishing gear procurement information and fishing ground information; (3) With the increase of voyage days and annual fishing days, R_G shows an increasing trend; (4) With the increase of fishing depth and the voyage carrying netting weight exceeding 18000 kg, R_G shows an decreasing trend. The measures recommended to reduce the netting loss rate of gillnetters are as follows: (1) The voyage days should not exceed 30 days and reduce the annual fishing days; (2) According to the gross tonnage of fishing boat, the voyage carrying netting weight should be more than 18000 kg; (3) The fishing depth and fishing area should be regulated according to the height of gillnet gear. We should specify the maximum height of gillnet gear according to the current and seabed topography of the fishing ground in waters near China; (4) The gross tonnage of fishing boat should be smaller than 200 gross tons.

Abstract:Hyriopsis cumingii is a unique freshwater breeding pearl mussel in our country. It has the reproductive biology characteristics that the fertilized eggs develop to the larvae in gills. The unique reproductive biology characteristics make the offspring produced by the mussels random in the process of artificial breeding and restrict the application of modern biological breeding techniques to mussels such as triploid induction, gynogenesis and gene editing. In this study, in order to do research on development of H. cumingii embryos and explore the in vitro culture of its early embryos, male and female gametes obtained by anatomy were used as materials for in vitro fertilization and in vitro culture with isoosmotic balance salt solution (BSS) suitable for freshwater mussels. And the morphological characteristics of each stage of embryo were observed with the required time for development recorded. Meanwhile, the embryos in its nurturing pouch of outer gill were continuously observed and the water temperatures were recorded day and night at the same time, then we calculated the embryonic developmental biological zero point and examined the reliability of the biological zero point. After that, effective accumulated temperature of each embryonic development stage was calculated according to the biological zero point confirmed. Results show that when the water temperature is (25 ± 1) °C, the anatomy of the sperms and eggs were mixed after diluting by BSS saline and fertilized eggs could develop to morula stage. The fertilized eggs developed to polar body emissions at 1.8 h, developed to 2-cell stage at 2.8 h, developed to 4-cell stage at 5.6 h, developed to 8-cell stage at 10.5 h, developed to 16-cell stage at 13.9 h, and developed to morula stage at 17.3 h after observing under optical microscope. And then embryos did not change, and some of embryos were deformed. The morphological characteristics of embryos at each stage under in vitro culture condition were similar to those in the gills. Meanwhile, when the average water temperature in the pond was 26.54 °C, 28.08 °C and 29.51 °C, it took respectively 10 d, 9 d and 8 d for embryos to develop into mature glochidium. And the biological zero point for embryonic development of H. cumingii was 14.81 °C which was confirmed reliable after examining. The effective accumulated temperature were 12.95 °C×d for embryos developing to cleavage stage, 25.99 °C×d for embryos developing to blastocyst stage, 42.27 °C×d for embryos developing to gastrulation stage, 69.21 °C×d for embryos developing to glochidium stage and 118.14 °C×d for embryos developing to cleavage stage mature mlochidium stage, respectively, according to the biological zero point calculated previously. The study tried to cultivate H. cumingii embryos under in vitro condition, which confirmed the feasibility to cultivate H. cumingii embryos under in vitro condition, and then we analyzed and calculated the biological zero point and effective accumulated temperature of early embryonic development. The results of this study can provide reference for the researches on artificial breeding and development of modern biological genetic breeding technology of H. cumingii.

Abstract:As a common machine learning method, BP neural network model is widely used in species distribution models to analyze the relationship between biological distribution and environmental factors. Compared with the traditional regression models, this model can flexibly deal with the nonlinear relationship between variables. However, there are substantial uncertainties in parameter setting as a result of its complex structure, which may affect the prediction and application of this model. This study considered approaches to optimize the model parameters, including the group method of data handling, genetic algorithm and adaptive algorithm, to improve initial weights and the number of hidden nodes of the model, respectively. Seven combinations of optimized BP models were implemented based on the survey data obtained from fishery resources and environment in Shandong offshore between 2016 and 2017. Our results showed that there were significant differences in the predictive performance of the seven optimization models. The predictive performance of the one-way and two-way optimization models was approximately the same. The root mean square error and the square of residual error were 0.35 and 1.94 respectively, which were smaller than the initial model's 0.52 and 2.40, and the maximum correlation coefficient was 0.45, indicating that the optimization effect of the model was the best. After the comparison and optimization, it was found that the resource density of Oratosquilla oratoria was basically different with the increase of bottom salinity while the resource density of O. oratoria was significantly different with the increase of bottom temperature. In addition, the increase of water depth in the optimal model compared with the initial model was a key environmental factor,which had an important effect on the resource density of O. oratoria. In this study, the parameter optimization method of the BP neural network model was further developed, which proved that the parameter optimization had important effect on the prediction performance of the BP model, and the model optimization was of great significance for the analysis of the relationship between resource density and environmental factors.

Abstract:The production of aquatic products is always increasing worldwide, due to their high nutritional value. However, a large number of by-products are produced during the consumption and processing of aquatic products, causing serious resources waste and environmental pollution. On this basis, the by-products of aquatic products were developed and applied in many fields according to their different characteristics. Among them, the by-product proteins have been widely recycled and utilized in practice due to its unique functions and high added value. Therefore, this article reviewed the typically physical, chemical and biological methods for recycling the proteins from aquatic products. Meanwhile, we also introduced the research progress in the high-value utilization of by-product proteins from fish, crustaceans and shellfish, as well as their future development trend, with the purpose of further enhancing the comprehensive utilization of aquatic by-products and promoting the high-quality development of aquatic product industry. Generally, the aquatic products from fishing or aquaculture often needed to be processed before their marketing. However, massive by-products were left after being processed, and they must undergo further treatment to obtain end-products with high added value. Among these by-products, proteins accounted for a large proportion and had higher nutritional value. The efficiency of traditional technology to recycle by-products of different aquatic products was limited, and the yield was very low, therefore, it was difficult to potently recycle a large number of by-product proteins from aquatic products. In the past century, the scientists in academic and industrial community made great efforts to fully recycle the by-product proteins of aquatic products, and achieved many end-products through physical, chemical and biological methods. With the advance and innovation of science and technology, the recycling conditions of by-product proteins from aquatic products have been greatly optimized, and the recyclable types of by-products have greatly expanded, as well as the proteins yield has been significantly improved. Therefore, the research hotspots of by-product proteins from aquatic products mainly focused on two aspects: innovative recycling methods and enhancing the added value of utilization. With the continuous development of molecular biology and polymer science, the researchers could modify the by-product proteins from aquatic products through physical, chemical and biological methods, which could not only improve and enhance their biological properties, but also broaden the applications of related products range. China's aquaculture output has ranked first in the world for 28 consecutive years. However, the research on the high-value utilization and industrial application of aquatic by-products needed to be further improved. Moreover, from the perspective of industrial development, whether it was the introduction and development of by-product development technology, or the formulation and management of related product standards, China is facing many practical problems and challenges. Consequently, it is necessary to focus on the current situation and development trend, carry out the next step of planning, which will introduce new vitality to the aquatic product processing industry. In the future, the commercial development of by-product proteins from aquatic products will bring more possibilities and development prospects. More and more foods and drugs prepared using by-product proteins will be approved and promoted, which will provide a driving force for the further development of aquatic products industry.

Abstract:The acquisition of energy and nutrients is the basis of sustaining the life activities of hermatypic corals, which mainly depends on the alimentation mode. Hermatypic corals are mixotrophic organisms, both autotrophic and heterotrophic. Photosynthesis by zooxanthellae is the basic form of autotrophy, but little is known about the heterotrophic feeding. To know more about it, three representative species in the Sanya Luhuitou Bay, Galaxea fascicularis, Pocillopora damicornis and Acropora muricata were selected in this study to test their feeding habits. Their feeding reaction to Artemia salina, copepod used in aquarium, yeast extract liquor and aquatic coral food were observed. What’s more, G. fascicularis’s feeding rate and digestion of A. salina were probed into. G. fascicularis ingests four kinds of food mentioned above, while A. muricata ingests none of them and P. damicornis only ingests A. salina and aquatic coral food. It’s reported for the first time that P. damicornis ingests A. salina by mesenterial filaments and it ingests aquatic coral food through the cooperation between polyps. Moreover, G. fascicularis’s feeding rate of A. salina and the density of A. salina nauplii well fitted into the Michaelis-Menten model. A. salina was completely digested by G. fascicularis in 3 h. The research can deepen our knowledge about the feeding of hermatypic corals, especially in the process of artificial coral breeding.

Abstract:The data obtained by tag-recapture method are commonly used to evaluate the fish population dynamics and the effect of stock enhancement. However, to ensure the accuracy of the recapture data, it is necessary to choose the appropriate fish size for tagging. Acanthopagrus latus was employed to conduct two indoor experiments. In the first experiment, the effects of T-bar anchor tags on growth, survival and tag retention of A. latus from four size groups (average body length: 5 cm, 7 cm, 11 cm, and 14 cm, respectively) were examined and the size-dependent effects on survival and tag retention were tested. In the second experiment, different mix ratios of tagged fish to untagged fish from two size groups (average body length: 7 cm and 14 cm, respectively) were set for simulated catching and the catching results were resampled. The differences in catching results between groups were compared, the differences between catching result and resampling result were also compared. The results show that: ① there was no significant difference in the specific growth rate between 7 cm, 11 cm and 14 cm groups. ② the survival rate was size-dependent. All the tagged fish of 5 cm group died in the first week after tagging. The survival rate of other size groups was 77.5%, 92.5% and 100.0%, respectively. The relational expression of logistic regression between body length and the probability of survival was: P=exp(0.099X−6.900)/[1+exp(0.099X−6.900)]. ③ the tag retention rate was high (97.5% in 7 cm group, 100.0% in 11 cm and 14 cm group) but seemed to be unrelated with fish size. ④ there was no significant effect of fish size on the catching result and the resampling result but significant effect of mix ratio of tagged to untagged fish on it. In summary, it is recommended that study objectives and cost should be considered when choosing the appropriate fish size for tagging stocking fishes like A. latus. If the probability of survival needs to be >50.0%, the tagged individuals with body length should be >7 cm. If >75.0%, the body length should be >8 cm. If >95.0%, the body length should be >10 cm. And it is worth further study to know about the appropriate proportion of tagged fish for tagging and releasing.

Abstract:The aim of this study was to investigate the effects of kelp enzymatic extract on the growth and hepatopancreas transcriptome of Ictalurus punctatus. We prepared different diets with enzymatic extract of kelp addition amount of 0 g/kg (S), 0.3 g/kg (KP3), 0.5 g/kg (KP5), 1.0 g/kg (KP10), 1.5 g/kg (KP15), 2.0 g/kg (KP20) to feed channel catfish in cage-culture for 60 days. Channel catfish [initial body weight (51.18±1.14)g] as exprimental object were randomly divided into 6 groups with 3 replicates per group and 40 individuals per replicate. The results showed that: ① Compared with the control group S, the addition of kelp enzymatic extract in diet had no significant effect on the suvival rate(SR) of channel catfish, the specific growth rate(SGR) increased by 1.20% to 5.39%, and the feed coefficient rate(FCR) decreased by −0.83% to 9.09%. A quadratic polynomial regression analysis was carried out with SGR and FCR as the targets respectively, and the optimal addition amount of kelp enzymatic hydrolysis extract in the feed was 0.98 and 0.96 g/kg. The condition factor(CF) and viscero-somatic index(VSI) decreased significantly in groups KP10 to KP20, and the hepatosomatic index(HSI) decreased significantly in group KP10. The moisture, crude fat, crude protein and ash of fish had no significantly difference in all groups. ② Height of duplicature in group KP3, height and width of duplicature and thickness of muscularis in group KP5, thickness of muscularis in group KP10 were all significantly higher than those in group S. ③ Total RNA was extracted from S and KP10 groups’ hepatopancreas. Transcriptome sequencing results showed that there were 81 significantly down-regulated genes and 199 up-regulated genes. GO functional classification analysis results indicated that differentially expressed genes(DEGs) were annotated to items such as DNA transcription, metal ion binding, membrane and membrane composition. According to the results of KEGG pathway analysis, we found that DEGs were mainly enriched in 12 related pathways including cell proliferation and differentiation, hormone regulation, lipid metabolism, carbohydrate metabolism, growth factor metabolism and so on. Our studies have indicated that adding kelp enzymatic extract to diet can promote the growth of I. punctatus, reduce feed coefficient, and have positive effects on the intestinal morphology and the glucose and lipid metabolic capability of liver and pancreas. The suitable addition amount was 0.96-0.98 g/kg.

Abstract:A variety of bacterial or viral diseases usually cause massive hemolysis in fish, and release a large amount of cytotoxic hemoglobin into the tissues. However, the damage mechanism of hemolysis to fish tissues is still not well known. Therefore, to explore the damage mechanism of hemolysis in fish to the tissues, Ctenopharyngodon idella was used to systematically study the damage mechanism of hemolysis to the liver. Firstly, the hemoglobin was injected into the C. idella, hematoxylin eosin (H.E) staining assay results showed that injection of hemoglobin caused the dead cells to increase obviously in liver, and Prussian blue staining revealed that more iron deposited in liver. Quantitative real-time PCR (qRT-PCR) detection results showed that the injection of hemoglobin activated the expression of iron metabolism related genes. In order to further explore the damage of hemolysis to the liver, we injected phenylhydrazine into C. idella, H.E and Prussian blue staining results showed that the hemolysis caused the liver cell necrosis and iron deposits to increase, and the content of hemoglobin and iron in the liver all significantly increased with time. Furthermore, the increase of iron also activated the expression of iron metabolism related genes. Then, we detected the expression of inflammatory cytokines in liver after the injection of phenylhydrazine, and qRT-PCR results showed that high dose of hemoglobin activated the expression of various cytokines, such as pro-inflammatory cytokines TNF-α, IL-1β and IL-6, anti-inflammatory cytokines IL-10, and chemokines IL-4 and IL-8. In order to further explore the oxidation damage of hemoglobin in liver, we tested the content of malondialdehyde (MDA) and lipid peroxide (LPO) and β - galactose glucoside enzyme in liver, and the results showed that the hemolysis significantly increased liver oxidative damage effect, at the same time, the qRT-PCR and enzyme activity test results revealed that the oxidative damage of hemolysis induced the occurrence of liver cell apoptosis. Finally, we also examined the expression of antioxidant enzymes in liver, and the results showed that in vivo hemolysis significantly activated the antioxidant system in liver. In conclusion, the present study revealed that massive hemorrhage in fish released the high oxidative activity of hemoglobin which activated the occurrence of inflammation, oxidative damage and apoptosis in liver, and simultaneously up-regulated the expression of antioxidant system. The results of this study will enrich the blood immunology of fish, and also provide theoretical reference for the healthy culture of C. idella.

Abstract:In order to explore the regulatory effect of microRNA (miRNA) on Macrobrachium nipponense chitinase 3A(MnCHT3A) gene, bioinformatics approach was firstly used to predict and screen the miRNA--miR-305-5p bound specifically to MnCHT3A. Using qRT-PCR, biochemical and histological methods, the regulation of miR-305-5p on target gene MnCHT3A was studied in vivo. The results showed that the expression change of miR-305-5p was negatively correlated with MnCHT3A during the molting cycle of M. nipponense. The level of miR-305-5p peaked at stage C and was the lowest at stage A, while the expression trend of MnCHT3A mRNA was opposite. After injection of miR-305-5p mimics or miR-305-5p inhibitor, the transcription level of MnCHT3A was decreased by 60% or increased by 166%, and the activity of MnCHT enzyme, meanwhile, was decreased by 39.53% or increased by 133%, respectively compared with the control group. Histological results showed that a three-layer cuticle structure of carapace in stage C was observed by means of H-E staining, namely, epicuticle, exocuticle and endocuticle from outside to inside. The images of chitin fluorescence staining showed the presence of chitin in the exocuticle and endocuticle. Results from scanning electron microscopy clearly showed the lamellar structure of the exocuticle and endocuticle. Compared with the control group, a thickening trend in the endocuticle with the lamellae as well as the blue fluorescence chitin stripe was observed in miR-305-5p mimics group. But in miR-305-5p inhibitor group, the culticular structure was disordered. Correspondingly, the blue fluorescence chitin stripe was not uniform and weakened in some areas. The results obtained above indicate that the target gene of miR-305-5p is MnCHT3A, and miR-305-5p can specifically inhibit the transcription of MnCHT3A in vivo.

Abstract:Aeromonas hydrophila is known as a common pathogen of human, animal and fish worldwide, which is of great significance to aquaculture and public health. Currently, antibiotics as a kind of high effective and widely used medicine are used in the prevention, control and treatment of A. hydrophlia infections. However, abuse of antibiotics caused the problem of drug-resistant strains. New sustainable drugs to control bacterial infection needed to be studied. In recent years, the active ingredients extracted from traditional Chinese herbal medicine have excellent antibacterial effects in vitro, not easy to develop drug resistance and being able to reverse the properties of bacterial resistance. And these characteristics merit attention. The objective of this study is to analyze the potential inhibitory mechanism of oridonin against A. hydrophila, to provide a theoretical basis for the control of drug-resistant A. hydrophila infection using oridonin as an alternative drug. The minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and the effect of growth curve were measured in this study. Morphological changes in A. hydrophila following oridonin treatment were determined with scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The effects of lactate dehydrogenase (LDH) and soluble proteins were detected by lactic dehydrogenase kits and polyacrylamide gel electrophoresis (SDS-PAGE). Electrical conductivity was analysed to study oridonin effects on cell membrane. Effect of oridonin on A. hydrophila cell membrane was measured based on electrical conductivity. Oridonin had definite inhibitory activity on A. hydrophila, and the MIC and MBC values of oridonin were 256 μg/mL and 512 μg/mL, respectively. After treating A. hydrophila (CW) with 2 MIC oridonin, SEM images showed the surface roughness and collapse of the bacteria, and TEM images showed that the structure of the bacteria was damaged, the cell membrane and cell wall were separated, and the cytoplasm showed vacuolation, while the control group showed no significant changes. 6 h later, the conductivity level of oridonin-treated A. hydrophila CW reached by 5.66% (P<0.05), indicating that oridonin changed cell membrane and wall permeability of CW strain. The results of content of LDH showed that oridonin reduced the formation of LDH by 20.8% (P<0.05). SDS-PAGE results show that the soluble protein content was lower compared with the control group, indicating that oridonin inhibited the protein metabolism of bacteria. It was shown that density and intensity of fluorescence decreased and DNA exosmosis level improved by (29.32±1.02) mg/L(P<0.01). Thus, the decrease of DNA content caused by oridonin affected the cell membrane and cell wall. The results demonstrated that oridonin could inhibit the growth of A. hydrophila significantly by increasing the permeability of cell membrane and affecting the metabolism of protein.