Blood cells play an important role in animal immune system. Accurate classification and functional analysis will facilitate the understanding of immunol response mechanism. In this research, hemocyte populations from Litopenaeus vannamei of 17-23 g weight were identified through micro-observation and flow cytometry, which referred to distinguishable intracellular partical structures and measurable side scattering characteristics of cells separately. Further analyses of verifying the response hemocyte groups and their quantitative changes in early stage of bacteria or virus infection were also carried out. Four hemocyte populations of non-granular, small-granular, intermediate-granular and large-granular hemocyte could be identified by micro-observation and flow cytometry, they count for (69.88%±4.71%)/(74.88%±3.97%), (9.04%±5.06%)/(20.75%±4.07%), (11.13%±7.02%)/(2.94%±0.72%) and (9.96%±1.31%)/(1.25%±0.58%) of the total hemocytes respectively. In both methods, non-granular hemocyte count for about 70%, but proportions of other 3 granular hemocytes were different depending on the method. Lipophilic tracer DID staining showed intracellular granules were covered by lipid membrane. Size similarity of granules in both intermediate-granular hemocyte and large-granular hemocyte inferred an endogenous mechanism, supporting granules were derived from golgi apparatus. The size distribution range of large-granule hemocytes was narrower and their cytoplasm was full of granules, indicating they might be in a mature stage. Further results from low intensity challenging of bacterial or virus pathogens showed that in the early time of Vibrio parahaemolyticus or WSSV infection, total cell counting reflected a similar trend of “down and up”; non-granular and small-granular hemocytes, the two major phagocytic populations, were main response groups. The low point of cell counting in V. parahaemolyticus infection experiment was observed at 24 h; but for WSSV infection, low counting period was between 36 and 48h, it should be related to virus need more time for entering cell and replicating. These support that a large number of hemocyte could be lost in a short period of time after infection. On the other hand, full of granules and showing little count fluctuation after infection, indicated both intermediate-granular hemocyte and large-granular hemocyte might participate in immune defense through secreting bioactive molecular other than phagocytosis. Above results indicated that hemocytes populations from different crustacean species could be distinguished with similar quantitative criteria and they responded to pathogen invasion in different ways after infection.
Serine protease (SP) is a kind of important and widely distributed proteolytic enzyme, which participates in a series of important physiological processes, including digestion, blood coagulation, embryonic development and immune response. But there are only a few preliminary reports about SP in crustaceans. Shrimp farming is constantly threatened by diseases. Further research on shrimp immune defense mechanism will provide clues for finding new ideas of disease resistance. In this study, the full-length cDNA sequence of a novel serine protease homologs (SPH) gene (Lv-SPH, GenBank accession number: KR020739) was cloned from hemocytes of Litopenaeus vannamei according to our previous transcriptome results. The sequence characteristics were analyzed by bioinformatics method. Tissue distribution and expression profile in response to virus infection were discussed by real-time fluorescence quantitative PCR. Bioinformatics analysis showed that the total length of the gene was 1 249 bp, the open reading frame (ORF ) region was 1 005 bp encoding 334 aa with 156 bp 5′-UTR and 88 bp 3′-UTR. The deduced amino acid sequence contained a signal peptide sequence with 16 amino acids at the amino end. SMART analysis showed that Lv-SPH contained a clip domain and a highly conserved SP domain (Tryp-SPc), which had two of the three active catalytic sites (His and Asp), and the third one is Gly. Lv-SPH was expressed in different tissues of L. vannamei. The main expression was in hemocyte, followed by hepatopancrease, heart and intestine. The lowest expression level was in muscle. Real time fluorescence quantitative PCR analysis showed that white spot syndrome virus (WSSV) could induce the up-regulated expression of Lv-SPH significantly. The relative expression of Lv-SPH increased significantly at 24 h and reached the highest level at 48 h, which was about three times that of the control group. Lv-SPH has typical characteristics of SPH family members and has tissue expression specificity. WSSV can significantly induce the up-regulated expression of Lv-SPH gene, indicating that it may be involved in the immune response process of L. vannamei induced by WSSV. The results provide a reference for further exploring shrimp innate immune regulation mechanism, and have potential application value in shrimp healthy culture and disease control.
Vibrio alginolyticus is one of the main pathogens that causes mass mortality in aquatic animals and infects humans. To reduce the application of antibiotics, alternative therapies have been proposed. One of the promising possibilities is to control diseases through the use of lytic bacteriophages. As few current bacteriophages against V. alginolyticus have been reported, the main objective of this study is to develop effective bacteriophages for controlling pathogenic bacteria. This study describes the isolation and characterization of lytic bacteriophages against a V. alginolyticus strain (VAHN1). The bacteriophages were isolated from Hainan shrimp pond and Fujian marine products by double-layer agar culture method, and V. alginolyticus VAHN1 was used as the host strain for bacteriophage isolation. The phages were classified and identified by transmission electron microscope (TEM), restriction endonuclease and phylogenetic tree analysis.The physiological and biochemical characteristics of the bacteriophages were determined, including optimal multiplicity of infection (MOI), lysis spectrum detection, pH stability, thermal stability, resistance to UV light, and sensitivity to chloroform and ether. In addition, the effect of bacteriostasis were also measured. The results showed that 2 lytic bacteriophages against V. alginolyticus were isolated, named as VAP9 and VAP21. The plaques of VAP9 and VAP21 were neat and transparent, with a diameter of 1.5-2 mm. The nucleotides of the phage VAP9 and VAP21 were all dsDNA, and all their heads were shown as icosahedral shape with about 55 nm and 65 nm diameter respectively under TEM. The tail of the phage VAP9 was approximately 65-70 nm in length and 15 nm in width, while the tail of the phage VAP21 was about 75-80 nm in length and 18 nm in width. The 2 bacteriophages are grouped under the Myoviridae family. The phage VAP9 and VAP21 had good tolerance to different physical and chemical environment. The survival rate of the 2 bacteriophages was greater than 43% at 60 °C for 2 h. The optimal pH of VAP9 was 6-8 and VAP21 was 7-11. The 2 bacteriophages tolerated peracetic acid in universal bactericidal concentration. The phages VAP9 and VAP21 were insensitive to chloroform and ether, with a certain resistance to UV light. The optimal MOIs of both VAP9 and VAP21 were 0.001. The cocktail of VAP9 and VAP21 was able to infect 95.2% (157 strains among 165 strains) of the V. alginolyticus strains and 50% (3 strains among 6 strains) of the V. parahaemolyticus strains used in this study. And they could not infect other species of tested bacteria except V. alginolyticus and V. parahaemolyticus. The phages VAP9 and VAP21 could effectively inhibit the growth of V. alginolyticus VAHN1 and have the same inhibitory trend on VAHN1, but the VAP21 strain had a stronger bacteriostatic effect than that of the VAP9 strain. Moreover, the inhibitory effect involved the cocktail of 2 bacteriophages on V. alginolyticus were better than that of a single phage. Alignment with the sequences of the conserved protein amino acid sequences of the phage VAP9 and VAP21 on National Center for Biotechnology Information (NCBI), it was showed that the 2 bacteriophages had low homology with other bacteriophages. Furthermore, phylogenetic and genome analysis revealed that the phages VAP9 and VAP21 have low homology with other bacteriophages and may be 2 novel Myoviridae bacteriophages that infect bacteria related Vibrio spp. This study did not only enrich the species resources of bacteriophages against V. alginolyticus, but it also laid a theoretical foundation for the development and application of Vibrio bacteriophages as microecological antimicrobial agents.
Toll-like receptor gene family is a class of conserved pattern recognition receptors, which plays an essential role in innate immunity providing efficient defense against invading microbial pathogens. Epinephelus akaara is one of the most important commercial marine fishes, and the main aquaculture industry in China is distributed along the coast of Fujian. The species is also considered a good model for studying immunity. Although TLRs have been extensively characterized in both invertebrates and vertebrates, a comprehensive analysis of TLRs in E. akaara is lacking. This research aims to study the systematic evolution, chromosome distribution, and expression regulation patterns of the toll-like receptor genes in different tissues of E. akaara, based on the published genome and transcriptomic data of E. akaara, this study analyzed the phylogenesis, chromosome distribution, and expression regulation patterns of toll-like receptor gene family in E. akaara tissues using bioinformatics methods including BLAST, phylogeny and synteny. The results showed that a total of 17 TLR genes were identified in E. akaara, which were divided into 5 subfamilies (TLR1, TLR3, TLR5, TLR7 and TLR11) and distributed on 11 of the 24 chromosomes. The 17 TLRs showed different expression patterns in different tissues. EaTLR1-2, EaTLR2-2, and EaTLR13-2 were mainly highly expressed in the spleen. EaTLR5M was highly expressed in the kidney, spleen, gills, and heart, but lowly expressed in other tissues. EaTLR5S was highly expressed in the kidney, liver, and spleen, but with low expression in other tissues. EaTLR18-1 and EaTLR18-2 were mainly expressed in the heart. EaTLR8 was highly expressed in all tissues except muscle and liver. EaTLR1-1, EaTLR2-1a, EaTLR2-1b, EaTLR3, EaTLR7, EaTLR9, EaTLR13-1, EaTLR21, and EaTLR22 were mainly highly expressed in the brain, spleen, kidney, and gill. In addition, EaTLR18-1 and EaTLR18-2 were both located on chromosome 9 with highly similar expression patterns. EaTLR2-1a and EaTLR2-1b were located on chromosome 20 and their expression patterns were also highly similar. In addition, EaTLR5M and EaTLR5S, which had high LRR domain similarity, were located on chromosome 14, and had high similarity in expression levels in different tissues. On the contrary, EaTLR7 and EaTLR8, which were located on chromosome 18, and EaTLR2-2 and EaTLR3, which were located on chromosome 4, had different expression patterns. These suggested that copies of TLR genes in the same chromosome may have similar expression patterns or functions. This work provided reference data for studying the evolution of the toll-like receptor system in fish, and laid a foundation for further research on the function of toll-like receptor genes in E. akaara.
To investigate the cause of an outbreak of unknown disease occurred in cultured largemouth bass (Micropterus salmoides) in a reservoir in Fujian Province, with a cumulative mortality of over 30%. In March 2021, the dominant bacterial colonies were isolated from the liver, spleen and kidney tissues of affected fish, the pathogenic bacteria was identified by artificial infection experiments, and its species was identified by physicochemical characteristics, 16S rDNA sequences, and mass spectrometry analyses. Further, virulence gene detection, drug sensitivity research and histopathological observation were carried out. A predominant bacteria strain Yersinia ruckeri was isolated from the spleen of the inffected M. salmoides. A set of virulence genes including YrP1+ - YhlA+ - YhlB+ - invF- - traC- - traL- - traN- were detected by PCR from in the isolated strain, with a LD50 of 3.8×105 CFU per fish by intraperitoneal injection. The antibiotic sensitivity test showed that the pathogen was sensitive to enrofloxacin, doxycycline hydrochloride, flumequine, neomycin sulfate and florfenicol. Histopathological observation found that Y. ruckeri infection caused significant damages to liver, spleen, kidney and other organs of M. salmoides. The main pathologic lesions showed obvious degeneration, necrosis and infiltration of the inflammation cells. It could be included that the current research is the first report of cultured M. salmoides infected by Y. ruckeri, which will be helpful for the well as the early diagnosis and better prevention of the disease in cultured M. psalmodies.
In order to explore the role of rhamnose-binding lectin (RBL) in the non-specific cell defense of bony fishes against pathogen infection. The head kidney monocytes/macrophages from Nile tilapia (Oreochromis niloticus) were isolated to conduct a bacterial stimulation experiment in vitro. The expression of Onrbl-1 was significantly up-regulated following challenges with two important pathogens, Streptococcus agalactiae and Aeromonas hydrophila. Meanwhile, OnRBL-1 recombinant protein could participate in the regulation of the expression of pathogen-induced cell inflammatory factors by quantitative real-time PCR (qRT-PCR), including significantly inhibiting the expression of il-6, il-8 and tnf-α, and promoting the expression of il-10 and tgf-β. In addition, the results of showed that OnRBL-1 could promote the phagocytosis, enhance the level of respiratory burst and the release of reactive oxygen species in monocytes/macrophages through flow cytometry analysis. Taken together, the results indicated that OnRBL-1 plays an important regulatory role in the non-specific cellular defense of monocytes/macrophages from O. niloticus. This study provides a reference for exploring the function of RBL-1 in host defense against pathogen infection, which helps to explain the action mechanism of bony fish RBL in the defense against pathogen infection and improve the basic theoretical system of its participation in immune response, with great significance in science.
Cryptocaryon irritans is a parasitic ciliate causing ‘white spot disease’ on marine teleosts that often results in huge financial losses to mariculture. So far, there is no safe and effective method to control the parasite infection. Calmodulin (CaM), as a sensor for Ca2+ signaling, regulates cell movement, cell division and invasion of some protozoan parasites to their hosts. To understand the role of calmodulin in the growth and development of C. irritans, the calmodulin gene (cicam) was cloned from the C. irritans trophozoite cDNA library, and the open reading frame (ORF) was synthesized after codon optimization. The recombinant plasmid pGEX-4T-1/cicam was constructed, then transformed into Escherichia coli and induced its recombinant expression with ZYM-5052 self-inducing medium. The recombinant protein rCiCaM was purified by glutathione agarose gel and thrombin, and obtain polyclonal antibodies by immunizing with the mouse BALB/c strain. The expression of cicam and its encoded protein CiCaM in each stage of the worm was examined by reverse transcription PCR and immunoblotting assay, respectively. The localization of CiCaM in the larvae was examined by indirect fluorescent antibody assay. The activity of recombinant protein rCiCaM binding recombinant actin-depolymerizing factor was initially investigated by blot overlay assay. The results showed that the open reading frame (ORF) of CiCaM was 450 bp, which encoded a polypeptide of 16.9 ku, consisting of 149 amino acids; the molecular masses of GST-rCiCaM and rCiCaM were 43 ku and 16.9 ku respectively, which corresponded with the predicted ones; the CiCaM gene expressed in all developmental stages of C. irritans, and the molecular mass of the native CiCaM corresponded to the predicted value; the CiCaM distributed all over the cytosol of C. irritans theronts, especially abundant around the four macro nuclei and the peripheral area of cytostome; rCiCaM could interact with rCiADF2 in a Ca2+-dependent way. This study enriched the knowledge of molecular biology of the pathogen C. irritans, which would provide a reference for the prevention and control of cryptocaryonosis.
Dendritic cells are the outposts of the immune system, which link innate immunity with adaptive immunity, and thus are considered as the most powerful antigen-presenting cells in vivo. To further investigate the functions of CD8α and CD207 in grass carp (Ctenopharyngodon idella) dendritic cells (DCs), the recombinant eukaryotic expression plasmids of CD8α and CD207 genes were constructed and successfully expressed in DCs of C. idella. The open reading frames (ORFs) of CD8α and CD207 were obtained from DCs of C. idella by RT-PCR and inserted into recombinant eukaryotic expression plasmid pcDNA3.1, respectively, to construct recombinant eukaryotic expression plasmids pcDNA3.1-CD8α and pcDNA3.1-CD207; after the sequencing was confirmed to be correct, the recombinant eukaryotic expression plasmids were transfected into DCs of C. idella using liposome method, and the expressions of CD8α and CD207 were verified by cell immunofluorescence technology and Western blot. The results showed that in the Western blot assay, the CD8α and CD207 proteins were expressed normally and significantly overexpressed after transfection of the recombinant eukaryotic expression plasmid, and the size was consistent with that of the predicted result. Cellular immunofluorescence showed that the CD8α and CD207 proteins were mainly expressed on the cell membrane of DCs. Hence, DCs of C. idella exhibit immunophenotypes and functions that are conserved in their mammalian counterparts. The recombinant eukaryotic expression plasmids of pcDNA3.1-CD8α and pcDNA3.1-CD207 were successfully constructed, which laid a foundation for further research on the action mechanism of CD8α and CD207 genes in DCs. Carrying out studies on the immunophenotype of DCs will play a positive role in analyzing the DCs-mediated immune regulation mechanism of C. idella. In the cultivation process, C. idella are easily infected by pathogens and induce inflammatory reactions. The development of DCs-based immunotherapy can provide new ideas for the prevention and control of C. idella related diseases, which has a wide application prospect in reducing diseases and enhancing the immune efficacy of vaccines for teleost fish.
Vitamin C-based carbon dots are a new class of nanomaterials with antibacterial properties, but their antibacterial activities against aquatic pathogenic bacteria have not been verified. For this reason, the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of VC-CDs against 12 aquatic pathogenic bacteria such as Aeromonas hydrophila, Vibrio parahaemolyticus, V. splendens and Nocardia sp. were determined using 3-fold serial dilutions. The bacteriostatic kinetic curves of the 12 aquatic pathogens were determined. In addition, based on the potential biocompatibility of nano materials, we used MTT method to detect the cytotoxicity of VC-CDs on CIK cells. Danio rerio embryos were used as test objects to determine the embryotoxicity after exposure to VC-CDs. The results showed that VC-CDs had significant inhibitory effect on 12 aquatic pathogenic bacteria, and the minimum inhibitory concentrations ranged from 20.6 to 61.7 μg/mL, basically reaching the same antibacterial effect as the antibiotic florfenicol. The survival rate of zebrafish embryos and CIK cells was almost 100% in the range of MIC. This study shows that VC-CDs have good bactericidal and antibacterial effects on the main pathogenic bacteria in aquaculture, and have good biocompatibility, which signifies the potential as a promising alternative to antibiotics in the prevention and control of bacterial diseases in aquaculture.
Species distribution and biomass are the basis for evaluating population dynamics and community structure in an ecosystem. Unfortunately, it is frequently full of challenges to capture the distribution of the rare species through traditional methods. Procypris rabaudi is a unique economic species in the upper reaches of the Yangtze River, the source of this species has declined dramatically in recent years. Environmental DNA technology is a sensitive, low-cost and non-invasive new technology for species monitoring. It has great potential application in detecting endangered and invasive species. In order to establish a real-time quantitative PCR (qPCR) method for the detection of P. rabaudi and distinguish it from other fishes in the Yangtze River, this study designed eDNA primers and a TaqMan probe based on 12S rRNA gene sequence in mtDNA. The sequence of the 12S rRNA gene was amplified by PCR and cloned into the pMD19-T vector to construct the standard plasmid, and a qPCR method was developed for detection of P. rabaudi using serially diluted standard plasmid as templates, then the sensitivity, specificity and application effects of the method were evaluated. The results showed that the cycle threshold value (Ct) of qPCR assay had a great linear relationship with the copy number of the standard plasmid. Amplification specificity analysis indicated that the method could specifically detect P. rabaudi. Then, eDNA was detected in the water samples in aquarium tanks with different numbers of P. rabaudi, the target DNA concentration and the number of P. rabaudi had a positive correlation of R2 was 0.957, the correlation curve between DNA concentration and the individual number was obtained: y = 131 546x + 77 623. In addition, after the removal of P. rabaudi, the copies of eDNA were negatively correlated with time, and its retention time in the water environment was about 17 days. In this study, we found that the DNA primer and Taqman probe could be applied to the qualitative detection of P. rabaudi in water with high specificity, as well as estimate fish density in tanks quantitatively. These results demonstrate that eDNA analysis method is a potential tool to reflect the biomass of P. rabaudi in different sampling sites, which can provide a basis for assessment of artificial release effect and resource management of P. rabaudi in future.
To understand the protection effect of Dachenyang spawning ground reserve, based on the survey data of fishery resources in the Dachenyang Spawning Ground Reserve in April (spring) and November (autumn) 2018, using relative importance index (IRI), diversity analysis, cluster analysis and canonical correspondence analysis (CCA) and other methods to analyze the characteristics of fish community structure in this sea area and its relationship with environmental factors. The results showed that: a total of 83 species of fish were caught, belonging to 9 orders and 51 families, including 2 dominant species of fish, 18 important species of fish, belonging to 5 orders and 11 families, and the dominant species was Amblychaeturichthys hexanema and Harpodon nehereus. Hierarchical clustering (CLUSTER) and non-metric multidimensional measures (NMDS) analysis showed that the spring and autumn fish communities can be divided into 3 groups spatially, and there are significant differences between the community groups (P<0.05). H. nehereus, Benthosema pterotum and Thryssa kammalensis are the main divergent species that affect the seasonal changes of community structure. ABC curve analysis showed that the fish community in spring was moderately disturbed (W=0.027); the fish community in autumn was in a state of severe disturbance (W=–0.185). Canonical Correspondence (CCA) analysis shows that water depth, temperature and salinity areimportant marine physical and chemical factors that affect the composition of fish species and the temporal and spatial changes of community structure in this sea area.Compared with the resources in the same period in 2015 and 2017, both biomass and abundance increased significantly in November 2018. This study found that the establishment of protected areas played a protective role in the recovery of fishery resources, but long-term resource protection was needed to restore marine resources to an ideal state.
Benthic bivalve clam (Meretrix Meretrix) is one of the most important economic shellfish and widely distributed in the coastal tidal flats of China. The coastal areas of Jiangsu and Liaoning Provinces are natural nursing ground for clams, and the amount of clams had reached more than ten thousand tons in the history. However, due to overfishing, the fishery resources of clams generally decline. Understanding the growth process of M. Meretrix in natural waters is of great theoretical significance and application value for the quantitative research on assessing the ecological capacity of M. Meretrix. Because the matter circulation and the energy flow are complex in marine ecosystems, it is difficult to evaluate the dynamic growth changes of benthic bivalve clam in natural waters through experiment approach. A mathematical model, as a research tool, provides an effective tool for the study of shellfish growth in varied water environments. In this study, an individual model of the clam, based on the dynamic energy budget theory, was newly constructed. The developed model was parameterized based on the physiological and ecological data of clam achieved in laboratory and field experiments, which were analyzed by linear and nonlinear regression methods. The relationship between shell length and wet weight of clam was verified based on the comparison between data observation and simulation. The change process of shell length and wet weight of soft tissue was simulated by using WHDEBSTD software, which was originally developed by the authors, in the field environment. The results show that the shape coefficient of the clam is 0.57, the Arrhenius temperature value is estimated to be 9278 K, and the volume-specific cost for structure value is 2056.3 J/cm3. The good agreement between measured and simulated results of shell length, total weight and soft body weight change can be found in earth pond culture of M. Meretrix. The average correlation coefficient R2 is about 0.996 and the average discrepancy between simulated and measured is 3.58%. In the coastal area of Rudong, the shell length and the soft body dry weight of M. Meretrix were observed as 3.12 cm and 0.48 g in June, and the simulated value were 3.2 cm and 0.476 g, respectively, which indicate that the numerical model could reproduce the growth of M. Meretrix in the natural sea. This study provides useful information for research on constructing the clam module in ecosystem model and evaluating the ecological capacity of clam in natural waters. However, some discrepancies can still be found between the simulated and the observed growth of clam, which might be caused by the difference between the sexes of the clam in terms of growth. And the hibernation state of M. Meretrix cannot be reproduced by the numerical model. Follow-up studies will focus on the sexual differences and the hibernation state of clams, so as to further improve the accuracy and stability of the model.
The fishery resources and ecological environment in Shandong offshore are under great pressure. With the impact of exploitation activities such as coastal engineering and overfishing in Shandong, the fishery resources in this area are in decline, and the number of fish of high economic value is significantly reduced. To effectively monitor and manage the fishery resources, it is necessary to understand fishery ecosystem, for which biodiversity is a crucial index in community ecology research to indicate ecosystem status. To understand the spatial distribution of coastal biodiversity, and the relationships between biodiversity indices and environmental factors, this study used several diversity indices, including number of species, Simpson index, Shannon index, and Pielou index, to analyze the spatial distribution of biodiversity based on investigation data of fishery resources in 2017 winter in Shandong offshore. Owing to a lot of non-linear and non-additive processes in fishery ecosystem, random forest model is used to assess the relationship between diversity indices and environmental factors. The results showed that the spatial distribution of diversity indices varied substantially in the Shandong offshore area, with a trend of higher diversity in the southern area of the Peninsula than that in the north, which included Yanwei fishery ground, Laizhou Bay, and the southern Bohai Sea. The southern Bohai Sea have the lowest level of species diversity. Random forest model could be properly fitted to the diversity data, and the rate of explained deviation reaches 77.46% and 45.66% for species number and the Shannon diversity index, respectively. However, the regression model of Pielou index had a low proportion of explained deviation. The bottom temperature, salinity, and water Depth had significant influences on the diversity indices, significantly correlated with species number, diversity index, and evenness index. The effect of bottom sediment type was not significant on species number and two diversity indices, but was high for the Pielou evenness index. The results were also compared for fishery resource surveys at different scales. The conclusion is that surveys of similar scale are necessary for long-term monitoring of biodiversity. This study systematically analyzes the diversity pattern of Shandong offshore ecosystem and provides scientific support for long-term marine ecosystem monitoring and specific marine management. We highlight the necessity of protecting biodiversity in order to restore fishery resources. Decisions could be made based on the level of biodiversity in terms of habitat restoration and the establishment of protected areas.
Complement and coagulation cascade are important components of innate immunity, in which complement protein 5a (complement component 5a, C5a), complement protein 5a receptor (C5a receptor, C5aR) and coagulation factor II (blood coagulation factor II, FII) play a key role in dealing with virus infection. In order to study their protein expression and interaction in Ctenopharyngodon idella infected with grass carp reovirus (GCRV), the prokaryotic expression system of C5a and FII protein was constructed, the C5aR-KLH conjugate against C5aR protein was constructed, and the protein was purified. Japanese big-eared rabbits were immunized to prepare polyclonal antibodies against the three kinds of proteins. The expression and interaction of the three proteins were detected by western blot, Co-immunoprecipitation (Co-IP) and pulldown test. Western blot results showed that C5a and C5aR proteins were expressed in liver, spleen, kidney, head kidney, intestine, gill and muscle of C. idella. FII protein was expressed in liver, spleen and intestine, but not in kidney, head kidney, gill and muscle. Expression of C5a, C5aR and FII proteins in liver tissues of C. idella infected with GCRV showed an upward trend with the progression of the disease. Co-IP results showed that C5a, C5aR and FII proteins interacted with each other after GCRV treatment. Pull down results showed that 28 candidate proteins such as C3 and RIG-I were identified by C5a pull down, and 24 candidate proteins such as transaldolase and macroglobulin were identified by C5aR pull down. This study provides a basis for further exploring the interaction and regulation of C5a, C5aR and FII, and for further exploring the mechanism of the three associated proteins in the complement and coagulation cascade system responding to GCRV infection.
This experiment was conducted to investigate the effects of dietary bile acid levels on growth, lipid deposition, lipid metabolism enzyme activities and related gene expression of juvenile Schizothorax prenanti. A total of 360 juvenile S. prenanti with an average body weight of (12.74±0.14) g were randomly divided into 4 groups with 3 replicates per group and 30 fish per replicate.The diets with different bile acid supplemental levels (0, 75, 150 and 300 mg/kg) were fed to juvenile S. prenanti for 70 days. The results showed that: with bile acid supplemental level raising, the weight gain rate(WGR) had a trend of increased first and then decrease(P<0.05). The WGR was the highest (226.63%) when the bile acid supplemental level was 150 mg/kg. At the same time, the activities of intestinal lipase (LPS), hepatopancreas hepatic lipase (HL), lipoprotein lipase (LPL) and total lipase (TL) were all firstly increased and then to stabilization (P < 0.05), conversely, the activities of fatty acid synthase (FAS) were was firstly decreased and then to stabilization (P < 0.05) with dietary bile acid levels raising. Meanwhile, with dietary bile acid levels raising, the liver LPL mRNA expression of S. prenanti increased first and then tended to stabilization (P<0.05), FAS mRNA expression decreased first and then tended to stabilization (P<0.05). The lipid content of liver, muscle and whole fish were all trended to stable after the first reduced with dietary bile acid levels raising (P < 0.05). As well as the survival rate and lipid deposition rate were not significantly different of S. prenanti (P > 0.05). Collectively, the results of above indicate that the suitable dietary bile acid levels can up regulation of LPL mRNA expression and down regulation of FAS mRNA expression of S. prenanti, enhanced the activities of fat metabolism enzymes, promote the digestion and utilization of feed fat, reduce lipid deposition and protect liver health effectively.
Mammalian cylindromatosis (CYLD) has been identified as a tumor suppressor and participates in innate immune signaling transduction in various cell types through negative regulation of NF-κB activation by deubiquitinating TRAF6 and NEMO. To investigate the role of CYLD in the immune response of large yellow croaker (Larimichthys crocea), the cDNA sequence of CYLD was cloned and identified, named LcCYLD. Gene expression profile was detected by real-time fluorescence quantitative PCR. Then the recombinant plasmid pTurboGFP-CYLD was constructed for subcellular localization and transfected into HEK293T cells. For further understanding of the function of CYLD in innate immune, the recombinant overexpression vector pcDNA3.1-CYLD was constructed and transfected into cells, and the activation capability of NF-κB, proinflammatory factors TNF-α and IL-1β were detected. Sequence analysis showed that the ORF of LcCYLD contained 2 754 bp, encoding 917 amino acids. The putative LcCYLD protein contained three conservative N-terminal CAP_GLY domain, a phosphorylation region, and a typical C-terminal UCH domain. Multiple alignments showed that CYLD was highly conserved among the analyzed species. Phylogenetic analysis showed that LcCYLD was clustered with bony fish and closely related to striped bass (Morone saxatilis). Gene expression analysis indicated that LcCYLD expressed in most examined tissues with the most predominant expression in the brain, followed by blood and intestine. However, the expression levels in other tissues are very weak. LPS and poly I:C stimulation significantly induced the transcriptional expression of LcCYLD. Subcellular localization showed that LcCYLD expressed both in cytoplasm and nuclease. Overexpression of LcCYLD could significantly inhibit the immuno-activation of NF-κB and proinflammation of cytokines TNF-α and IL-1β after LPS and poly I:C challenge. These findings suggested that LcCYLD could negatively regulate NF-κB activation. The present study might be helpful for better understanding the function of LcCYLD in innate immune signaling transduction of L. crocea.
To investigate the optimal supplementation of dietary exogenous protease in plant-based diets of red swamp crayfish (Procambarus clarkii), crayfish [initial mean weight (9.25±0.20) g ]were fed six isonitrogenous and isoenergetic diets which were formulated to contain graded protease levels (0, 0.1, 0.2, 0.4, 0.8 and 1.6 g/kg protease, respectively) for 8 weeks. Each diet was fed to triplicate tanks with 15 per crayfish in each tank. The results showed that, with the increase of dietary protease supplementation: ① There were no significant difference in survival rate, hepatosomatic index and flesh content among groups. The weight gain rate firstly increased and then decreased, and reached the maximum when dietary protease supplementation level was 0.2 g/kg. The feed conversation ratio reached the lowest value when dietary protease supplementation level was 0.4 g/kg. The protein efficiency and protein deposition rate reached the highest in shrimp fed the diet with 0.4 and 0.2 g/kg protease, respectively. Broken-line model analysis in terms of the weight gain rate, feed conversion ratio and protein efficiency indicated that optimal dietary protease supplement of crayfish was 0.16, 0.24 and 0.23 g/kg, respectively.② The crude protein contents in muscle and whole shrimp firstly increased and then decreased, and the maximum occurs at the shrimp fed the diet with 0.2 g/kg protease. ③ The activities of protease, lipase and amylase in intestine and hepatopancreas all reached the maximum at the shrimp fed the diet with 0.2 g/kg protease, which was significant higher than the control shrimp. ④ The maximum content of total protein in serum were observed in shrimps fed the diets with 0.4 g/kg protease; the activities of glutamic oxaloacetic transaminase and glutamic pyruvic transaminase firstly decreased and then increased, with the lowest value occurs at the shrimp fed the diet with 0.4 g/kg protease; the activities of alkaline phosphatase and acid phosphatase firstly increased and then decreased, and reached the maximum in 0.2 g/kg group and 0.4 g/kg group, respectively. The content of malondialdehyde had the minimum value at the shrimp fed the diet with 0.2 g/kg protease. ⑤ The activities of alkaline phosphatase and acid phosphatase in hepatopancreas firstly increased and then decreased, and reached the maximum at the shrimp fed the diet with 0.2 g/kg protease, which was significantly higher than the control shrimp. In conclusion, under this experimental condition, the appropriate supplementation of dietary protease in red swamp crayfish is from 0.16 to 0.4 g/kg.
The pharyngeal myxosporidiosis caused by Myxobolus honghuensis is one of the most severe parasitic diseases in the culture of Carassius auratus gibelio. In order to reveal the tissue distribution and differences of M. honghuensis in the diseased and asymptomatic C. auratus gibelio, therefore, this study was carried out. This study has collected and examined the muscle, pseudobranch, gills, head kidney and other 11 organs by microscopic, one-tube semi-nest PCR and histopathological section. Microscopical exam showed that the sporocyst and mature spores were most detected in the pharynx, pseudobranch and gills of diseased fish, while the mature spores were enclosed with yellow nodal and most detected from pseudobranch and first efferent branchial artery in the asymptomatic gibel carp. PCR results showed M. honghuensis could be detected in the most tested organs in diseased fish except muscle, and pseudobranch had the highest infection rate (100%). In asymptomatic C. auratus gibelio, positive results were detected in pharynx, pseudobranch, head kidney, kidney, spleen and ovary, and the highest infection rate (26.7%)also was from the pseudobranch. Tissue sections of the diseased C. auratus gibelio of microscope showed M. honghuensis forming numbers of sporocysts between the pharynx wall and skull, and some near the pharyngobranchials. The pseudobranch was seriously destructed by the sporscysts, and the sporogonic cells of M. honghuensis could be observed near the left pseudobranch filament. Based on the microscopic exam and PCR test, this study firstly found that the pseudobranch may be the main parasitic and sporogonic site of M. honghuensis to parasite and mature development, and revealed the infection variation of M. honghuensis between the diseased and asymptomatic C. auratus gibelio.
Fish mucosa (mainly skin, gill, intestine, etc.) and their symbiotic microorganism play a key role in regulating the homeostasis of the fish mucosal microenvironment and promoting fish health. In the past 10 years, fish intestinal mucosal microbes have attracted much attention because of their important role regulating fish nutrition and health. However, research on symbiotic microbes in other mucosal organs of fish (such as skin and gill) has been relatively delayed. In order to better understand the relationship between fish mucosal microbes, mucosal microenvironment homeostasis and their effects on fish health, this article will review the research progress and the existed problems of fish mucosal microorganism, in terms of the community structure, influencing factors, research strategies and application value of fish mucosal microbes based on the literature published in recent years. It aims to provide new ideas and strategies for the effective regulation of mucosal microorganisms of fish and their applications in production and practice.
Fish intends to use different habitats in different life cycle stages. A comprehensive understanding of habitat selection strategy by fish plays key roles in polulation protection, utilization, target species selection as well as on stock enhancement in marine ranching programs. Based on the bottom trawl survey in summer (August, 2016), autumn(December, 2016), winter (March, 2017) and spring (May, 2017) in Eastern Ma’an Archipelago, we examined 3496 Larimichthys polyactis individuals collected from 19 sites. Biological characteristics of small yellow croaker, including age structure, sex ratio, sexual maturity and the food composition were statistically analyzed using indices such as resource density, relative importance index and GIS spatial analysis. Regression analysis was explored to reveal the relationship between the biological parameters and environmental factors. Our results showed that the population of small yellow croaker in the Eastern Ma’an Archipelago expressed a strong seasonal variation and spatial difference:the population density of small yellow croaker was the highest in summer. There was a higher population density in the areas adjacent to islands than that in the areas off islands. In terms of age structure, 1-year-old small yellow croaker dominanted allmost all seasons except for summer(0-year-old small yellow croaker accounted for 75. 7%). Contrary to spring and autumn, the preys richness for small yellow croaker in areas adjacent to islands was significantly higher than that in areas off islands in summer. Regression analysis indicated that the primary productivity in the areas off islands was the most significant factor(p<0. 05). Correlation analysis revealed that higher abundance of phytoplankton contributed to higher population density of small yellow croaker. However no such significant correlation was tested in the areas adjacent to islands. The research revealed that in Eastern Ma’an Archipelago, large scale of reefs and artificial habitats, provide suitable habitats for small yellow croaker as feeding, breeding and sheltering ground. Small yellow croaker in the areas adjacent to islands didn’t show obvious preference for specific environmental factors except for the food factor, but showed a certain preference for the habitats combined by various environmental factors in the areas adjacent to rocky islands.
To accurately identify the pathogen of Siniperca chuatsi, the experiments were based on morphological comparisons, combined with parasitic characterization and molecular phylogenetic methods to carry out the study and observe the ultrastructure. The results showed: ① Fusiform or dumb-bell white sporangia, about 1.9-6.8 mm in length, were observed in the fins, skin, oral cavity, gills, the outer and the inner layer of the operculum of the diseased fishes; ② Endospores of (9.8±1.8) (7.3~13.8) μm in diameter; ③ The ring-like cytoplasm and circular refractive bodies of about (6.5±1.2) (3.9-8.3) μm in diameter. It was identified as Dermocystidium. The measured values of this species were different from those of other Dermocystidium and were in general agreement with those of the undetermined species (HB) parasitizing the gills of Siniperca chuatsi; it was parasitized by similar sites as Dermocystidium from Guangdong and Henan provinces (GD and HN) and Dermocystidium sp. (HB). Molecular sequence alignment and phylogenetic results showed that this species shared over 99% similarity with Dermocystidium sp. (HB and HN), Dermocystidium anguillae , D. fennicum, and D. salmonis in SSU rDNA sequences. Phylogenetic analysis indicated that D. anguillae sampled from Guangdong (GD) and Henan province (HN) branched closely with the present species, then D. salmonis, D. anguillae, and D. fennicum. We concluded that Dermocystidium sp. infecting Siniperca sp. are conspecific and we revised the records of the D. anguillae (GD and HN) isolates of Siniperca chuatsi; we named Dermocystidium in this study together with the isolates (HB, GD and HN) as Dermocystidium sinipercae sp. n.. The intraspecific morphological variation of Dermocystidium sinipercae sp. n. may be related to the sampling time and developmental environment; spore proliferation and differentiation processes were recorded and described in ultrastructural observations. This study confirmed that Dermocystidium sinipercae sp. n. has been distributed in many parts of China, emphasizing the urgent need to implement quarantine measures for the origin of Siniperca chuatsi.
To explore the pathogenic mechanism of nocardiosis in Channa argus, we studied the pathogenicity and drug sensitivity of the pathogen and the immune resistance of C. argus by isolation and identification of the pathogen, histopathology and gene expression analysis. The results showed that the diseased C. argus was mainly infected with a pathogen strain SDAT 0011. The strain SDAT 0011 was identified as Nocardia seriolae using the morphologic structure and staining observation, PCR amplification of species-specific primers, sequence analysis of 16S rRNA gene, and physiological and biochemical tests. The challenge experiments showed that the isolated strains were pathogenic on C. argus and the mortality rate of the 1 × 105 − 1 × 108 CFU/mL injection groups was 100%. In addition, the diseased C. argus exhibited numerous marked white nodules (round/ovoid), ranging from 0.1 to 0.2 cm in diameter on internal organs, especially the spleen, kidney, and liver. Histopathological observation and analysis showed that the structure of chronic granuloma was visible, with many lymphocytes, damaged or dead cells in the center. The antibiotic susceptibility assays of the strain SDAT 0011 showed that the strain was sensitive to rifampin, but resistant to penicillin, cefradine, and ampicillin, etc. Furthermore, the expression levels of toll-like receptors 2 gene (TLR2), toll-like receptors 13 gene (TLR13), and C-C chemokine receptor type 9 gene (CCR9) after infection were analyzed by real-time fluorescence quantitative PCR (qRT-PCR). In our study, following N. seriolae challenge, TLR2 and TLR13 were significantly up-regulated, while CCR9 was significantly down-regulated at all examined time points in the spleen and head kidney. These results implied that some genes of the toll-like receptor signaling pathway and the chemokine signaling pathway have been activated in the early stages of infection to resist Nocardia infection. The results of this study will lay a foundation for the treatment of N, seriolae and studying it pathogenesis..
In order to understand the regulation mechanism of 17β-HSDs on gonadal development and reproductive endocrine of mollusks, this paper has reviewed the types and functions of 17β-HSDs, classified all the subtypes reported so far, and summarized the cloning, expression, function and mechanism of 17β-HSDs in mollusks. In spite of some progress made in 17β-HSDs regulating the reproductive process of mollusks, many underlying problems remain unsolved and further investigations are needed: ①discovering new subtypes of 17β-HSDs in mollusks, ②analyzing the characteristics of temporal and spatial expression, ③investigating the structure-activity relationship, ④clarifying the impact of environmental pollutants and making the corresponding countermeasures, ⑤guiding the practice and application of aquaculture industry. This review has synthesized former research of other species, which offers reference and guidance for further study on the mechanism and application of 17β-HSDs in regulating gonadal development and reproductive endocrine of mollusks.
Pacific oyster, Crassostrea gigas and Fujian oyster, C. angulata are economically important shellfish in the northern and southern parts of China. In order to analyze the dynamic growth of the two oysters, individual growth models to the C. gigas and C. angulata in the mariculture area of Sanggou Bay and Shenhu Bay were applied, based on Dynamic Energy Budget (DEB) theory. In this model, seawater temperature and the concentration of Chl. a were used as forcing variables, and the parameters were estimated from field measurements, model calibration and published data. The sets of data used to validate the model came from two long-term growth experiments performed on C. gigas and C. angulata. Results showed that: ① the DEB model developed here displayed good growth simulations and a significant correlation between the simulated and the observed values; ② parameters of upper boundary of tolerance range (TH), lower boundary of tolerance range (TL) and half-saturation constant for food (FH) in the two oysters were different probably because of the different physicochemical environment, diet composition and the selective ingestion; ③ C. gigas showed slow-growing in winter (limited by water temperature and food), while C. angulata tended to be continuous-growing (mainly limited by food) during the simulated period. These results will provide a scientific basic for the subsequent development of ecological model and the assessment of oysters’ carrying capacity.
The scallop (Pationopecten yessoenisis) is one of the most important aquaculture species in China. The main cultured areas are distributed in Chang-Shan archipelagos of Liaoning. But since 2007, the raft culture scallops P. yessoenisis outbreak massive mortality in ChangHai, Liaoning. The moribund scallops exhibited severe disease signs including brown deposit on the inner shell. So the cause of brown deposit symptom and the relationship with death of P. yessoenisis was analyzed. The epidemiological investigation and experiment of avoiding bite were used for research the preliminary cause and formation of brown deposit symptom. The infrared spectroscopy scanning also was used for studying the component of brown deposit. The result show that the cumulative mortality of P. yessoenisis cultured in aquaculture cage for avoiding bite was 87.60%, the proportion of brown deposit symptom was 74.50%. So biting or mechanical abrasion between scallops was not the cause of brown deposit symptom; the FITR spectra of brown deposit was consistent with the FTIR of BSA, and had protein characteristic peaks of amide Ⅰ and amide Ⅲ, the main component of brown deposit was protein; the brown deposit symptom often happened in mid late June and early July when the temperature of seawater was 17 °C. 2017-2019, the proportion of brown deposit symptom was 85.7%, 1.54% and 10.9% respectively, the cumulative mortality of P. yessoenisis cultured in ordinary cage was 90.40%, 49.20% and 48.16%, the proportion of brown deposit symptom was significantly associated with the cumulative mortality of P. yessoenisis. So pathogenic infection maybe the cause of brown deposit symptom, and it had relationship with the massive mortality of P. yessoenisis.
In order to explore the effect of salinity stress on the intestinal gene expression level of Cherax quadricarinatus, Illumina Hiseq 2500 high-throughput sequencing platform was used to conduct bidirectional sequencing of intestinal tissues of C. quadricarinatus under different salinity conditions (0, 5, 10 and 15 psu). After quality control and assembly of the obtained raw data, 76 534 unigenes was obtained. Blast in NR, NT, KO, Swissprot, PFAM, GO and KOG databases, 37 378 unigenes could be annotated. The amount of gene expression was estimated according to FPKM (Fragments Per Kilo bases Per Million Fragments). Based on the protein annotation results of NR and Pfam databases, 448 362 unigenes was functional annotation. According to the relationship between KO functional annotation and Pathway, 9 483 unigenes was annotated and classified into 34 pathways. Gene expression differences were analyzed by DEGseq. 0 psu was used as the control group, and the screening conditions for significantly different genes were set as Q value<0.05 and multiple |Fold Change|>2. The 5, 10 and 15 psu groups obtained 2 733, 91 and 236 differentially expressed genes respectively, among which 2 068 genes were significantly up-regulated and 665 genes were significantly down-regulated in the 5 psu groups with the most differentially expressed genes. The KEGG pathway enrichment analysis of all differentially expressed genes showed that 5, 10 and 15 psu groups were enriched to 265, 80 and 120 pathways respectively, and only 10 psu and 15 psu groups had a significantly enriched pathway respectively, all of which were legionella pathways. After identification, legionella pathway was significantly down-regulated, which was consistent with the previous results of enterobacteriaceae. The results showed that red chelicerae could protect the intestinal tract from infection by exogenous pathogenic bacteria by inhibiting the relevant pathways and abundance of potential pathogenic bacteria under the condition of increased salinity. In addition, changing in differential genes (eEF1α、udp、UPP、ACTB_G1、TUBA, TUBB、PFN、CALM) suggest that increased salinity seriously affects the cellular morphology, intracellular material transport, and intracellular signal transduction in the gut of red chelicerae, and activates pyrimidine remediation pathways to repair genetic damage caused by uracil nucleotide imbalance. The results provide important reference materials for the saline-alkali or brackish aquaculture of C. quadricarinatus in the future, and provide reference for the further study of the intestinal immune regulation mechanism of C. quadricarinatus under salinity stress.
To analyze the effect of the Cyprinus carpio Slc15a2 gene on the immune response mechanism during Aeromonas hydrophila infestation, this experiment prepared rat-derived Slc15a2-1 and Slc15a2-2 polyclonal antibodies, and changes in protein expression of Slc15a2-1 and Slc15a2-2 in C. carpio sera following A. hydrophila infection was detected by enzyme linked immunosorbent assay (ELISA). The results showed that ① compared with the blank control The maximum protein expression levels of the two copies were increased by 4.81 and 2.48 times, respectively, after infection. The protein expression of Slc15a2-2 was consistently higher from 0 h to 48 h compared to that of Slc15a2-1. ② Overall, there was a similar expression trend between the two copies of Slc15a2, with both increasing, then decreasing, then increasing and then decreasing again. However, Slc15a2-1 reached its first expression peak at 3 h, while Slc15a2-2 reached its first expression peak at 6 h. ③ Slc15a2-1 and Slc15a2-2 protein expression showed complementary expression trends during the four periods from 6 h to 24 h. That is, the expression of one copy decreased significantly and the expression of the other copy increased significantly during the same period, but the overall expression level remained high. The results indicate that the polyclonal antibodies prepared in this experiment have high affinity and specificity, and the overall similar expression trend between the two copies after infection with A. hydrophila suggests that they may have similar gene functions. Slc15a2-2 had significantly higher expression between 0 h and 48 h than Slc15a2-1, possibly as the major gene in the copy. The two copies showed complementary expression from 6 h to 24 h, and the co-expression was maintained at a high level, enabling an effective response in the face of pathogen attack. This study may provide some basis for insight into the regulatory mechanism of the immune response of common carp Slc15a2 gene.
The beak was the main feeding organ of cephalopods which contained abundant biological information and was widely used in cephalopod biology research. [Object] In this paper, the characteristics of pigmentation stage in the beak were studied, which provided the basis for the study of fishery ecology of Berryteuthis magister shevtsovi; [Method] Based on the 303 schoolmaster gonate squid samples which collected in the Japan Sea, we measured the basic biological data which included the mantle length (ML), body weight (BW), sexual maturity stage (SMS), the external morphological parameters (lower hood length, LHL; lower rostrum length, LRL; lower lateral wall length, LLWL; lower wing length, LWL) of the beak and stomach stage (SS), divided and determined the beak pigmentation. The relationships between the beak pigmentation and the ML, BW, SMS, the external morphological parameters of the beak and SS were analyzed by artificial neural networks, and the median values of the samples were fitted linearly; [Results] The results indicated that the ML contributed the most to beak pigmentation stage, which was 22.9%, followed by the LHL, SMS, BW, LRL, LWL, which contributed 16.5%, 14.4%, 11.9%, 11.7% and 11.6% respectively, while the LLWL and SS contributed less to beak pigmentation stage, which were 6.3% and 4.7% respectively. The analysis of covariance (ANCOVA) results showed that there was no gender difference between the beak pigmentation and ML, BW, sexual maturity stage and stomach stage (P=0.611, P=0.786, P=0.132, P=0.515). The relationships between beak pigmentation and ML (R2=0.9870), BW (R2=0.9148) and the external morphological parameters of the beak (R2=0.9082, R2=0.8768, R2=0.8329, R2=0.7807, R2=0.8938, R2=0.6877, R2=0.7474, R2=0.7787) were significantly relevant. The beak pigmentation stage also increased with the increasing of sexual maturity stage, but there was no significantly relevant between the beak pigmentation and stomach stage. This research studied the relationships between various growth factors and beak pigmentation stage, in order to provide scientific basis for further study on fishery ecology and rational development of schoolmaster Berryteuthis magister shevtsovi; [Discussion] The relationships between ML, BW, the external morphological parameters and beak pigmentation stage were significantly relevant, so that we can evaluate the beak pigmentation stage by this growth factors.
Catalase (CAT) is the main member of antioxidant enzyme system, which plays an important role in maintaining redox homeostasis and resisting pathogen infection. In order to study the function of CAT gene in mollusc under pathogen infection, the full-length cDNA sequence of CAT in Sinonovacula constricta was cloned by RACE approaches, and designated as ScCAT. The full-length cDNA of ScCAT was 2840 bp and encoded a polypeptide of 508 amino acid residues. Sequence analysis showed that ScCAT protein contains a CAT core domain (25-410), a catalase proximal active site signature (61FNRERIPERVVHAKGAGA78) and a proximal heme-ligand signature sequence (351RLFSYPDTH359). Multiple sequence alignment and phylogenetic tree analysis confirmed that ScCAT belongs to the CAT family, and was more closer to invertebrate Meretrix meretrix. Tissue distribution analysis revealed that ScCAT was constitutively expressed in all examined tissues, and the highest expression was found in the hepatopancreas (85.67-fold, P < 0.01), followed by gill (50.09-fold, P < 0.01), and the lowest level was detected in hemocytes (0.76-fold) compared to that of adductor muscle, respectively. After the razor clams were challenged by Vibrio parahaemolyticus, the mRNA level of ScCAT was significantly increased in the hepatopancreas, and reached the highest level at 12 h compared with control (3.56-fold, P < 0.01). Moreover, the ScCAT protein activity in hepatopancreas and gills were significantly increased after V. parahaemolyticus challenge, with the higher magnitude in hepatopancreas. In addition, the recombinant protein was expressed in Escherichia coli, and the purified ScCAT showed highly catalase activity. All these results showed that ScCAT was an important antioxidant enzyme, which participated in the immune response of S. constricta.
Island and reef lagoon is an important environmental field for the evolution of island and reef biodiversity, and plays an important role in the material circulation and energy flow of island and reef water ecosystem. The ecological function of microzooplankton is one of the important research contents to analyze the composition, flow direction and energy flow efficiency of primary productivity of lagoon ecosystem in Nansha Islands waters. In this paper, microzooplankton and ecological environment were investigated in the surface waters of Zhubi Reef, Meiji Reef and Yongshu Reef lagoon, the key islands and reefs of Nansha Islands, and the community structure and its relationship with environmental factors were studied, and the grazing pressure of microzooplankton was studied by dilution culture experiment. There are 20 species of microzooplankton in the investigated waters, with the total abundance ranging from 320 to 1,460 ind/L, among which the abundance of Ciliated ciliate is the highest. The abundance of Ciliated ciliate is highest in the western waters of Zhubi Reef lagoon (ZB-1), while the abundance of Aloricate ciliate appears in the middle waters of Yongshu Reef lagoon (YS-3), and the maximum value of Nauplius larva appears in the northern waters of Meiji Reef lagoon (MJ-2). The cluster analysis shows that the community similarity of microzooplankton in the middle area of lagoon is relatively high, and dissolved oxygen is the most important factor affecting microzooplankton community structure in these islands and reefs, and it has the most obvious influence on MJ-3 station. The growth rate of phytoplankton in the lagoon waters of the three major islands and reefs is 0.22-1.36 d-1. The grazing rate of microzooplankton ranged from 0.22 to 0.60 d-1, and the micro-zooplankton consumed about 20.5%-45.1% of the existing phytoplankton every day, equivalent to 37.1%-222.9% of the primary productivity. Studies have shown that different environmental and biological factors on the three major islands and reefs of Nansha Islands have influenced the different grazing pressures of microzooplankton, thus forming different evolution mechanisms of microzooplankton biodiversity on each island and reef. This study provides data support for the energy flow of microbial loops in the lagoon ecosystem under the ecological effects of islands and reefs.
Scophthalmus maximus, as an economic fish adapted to cold water at lowtemperature, high temperature severely affects its growth and survival. Scolars have confirmed that heart function is an important factor in setting the upper limit of heat range for fish. The present study aimed to investigate in heart damage and mechanism the effects of thermal stress on physiologic, biochemical response and apoptosis gene expression multiple levels. In this study, we investigated the characterization and mechanism response to thermal stress in the heart, using H.E staining, electron microscopic observation, enzyme activity detection and qPCR. The results showed that the aggravated degrees of swelling and breakage of myocardial fiber, dilatation of interstitial space, inflammatory cell infiltration, mitochondrial structure destruction and other tissue damage with the elevated temperature, but the tissue damage was significantly reduced at 24 °C-24 h. CK activity increased significantly with the escalation of heat stress; LDH, SOD activity and MDA content reach their peak at 24 °C. Expression levels of Bax and Caspase-3 decreasing significantly after thermal tress, and the tendency were similar to them. However, the expression level of Bcl-2 gradually increased. These results indicated that the myocardium could reduce the expression of Bax and Caspase-3 genes and promote the expression of the anti-apoptotic gene Bcl-2 to reduce the loss of myocardial cells to reduce thermal stress damage when it suffered a lesser degree of heat stress. This suggested that turbot suffered thermal stress, which lead the heart defense enzymes to exert resistance to maintains body homeostasis. The organism defense system itself is damaged because the heat stress exceeds its own physiological regulation threshold when the heat stress intensifies to 28 °C, which caused severely damages of heart structure and even lead to death in turbot. The results showed that thermal stress could cause myocardial damage of turbot, and the body maintains homeostasis via regulating the activity of defense enzymes and apoptosis pathway related genes. This study provides a theoretical basis for subsequent research of the physiological adaptation mechanism of turbot and other fishes heart to thermal stress. At the same time, it provides more trait indicators for the high temperature tolerance traits to improve the breeding accuracy of marine fish.
Coilia nasus belongs to genus Coilia, family Engraulidae, order Clupeiformes, which is migratory fish with high nutritional and economic value in China. C. nasus has both migratory and land-locked populations, which is an excellent model for studying reproductive migration and adaptive evolution. This study identified and analyzed the expansion and shrink of gene families in C. nasus, which in order to explore the special mechanism of C. nasus caused by changes in the core gene family in the evolutionary process. Comparative genomology was used to identify the C. nasus’s gene families by using the genomes of Danio rerio, Gasterosteus aculeatus, Oryzias latipes, Takifugu rubripes and Tetraodon nigroviridis. Then CAFE V4.2 software was used for gene family expansion and contraction analysis. Finally, GO, KEGG and other database were applied for annotations and pathway analysis of the significantly expansion gene families’ genes. 11 872 gene families were identified in C. nasus, including 16 470 genes. Besides, 2 963 unclustered genes were identified. Compared with the other 5 species, 150 gene families containing 419 genes were found unique to C. nasus. In addition, 1 200 gene families of C. nasus expanded and 7 543 gene families contracted. P values of 39 expansion gene families (including 508 genes) and 36 contraction gene families (including 21 genes) are less than 0.05. 508 genes of significantly expansion gene families were utilized for pathway analysis. Metabolic process, catalytic activity, cell and cell part are the top GO terms. Tight junctions, adrenergic signaling in cardiomyocytes, cardiac muscle contraction, cell adhesion molecules (CAMs), etc are the top KEGG pathways. Tight junctions and cardiac muscle contraction pathways are related to osmoregulation. GnRH signaling pathway and olfactory transduction pathway are involved in reproductive migration behavior. Furthermore, we mapped 508 genes of significantly expansion gene families on the genome, and found that chromosome 23 had much more genes than other chromosomes. And the genes of significantly expansion gene families located on chromosome 23 were mostly histone genes, which were related to the regulation of chromatin structure. Pathway analysis of the expansion and exclusive gene families of C. nasus indicate that the osmotic regulation, signal transduction, metabolism, gonad development and olfactory transduction may have undergone adaptive evolution, and the regulation of its chromatin structure may also have been altered. These results strengthened our understanding of the adaptive evolution of C. nasus.
Shellfish farming is an important part of China's aquaculture industry. With the continuous expansion of the scale of aquaculture, the unreasonable operation of aquaculture industry has caused a greater burden on the environment, caused the change of microbial community structure in aquaculture water, and caused certain harm to the health of shellfish aquaculture. Meanwhile, it has also caused huge economic losses to the shellfish aquaculture industry, which seriously affected the sustainable development of shellfish industry. In order to study the open aquaculture waters of offshore shellfish, the distribution of bacterial populations, and the response of microbial community diversity to environmental factors, the relationship between the incidence of aquatic animals, we collected sea water samples from different positions in an open aquaculture water area in Huangdao from July to November 2019 to analyze different water quality factors (water temperature, salinity, pH, dissolved oxygen, chlorophyll a, ammonia nitrogen, phosphates, nitrates and nitrites), and using high-throughput sequencing methods to analyze the differences in the microbial community structure and diversity of water samples and Chlamys farreri tissue samples in different months, and discuss the correlation between environmental factors and microbial community structure. The results showed that 42 phyla and 94 classes were detected in the bacterial community structure of water samples. It is composed of Proteobacteria, Bacteroides, Cyanobacteria, Actinomycetes, Firmicutes, etc. Among them, Proteobacteria is the dominant phyla, mainly including γ-Proteobacteria and α-Proteobacteria. The results of the diversity analysis of bacterial OTU levels in different months were November>September>July>October>August; the diversity analysis of Vibrio OTU level showed that the diversity of Vibrio increased first and then decreased, and the trend was consistent with the change of water temperature. The diversity of the culture area in August was higher than that of the control area. RDA analysis showed that the chlorophyll content (μg/L) and nitrite (mg/L) concentration had a significant impact on the species composition and community structure at the phylum level. Phosphate content (mg/L) and DO (mg/L) have a significant impact on the community structure of Vibrio. A total of 45 phyla were detected in the bacterial community structure of tissue samples, and the Tenericutes is the dominant phyla, mainly including Mycoplasma, which has a significant positive correlation with the phosphate content (mg/L). The results of the isolation and identification of the dominant bacteria of Vibrio showed that the dominant Vibrio in the culture environment from July to November were V. alginolyticus and V. harveyi. Studies have shown that aquaculture activities have a certain impact on the microbial community structure in the water by changing the content of chlorophyll and nitrite in the water. At the same time, changes in phosphate and DO have a certain impact on the community structure of the Vibrio genus. The results of this study can provide certain theoretical support for the occurrence of shellfish diseases.
To elucidate the tissue tropism and spreading routes of Haliotid herpesvirus 1 (HaHV-1) in susceptible Haliotis diversicolor aquatilis after infection, an in situ loop-mediated isothermal amplification (LAMP) method for HaHV-1 was developed in this study based on LAMP and in situ hybridization techniques. The developed in situ LAMP method was then applied on pedal nerve branches, mantle, hepatopancreas and gill of specimens collected across the experimental infection process. For the establishment of the HaHV-1 in situ LAMP detection method, we firstly optimized LAMP primers for specific detection of HaHV-1, improving the specificity and stability of the LAMP reaction on slides. We then optimized the time for color development (about 60 min) on pedal nerve branches, which was identified as the most suitable target for pathological investigation and in situ LAMP detection. Finally, the HaHV-1 in situ LAMP method was established after color development with immunoenzymatic labeling technique. The results showed that the incorporation of the designed loop primers yielded stabile results during LAMP amplification. The developed in situ LAMP detection method was applied on major tissue collected at 24、36、48、60 and 72 h across the experimental infection process. The in situ LAMP signals were firstly detected at 36 h on pedal nerve branches, and at 48 h on mantles after infection. The viral signals detected at mantle were confined to the peripheral nerves scattered throughout the mantle. Finally, in situ LAMP signals were identified occasionally in connective tissue of hepatopancreas of samples collected at the end of the experimental infection. The viral infection signals were always accompanied by tissue lesions and infiltrated haemocytes infected with HaHV-1. The in situ LAMP method developed in this study achieved the goal of rapid detection and definite diagnosis of HaHV-1, and is helpful for investigating the tissue tropism and pathological characteristics in different susceptible species. The detection method constitutes a potentially valuable tool for the diagnosis of HaHV-1 infection, and studies associated with tissue tropism, invasion pathways and pathogenic mechanism of HaHV-1.
Micropterus salmoides is a high-quality freshwater fish among the main aquaculture species in my country. In recent years, diseases of M. salmoides have occurred frequently, which has caused a great impact on my country's aquaculture industry. Largemouth bass ranavirus (LMBV) is a pathogen that seriously harms the M. salmoides farming industry. In order to explore the pathological changes of M. salmoides after infection with LMBV, the relationship between the dynamic changes of virus content in the body and the occurrence of diseaseases. The typical symptom of diseaseased fish is muscle necrosis, commonly known as rotting. In this study, 723 diseased M. salmoides samples collected from 2019 to 2021 in Guangdong Province and surrounding areas were classified and sorted, and their liver, spleen and kidney tissues were collected for quantitative PCR detection.. The results showed that the rate of positive LMBV was 63.62%. In addition, the incidence and detection rate are higher in the summer high temperature season. LMBV positive samples were selected to inoculate CPB cells, and a total of 93 strains of LMBV were obtained. Through PCR amplification and sequencing analysis of the isolates MCP, ATPase, DNA polymerase and methyltransferase, the results showed that these genes are highly conserved in LMBV isolates. The identity of the MCP and ATPase gene is 100.0%, and the methyltransferase gene identity is 99.7%-100.0%, and the DNA polymerase gene identity is 99.8%-100.0%, indicating that the variation between LMBV strains is minimal. The representative strain was selected to inject 100 μL 3.72×106 TCID50/mL LMBV into the pectoral fins of M. salmoides to artificially infect the LMBV. The clinical symptoms show similar symptoms to those infected with LMBV in the natural state, including darkening and darkening of the fish body, surface ulceration, and liver atrophy, etc. Four-tailed fish were randomly selected at 0 h 4 h, 8 h, 12 h, 1 d, 2 d, 3 d, 4 d, 5 d, 6d after infection, and liver, spleen, kidney, stomach, intestine, brain, heart and gill tissues were taken to make paraffin sections with HE staining for histopathological changes analysis. qPCR was used to detect the tissue distribution and kinetics of the virus. The results showed that the viral load of LMBV gradually increaseased within 5 days after infection, and the virus was distributed in the heart, liver, spleen, kidney, stomach, intestine, brain and other tissues, and the highest viral load in the heart was 9.5×105 copies/mg. The minimum viral load in brain tissue is 2.9×102 copies/mg. The histopathological results after virus infection showed that LMBV can cause a variety of tissue necrosis of M. salmoides, among which liver, kidney and heart diseases become more serious. Typical pathological features include disorder of liver cell arrangement, dilatation of liver sinusoids, nucleus evacuation, renal tissue cell evacuation, Macrophage rupture, necrosis of ventricular membrane fibers, etc.
γ-aminobutyric acid (GABA) is an important inhibitory neurotransmitter in the central nervous system in mammals. In marine invertebrates, GABA has been reported to induce larval settlement and metamorphosis in many species. The pharmacological experiment was conducted to study the effects of GABA and its receptor inhibitors Bicuculline (GABAA receptor) and CGP52432 (GABAB receptor) on larval settlement and metamorphosis of M. coruscus. GABA receptor-associated protein (GABARAP) and GABAB2 receptor (GABAB2R) genes have been found in the transcriptome data of different developmental stages of M. coruscus. GABAB2R was highly significantly expressed in the pediveliger stage than in other development stages. A similar expression pattern was also observed using the real-time fluorescent quantitative PCR experiment, suggesting that GABAB2R may be involved in the larval metamorphosis. The results showed that 10-4 mol/L GABA induced 27.2%±3.0% of the pediveliger larvae metamorphosis. Bicuculline and CGP52432 significantly inhibited larval metamorphosis compared to the control larvae. The higher concentrations of antagonists showed more pronounced effects than lower concentrations. In addition, both GABA and its receptor antagonists inhibited the swimming behavior of mussel larvae. Both GABAA and GABAB receptor antagonists inhibited the larval settlement and metamorphosis of M. coruscus, suggesting GABA receptors may be involved in mediating larval settlement and metamorphosis of M. coruscus. The present study contributes to further exploring the mechanism of the GABAnergic signaling system regulating larval settlement and metamorphosis of M. coruscus.
Shrimp iridovirus disease is an acute and infectious disease that has widely broken out in crustaceans in China in recent years. Its pathogen is Decapoda iridescent virus 1 (DIV1) that belongs to a new genus Decapodiridovirus of family Iridoriridae. The DIV1 has a high transmission risk, wide host range, and extensive mortality, leading to serious economic losses in cultured shrimp in China. Some references have been reported on the etiology, pathology, epidemiology, detection and diagnosis of Shrimp iridovirus disease, but little is known about the molecular mechanism of viral infection and the host responses to DIV1. Here, we provided an overview of the latest research progresses on the discovery of DIV1, classification status, morphological characteristics. and chemical composition and physicochemical properties, and introduced the genomic information, pathogenicity, host ranges, route of transmission, detection and diagnostic technology, as well as the shrimp's responses. The disease prevention and control strategies and future studies on Decapoda iridovirus 1 were also suggested. This information will promote understanding of the pathogenic mechanism of DIV1, which will benefit virological research and the effective control of shrimp iridovirus disease.
Prolyl endopeptidase (PEP) is a serine protease which has been implicated in many biological processes, such as learning and memory, cell proliferation and differentiation, and glucose metabolism. A small number of reports have also suggested PEP participates in mammalian reproductive process by specifically degrading peptide hormones and gonadal hormones containing proline residues. Compared with mammals, research on PEP of mollusks is relatively scarce at home and abroad. This observation led us to investigate PEP and whether it is related to reproductive development of mollusk. We analyzed the natural PEP of Haliotis discus hannai and the protein expression level changes during gonad development. Fluorescence quantitative PCR (qRT-PCR) and enzyme activity analysis were used to detect the expression of the PEP in tissues of H. discus hannai. The enzyme activity and the expression level of PEP in H. discus hannai gonad, followed by the muscle. PEP was purified from the gonad by column chromatography with a molecular weight of 82 ku. The isoelectric point of PEP was 5.5 by two dimensional electrophoresis, it is similar to the isoelectric point of predicting PEP of abalone. The 182 peptides were obtained by LC/MS/MS analysis, which were consistent with the H. discus hannai prolyl endopeptidase of NCBI (KY214290). The analysis of enzymatic properties showed that the optimum temperature and pH of PEP are 25 °C and 6.0, respectively, high enzyme activity can be maintained at 15-25 °C and pH 5-8. Circular dichroism was used to analyze the effect of temperature on structure of PEP. The results showed that the secondary structure of PEP changed significantly as the temperature increased. When the temperature increases from 25 °C to 95 °C, the content of α-helix structure decreases from 12.5% to 5.8%, and the random coil increases from 24.4% to 28%, when the temperature was lowered from 95 °C to its optimum temperature (25 °C), the content of α-helical structure decreased by 1%, β-folding decreased by 4.6%, β-turn and random coiling structure increased by 1.5% and 2.4%, respectively, compared with the initial secondary structure at the same temperature. The secondary structure could not be restored, indicating that the thermal denaturing of PEP was irreversible. The fitting thermal denaturation temperature was 51.4±0.2 °C. In order to further analyze the protein and gene expression level of PEP in the development of H. discus hannai gonad, we divide abalone gonad development into different growth stages (preliminary, early, middle and late growth stages), the results of Western blot and qRT-PCR analysis showed that PEP could be detected in all stages of male and female. The expression level is highest in the middle stage of maturity of male gonad and the late stage of maturity of female gonad. These results indicated that the difference of PEP at developmental stages of H. discus hannai gonad demonstrated that it may be involved in the process of gonad development. The current study analyzed PEP of mollusks and speculated that PEP is associated with gonad development in H. discus hannai. It provides a theoretical reference for further research of the function of PEP in H. discus hannai gonad development.
Mannan oligosaccharide is a new kind of feed additives. However, the effects of manna oligosaccharides on fish flesh quality has not been well studied. In this study, 315 healthy juvenile Oreochromis niloticus with an average body weight of (2.2±0.2) g were randomly divided into three treatment groups: control group (35% carbohydrate), high carbohydrate diet group (HC, 45% carbohydrate) and high carbohydrate diet supplemented with mannan-oligosaccharide (HM, 45% carbohydrate, 0.5% mannan oligosaccharide). Three tanks for each group, and 35 fish reared in each tank. After the feeding trial for 10 weeks, the growth indicator, muscle nutritional composition and flesh texture were determined. Compared with the control group, the hepatosomatic index, muscle cohesiveness, the myofiber number, the content of inosinic acid and triglyceride, the proportion of delicious amino acids, and the relative expression of CAST gene, which could improve muscle growth, in the fish of HC group were significantly increased; the muscle hardness, gumminess and chewiness, myofiber diameter, cooking loss, the proportion of essential amino acids, the contents of fatty acids, diglyceride and phosphatidylinositol, and the relative expression of MyoG gene, which functions in the differentiation of muscle fibers, in the fish of HC group were significantly decreased. Compared with the HC group, muscle springness, the proportion of essential amino acids, contents of fatty acids and diglyceride and the CAST gene relative expression were significantly increased in O. niloticus of HM group; the muscle adhesion, the number of myofiber, the contents of inosinic acid, phosphatidylethanolamine and phosphatidyl choline, and the MyoG gene relative expression in Nile tilapia of HM group were significantly decreased. Compared with the control group, the weight gain rate, muscle cohesion and contents of triglyceride and fatty acids were significantly increased in fish of the HM group; the hepatosomatic index, muscle hardness and chewiness, content of phospholipid in the fish of HM group were significantly decreased. The above results indicated that with the addition of manna oligosaccharides in high carbohydrate diet, the meat yield of O. niloticus was greatly improved, the flesh quality was improved due to the elevated water holding capacity, texture and the nutritional value of amino acids, however the decreased phospholipid content should not be neglected.
In our study, a complete open reading frame of PyMFG was obtained by RT-PCR based on the sequence of differentially expressed genes (Contig-21827) screened by transcriptome sequencing of Pyropia yezoensis. Sequence analysis showed that the ORF of the PyMFG was 1 224 bp in length and encoded a polypeptide fragment of 407 amino acids with a molecular weight of 46.24 ku, and theoretical pI of 9.08. Domain analysis revealed that the protein contains a conserved TEA domain and a YAP domain, which belongs to the TEA-ATTS superfamily domain. We found that the protein and fungal conidia forming proteins clustered into one large branch with close genetic relationship through multiple sequence alignment and phylogenetic analysis. In addition, the comparison of the ability of releasing monospores and qRT-PCR analysis between different strains indicated that the expression trend of PyMFG in PY26W and PY26W' was highly consistent with macroscopic statistical results, while the trend in PY26R and PY26R' were exactly existed difference. By inference, the gene type and molecular mechanism regulating the formation and release of monospores in the parental (PY26W) and partial parental strain (PY26W') and the female parental (PY26R) as well as partial female strain (PY26R') existed certain difference, leading to the divergence between the macroscopic statistical results of monospores and the qRT-PCR results.
The excavation and utilization of excellent germplasm resources are pivotal in ensuring the supply of China’s high-quality aquatic protein. As Larimichthys crocea owns China’s highest production in marine aquaculture, and its germplasm resources serve as an indispensable material basis for the healthy and sustainable development of L. crocea breeding industry, it is of great strategic significance to carry out the collection, preservation, identification, evaluation, protection and utilization of L. crocea germplasm resources systematically. This paper has reviewed the history and present situation of germplasm protection and utilization of L. crocea, and analyzed the main current problems from the aspects of scientific research, breeding technology system, industry, and talents. Besides, based on the scientific and technological innovation of L. crocea seed industry and the creation of new varieties, the development direction creating major new species with such traits as disease resistance, stress resistance, high yield, high quality, and high compound feed conversion ratewas proposed to substantially expand the coverage of improved L. crocea and provide a fine bread guarantee for the healthy development of L. crocea industry.