Abstract:As an important incretin, GIP (glucose-dependent insulinotropic polypeptide) is involved in regulation of glucose uptake and lipid deposition. To investigate the role of Gip in Cyprinus carpio, the gip was cloned from the foregut by RT-PCR (reverse transcription-polymerase chain reaction). The sequence was analyzed by bioinformatics and the mRNA expression was detected by Real-time PCR. The result showed that the ORF (Open reading frame) of C. carpio gip is 324 bp, which encodes 107 amino acids. Based on amino acids sequence, the structure of Gip precursor protein contains four domains: signal peptide, N-terminal domain, mature Gip peptide and C-terminal domain. The result of sequence alignment and phylogenetic tree showed that the identity of C. carpio Gip protein was the closest to that of D. rerio. The Real-time PCR result showed that the gip was widely expressed in multiple tissues, and had higher expression in the intestine and pituitary of C. carpio. In OGTT experiment, the gip expression was significantly increased in C. carpio foregut and brain after glucose treatment. In the fasting and refeeding experiment, the gip expression in the foregut was markedly decreased after fasting for 7 days, and was significantly increased after refed. In the feeding experiment, the gip expression level was increased in the high glucose and high lipid groups. In this study, C. carpio gip was cloned, and the expression level was regulated by nutrient status. The results provide the data base for illustrating Gip biological functions and theoretical foundation for investigating the regulation of glucose and lipid metabolism in fish.

Abstract:The olive flounder (Paralichthys olivaceus) occupies an important position in the mariculture. It has proved as an useful model for study of caudal neurosecretory system (CNSS). The CNSS is a unique neurosecretory structure of fish that may be involved in thermoregulation and reproduction. Previous studies suggest that follicle-stimulating hormone (FSH) signaling is involved in the temperature-dependent sex determination and reproduction in olive flounder. However, a direct effect of FSH on Dahlgren cells remains underexplored. Here, we examined the electrophysiological response of Dahlgren cell population of the CNSS to FSH and the transcription of related key genes of CNSS. In addition, the gonadosomatic index (GSI, %), the expression level of fshβ in pituitary, and FSH receptor gene (fshr), corticotropin-releasing hormone gene (crh), urotensin Ⅰ gene (UⅠ) and urotensin Ⅱ gene (UⅡ) in CNSS during the breeding season were also analyzed. We found that FSH increased overall discharge frequency and may have changed the discharge pattern from silent to burst discharge in a subpopulation of Dahlgren cells. The effect of FSH on Dahlgren cells discharge activity was blocked by the FSH-receptor binding inhibitor (FRBI-8). The mRNA levels of fshr did not differ between FSH treatment and control group. While treatment with FSH stimulated the expression of L-type voltage-dependent calcium channel (L-Ca²⁺). In addition, a positive correlation was observed between the fshβ mRNA levels and GSI or UⅡ mRNA levels during the breeding season. These findings proved for the first time that FSH could act as a modulator within the CNSS and enhanced our understanding of the physiological role of the CNSS in reproduction and seasonal adaptation.

Abstract:Changfeng silver carp (Hypophthalmichthys molitrix) is a new variety of silver carp bred by the Yangtze River Fisheries Research Institute of the Chinese Academy of Fishery Sciences. It has many advantages, such as a quick growth rate, a high body size, and a neat appearance, which are associated with more excellent growth characteristics than those of common H. molitrix. And growth traits are important economic traits of aquaculture species. However, the study on H. molitrix at home and abroad mainly focuses on developing microsatellite markers, analyzing genetic structure, and establishing the genetic linkage map. Few molecular markers associated with H. molitrix growth traits have been considered, and it is not known whether these markers can be used in the selection and breeding of Changfeng H. molitrix. This study aimed to screen out the molecular markers related to the growth traits of Changfeng H. molitrix to guide the germplasm identification and selective breeding. In this study, 15 polymorphic microsatellite markers were used to analyze the genetic diversity and growth trait association of Changfeng H. molitrix at 6, 17 and 36 months. The results show that a total of 51 alleles were detected at 6 months, and the average number of alleles was 3.40; a total of 42 alleles were detected at 17 months, and the average number of alleles was 2.80; A total of 46 alleles were detected at 36 months, and the average number of alleles was 3.07. The observed heterozygosity (H_o) and expected heterozygosity (H_e) of each month-old group Changfeng H. molitrix were higher than 0.5. The average PIC values were 0.555, 0.445, and 0.490 respectively at each age; indicating that the population was highly genetically diverse. The results of associations between microsatellite markers and growth traits revealed that locus BL55 and BL109 were significantly associated with body height at 6-month-old (P<0.05); S65 was found significantly associated with body height at 17-month-old (P<0.05); locus BL55 was significantly associated with total length (P<0.05), and BL106-2 and BL116 were significantly associated with body weight at 36-month-old (P<0.05). The different growth traits associated with different months of age of the same marker may be due to the different growth trends of Changfeng H. molitrix. Locus BL55 on chromosome 15 has the same allele containing DNA fragment length of 231 bp at different month ages, and it is preliminarily speculated that the genotypes 225/231, and 231/245, will have negative and positive effects, respectively. To further examine whether this locus can be used for molecular marker-assisted breeding in H. molitrix populations, two wild populations of H. molitrix from Yangtze River and Xiangjiang were selected for analysis. The results showed that the allele of the 231 bp fragment existed in both the Yangtze River and Xiangjiang populations. This result preliminarily confirmed that the marker BL55 was determined as a candidate marker related to growth traits, which has reference value for genetic improvement and selective breeding of H. molitrix.

Abstract:Genetic diversity is the cornerstone of species survival and evolution. For most aquatic animals, keeping the high genetic diversity is the guarantee of population life ability. Groupers are important economic fishes for mariculture due to delicious taste and rich nutrition. With the rapid development of grouper breeding technology, the artificial culture of grouper has become the second marine fish industry of China in 2019. The hybrid grouper [Epinephelus fuscoguttatus(♀)× E. lanceolatus(♂)] is the maximum yield aquaculture grouper with heterosis of growth and disease resistance. In recent years, more and more culture groups showed growth phenotypes variation and frequent diseases. The decline of genetic diversity and inbreeding depression are considered to be the common reasons for the decline of aquaculture bio-economic traits. In order to explore whether the decrease of population genetic diversity leads to the decrease of breeding traits of hybrid grouper, in this study, 27 EST-SSRs markers were used to analyze the genetic diversity and population genetic structure of 6 groups of the hybrid grouper in the main seed production area. The results showed that, the average number of alleles (N_a) ranged from 6.666 7 to 8.5556, the average number of effective alleles (N_e) from 3.511 6 to 3.959 0, and the average observed heterozygosity (H_o) ranged from 0.764 6 to 0.7943, the average expected heterozygosity (H_e) from 0.7091 to 0.750 4, average polymorphism information content (PIC) ranged from 0.662 2 to 0.706 4. The results of molecular analysis of variance (AMOVA) showed that the variances among the groups, individuals within the group, and all individuals of the cultured groups of hybrid grouper were all at a significant level (P<0.01), and principal coordinate analysis (PCoA) showed the difference mainly came from the individual. F_st between groups, genetic distance and cluster analysis showed that the HB and XC groups first clustered together, then clustered together with WT and HW groups, GC and QT groups first clustered together, FP groups were independent. Genetic structure analysis showed that all samples were divided into 5 theoretical groups (K=5). HB group was composed of FP, WT, XC and GC groups. The results of this study showed that the hybrid grouper population still had high genetic diversity, with clear grouping. The possibility of decline of hybrid varieties affected by parental inbreeding was not high. The variation of pathogenic organisms and irregular breeding management may be the causes of frequent diseases. Therefore, it is necessary to strengthen the management of non-toxic parent screening, aquaculture water and diseased fish, and cut off the vertical and horizontal transmission of viruses to achieve effective disease prevention and control. This study provides a theoretical basis for further genetic breeding and disease control of grouper.

Abstract:The number of chromosomes of Misgurnus anguillicaudatus (Cobitidae; Cobitinae; Misgurnus) is (2n=50) and that of Paramisgurnus dabryanus (Cobitidae; Cobitinae; Paramisgurnus) is (2n=48). Hybridization between M. anguillicaudatus (DD) and P. dabryanus (PP) can successfully obtain hybrid progeny(DP, PD) with karyotypes (2n=49), but the testes and ovaries of the hybrid progeny showed obvious developmental retardation. Self-crossing and backcrossing experiments of DP (DD♀×PP♂) showed that male DP were infertile. The sperm morphology and vitality of the loach M. anguillicaudatus (DD)， large scale loach P. dabryanus (PP), hybrid F₁ generation (DP) and (PD) were analyzed through computer assisted sperm analysis system (CASA), optical microscope, scanning electron microscopy (SEM), transmission electron microscopy (TEM) and flow cytometry technology in order to explore the reason of male sterility in F₁ generation of hybrid between M. anguillicaudatus and P. dableurus. The results showed that the sperm motility rates of DD and PP were 76.50%±0.70% and 75.17%±8.60% respectively at 30 s of activation, which were significantly higher than DP (3.65%±1.75%) and PD (2.68%±0.63%) (P<0.01), and the related motion parameters such as VAP, VCL, VSL of hybrid loach are significantly lower than DD and PP (P<0.01), which reveals that the the sperm of the hybrid loach are particularly inactive. Ploidy identification was carried out on the sperms of four species of loach in the sperms of DD and PP by flow cytometers. It was found that the sperms of DD and PP were mostly 1N cells, while there were a large number of 2N and 4N and a small number of 8N cells in the sperms of DP and PD except for the normal haploid sperm. The spermatozoa in DD and PP testes were dense, intact and uniform in length (33.71±2.12) and (34.28±1.83) μm, respectively, by SEM and TEM. The head length of sperm in the testis of DP and PD was significantly longer than that of DD and PP (P<0.05), while the tail length, midlength and full length of sperm in the testis of DP and PD were significantly shorter than those of DD and PP (P<0.05). DP and PD in the testis displayed a large number of abnormal sperm, such as multitailed sperm, binuclear sperm and big-head sperm. Head size showed obvious polymorphism, according to the head size measurements to estimate its ploidy, including normal sperm haploid sperm, at the same time have diploid and tetraploid sperms, or even higher haploid sperm. The abnormal sperm of these hybrid loach provided important evidence for its low sperm motility and male sterility.

Abstract:Habitats distributed in the Laizhou Bay were critical for the accomplishment of the life cycles of fish and productive fisheries, in the Bohai Sea or even in the Yellow Sea. Based on a pooled analysis of the historical data available for almost 40 years, and combined with the field survey of current fish habitat conditions, a long-term data set of early life resources surveys to marine Osteichthyes, carried out in the Laizhou Bay was built. Mathematical statistics and time series analysis were used to interpret the recruitment characteristics and long-term trends in early life resources assemblage structure, biodiversity, and synchrony. The analysis showed that the early life resources assemblage structure in the Laizhou Bay changed continuously. The seasonal differences in community composition, abundance index, predominant taxa, and species diversity to eggs and larvae fish assemblage were evident. The abundance index and taxa number of egg and larval fish assemblages declined to an all-time low around the early 2010s, after which it showed signs of recovery recently. The taxa number of egg and larval fish decreased from 44 in the 1980s, 34 in the early 1990s, 40 in the late 1990s, and 35 in the 2000s to 24 in the early 2010s; from the middle and late 2010s, it showed a certain degree of recovery, and recently increased to 38 species. The recent number of the pelagic eggs taxa is only sixty percent of that in the 1980s, and with its abundance less one-third of the 1980s. The number of the pelagic eggs taxa had fell to an all-time low around the early 2010s, with taxa number less than half of the 1980s; while, the abundance of the pelagic eggs fell to an all-time low around the late 2000s, with its abundance only one-twentieth of the 1980s. The number of larvae fish taxa decreased first and then increased in each season (except in winter), while the abundance of fish larvae first increased and then decreased, however, both showed an upward trend in winter. The recent number of the larvae fish taxa is only three-fourths of that in the 1980s, and with its abundance less than ninety percent of the 1980s. The number of the larvae fish taxa fell to an all-time low around the early 2010s, with the number of taxa only half of the 1980s; while, larvae fish abundance reached a historical peak around the late 2000s, with its abundance about 1.9 times as much in the 1980s. Small pelagic and benthopelagic taxa with short life span, rapid maturation, strong coupling to lower trophic levels, were the main components by relative abundance detected in the eggs and fish larvae collected across different survey periods. The dominant taxa substitution was obvious, otherwise, the substitution rate was significantly accelerated in recent years. A significant amount of interannual and decadal variability remained in the species biodiversity index during the same survey season. Habitat and temperature adaptation studies of the spawning stock showed that the number of taxa with different habitat types and different temperature adaptation types took on a tendency of first decreasing and then increasing, profiling in the survey seasons across different survey times; annual percentage of the continental shelf pelagic-neritic fish taxa increased, combined with the continental shelf demersal and benthopelagic taxa decreased. Compared with the survey result in the1980s, the taxonomic composition and abundance of the egg and larval fish assemblage changed considerably. These variations were determined by a complex array of continuously changing conditions interacting with the demographic variables of each species, which was the concrete embodiment of high turnover in fish community structure and decline of fishery resources under long-term effects of overfishing and environmental change. Systematically summarizing long term trends in early life resources assemblage structure, biodiversity, and synchrony in the Laizhou Bay, and looking at how communities have changed in the past can help us understand the mechanism of spawning habitats stability, which could provide scientific evidence on laying down certain measures for the conservation and management of fishery resources in the Laizhou Bay, and the following impact assessment. Such as fishing, pollution, mariculture, and large-scale coastal engineering projects on the relative effects of climate and anthropogenic-induced factors on marine fish populations.

Abstract:To construct recombinant Lactobacillus casei with ompA and flaA of Aeromonas hydrophila and to detect the growth and immunogenicity of their expression products to common carp, Cyprinus carpio, the target genes were cloned into Lactobacillus Shuttle expression plasmid pPG612, and electric-transfered into L. casei. Fish were fed with dietary mixed with different recombinant L. casei for 56 days, and we weighed and collected serum and tissues, and analyzed the growth and related immune index changes; feeding at 56 days after the immunization, the growth index results showed that the recombinant L. casei had no significant difference compared with the control group, which showed that it had no effect on growth, immune indexes. Results showed that level of IgM in serum was significantly increased, alkaline phosphatase (AKP), lysozyme (LZM), superoxide dismutase (SOD), catalase (CAT), Complement 3 (C3) and Complement 4 (C4) were also significantly increased, qRT-PCR detection found that the expression levels of Interleukin 1β (IL-1β), Interleukin 8 (IL-8), Tumor Necrosis Factor α (TNF-α), Nuclear factor-kappa B (NF-κB), Toll-like receptor 5 (TLR) and myeloid differentiation factor 88 (MYD88) in liver, spleen, kidney and intestinal tissues after immunization increased significantly to varying degrees; the expression of TLR5 in the Lc-mcs-flaA group was higher than that in Lc-mcs-ompA groups; after challenge, the immune protection rates of Lc-mcs-ompA and Lc-mcs-flaA were 59% and 54%, respectively, and the survival rate was significantly higher than control group. The constructed recombinant L. casei Lc-mcs-ompA and Lc-mcs-flaA immuned carp could stimulate the body to activate immune response, thereby increasing its survival rate. It is an oral solution for preventing fish Aeromonas hydrophila infection. The research and development of immune preparations laid the foundation and provided scientific theoretical basis.

Abstract:Singapore grouper iridovirus (SGIV), one of the most important viral pathogens in grouper, has caused great economic losses to grouper industry. To evaluate the risk of iridovirus disease in grouper aquaculture, a risk assessment model of grouper iridovirus disease was established using Delphi method and Analytic Hierarchy Process (AHP) in this study. The evaluation index system consisted of 1 target level (risk of grouper iridovirus disease), 5 criterion layers (including water quality, grouper health status, feeding management, aquaculture mode and aquaculture environment), and 20 risk factors at the index level (including water temperature, pH, dissolved oxygen, ammonia nitrogen, iridovirus infection, bacterial infection, etc.). The weight values of 5 criterion-level risk factors was W= {0.1298, 0.3672, 0.1733, 0.0327, 0.2971}. The higher weight values of risk factors, including virus infection(0.2485), incidence in neighboring areas(0.1384), water temperature(0.1123) and aquaculture density(0.1050), respectively, suggested that these factors might be crucial for the incidence of grouper iridovirus disease in aquaculture. Consistently, the infection experiments in laboratory confirmed that virus dose, density, and temperature all significantly affected the virus replication and the mortality of the fish. Thus, it was proposed that our risk assessment model could be used to assess the risk of iridovirus disease in grouper aquaculture.

Abstract:The immunomodulatory effect of oligochitosan has been demonstrated in several fishes. However, the underlying mechanisms of oligochitosan (OCS) on immune regulation responses are not well characterized. Here, to investigate the effect of OCS on the non-specific immunity and the mechanism of action in Epinephelus fuscoguttatus(♀)×E. lanceolatu (♂), the feeding trial in this study was conducted in quadruplicates with the control diet supplemented with OCS at different concentrations (0, 200, 400 and 800 mg/kg) for four weeks, in order to explore the effects of OCS on the immunomodulation of grouper. Subsequently, the effects of OCS on the immune and antibacterial enzyme activities were examined. The results showed that feeding OCS could significantly improve the enzymatic activities of glutathione peroxidase, lysozyme, catalase, alkaline phosphatase, glutamate transaminase and glutamate transaminase in grouper, but had no significant effect on superoxide dismutase. The addition of OCS could significantly increase the weight of the head kidney of grouper. Quantitative real-time PCR (qRT-PCR) was used to further address the immunomodulation stimulated by OCS, the results showed that the expression levels of IL-6 and IL-1β were significantly up-regulated from head kidney by the addition of lower concentrations of OCS, by contrast, addition of higher concentration of OCS inhibited the expression of IL-6, IL-1β and TNF-α via NF-κB pathway. It means that OCS has a bidirectional effect on inflammation in the grouper. The immunoprotective effect of OCS on grouper was detected by the Vibrio harveyi challenge test. When the groupers were challenged with V. harveyi, the H. E staining results showed that the morphology of head kidney in groupers fed with different concentrations was greatly improved compared with the control group, especially in 800 mg/kg OCS concentration group. Also inflammatory cytokines such as IL--6, TNF-α, IL--1β and NF-κB pathway genes significantly declined in qRT-PCR from kidney of groupers, indicaing that OCS could inhibit the inflammation response of mRNA expression inflammatory factors via NF-kB signaling pathway. Additionally, it significantly inhibited the expression of apoptosis-related genes like Fas, FADD, Bax and Caspase-3 activity. Taken together, appropriate addition of dietary OCS could improve the growth and innate immunity of grouper so as to play a role in the immune protection of the grouper against the infection of V. harveyi. Our results shed a new light on the application of OCS in aquaculture, and paved a new way for the prevention and control of the diseases of grouper.

Abstract:To identify the pathogen causing hemorrhagic disease of Rana nigromaculata tadpoles and screen out sensitive drugs to control the disease, in this study, a strain KD-CXB-1 was isolated from diseased tadpoles, and healthy tadpoles showed hemorrhagic symptoms similar to those of naturally infected ones after being artificially infected with strain KD-CXB-1. Meanwhile, a dominant strain consistent with the morphological, physiological and biochemical characteristics of strain KD-CXB-1 was isolated from the dead tadpoles artificially infected. These results indicated that strain KD-CXB-1 was the pathogen causing hemorrhagic disease of R. nigromaculata tadpoles. Moreover, based on strain KD-CXB-1, several experiments were performed including morphological observation, gram staining, physiological and biochemical tests, 16S rDNA gene sequencing, construction of phylogenetic trees, and drug susceptibility. The morphological observation showed that the KD-CXB-1 was a rod-shaped Gram-negative bacterium with the length of about 1 μm and rough surface. Strain KD-CXB-1 was identified as Aeromonas hydrophila by 16S rDNA sequencing and physiological and biochemical tests. Drug susceptibility tests indicated that strain KD-CXB-1 was highly sensitive to 10 drugs such as florfenicol, Syzygium aromaticum and Caesalpinia sappan; moderately sensitive to Prunus mume, Rhus chinensis and Scutellaria baicalensis, and insensitive to 8 drugs such as amoxicillin, Rheum palmatum and penicillin. In conclusion, our study proved that A. hydrophila was the pathogen causing hemorrhagic disease of R. nigromaculata tadpoles, and the highly sensitive drugs screened out by drug susceptibility tests may provide theoretical reference and practical basis for the effective control of hemorrhagic disease of R. nigromaculata tadpoles.

Abstract:Vibrio harveyi is a pathogen common in the marine fish culture of China. To explore the pathogenic mechanism of V. harveyi and develop effective methods for prevention and control V. harveyi infection, we extracted extracellular products (ECPs) from the high virulent strain H2LB1 and the low virulent strain H1SAI3 of V. harveyi. The proteins of ECPs were analyzed and identified by using the label-free proteomics method. According to the result of protein identification, we selected the high virulent strain-specific protein HutZ for further analysis. Firstly, qRT-PCR technology was used to detect the expression difference of of V. harveyi gene HutZ under culture conditions with different iron sources. Secondly，the gene HutZ was cloned to the expression vector，and recombinant HutZ protein was successfully expressed and purified. Then the purified recombinant HutZ protein was incubated with hemin, and the spectrum was scanned at 300-700 nm. Finally, Cynoglossus semilaevis were immunized with purified recombinant HutZ protein twice. After immunization, the serum titers of 1-7 weeks were detected by the indirect hemagglutination method, and the challenge experiment was performed after 4 weeks of immunization. The results showed that the expression of gene HutZ is related to the iron source; HutZ protein affected the absorbance of hemin; the antibody titer of C. semilaevis against HutZ protein reached up to 1∶128, and the immune protection rate was as high as 73.3%. These results indicated that HutZ protein is a vital factor for the iron uptake of V. harveyi and potential candidate antigen for developing effective subunit vaccines to prevent the V. harveyi infection.

Abstract:As an important economic shellfish in coastal area of China, abalone is susceptible to bacterial infection, especially Vibiro parahaemolyticus. Similar to other invertebrates, Haliotis discus hannai resists pathogen infection by innate immune response. Tissue inhibitors of metalloproteinases (TIMPs) are endogenous protein regulators of the matrix metalloproteinases (MMPs) family. As MMP-1 was engaged in the innate immunity of abalone, the involvement of TIMP in the regulation of MMP-1 expression and activity as well as the immune responses merits further investigation. In this study, the role of TIMP in the innate immune responses of H. discus hannai against V. parahaemolyticus infection and the interaction between TIMP and MMP-1 were investigated. The full length cDNA sequence of TIMP was obtained, and the expressions of TIMP in different tissues of H. discus hannai were analyzed post Vibrio infection. The cloned TIMP cDNA sequence was 2291 bp in length. NetNGlyc 1.0 Server and Netoglyc 4.0 Server were used to analyze the glycosylation sites of TIMP. The results showed that TIMP had three potential N-glycosylation sites, namely Asn at positions 47, 77 and 152, and a potential O-glycosylation site, namely Thr at position 108. Multiple alignment of TIMP amino acid sequences from different molluscs showed that the similarities of TIMP in H. discus hannai with those of H. diversicolor, Crassostrea gigas and Tegillarca granosa were 76%, 18.9% and 19.3%, respectively. In the early stage of V. parahaemolyticus infection, the expression of TIMP in hemocytes and gill tissues were significantly up-regulated. To study the interaction between TIMP and MMP-1 in the anti-Vibrio immunity of abalone, RNA interference technology was used to knock down the expression levels of TIMP and MMP-1. After the silencing of MMP-1 gene in abalone, TIMP expression was significantly up-regulated. When TIMP expression was inhibited, MMP-1 expression decreased significantly. These results indicated that MMP-1 expression was positively regulated by TIMP, while TIMP expression was negatively regulated by MMP-1 in abalone. In conclusion, the present study will help to reveal the interaction between TIMP and MMP-1 in abalone innate immunity against V. parahaemolyticus infection. It also provided new insights into the prevention and control of pathogens in abalone culture.

Abstract:To investigate the effects of addition of EM on the water quality and microbial community structure in polyculture of Eriocheir sinensis and Micropterus salmoides , one-month EM treatment experiment was conducted with period Ⅰ (April 10th), period Ⅱ (April 20th), period Ⅲ (April 30th), and period Ⅳ (May 10th) under the analysis of water indexes and microbiome structure. The 16S rRNA high-throughput sequencing results showed that the dominant phyla were Proteobacteria, Actinobacteriota, Firmicutes and Cyanobacteria during the whole experiment period. Meanwhile, the relative abundance of Cyanobacteria in the control group was significantly higher than that of the experimental group in period Ⅲ and Ⅳ. Compared with the control group, Limnohabitans (20.34%) was the most dominant at genus level in period Ⅰ, whereas Exiguobacterium occupied the most abundant position in other three periods (56.33%, 38.11%, 17.88%). However, Rhodoferax (10.37%), Exiguobacterium (47.67%), and Mycobacterium (36.01%, 42.27%) were the most abundant in each period of the control group. The Sobs and PD indexes of the experiment group were significantly higher than those of the control group in period Ⅲ and Ⅳ. Compared with the control group, the total nitrogen (TN) of the experiment group was significantly decreased during the whole monitoring periods, moreover, the content of ammonia nitrogen decreased significantly in period Ⅱ, Ⅲ and Ⅳ. Correlation analysis of environmental factors showed that total nitrogen had the greatest impact on the bacterial community, and Exiguobacterium as the dominant probiotics in the experimental group were positively correlated with TP and negatively correlated with TN, NH₄⁺-N, NO₃^--N, NO₂^--N. Studies have shown that the addition of EM could improve the water quality of aquaculture water, effectively inhibite Cyanobacteria, significantly optimize the bacterial structure, and had significant in-situ water remediation function. It may promote green and sustainable development of aquaculture.

Abstract:Freshwater shellfish resources are abundant in China. More than 470 species of freshwater shellfish have been recorded and are widely distributed in lakes, rivers and intermountain wetlands in China. Among them, the unique germplasm resources are very rich where the proportion of specific shellfish species in all major river systems in China is higher than 50%. Freshwater shellfish not only play an important role in the ecosystem, but also have great economic value. The resources of freshwater pearl mussel Hyriopsis cumingii, Cristaria plicata, as well as some edible shellfish, Sinotaia quadrata, Corbicula fluminea, etc. are fully exploited. However, due to the wide distribution and diverse habitats of freshwater shellfish, some other freshwater shellfish are only investigated and identified. On the whole, the germplasm resources assessment of freshwater shellfish in China is not systematic enough, and the protection, development and utilization of freshwater shellfish have not been paid enough attention. Therefore, in this paper, the species diversity and regional distribution of freshwater mollusks, the sustainable development and utilization of freshwater shellfish germplasm resources, the status quo of introduced freshwater shellfish germplasm resources and the current protection measures of freshwater mollusks germplasm resources in China were summarized, and then, some suggestions on the protection and sustainable utilization of freshwater shellfish germplasm resources were put forward. It is expected to promote the synergistic development of protection, exploitation and utilization of freshwater shellfish resources in China.