Abstract:In order to investigate the role of the KLHL10 gene in the sex development of Hyriopsis cumingii, the full length of its cDNA was cloned using RACE, and KLHL10 expression levels in six tissues (gonad, gill, adductor muscle, foot, mantle, and hepatopancreas) were determined. Further, the quantitative real-time PCR was performed to compare expressions levels between 1-8 months of age and 12-, 24-, and 36-month-old H. cumingii, and its function was explored using RNA interference. The results showed that the full length of the KLHL10 gene was 2 361 bp, and the 5' non-coding region was 93 bp, the 3' non-coding region was 447 bp, and the open reading frame was 1 821 bp which encoded 606 amino acids. qRT-PCR results showed that the gene was highly expressed in the testis, and was expressed at the highest level at 6 months of age in early stage. Moreover, in 12-, 24-, and 36-month-old individuals, KLHL10 gene expression levels in the testis were higher than those in the ovaries(P<0.05). At the same time, the double-stranded RNA (dsRNA) interference experiments were also performed using three dsRNA strands. The results showed that the expression of KLHL10 in gonad was effectively reduced. According to the above results, it is speculated that the KLHL10 gene is a male-related gene in H. cumingii, which may participate in sex differentiation and testis development of H. cumingii.

Abstract:The transcription factor oct4 plays an important role in maintaining the pluripotency of stem cells, early embryonic development and reprogramming of somatic cells. Construction of the eukaryotic expression vector of oct4 gene from Megalobrama amblycephala (Maoct4) can provide a practical tool for further study on the mechanism of the gene. The target gene fragment was obtained from the pCS2+Maoct4 vector containing the ORF sequence of the Maoct4 gene, and was cloned into pCVpr to construct the infusion and recombinant vector of pCMV-Maoct4-Red. After enzyme digestion and sequencing identification, the recombinant vector was transfected into HepG2 cells by liposome, and the expression and localization of the red fluorescence in HepG2 cells were observed in the nucleus, and the fusion protein was also detected by Western blot using MaOct4 antibody. Therefore, the fluorescent eukaryotic expression vector of pCMV-Maoct4-Red was successfully constructed, which could be effectively localized in the nucleus in eukaryotic cells,which would lay the foundation for further study on the function of the Maoct4 gene.

Abstract:In order to explore the role of miR-192 in response to carbonate alkalinity stress in Nile tilapia, real-time PCR, bioinformatics, dual-luciferase reporter assay and miRNA inhibition in vivo were used. The results were as follows:(1) The real-time PCR experiments showed that the expression of miR-192 was significantly down-regulated, while solute carriers16A7,SLC16A7 was significantly up-regulated in gills of Nile tilapia after 6 hours acute alkalinity stress (6 g/L NaHCO₃)(P<0.05); (2) Bioinformatics analysis revealed that SLC16A7 might be the target gene of miR-192; (3) Dual-luciferase reporter assay showed that miR-192 directly regulated SLC16A7 by targeting its 3'UTR; (4) Inhibition of miR-192 significantly increased the mRNA level of SLC16A7 in vivo (P<0.05). In conclusion, miR-192 was involved in the adaptation of Nile tilapia to alkalinity stress,and SLC16A7 was the direct target gene of miR-192. This study provides a basis for exploring the mechanism of miRNAs in the response to alkalinity stress in Nile tilapia.

Abstract:The genetic diversity and structure of Protosalanx hyalocranius in four freshwater lakes in Jiangsu Province were assessed. Based on PCR amplification and sequencing technologies, the mitochondrial Cytb gene sequences from 160 individuals of four freshwater lake populations (Taihu Lake, Gaoyou Lake, Hongze Lake and Luoma Lake) were analyzed. The results showed that 25 variable sites were detected in 160 Cytb gene sequences, containing 11 singleton mutation and 14 parsimony informative sites. A total of 23 haplotypes were defined in all 4 populations and overall haplotype diversity (H_d) and nucleotide diversity (π) were 0.878±0.014 and 0.002 1±0.000 1, respectively. Therefore, the genetic diversity of P. hyalocranius showed the pattern of high haplotype diversity and low nucleotide diversity. In the four populations, the Gaoyou Lake and Taihu Lake populations had the highest haplotype diversity (0.871±0.031) and nucleotide diversity (0.002 9±0.000 1), respectively, and the Hongze Lake population had the lowest haplotype diversity (0.755±0.455) and nucleotide diversity (0.001 3±0.000 1). The result of AMOVA showed that 15.13% molecular variation was among populations and 84.87% within populations, and most molecular variation occurred within populations. The pairwise fixation index (F_st) revealed that there were significant differences between Taihu Lake population and Gaohu Lake, Hongze Lake and Luoma Lake populations, but there were no significant differences among Gaohu Lake, Hongze Lake and Luoma Lake populations. It is concluded that the Taihu Lake population and the other three lake populations should be managed and protected as different evolutionary units. Both the mismatch distribution analysis and the neutrality tests indicated that there was a recent population expansion in P. hyalocranius, which might have occurred 103 000-41 000 years ago during the late Pleistocene. Despite the high genetic diversity, P. hyalocranius already showed obvious signs of decline. More attentions should be paid to the management of fisheries in order to protect the germplasm resources of P. hyalocranius and to maintain the sustainable development.

Abstract:In order to investigate the response of male and female Nile tilapia (Oreochromis niloticus) to continuous high temperature habitat, the morphological characteristics of female and male fish were continuously monitored in the 36℃ experimental group and the 28℃ control group, and the brain, back muscles and gill tissues of female and male fish treated for 70 days were analyzed by TUNEL staining method, and back muscles were selected for transcriptome sequencing. The results showed:The growth rate of the high-temperature treatment group was significantly slower than that of the control group, and the male fish grew faster than the female fish; TUNEL staining showed that the signal of apoptosis was the strongest in back muscle tissues, but weaker in other tissues; The results showed after high temperature treatment, 3 405 DEGs were identified in females and 4 645 DEGs were identified in males, with more up-regulated genes than down-regulated genes. Through the KEGG pathway cluster analysis, female fish were mainly involved in the cell cycle, purine metabolism and DNA replication and other pathways, and male fish were significantly enriched in the pathways of adrenaline signaling, myocardial contraction and tight connection in cardiomyocytes. The research results provide basic information for further exploring the adaptation mechanism of different sex Nile tilapia to high temperature environment.

Abstract:The effects of low salinity on the growth performance, serum biochemical indices, and non-specific immunity of Maccullochella peelii peelii were evaluated. After acclimating to different salinities, Maccullochella peelii peelii was cultured in four experimental groups for 60 days with salinities of 0, 3, 6, and 9, respectively. The results showed that:the final body weight was significantly decreased with the increasing salinity (P<0.05); Additionally, the feed conversion ratio attained an optimum value when the salinity was 0 (1.11±0.11); and the survival rate was the highest when the salinity was 3 (98.33%±2.89%). The serum indices showed that:Creatinine levels followed a decreasing trend with the increasing salinity; Urea nitrogen content increased with the increasing salinity levels, i.e. in the salinity 9[(7.00±0.26) mmol/L], which was significantly higher than that of group 0 (P<0.05). Notably, there were no significant differences in other indicators evaluated. The activities of non-specific immune enzymes were significantly affected by the salinity. The activities of alkaline phosphatase and lysozyme were the highest when the salinity was 3. Also, the activity of superoxide dismutase was the highest when the salinity was 6, which was significantly higher than that in other groups. The catalase activities of groups 0 and 6 were significantly higher than that in other groups. In summary, the water salinity level of 3 was found to be suitable for the cultivation of Maccullochella peelii peelii.

Abstract:The changes of individual morphology and oocyte characteristics of conger eel during the development of ovary were studied; meanwhile, the nutritional components of ovary, liver and muscle were investigated. The results showed that the ovary development of conger eel could be divided into 5 stages:the single-layer follicular cells stage, the oil drop stage, the yolk filled stage (early, middle and late), the nucleus polarized stage, and the mature oocyte stage. The diameter of the oocyte, nucleus, oil drop, and the yolk globule gradually increased with the development of the oocyte. The initial location of oil drops was around the nucleus, and preceded the yolk sphere. The oil drops were distributed in a ring shape and gradually extended toward the cell membrane through the oocyte development. The initial occurrence site of yolk sphere was in the cytoplasmic layer around the cell membrane, which was distributed in a ring shape as the oocyte developed and extended toward the nucleus. The gonadosomatic index, hepatosomatic index, ocular index, and anal length index all showed positive correlations during development. The digestive gut index was negatively correlated with gonadosomatic index, hepatosomatic index, ocular index, and anal length index. The changes in nutritional composition indicated that the fat storage location is in muscle, and the fat transportation plays a critical role during the ovary development. The results of this study give an essential reference for the artificial reproduction of conger eel.

Abstract:The allometric growth pattern of Schizothorax waltoni Regan at the age of 0-90 days was studied by experimental ecology. The results showed that the inflection point of larvae growth from slow to fast was 28 days old. Most of the external organs of larvae of S.waltoni Regan had the characteristics of allometric growth. Head and tail grew faster than trunk. Head length, anterior anus length and posterior anus length showed inflection points at 24-25 days, 36-37 days and 37-38 days respectively. Head height and body height presented growth inflection points at 40-41 days and 49-50 days respectively; eye diameter presented growth inflection points at earlier 18-19 days, which promoted the full development of eyes, so as to improve the ability of early larvae to eat exogenous food at the opening stage. Snout length presented growth inflection points at 42-43 days, which promoted the full development of mouth to adapt to different conditions. The growth inflection points of pectoral fin, dorsal fin, pelvic fin, caudal fin appeared at 18-19 days, 39-40 days, 43-44 days, 46-47 days and 37-38 days respectively. The relative total length of pectoral fin and pelvic fin showed positive allometric growth before and after inflection point, but the growth index after inflection point decreased significantly. The swimming ability of larvae of S.waltoni Regan greatly improved. The allometric growth pattern of larvae of S.waltoni Regan guarantees the full development of important functional organs, effectively guarantees the survival ability of larvae after the beginning of exogenous nutrition, and has important guiding significance for early wild resource protection and seedling production.

Abstract:EST-SSR markers were developed based on the transcriptome of Pyropia haitanensis and DNA fingerprinting was constructed using polymorphic SSR markers for the germplasm identification of the new strain SW-81 of Pyropia haitanensis. Experimental results:9 033 microsatellite loci were detected in the transcriptome data, of which the trinucleotide repeat motif was the absolute dominant type, accounting for 95%; Designing primers for microsatellite loci by Primer 3.0 software, and 187 EST-SSR primers were synthesized randomly to analyze the effectiveness, and the 92 pairs of effective primers were finally obtained, of which 42 pairs were polymorphism EST-SSR markers, with the polymorphism amplification rate 22.46%; DNA fingerprints were constructed using the polymorphism EST-SSR marker, which was suitable for 5 strains of P. haitanensis, providing an effective molecular detection method for the germplasm identification of the new strain SW-81. The results above showed that there were abundant microsatellite loci in the genome of P. haitanensis, and the gene sequence of the same loci varied among different strains, showing the polymorphism of amplification products. The polymorphism analysis based on EST-SSR marker can provide an effective means of molecular detection for the management and identification of germplasm resources.

Abstract:The effects of citric acid supplementation in diets were evaluated on the growth, nutrient utilization, and nitrogen (N) and phosphorus (P) secretion of largemouth bass (Micropterus salmoides). Three diets were prepared with the addition of monocalcium phosphate (MCP) at 5, 10 (low-P diets), and 15 g/kg (positive control) (P5, P10, P15), then 10 g/kg of citric acid was added into the two low-P diets of P5 and P10. The five diets were fed to juvenile fish[initial body weight (16.0 ±0.16) g] for 60 days. The results showed that the weight gain (WG), whole body P, crude protein retention, P digestibility, vertebral P and plasma P significantly increased, and N excretion, whole body crude lipid decreased with the increasing MCP level (P<0.05); P15 group showed the best performance among all the groups. The addition of citric acid in P5 diet significantly increased WG (+5.6%) and serum P level, and reduced FCR (-0.05) and N excretion (P<0.05), which reached the similar levels to the P10 group (P>0.05). The addition of citric acid in P10 diet numerically improved the growth performance with similar levels to the P15 group (P>0.05). The supplementation of citric acid in both P5 and P10 diets significantly promoted P digestibility and reduced P excretion (P<0.05). In summary, the addition in 10 g/kg citric acid in low-P diets can improve the growth and the utilization of P and diet in largemouth bass.

Abstract:The content of calcium in the gill and carapace of juvenile Eriocheir sinensis during the molting cycle was determined, and the histological structure of gill and carapace in different molting stages was observed by paraffin section and electron microscope. The results showed that the content of calcium in gill and carapace was the lowest in the ecdysis stage. The results of microscopic observation showed that the inner and outer cuticles at the end of gill lobes were separated, and the lumen of marginal vessel was not completely recovered. The cavity of marginal vessel returns to normal in the postmolt stage. The results of ultrastructural observation showed that in the ecdysis stage, the content in the cavity of gills was less, the staining was shallower, and the number of mitochondria and microvilli was large. In the postmolt stage, the contents in the cavity of gills increased, the staining deepened, and the number of mitochondria and microvilli was larger. The carapace was the thinnest in the ecdysis stage, and only had two layers of structure, the epicuticle and the exocuticle. The endocuticle did not form until the postmolt stage. In conclusion, the calcium content and tissue structure of Eriocheir sinensis are closely related to the molting stages.

Abstract:From May to November 2019, the water quality of four freshwater crab culture ponds (T1, T2, T3 and T4) and river supply sources were monitored, and based on nitrogen and phosphorus load and tailwater discharge standard, two models were used to estimate the carrying capacity and the optimal stocking density. The results showed that:During the entire breeding cycle, the TN of the experimental crab ponds within the surface water quality standard of Class Ⅲ, and the TP exceeded the standard in the early stage of aquaculture, and was better than the Class Ⅲ water quality standard at the end; according to the phosphorus emission standard, the carrying capacity of T1, T2, T3 and T4 in a culture cycle is 4 835, 3 894, 5 035 and 4 546 kg, respectively, and the stocking density is 12 012, 12 249, 12 154 and 16 770 ind./hm², respectively; according to the nitrogen emission standard, the carrying capacity of T1, T2, T3 and T4 in a culture cycle is 3 786, 2 999, 3 948 and 3 401 kg, respectively, and the stocking density is 9 406, 9 432, 9 530 and 12 547 ind./hm², respectively, T1, T2, T3 can increase by more than 50%, T4 can only increase by 19.75%. Combined with the actual situation, the culture capacity of river crab in the experimental pond was 2 006-2 598 kg/hm², and the cultivation density was 10 229-13 296 ind./hm²; compared with the phosphorus emission standard, the carrying capacity estimated based on the nitrogen emission standard is lower, which may be related to the excessive nitrogen content in the water supply of the aquaculture pond.

Abstract:The concentrations of heavy metals (Cu, Zn, Pb, Cd, Cr, Hg, and As) in the sediment which were collected from 17 ponds were determined to clarify their distribution characteristicsand evaluate their ecological risk in aquaculture ponds in Shanghai.Their distribution and sources were analyzed using multivariate statistics, and the ecological risk of heavy metal was assessed by three different methods.The results showed that the average concentrations of heavy metals in the surface sediment were as follows,Zn(58.36±23.41)mg/kg, Cr(41.96±11.92)mg/kg, Cu(20.61±7.76)mg/kg, Pb(10.87±5.83)mg/kg,As(7.81±2.12)mg/kg, Cd(0.13±0.05)mg/kg, Hg(0.055±0.03)mg/kg. There were no significant difference of the annual values of Cu,Zn,Pb,Cr, and As among the sediment from different aquaculture species(P>0.05),while the values of Cd and Hg in the sediment of shrimp and crab ponds were higher than fish ponds obviously (P<0.05). The order of geo-accumulation index (I_geo) of the heavy metals in the sediment were as follows, Cd > As > Cu > Zn > Cr > Hg > Pb, and more than 95%of the samples belong to unpolluted class. The potential ecological risk index (RI) of the heavy metals were 56.94, 76.51, and 69.65 in the sediment of fish, shrimp and crab ponds, respectively. They were all ranged to the low ecological risk grade.Evaluations of consensus-based sediment quality guidelines (CBSQGs) showed that the mean probable effect concentration quotients (M_PEC-Q) of samples were all lower than 0.5, which means that the combined effects of heavy metals in the sediment were not toxic.

Abstract:The samples of Pennahia pawak were collected at the Behai and Dongfang fishing ports along the coast of Beibu Gulf from 2008 to 2009 and 2018 to 2019. Samples were measured for biological data.Length frequency analysis (ELEFAN) technique with FiSAT Ⅱ software was used to analyze the growth, mortality parameters and exploitation rate to assess the resource utilization status in the two periods. The results indicated that the dominant body length was 11-17 cm and 10-15 cm, with the body length decreasing from 14.83 cm to 12.59 cm in the two periods.The dominant body mass were 30-100 g and 10-80 g, with the body mass decreasing from 85.88 g to 52.91 g. The average of body length and body mass decreased by 15.1% and 38.4%, respectively.The allometric parameter b in the two periods was 2.84 and 3.00, P.pawak tended to isometric growth.L_∞(asymptote length) declined 8.7% from 24.15 cm to 22.05 cm while L_c(first catchable size) declined 18.1% from 12.56 cm to 10.29 cm. The growth parameter K decreased from 0.39 to 0.32. The total mortality coefficient decreased from 1.39 to 1.17. The natural mortality coefficient of P. pawak was 0.98 and 0.90.The fishing mortality coefficients was 0.41 and 0.28. The exploitation rate decreased from 0.29 to 0.24, respectively. The population structure of P.pawak in the Beibu Gulf showed a tendency to miniaturize and it was not over-exploited.

Abstract:The seasonal sampling was conducted in the West Lake section of Qiantang River from 2016 to 2018. A total of 31 species of fish belonging to 27 genera, 9 families and 7 orders were collected, among which the most species were Cyprinidae (21 species), accounting for 67.74% of the total species. According to ecological types, freshwater settlement, middle and lower layers, omnivorous fish are the main species. The dominant species were Carassius auratus, Hypophthalmichthys molitrix, Coilia nasus, Aristichthys nobilis, and Cynoglossus gracilis. Compared with the historical records, the decline of fish resources is more obvious, only the river and sea migratory fish and estuarine fish species decreased by 16 species, and there is the problem of fish miniaturization and singularity. The Jaccard similarity coefficient shows that the fish species composition is at a moderate dissimilarity level between any two years. The ABC curve also shows that the fish community was stable in 2016 and 2018, while it was disturbed in 2017.The Hypophthalmichthys molitrix, Carassius auratus and Hypophthalmichthys nobilis are the most important fish resources in this section, accounting for 49.65%, 20.14% and 16.19% of the total catch weight respectively.Although most of the traditional indigenous economic fishes in the Qiantang River are supplemented by long-term breeding and releasing, no dominant population has been formed in this section.Although the fish diversity index fluctuated seasonally and annually, the changes did not reach a significant level, and the seasonal changes of the fish community structure were related to the living habits of the fishes and the hydrogeological characteristics in the water to a certain extent.It is suggested to strengthen the protection of fish resources in this section of the river from the aspects of strengthening law enforcement, scientific breeding and releasing, improving habitat conditions and so on.In view of the lack of investigation into the fish community structure in the Qiantang River estuary section, it is suggested to carry out a comprehensive survey on the fish community and fish habitat in the Qiantang River estuary section continuously, so as to provide scientific basis for the formulation of detailed fish protection measures.

Abstract:The neon flying squid (O. bartramii) is a short-lived species which is an important fishing and development target in the Northwest Pacific. In this paper, we used the grey correlation clustering method to cluster catch per unit effort (CPUE) of neon flying squid from May to December,1998-2017, and analyzed the effects of El Niño and La Niña events with different intensities and environmental factors on CPUE. The results show that the CPUE difference between the annual latitude groups and the annual longitude groups is more obvious. The monthly latitude groups are more seasonal than the monthly latitude groups. The groups with strong La Niña and weak La Niña events are significant. The higher CPUE of the annual latitude groups, the higher the sea surface temperature anomaly (SSTA) and the lower the chlorophyll a concentration anomaly (CHLA). The studies have shown that abnormal weather events of different intensities have different effects on CPUE in neon flying squid. Strong La Niña events can increase CPUE of neon flying squid, weak La Niña events can slightly reduce CPUE, and El Niño events above moderate intensity can cause CPUE to decrease significantly.

Abstract:In this study,the samples of Ommastrephes bartramii and Gonatopsis borealis were collected by the Chinese squid jigging vessels during September to November in 2018 in the high seas of north Pacific Ocean (41°05'N-43°45'N,154°05'E-159°43'E).Their beaks'characteristics were measured and analyzed. The results showed that the two species had extremely significant differences in mantle length and body mass. The principal component analysis showed that upper crest length(UCL) was the best for representing the beaks' external morphological features of O.bartramii, and upper crest length(UCL) and lower lateral wall length(LLWL) were the best for representing the beaks' external morphological features of G.borealis. The AIC(Akaike's information criterion) showed that:The relationships between ML and UHL, LHL, LLWL, LWL were best described by power function for O.bartramii, and relationships between ML and UCL, ULWL, UWL, LCL were best described by logarithm function for O.bartramii; The relationships between ML and UHL, LRL and LLWL were best described by power function for G.borealis,relationships between ML and UCL and LCL were best described by exponential function for G.borealis, the relationships between ML and URL, ULWLand LHL were best described by logarithm function for G.borealis. Stepwise discriminant analysis showed that the correct discrimination rates of upper and lower beaks were 92.70% and 84.50%, respectively. Meanwhile,the discriminant equations were built. The results indicated that the external shapes of beaks can be used to identify these two kinds of squids.

Abstract:Vibrio parahaemolyticus transcriptional regulator vtrB gene knockout and complementary strains were constructed, and the role for VtrB in bile resistance was also explored. Two pairs' primers were designed according to the vtrB gene in GenBank. The upstream and downstream flanking DNA fragments of vtrB gene were fused by PCR, and then cloned into the suicide plasmid pDS132. The recombinant suicide vector was transferred from E. coli S17-1 λpir to V. parahaemolyticus for homologous recombination. The vtrB gene mutants were screened by 10% sucrose. Complement strain C-ΔvtrB was constructed by transferring pBAD33-vtrB into ΔvtrB mutant strain. The survival rates of wild type, ΔvtrB mutant and C-ΔvtrB strains cultured in media containing 2% deoxycholate (DOC) were determined. Based on the principle of homologous recombination, the PCR amplification and transcription level detection results of the vtrB gene showed that V. parahaemolyticus vtrB gene mutant ΔvtrB and its complementary strain C-ΔvtrB were successfully constructed. The survival rates of the ΔvtrB strain was 17%, 4% and 1% in the presence of 2% DOC for 20 min, 40 min and 60 min, respectively. This was a significantly lower level of survival than that of the wild type and C-ΔvtrB strains (P<0.001). The results indicated that the VtrB is helpful to the bile resistance of V. parahaemolyticus.

Abstract:Euglena gracilis was used as raw material. EGPS was separated and purified using DEAE cellulose colum and Sephadex G-100. EGPS1-A, EGPS1-B, EGPS3-A were obtained. On this basis, the purity identification and antioxidant activity of polysaccharide fractions were analyzed. The polysaccharides with better antioxidant activity were determined by molecular weight and monosaccharide composition. The purity identification results showed the purity of the three polysaccharides was high. Infrared detection confirmed that the purity of the three polysaccharides had characteristic absorption peaks of polysaccharides. The results of antioxidant activity showed EGPS1-A had the highest antioxidant activity among the three polysaccharides. The molecular weight of EGPS1-A was 136.8 ku and it consisted of 9 kinds of sugars such as mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, xylose, arabinose, fucose, and the composition ratios were 0.072:0.430:0.049:0.010:0.260:0.620:0.530:0.180:1.010.