Abstract:The juvenile yellowtail kingfish (Seriola aureovittata) has strong physiological metabolism and the energy during the growth and development stage, and thus its nutrient requirements are significantly different from those of other carnivorous farmed fish. To identify the digestion and gastric emptying characteristics of S. aureovittata, and determine the optimal feeding interval, in the present study, the changes of contents weight, digestive enzyme activity in liver and intestine and pituitary appetite-related genes expression were evaluated during gastric emptying of the juvenile S. aureovittata (63.96±5.63) g. The degree of fitting of the gastric emptying curve was analyzed using a linear model, square root model, and cubic root model. The results showed that the wet weight of the gastric contents gradually decreased after the feeding and reached 0 at 18 hours, which was a type of linear descending gastric emptying. Hepatic amylase, lipase and chymotrypsin activities increased first and then decreased, followed by a rise and then decreased the "M" type of change trend during gastric emptying. Amylase activity increased significantly from 0 to 6 h after feeding (P<0.05), and lipase and chymotrypsin activity increased significantly from 0 to 9 h (P<0.05). The activities of the three enzymes significantly decreased from 9 to 12 h (P<0.05), and then increased from 15 to 18 h (P<0.05), with the highest value observed at 18 h. Intestinal amylase activity showed the similar results like hepatic amylase, whereas increased at 12 h with the highest value, subsequently significantly decreased until 24 h. The intestinal lipase and chymotrypsin activity increased significantly from 0 to 12 h after feeding (P<0.05), the highest value observed at 12 h, and then gradually decreased. The expression of neuropeptide Y (npy) and orexin (ore) mRNA levels were increased from 12 to 15 h and from 9 to 15 h (P<0.05), respectively. The highest values of npy and ore mRNA was that at 15 h, and then significantly decreased until gastric emptying completed (P<0.05). The residual percentage of gastric contents was negatively correlated with gastric emptying time and the expression of npy mRNA (P<0.05), while npy mRNA was positively correlated with gastric emptying time (P<0.05). In conclusion, the digestive enzyme activity in the liver and intestine of juvenile S. aureovittata was decreased to the lowest from 12 to 15 h after feeding. The best model cube root model for fitting the gastric emptying test data of juvenile S. aureovittata showed 80% gastric emptying time (basic recovery of appetite) and 100% gastric emptying time (complete recovery of appetite) were approximately 14.5 and 19 h, respectively. Meanwhile, the mRNA levels of npy and ore in pituitary of juvenile S. aureovittata obtain the highest value at post-feeding 15 h. Therefore, the optimal interval for feeding is recommended to be fed 14.5 h based on the characteristics of gastric emptying, hepatic and intestinal digestive enzyme activities and pituitary appetite-related gene expression. The relevant results not only help to clarify the optimal feeding interval of S. aureovittata, but also provide theoretical basis and technical support for research on its feeding law.

Abstract: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 were functional annotation. According to the relationship between KO functional annotation and Pathway, 9 483 unigenes were 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, with the 5 psu group having 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.

Abstract: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 confirm that ScCAT belongs to the CAT family, and is 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. 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 show that ScCAT is an important antioxidant enzyme, which participats in the immune response of S. constricta.

Abstract: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 (GABA_A receptor) and CGP52432 (GABA_B receptor) on larval settlement and metamorphosis of M. coruscus. GABA receptor-associated protein (GABARAP) and GABA_B2 receptor (GABA_B2R) genes have been found in the transcriptome data of different developmental stages of M. coruscus. GABA_B2R was significantly highly 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 GABA_B2R 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 GABA_A and GABA_B 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 GABAergic signaling system regulating larval settlement and metamorphosis of M. coruscus.

Abstract:The beak is the main feeding organ of cephalopods which contains abundant biological information and is widely used in cephalopod biology research. 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. Based on the 303 schoolmaster gonate squid samples 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. The results indicated that the ML contributed the most to beak pigmentation stage, which was 22.90%, followed by the LHL, SMS, BW, LRL, LWL, which contributed 16.50%, 14.4%, 11.90%, 11.70% and 11.60% respectively, while the LLWL and SS contributed less to beak pigmentation stage, which were 6.30% and 4.70% respectively. The analysis of covariance (ANCOVA) results showed that there was no gender difference in the relationship 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 significant relevance 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 Berryteuthis magister shevtsovi. 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 these growth factors.

Abstract:Fish tends 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 performed 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, when 0-year-old small yellow croaker accounted for 75. 7% of the toal. 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 constituted by various environmental factors in the areas adjacent to rocky islands.

Abstract: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 C. ciliate is highest in the western waters of Zhubi Reef lagoon (ZB-1), while the abundance of A. 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 was relatively high, and dissolved oxygen is the most important factor affecting microzooplankton community structure in these islands and reefs, with its most obvious influence on MJ-3 station. The growth rate of phytoplankton in the lagoon waters of the three major islands and reefs was 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. This study shows 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.

Abstract:Shellfish farming is an important part of China's aquaculture industry. With the continuous expansion of the scale of aquaculture, the unreasonable operation of the 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 the shellfish industry. In order to study the open aquaculture waters of offshore shellfish, the distribution of bacterial populations, the response of microbial community diversity to environmental factors and its relationship with the incidence of diseases in aquatic animals, we collected sea water samples from different locations 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), used 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 discussed 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.

Abstract: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, relative importance index (IRI), diversity analysis, cluster analysis, canonical correspondence analysis (CCA) and other methods were used 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 were 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 were 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 are important 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.

Abstract: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 contraction of gene families in C. nasus, in order to explore the special mechanism of C. nasus caused by changes in the core gene families 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 expanded 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 expanded gene families (including 508 genes) and 36 contracted gene families (including 21 genes) are less than 0.05. 508 genes of significantly expanded 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 expanded 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 have strengthened our understanding of the adaptive evolution of C. nasus.

Abstract: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: ① discovering new subtypes of 17β-HSDs in mollusks; ② analyzing the temporal and spatial characteristics of expression; ③ investigating the structure-activity relationship; ④ clarifying the impact of environmental pollutants and making the corresponding countermeasures; ⑤ guiding the practice and application of aquaculture industry. This review has referred to former research on 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.

Abstract: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 had 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.

Abstract: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 existing 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.

Abstract: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 number of which 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. Subsequently the sensitivity, specificity and application effects of the method were evaluated. The results showed that the cycle threshold value (C_t) 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, and 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 the future.

Abstract: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 of the C. gigas and C. angulata in the mariculture area of Sanggou Bay and Shenhu Bay were constructed, 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 (T_H), lower boundary of tolerance range (T_L) and half-saturation constant for food (F_H) 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 basis for the subsequent development of ecological models and the assessment of oysters’ carrying capacity.

Abstract: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 experiments. A mathematical model, as a research tool, provides an effective tool for the study of shellfish growth in varied water environments. In this study, a new model of the clam, based on the dynamic energy budget theory, was constructed. The developed model was parameterized based on the physiological and ecological data of clam obtained 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 fact that 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.

Abstract: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 increasing first and decreasing later(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 increased at first and then leveled off (P < 0.05), conversely, the activities of fatty acid synthase (FAS) were decreased at first and then leveled off (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 level off (P<0.05). The lipid content of liver, muscle and whole fish were all trended to level off 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 upregulate LPL mRNA expression and downregulate FAS mRNA expression of S. prenanti, enhance the activities of fat metabolism enzymes, promote the digestion and utilization of feed fat, reduce lipid deposition and protect liver health effectively.

Abstract: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 occured 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 significantly 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 0.40 g/kg.

Abstract: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, and 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 fish with LMBV. The samples showed similar symptoms to those infected with LMBV in the natural state, including darkening of the fish body, surface ulceration, and liver atrophy, etc. Four fish were randomly selected at 0 h 4 h, 8 h, 12 h, 1 d, 2 d, 3 d, 4 d, 5 d, 6 d 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 was 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.

Abstract:Scophthalmus maximus is an economic fish adapted to cold water at low temperature and 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 the damage of thermal stress on turbot heart and its mechanism in terms of physiologic, biochemical response and apoptosis gene expression 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 ℃-24 h. CK activity increased significantly with the escalation of thermal stress; LDH, SOD activity and MDA content reached their peak at 24 ℃. Expression levels of Bax and Caspase-3 decreased significantly after thermal tress, while, 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 thermal stress causes 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 ℃, causing severely damages of heart structure and even leads to death in turbot. The results showed that thermal stress causes 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's and other fishes' heart against 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.

Abstract: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. 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 most of the 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 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, and revealed the infection variation of M. honghuensis between the diseased and asymptomatic C. auratus gibelio.

Abstract:Blood cells play an important role in animal immune system. Accurate classification and functional analysis will facilitate the understanding of immune response mechanism. In this research, hemocyte populations from Litopenaeus vannamei of 17-23 g in 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, accounting 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 accounted 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 at 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 48 h, which should be related to the longer time required by virus 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, 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 can be distinguished with similar quantitative criteria and they respond to pathogen invasion in different ways after infection.