PAPERS

• Evolution, target genes certification and expression analysis of microRNA-137 in Japanese ornamental carp (Cyprinus carpio var. koi)

  LI Junru, MA Xiao, WU Limin, LIU Huifen, SHI Xi, SONG Hongmei, TIAN Xue, LI Xuejun

  2021,45(6):831-845, DOI: 10.11964/jfc.20201012438

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  MicroRNAs (miRNAs) play an crucial role in post-transcriptional regulation of most transcripts in almost all animal species. Researchers have discovered that many miRNAs and a subset of their targets are conserved across deeply diverged phyla. miR-137 has been identified to function in melanogenesis in mammals. Considering the relation of miR-137 with melanin synthesis and conservation in species evolution, it might exert efforts in fish pigment synthesis. However, the function of miR-137 in fish color formation is still unclear. The Japanese ornamental carp (C. carpio var. koi) is famous for various colors and widely cultured all over the world. Due to its fast establishment of various colors under cultivation, C. carpio var. koi is a distinctive specie for genetics research in pigmentation. In order to explore the role of miR-137 in the color formation of C. carpio var. koi, the precursor sequences of miR-137 in 14 fish species were aligned and phylogenetic tree was constru-cted by Maximum Likelihood Estimate. Besides, the expression levels of miR-137 in different developmental stages and tissues also detected. Then, the target genes of miR-137 were predicted and analyzed by GO and KEGG analysis. In addtion, the luciferase reporter was used to verify the availability of miR-137 with its targets, and the expression levels of miR-137 and target genes in different development stages and tissues were checked and the correlation analysis between them was also conducted. The results showed that the precursor sequence similarity of miR-137 between C. carpio var. koi and C. carpio was the highest, and miR-137 sequences exhibited high conservation among all fish species. A great number of predicted target genes were enriched in melanogenesis, pigment cell differentiation, pteridine-containing compound metabolic process and so on. Real time PCR results showed the expression of miR-137-3p Feached the peak on 11 dph and rapidly dicreased in the following stages. miR-137 was expressed in all kinds of tissues, especially significantly in eye and muscle. Besides, the skin and fins, pigment cell enriched tissues, also had a higher expression and the expression of miR-137 in white tissues was higher than the red. The luciferase reporter analysis certified that miR-137 bound with the mitfa 3′-UTR to inhibit its expression but did not present significant inhibitory effect on sprb, and miR-137 was negatively correlated with the expression of mitfa and sprb in different developmental stages, but not in tissues. All the above results confirmed miR-137 was highly conserved during fish species evolution and displayed correlation with the koi color formation, in which miR-137 might regulate mitfa involved in color formation. This study provides basic research data for further exploring the role of miR-137 in C. carpio var. koi color formation.

• Cloning and expression analysis of autophagy genes ATG13 and ATG101 in Macrobrachium nipponense under hypoxic stress

  SUN Shengming, CHEN Yinxiang, ZHENG Cheng, ZHAO Qianqian, XUE Cheng

  2021,45(6):846-861, DOI: 10.11964/jfc.20200612316

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  In order to study the regulation of autophagy-related gene 13 (ATG13) and ATG101 in Macrobrachium nipponense under hypoxia stress, the full-length cDNA sequences of ATG13 and ATG101 of M. nipponense were cloned for the first time by RACE PCR through cloning, and the cell autophagy genes ATG13 cDNA had 2 043 bp (NCBI ID MT084347), including 211 bp 5′ untranslated regions (UTR), 449 bp 3′ UTR and 1 383 bp open reading frame (ORF), which encodes 460 amino acids; ATG101 cDNA is 1 051 bp long (NCBI ID MT084348), including the 5′ terminal non-translation region of 18 bp, the 3′ UTR of 373 bp and the open reading frame of 660 bp. The open reading frame encodes 219 amino acids. Based on bioinformatics analysis, the amino acid similarity ratio showed that the autophagy gene ATG13 of the biogas shrimp was rich in highly conserved LC3 functional domain (LIR). Phylogenetic tree analysis showed that the ATG13 gene of M. nipponense was closely related to the autophagy related genes of Litopenaeus vannamei. The results showed that ATG13 and ATG101 were highly expressed in hepatopancreas and brain tissues, but low in muscles. The difference of ATG13 and ATG101 expression in hepatopancreas tissues under hypoxia stress was tracked by real-time fluorescence quantitative PCR (qRT-PCR). The expression of ATG13 and ATG101 in the experimental group was significantly higher than that in the control group at 6 h and 12 h, but there was no significant difference between the experimental group and the control group at 12 h after the recovery of normoxia, which were similar to M. nipponense ATG13 and ATG101 protein expression abundance using Western blot. The results of ultrastructure observation by transmission electron microscope (TEM) showed that autophagic vacuoles began to appear in lysosomes of hepatopancreas after 6 h and 24 h of hypoxia, suggesting that acute hypoxic stress could induce the formation of autophagosomes. The results of this study may provide a theoretical reference for understanding the regulatory mechanism of M. nipponense in response to hypoxic stress.

• Proteomics of liver tissue of large yellow croaker (Larimichthys crocea) under high temperature stress

  ZHANG Xiaoying, CAI Xiaofang, LIU Xiande, ZHAO Hongbo, SONG Puqing, ZHANG Jing

  2021,45(6):862-870, DOI: 10.11964/jfc.20200812374

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  In order to explore the changes in protein expression of Larimichthys crocea under high temperature stress, tandem mass tag (TMT) combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to perform proteomic analysis on the liver tissues between the high temperature tolerant group and the high temperature sensitive group of L. crocea. A total of 3 369 proteins were identified, of which 687 were differentially expressed proteins, including 281 up-regulated proteins and 406 down-regulated proteins. 13 randomly selected proteins were validated with parallel reaction monitoring (PRM) assays, which showed that they were consistent with the TMT-labeled proteomic analysis results. Subsequently, through bioinformatics analysis, it was found that high temperature stress had a significant impact on the protein folding and energy metabolism of L. crocea. Among them, heat shock protein 70(HSP70), calreticulin(CRT), and glucose-regulated protein(GRP) are involved in regulating the correct folding of proteins. Pyruvate kinase(PK) is involved in energy supply under high temperature conditions. It is speculated that HSP70, CRT, GRP and PK play an important role in the response of L. crocea to high temperature stress. This study is helpful to further analyze the high temperature adaptation mechanism of L. crocea, and provides reference for breeding high temperature resistant varieties of L. crocea.

• Characteristics of submarine water temperature distribution of Trichiurus haumela in the East China Sea and Southern Yellow Sea with the improvement of the analysis methods

  LIU Yong, CHENG Jiahua, JIA Shiguang

  2021,45(6):871-886, DOI: 10.11964/jfc.20200312208

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  The study of the relationship between fishery organisms and water temperature is an important part of fishery scientific research, and the analysis of the suitable water temperature range of fish is an important aspect of this content. In this study, based on the distribution data of Trichiurus haumela resources obtained from a large-scale survey in four quarters from 2014 to 2015, the water temperature range analysis method was improved on the basis of previous studies to avoid some subjective factors in the analysis. A new biological subject temperature range analysis method was created and compared with the traditional frequency method and the previous biological subject method. The results show that the proportion of sites covered by the frequency method is relatively good, but the biomass coverage rate is relatively poor; in the previous biological main body method, the cumulative biomass coverage rate was the highest, and the site coverage rate was also relatively good. The disadvantage is that there are many subjective factors added in the operation process, and the distribution range is discontinuous and contains individual narrow range. In the new method, because its temperature range is relatively concentrated and short, the coverage of the site and biomass is not very high, but the biomass density in the determined temperature range is the highest, and the minimum range which can accommodate the most biomass is found and the distribution range is continuously concentrated. The characteristics of the biological spatial distribution will directly affect the results of the temperature range analysis method, for example, the aggregation degree of the spatial distribution of the belt fish is obviously lower than that of Larimichthys polyactis, that is, the spatial distribution of the belt fish is relatively uniform. As a result, one of the branches of the frequency distribution is consistent with its biological main body distribution, while the small yellow croaker has no such results, and there is a great difference between its frequency distribution and biomass distribution. The analysis also found that the populations with higher biomass are generally distributed in the waters with higher water temperature, which may be related to the good nutritional status of the population and the demand for rapid growth. The uneven distribution and aggregation in biological space may be related to the food needs and predation characteristics of species. According to the temporal and spatial distribution characteristics of the high biomass population of T. haumela, it is analyzed and inferred that the population may correspond to the reproductive fish schools of spring and summer and autumn.

• Age, growth and population structure of swordtip squid (Uroteuthis edulis) in the East China Sea based on statolith age information

  LI Nan, YU Jun, FANG Zhou, CHEN Xinjun, ZHANG Zhong

  2021,45(6):887-898, DOI: 10.11964/jfc.20200212154

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  The Uroteuthis edulis is one of the important economic squid resources in the East China Sea. Studying the composition, age, and growth pattern of population is the basis for understanding its life history. Based on the samples of the East China Sea U. edulis collected by the trawl nets from September to March of 2017 and 2018, this study used the age information of statoliths to study the sex and year differences in population structure and growth patterns. The results showed that there were differences in population mantle length and body weight composition between years, but there was no significant difference between genders. It was found by reading the age of statoliths and performing backward calculation that the two-year dominant age group was 190-220 d, and there were significant differences in age composition of male during the year. The backward calculation results showed that the peak hatching period was from March to May and August. The spring and summer stocks are the dominant hatching groups. The mantle length and body weight growth of the spring stock were best fitted with a Logistic curve, and the summer stock were best fitted with a von-Bertalanffy and a Logistic curve. In 180-270 d, the mantle length and body weight growth rate of spring stock was faster than that of summer stock. After 240 d, the male mantle growth rate was faster than that of female, and the body weight growth rate was slower than that of female. The age of the 50% maturity of the spring stock is younger than that of the summer stock, and the male is younger than the female. The results showed that different spawning populations existed in two years, and the growth pattern also had difference between sexes. Therefore, this study can provide a scientific basis for comprehensive understanding of the life history characteristics of the U. edulis.

• Effects of four microalgae diets on growth, nutritional composition, pearl producing function, shell nacre color and trace element content of Hyriopsis cumingii

  CHEN Qiang, JIANG Xiamin, PENG Ruibing, HAN Qingxi, SHENG Peng, XUE Ruiping

  2021,45(6):899-909, DOI: 10.11964/jfc.20191212114

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  China accounts for more than 95% of global freshwater pearl production, of which 80% is Hyriopsis cumingii. In high-density H. cumingii culture, large amounts of organic manure (duck manure, bird manure, soybean milk) and chemical fertilizers are used to produce sufficient quantities of feed. This method results in a large amount of feces, pseudo feces, and residual feed being discharged, causing eutrophication and negative effects on the benthic environment. Water pollution can result in frequent disease outbreaks and disease control drugs can further pollute the aquatic environment. Development of intelligent culture facilities, including high-quality nutritious diets for H. cumingii, is an urgent need. To dertimine the optimal microalgae diet for massive mussel culture, this experiment was conducted to investigate the effects of different microalgae diets (Cyclotella sp., Chlorella pyrenoidosa, Scenedesmus dimorphus, Monosporidium contortum, and mixed four microalgae) on the growth, pearl producing function, nutrition composition and shell nacre color of H. cumingii, using a single factor experiment method. The results showed that the growth of the mussel was significantly different with different microalgae diets. The mussels fed Cyclotella sp. and S. dimorphus showed weight growth (WG) of 9.94% ± 0.51% and 9.42% ± 0.89%, respectively, which were significantly higher than those of the other three groups; mussels fed M. contortum had the lowest WG 5.23% ± 0.30%. Similar results were obtained for shell length growth (SLG), with mussels fed Cyclotella sp. and S. dimorphus showing the highest values 1.99% ± 0.31% and 2.07% ± 0.53%, respectively. The group fed Cyclotella sp. showed the highest shell height growth (SHG) 6.55% ± 0.62%. The highest shell width growth (SWG) was also observed in the group fed Cyclotella sp. (5.31% ± 0.71%) while the group fed M. contortum group showed the lowest SWG (3.33% ± 0.91%); there was a significant difference between these two groups. Pearl weight growth (PWG) also differed significantly among the different treatment groups. The highest PWG was observed in mussels fed Cyclotella sp. (16.23% ± 1.23%). There were no significant differences among the groups fed C. pyrenoidosa (11.40% ± 2.09%), S. dimorphus (13.40% ± 2.00%), and mixed microalgae (11.80% ± 1.40%); the lowest PWG was observed in the group fed M. contortum (6.35% ± 1.90%). In summary, Cyclotella sp. and S. dimorphus were the optimal species for H. cumingii growth. Feeding different microalgae did not have a significant effect on the shape of the pearls (probability of producing round and near-round pearls), but did have a significant effect on the probability of producing round and oval pearls. The probability of a pearl shape being round was the highest in both the Cyclotella sp. and S. dimorphus groups (26.67% ± 4.71%) and was significantly higher than that in the M. contortum group. However, the S. dimorphus groups was significantly more likely to have oval round shape (16.67% ± 4.71%) than the other four groups. Different microalgae had significant effects on shell nacre color. The L* values for the Cyclotella sp. (64.89±1.97) and C. pyrenoidosa (65.18±2.24) groups were lower and significantly lower than the other three groups. And with the highest L* value in the mixed algae group (68.94±1.71), which was significantly higher than the other four groups. The mixed algae group had the highest dE* value (69.08±1.68), which was significantly higher than the other four groups. The smallest dE* value was found in the Cyclotella sp. group (65.23±1.89), which was not significantly different from the C. pyrenoidosa group (65.47±2.19). There was no difference between the S. dimorphus group (67.32±2.88) and the M. contortum group (67.52±2.46), but significantly higher than the Cyclotella sp. and C. pyrenoidosa groups. The largest C* value was found in the C. pyrenoidosa group (6.03±0.98), which was slightly higher than that in the Cyclotella sp. group (5.44±1.36) and significantly higher than those in the S. dimorphu, M. contortum and mixed groups. The trace element content of the mantle was significantly affected by diet. The highest Zn content was observed in mussels fed M. contortum [(170.33 ±3.86) mg/kg], followed by S. dimorphus, and Cyclotella sp. [(120.33 ± 2.05) and (122.33 ± 4.99) mg/kg]. The mixed algae group had the lowest Fe content (169.33± 22.13) mg/kg, and the others had no significant difference. The content of Mg and Mn had same trend, M. contortum > S. dimorphus > Cyclotella sp.= C. pyrenoidosa > mixed algae. The Ca content of the foot was the highest [(12 637.30 ± 624.39) mg/kg] for group fed Cyclotella sp., followed by the group fed M. contortum [(8 019.00 ± 513.72) mg/kg]. The Ca content of the foot for these two groups was significantly higher than those of the other groups. The highest Ca content in the mantle was observed in the mussels fed S. dimorphus (25 049.30 ± 1 320.36) mg/kg, followed by the group fed M. contortum (24 903.70 ± 359.06) mg/kg. The groups fed Cyclotella sp., C. pyrenoidosa, and mixed algae showed significantly lower Ca content in the mantle. In conclusion, Cyclotella sp. may be the best diet choice for intensive H. cumingii, and followed by S. dimorphus.

• Optimum lysine requirement of juvenile Pacific white shrimp, Litopenaeus vannamei when fermented soybean meal partially replacing fish meal in the feed

  GUO Xu, DAI Xilin, HUA Xueming, YU Zhongli, GAO Xuezhong, WEI Xiang, XU Lin

  2021,45(6):910-919, DOI: 10.11964/jfc.20201112508

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  The fish meal remains a high price with the worldwide shortage. Seeking different plant protein sources replacing fish meal to reduce the cost of feed has become one of the hotspots in shrimp nutrition research. However, most plant protein sources face the problem of amino acid imbalance after substituting fish meal, which affects the growth performance and feed utilization of the shrimps. Supplementing crystalline amino acids in feed is still the most effective and practical method to achieve amino acid balance of feeds. This study intended to explore the effects of different levels of lysine in feed on the growth performance, body composition and blood indices of Pacific white shrimp L. vannamei after replacing fish meal with fermented soybean meal at an optimal level, and to estimate the optimal dietary lysine requirement of juvenile L. vannamei. Five isonitrogenous and isolipidic practical diets(containing 41.5% crude protein and 7.5% crude lipid) were formulated with dietary supplemental lysine levels of 0%, 0.25%, 0.50%, 0.75% and 1.00%, and the measured values of dietary lysine contents were 2.43%, 2.65%, 2.87%, 3.06% and 3.24%, which were recorded as Lys0, Lys25, Lys50, Lys75 and Lys100. The L. vannamei with an average initial body weight about [(2.0±0.1) g] was randomly divided into 5 groups with 3 replicates per group and 50 shrimp per replicate. The feeding experiment lasted for 56 days in indoor cement pools. The results showed that, with the increase of feed lysine level, the weight gain rate and specific growth rate increased first and then decreased, reaching the highest in the Lys75 group with the lowest feed conversion ratio. The survival rate was not affected by feed lysine level. Muscle in Lys75 group had the highest crude protein and crude fat content, while crude ash and moisture content were not affected by feed lysine level. A variety of essential and non-essential amino acids in the muscle showed a trend of first increasing and then decreasing with the increase of feed lysine level. The turning point was in the Lys50 or Lys75 group. With the increase of feed lysine level, the activities of alanine aminotransferase and aspartate aminotransferase in serum increased first and then decreased, while the triglyceride content gradually increased, and the superoxide dismutase activity of Lys75 group was significantly lower than the other four groups. Based on the quadratic regression model of SGR and FCR with dietary lysine content, The optimal lysine requirements of L. vannamei were 2.88% (6.94% of dietary crude protein) and 2.89% (6.96% of dietary crude protein), respectively.

• Effects of four sugars as carbon sources on intestinal digestive enzymes and intestinal flora of Litopenaeus vannamei

  JING Bingyan, YANG Chao, GUO Ran, ZHU Yuzheng, LI Xuehe, ZHANG Yu, WANG Xiaoming, LIANG Beining, GAO Jiapeng

  2021,45(6):920-928, DOI: 10.11964/jfc.20200712334

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  The purpose of this study was to investigate the effects of four common sugar sources on the growth, digestive enzymes and intestinal flora of Litopenaeus vannamei. The selected initial weight of L.vannamei was (0.36±0.02)g. Four kinds of common sugars (glucose, fructose, starch and sucrose) were selected from a variety of sugars as the sugar source to prepare the feed. Four treatments were carried out in total, three replicates for each treatment, 40 prawns for each replicate. The breeding period was 56 days, and the added amount of each sugar source was 20%. The test results show that, ① The specific growth rate and protein efficiency of L.vannamei were significantly different, the starch group was significantly higher than the other three groups, but there was no significant difference in the liver body ratio and the meat content rate between the groups. The whole body nutrition of shrimp, the crude fat of sucrose group was significantly lower than the other three groups, the ash of glucose group was significantly higher than the other three groups, and there was no significant difference between crude protein and moisture. ② There were significant differences in digestive enzyme activities among different groups of L.vannamei, the activity of lipase in starch group and sucrose group was significantly higher than that in glucose group and fructose group. There was no significant difference between starch group and sucrose group, while the activity of pepsin in starch group was significantly higher than that in other three groups. ③ There was no significant difference in OTU, Ace and Chao indexes among the groups; Shannon index in starch group and sucrose group were significantly higher than glucose group; Simpson index starch group was significantly lower than sucrose group, and sucrose group was significantly lower than glucose group. At the level of phylum, the relative abundance of Proteobacteria in sucrose group was significantly higher than that in glucose group, while that in Bacteroidetes and Actinobacteria in starch group was significantly higher than that in glucose group, but there was no significant difference between the groups. At the class level, there was significant difference in the intestinal microflora of L. vannamei between the treatment groups. The Flavobacteriia, Gammaproteobacteria, Actinobacteria, Cytophagia in the intestinal tract of L. vannamei in starch group were significantly higher than those in sucrose group and glucose group.In summary, the absorption and utilization of starch and sucrose by L.vannamei is better than that of glucose and fructose. Meanwhile, starch and sucrose have certain influence on the diversity of intestinal flora of L.vannamei. Starch and sucrose are the suitable sugar sources in L.vannamei feed.

• Isolation, identification and pathogenicity of Flavobacterium psychrophilum from Oncorhynchus mykiss

  CHAI Jingru, WANG Di, LU Tongyan, CAO Yongsheng, LI Shaowu

  2021,45(6):929-939, DOI: 10.11964/jfc.20200512260

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  As the causative agent of bacterial cold-water disease (BCWD), Flavobacterium psychrophilum mainly infects juvenile salmonids and causes great economic loss in salmonid aquaculture worldwide. F. psychrophilum is characterized by a broad geographical distributions and hosts, inducing typical clinical signs of caudal peduncle erosion, skin ulcerations, pale gills, enlarged spleen, ascites and spiral swimming behavior in diseased fish. Although studies have been carried out on the epidemiology and pathogenicity of this bacterium in many countries, there is still no report on F. psychrophilum strains isolated from cultured salmonids in China. In the present study, two pathogenic bacterial strains (CH06 and CH07) were isolated from the muscle lesions of diseased rainbow trout (Oncorhynchus mykiss) using a polyphasic approach. The results indicated that CH06 and CH07 isolates had an egg-like appearance on TYES agar, producing flexirubin-like pigments, and positive for oxidase and catalase. It can hydrolyze gelatin and casein, but cannot hydrolyze starch or use fructose, galactose and esculin. The results of 16S rRNA alignment suggested that the homology of CH06 and CH07 isolates with F. psychrophilum type strain NBRC 15942 strain were 99.35% and 99.42%, respectively. Based on the biochemical and molecular characteristics results, CH06 and CH07 isolates were identified as F. psychrophilum. The multiplex PCR method was used to identify the serotypes of CH06 and CH07 isolates as type 1 (Fd type). MLST analysis indicated that the genotypes of CH06 and CH07 isolates were ST-12 and ST-78, respectively, both belonging to CC-ST10. The artificial infection results indicated that CH06 and CH07 isolates were highly pathogenic to juvenile O. mykiss with the LD₅₀ values of 7.1 × 10⁵ and 1.1 × 10⁵ CFU/mL, respectively. The dosage of the challenge was inversely proportional to the time when the clinical signs appeared. F. psychrophilum was successfully re-isolated from muscle, spleen of artificially infected fish. Histopathological changes showed that the hepatocytes of the diseased fish were swollen and vacuoles degenerated. Some hepatocytes were lysed and necrotic and the nucleus was dissolved. The spleen was congested and hemorrhagic with reduced amounts of the lymphocytes and increased amounts of the red blood cells and the hemosiderin. The muscle fiber gap was widened and broken, with uneven bending. Parts of the muscle cell sarcoplasmic dissolution was honeycomb-like. CH06 and CH07 isolates exhibited slightly different resistance profiles to 10 antibiotics. They were both highly susceptible to ampicillin and trimethoprim-sulfamethoxazole, while CH06 isolate was resistant to enrofloxacin and florfenicol and CH07 isolate was moderately susceptible to enrofloxacin and florfenicol. This is the first report on the biological characteristics of F. psychrophilum isolates recovered from cultured O.mykiss in China, and it is expected to provide scientific support for the prevention and control of BCWD in salmonid aquaculture.

• Pathogenicity of Anguillid herpesvirus to Anguilla anguilla

  CHEN Qiang, LI Yingying, YANG Jinxian, SONG Tieying, GE Junqing

  2021,45(6):940-947, DOI: 10.11964/jfc.20200812370

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  Anguillid herpesvirus (AngHV) is an important viral pathogen of cultured Anguilla anguilla, and also an important factor in the decline of wild A. anguilla populations. Since the 1990s, A. anguilla culture industry has developed rapidly in China, accompanied by frequent outbreaks of an eel epidemic disease with symptoms of “skin mucus sloughing, red head, and septicemia”, which was named eel “mucus sloughing and hemorrhagic septicemia disease”. The disease is the most common and harmful infectious disease of the cultured juvenile A. anguilla, and occurs every year in the eel farms in Fujian Province, causing huge economic losses to the breeders. We detected and isolated AngHV from the diseased A. anguilla samples. In order to determine the pathogenicity of AngHV, an AngHV strain (NA16108) isolated from “mucus sloughing and hemorrhagic septicemia disease” samples was used to study its pathogenesis to juvenile Anguilla anguilla. 15-20 g body weight A. anguilla were intraperitoneally injected with 10⁶ PFU of NA16108, and clinical symptoms, histopathological changes, viral replications in the major organs and tissues, etc, were used to evaluate its pathogenicity. The results showed that the body surface of the AngHV-injected A. anguilla had the symptoms of mucus exfoliation, fin hemorrhages, red head, etc. The diseased A. anguilla had pathological changes of mucus increasing and hemorrhages of gill, discoloration of liver and swelling of spleen and kidney, etc; further histopathological analysis showed that the skin had exfoliation of mucus and mucosa flat epithelial cells; gill had secondary lamellar hyperplasia, blood vessel hemorrhages, swelling and necrosis of respiratory cells; spleen had necrosis and aggregation of melanocytes, and kidney had necrosis wall cells and narrowing lumen of renal tubular, which are consistent with the clinical characteristics of A. anguilla "mucus sloughing and hemorrhagic septicemia disease". The mortality analysis showed that AngHV infected A. anguilla began to die from the 4th day post injection, and the cumulative mortality on the 7th and 14th day reached up to 26.7% and 56.7%, respectively. However, the eels in the control group had no obvious pathological symptoms during the experimental period. Quantitative real-time PCR analysis showed that AngHV could be detected in the liver, spleen, kidney, intestine, gill and skin mucus of AngHV infected A. anguilla with higher copy number in the gill and skin mucus, and AngHV was not detected in the corresponding tissues of the control group. Viral isolation experiment showed that AngHV could be re-isolated by EO cell line from the main internal organs of the injected A. anguilla, indicating that the injected A. anguilla had a systemic infection of AngHV. In addition, we found that the quantity of Pseudodactylogyrus in the challenged group increased rapidly, reaching to about 200 and 800 per A. anguilla on the 7th and 14th day post viral injection, respectively, however, the quantity of Pseudodactylogyrus in the control group did not change significantly, and remained within 100 per A. anguilla. The results indicated that AngHV is the pathogenic agent of "mucus sloughing and hemorrhagic septicemia disease", which laid foundation for further study on the pathogenesis and prevention and control of the disease.

• Molecular cloning and characterization in immune response of a novel C-type lectin gene (LvLc2) from Litopenaeus vannamei

  HAO Shaoyan, YIN Siyu, GONG Chen, ZHANG Yichen, GENG Xuyun, LIU Yichen, SUN Jinsheng

  2021,45(6):948-957, DOI: 10.11964/jfc.20210112595

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  Abstract:

  Litopenaeus vannamei is the largest economic shrimp breed in the world at present. However, with the expansion of shrimp culture, diseases and other problems are exposed gradually. Shrimp culture industry was affected seriously by diseases all over the world. Shrimp belongs to invertebrate, and has no typical acquired immune system. They mainly rely on the innate immune system for defense and protection when pathogens invade. In innate immune response, crustaceans recognize non-self components through pattern recognition receptors (PRRs). The components recognized by PRRs are mainly constitutive and conserved polysaccharide molecules on the surface of microorganisms, which are not produced by eukaryotic cell hosts and are necessary for the survival of pathogenic microorganisms. They are called pathogen associated molecular patterns (PAMPs). The recognition and binding of PRRs to PAMPs is the beginning of humoral immunity. The recognition of PRRs to pathogens can directly or indirectly activate proteases, hemocytes and intracellular signal pathways related to humoral immunity. A variety of immune defense reactions were triggered. It produces antibacterial substances and effective molecules to inhibit and eliminate pathogens. As a pattern recognition receptor and terminal effector, C-type lectin participates in the process of “non-self” recognition and elimination of invasive pathogens, which is crucial in the innate immune defense mechanism of crustaceans. In this study, a novel C-type lectin gene (LvLc2, GenBank accession number: KR020738) was cloned from hemocytes of L. vannamei according to our previous transcriptome results. Bioinformatics analysis showed that the ORF region of LvLc2 gene was 465 bp encoding 154 aa with 11 bp 5'-UTR and 126 bp 3'-UTR. The predicted molecular weight was 17.16 ku and the theoretical isoelectric point was 4.54. The deduced amino acid sequence contained a signal peptide sequence with 17 amino acids at the amino end and a conserved carbohydrate recognition domain (CRD) at the carboxyl end, in which four conserved cysteines formed two disulfide bonds. The motif associated with PAMP binding specificity is EPS (Glu¹¹³-Pro¹¹⁴-Ser¹¹⁵). Blast homology analysis showed that LvLc2 had high similarity with mannose receptor C-type lectin from different species, and 38% similarity with the mannose receptor of Rimicaris exoculata. LvLc2 gene was expressed in different tissues of L. vannamei. The highest expression was in intestine, followed by stomach, hepatopancrease, hemocytes, and the lowest expression level was in muscle. After shrimps were challenged by LPS and PGN, the expression profiles of LvLc2 gene in hemocytes were analyzed by qPCR. Compared with the control group, the expression of LvLc2 showed a slight down-regulated expression profile at the initial stage of LPS injection (0.5 h), and then reached the highest value at 12 h after LPS injection, which was about 2.2 times of the initial expression level. At 24-72 h after injection, the expression of LvLc2 basically returned to the initial level, and there was no significant change. At the early stage after PGN injection, the expression of LvLc2 fluctuated slightly. At the 48th hour, the expression of LvLc2 was significantly up-regulated, which was about 4.5 times of that in the control group and then fell back to slightly lower than the initial level. Different pathogen-associated molecular patterns (PAMP) could induce response changes of LvLc2 gene after stimulation, which indicated that it had a potential broad-spectrum response pattern. At the same time, it had different expression profiles for different pathogens, which showed the differences of recognition, combination and indirect immune effects. The research of its characterization and function are helpful to better understand the mechanism of shrimp C-type lectin during the process of pathogen infection.

• Effects of freshness-locked performance during the quality determination period on the iced Takifugu rubripes freshness quality

  LENG Hanbing, LIU Junrong, YI Hongli, WANG Xuanfei, TIAN Yuanyong, XU Tanye

  2021,45(6):958-970, DOI: 10.11964/jfc.20200612312

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  Abstract:

  Market demand for farmed fish products is growing rapidly, However, not all aquaculture sectors produce the premium quality at end of the market. To produce farmed seafood with consistent optimum quality, solutions of stress minimizing and deterioration delaying are worth studying. To explore mechanism and feasibility of “lock in freshness” of farmed fish chilled products, farmed Takifugu rubripes was taken as raw material. The experiment was performed in two post mortem stages, including a quality determination period (QDP) corresponding to on-site handling and post quality determination period (P-QDP) corresponding to the ice storage. The delayed effect of the QDP chilling treatment on the P-QDP was investigated. On-site handling was carried out using humane slaughter followed by instant bleeding in –1 °C sea water with 20 minutes, gutted and rinsed fish were individually wrapped and stored with flake ice surrounded. Meanwhile, the Control group as chilling-free was compared to the Treatment group with chilling at the QDP. The chilled T. rubripes postmortem biochemical metabolism was measured by rigor index, pH, lactic acid content, ATP and ATP-related compounds and K-Value. Meanwhile, the quality was analyzed by muscle ultrastructural changes in two treatments. Treatment temperature after chilling in quality determination period dropped rapidly from 10.9 to 2.7 °C in 20 minutes, which has a significant effect of chilling. Treatment temperature was increased dramatically from 2.7 to 8.5 °C in 1 hour because of the special strengthening body washing process of T. rubripes after being processed. And the fish temperature was reduced to 0.5 °C after 12 h iced storage. The delayed effect of the QDP chilling treatment on the P-QDP can be clearly observed by the muscle ultrastructure. Regardless of the Treatment or the Control, in subsequent chilling storage, muscle tissue degraded gradually. Notable, distinct difference can be observed between the Treatment and the Control. The phenomenon of cell structure degraded of the Control occurred compared with the Treatment on day 0. And intermyofibrillar spaces increased. Sarcomeres were non-parallel and swollen sarcoplasmic reticulum was obvious. Especially on day 4, sarcomeres were altered significantly, mainly at the Z-line and I-band level disruptions. Structure was obscured and destroyed more significantly than the Treatment. But the results showed that traditional biochemical indicators could not be detected for sensitive difference of the two treatments. The Treatment biochemical metabolism rate kept pace with the Control, which the rigor mortis progression was similar and rigor state was maintained 4-5 days. Lactate acid, pH, nucleotide metabolism rate and K-value had no significant difference, and K-value was 15% in 8^th day chilling storage. There was a quality determination period after fish was captured, which had the delayed effect of chilled fish quality in the post quality determination period. The degradation of muscle cell could be prolonged by QDP-chilling, which had significant effect of “lock in freshness” on quality of chilled fish. In this way, the image of seafood quality of domestic fisheries could be improved fundamentally, providing significant practical value in China.

• Indicative significance of rheotaxis behaviour of three familiar species of fish from Yuanshui River by hydropower cascade development

  SONG Bolan, SU Qiangen, LI Tianbao, LIU Liangguo

  2021,45(6):971-981, DOI: 10.11964/jfc.20200412222

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  Abstract:

  To understand the physiological and ecological adaptation mechanism of fish movement and the evolution of their movement mode from Yuanshui River, a branch of Dongting Lake water system, we investigated the swimming ability, rheotaxis behaviour and metabolic characteristics of local fish of different species and habits by the hydropower cascade development in this study. The rheotaxis behaviour and swimming metabolic characteristics of three familiar fish (Carassius auratus, Siniperca kneri, Pelteobagrus fulvidraco) from Yuanshui River were tested using a self-manufactured apparatus for measuring swimming ability and metabolism of fish by adjusting water velocity in the laboratory. The classical increasing velocity method and video counting method were used to study these data. The results showed that the relative induction velocities of C. auratus (15.84±3.60 g), S. knerii (27.10±7.41 g), and P. fulvidraco (17.50±4.78 g) were, respectively, (0.79±0.14) BL/s, (1.16±0.07) BL/s and (0.75±0.09) BL/s. The relative preferred swimming speed of the three fishes were, (2.31±0.11) BL/s, (1.86±0.04) BL/s and (1.60±0.03) BL/s, respectively, and the critical swimming speed were, (3.43±0.37) BL/s, (2.58±0.32) BL/s and (2.68±0.13) BL/s, respectively. Under the same conditions, the preferred swimming velocity, critical swimming speed, respiratory frequency (RF) and oxygen consumption rate (OCR) of P. fulvidraco were significantly lower than those of the two other fishes. At different flow rates, the RF, OCR and tail beat frequency (TBF) of the three species of fish all increased significantly with the increase of flow rate, and it showed a same tendency to first increase and then decrease. The RF of all fishes had peak values at the flow rate of 3.0 BL/s.The TBF and OCR values of C. auratus and S. kneri peaked at the flow rate of 2.0 BL/s, while that of P. fulvidraco was at the flow rate of 1.5 BL/s. The rheotaxis rate of C. auratus increased from 64.1% under the condition of still water to 100.0% at the flow rate of 4.0 BL/s, while S. kneri and P. fulvidraco increased respectively from 60.2% and 58.9% under the condition of still water to 100.0% at the flow rate of 2.0-3.0 BL/s. The swimming states of the three species of fish were obviously affected by the flow rate. Under the condition of still water, the swimming states of C. auratus were mainly in “moving forward against the current” and “downstream”. S. kneri under the condition of still water and most of the flowing water were at rest against the current. P. fulvidraco under the condition of still water and at lower flowing rate (<1.0 BL/s) were also mainly at rest against the current. With the increase of flow rate, the proportion of rest against the current decreased rapidly, and the proportion of “moving backward against the current ”increased continuously. Taken together, our findings indicated that the differences of the rheotaxis behaviour of three familiar fishes from Yuanshui River were closely related to their living habits, morphological characteristics, feeding habits, feeding patterns, and their living water environment.

ROUNDUPS

• Research progress of matrix proteins in Hyriopsis cumingii

  BAI Zhiyi, YUAN Li, LIU Xiaojun, LI Jiale

  2021,45(6):982-991, DOI: 10.11964/jfc.20200612302

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  Abstract:

  Shell and pearl are highly ordered biominerals formed by calcium carbonate under the control of organic matrix, composed of shell matrix proteins, sugars and lipids. Shell matrix proteins play significant roles in the formation of shells and pearls. It is very important to study the matrix proteins of Hyriopsis cumingii for revealing the formation mechanism of pearls and cultivating high quality pearls.In this paper, 26 matrix proteins related to the formation of the cuticle, prism layer and nacreous layer of H. cumingii were introduced. The structural characteristics of these matrix proteins were analyzed, including the amino acid composition, primary structure and advanced structure. The roles of these matrix proteins in biomineralization were also explained, including participating in the deposition of calcium carbonate, the formation of organic framework, the regulation of crystal morphology, the regulation of shell coloring and the correlation with pearl weight. The aim of this review is to provide basic references for further improving of pearl quality.

• Development status and prospect of Chinese deep-sea cage

  SHI Jiangao, YU Wenwen, LU Bencai, CHENG Shiqi

  2021,45(6):992-1005, DOI: 10.11964/jfc.20200612314

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  Abstract:

  Deep-sea cage is a new type of aquaculture model, which has the advantages of advanced technology, environmental friendliness and high quality of marketable fish. Therefore, it has a great significance to the promotion of green development strategy of aquaculture. Based on a brief introduction to the development of common cages, offshore cages, as well as deep-sea cages, this article provides a detailed overview of the present situation and research progress of deep-sea cages before and after 2017 in China, and prospects for future development directions. Before 2017, deep-sea cages were in its infancy. Nearly 10 types of deep-sea cages with simple structures, such as assembly type metal cages facilities in deep-sea lagoon, were developed and mainly distributed in Hainan, Guangdong and Guangxi, which provided technical reserves for the subsequent development of the deep-sea cage. Since 2017, deep-sea cages have entered a stage of rapid development. More than 10 types of deep-sea cages with complex structures have been developed, including the Deep Blue No. 1 full-submersible deep-sea farm. They ushered in the modernization of Chinese deep-sea cages. So far, the research on the definition of deep-sea cages, cage materials, and its supporting patents have been reviewed. As a new type of aquaculture model, deep-sea cages have obvious comprehensive advantages and will be a useful supplement to Chinese aquaculture industry in the future. According to the requirements of the green development strategy of the aquaculture industry, the deep-sea cages will be developed in the direction of offshore, large-scale, intelligent, and intensified in the future to expand the aquaculture area, to improve production efficiency, increase technological content, reduce culture costs, and upgrade the industry level. Although Chinese deep-sea cage aquaculture industry has made great progress, there is still a huge gap compared with foreign advanced technology. The related work has a long way to go, which is worth our efforts to study deeply.