Growth, Mortality and Stock Assessment of Squaliobarbus curriculus (Actinopterygii: Cypriniformes: Xenocyprididae) from Lanxi Section of Qiantang River, China

Population dynamics parameters and stock status of Squaliobarbus curriculus (Richardson, 1846) were analyzed from May to September 2021 in the Lanxi section of Qiantang River. FiSAT II software program was used. The growth coefficient K = 0.21 year–1, asymptomatic length L∞ = 39.48 cm, and age at theoretical zero-length t0 = –0.74 years were estimated. The von Bertalanffy growth function was calculated as Lt = 39.48[1 – e–0.21(t + 0.74)]. The growth curve for weight had an inflection at 5.86 years, corresponding to 29.61 cm in standard length and 372.29 g in weight. The natural mortality rate (M), the fishing mortality rate (F), and the total mortality rate (Z) were calculated as 0.51, 0.61, and 1.12 year–1, respectively. The exploitation ratio (E) was 0.54, which is greater than the value of 0.5 suggested by Gull (1971), indicating a probable state of overdevelopment. The annual average stock number and biomass of S. curriculus in the Lanxi section of Qiantang River were 31.86 × 106 individuals and 3656.82 t, respectively.

Replacing fishmeal with soybean meal affects survival, antioxidant capacity, intestinal microbiota, and mRNA expression of TOR and S6K1 in Macrobrachium rosenbergii

Using alternative plant-derived dietary protein to replace fishmeal,combined with practical evaluation indexes,is a recent focus for aquaculture practices.An 8-week feeding experiment with giant freshwater prawn Macrobrachium rosenbergii post-larvae was conducted to determine the eff ects of replacing fi shmeal(FM)with soybean meal in the feed,in terms of growth performance,antioxidant capacity,intestinal microbiota,and mRNA expression of target of rapamycin(TOR)and ribosomal protein S6 kinase B1(S6K1).Four isonitrogenous diets with isocaloric value were prepared to contain 100%,75%,50%,or 25%FM as the protein source(dietary treatments FM100,FM75,FM50,and FM25,respectively).Each diet was fed to post-larval prawns(mean weight 0.045±0.002 g)twice a day in four replicates.No signifi cant diff erence in weight gain was observed among all groups,but the survival rate of prawns fed the FM50 and FM25 diets was signifi cantly lower than that of prawns fed the FM diet.The mRNA expression of both TOR and S6K1 were the lowest in hepatopancreas of prawns fed the FM25 diet.Superoxide dismutase activity of prawns fed the FM25 diet was significantly lower than that of prawns fed FM50.In contrast,the malondialdehyde content was signifi cantly higher in prawns fed FM25 as compared with those fed FM75.The proportion of fishmeal in the diet did not affect the composition of core(phylum-level)intestinal microbiota,but greater fishmeal replacement with soybean meal had a potential risk to increase the relative abundance of opportunistic pathogens in the gut when considered at the genus level.These results suggest that fishmeal replacement with soybean meal should not exceed 50%in a diet for post-larval M.rosenbergii.

Isolation of CYP17 Ⅰ, 3β-HSD and AR Genes from Spotted Sea Bass (Lateolabrax maculatus) Testis and Their Responses to Hormones and Salinity Stimulations

In this study, three cDNA sequences corresponding to cytochrome P450 C17(CYP17 Ⅰ), 3β-hydroxysteroid dehydrogenase(3β-HSD) and androgen receptor(AR) were isolated from spotted sea bass(Lateolabrax maculatus). The mRNA abundances of CYP17 Ⅰ and 3β-HSD increased from stage Ⅱ to stage V with a significant increase at stage Ⅴ, and the highest abundance of AR mRNA was detected at stage Ⅲ in testicular development cycle. CYP17 I, 3β-HSD and AR transcripts were obviously abundant in steroidogenesis tissues such as testis, brain, head kidney among others. Strong and positive signals were observed mainly in interstitial cell regions of L. maculatus testis as were measured with in situ hybridization method. Significant increases of CYP17 Ⅰ and3β-HSD transcripts were detected after 12-48 h hCG(human chorionic gonadotropin) and GnRHa(gonadotropin-releasing hormone analogue) treatments. However, an opposite relationship was found for AR in testis at the same time. In addition, decreasing trends of CYP17 Ⅰ and 3β-HSD mRNA were observed in testis of L. maculatus in freshwater group(FW) from day 2 to day 6, and mRNA abundance of AR increased in brackish water(BW) group from day 4 to day 8. These findings revealed that these three steroid synthesis genes are import for testicular development, hormone and salinity treatment, and provided also an insight into the mechanism of reproductive endocrine of L. maculatus.

Development of Nucleic Acid Sequence-Based Amplification Assay for Detection of Macrobrachium rosenbergii Nodavirus

A nucleic acid sequence-based amplification（NASBA）assay was established for the detection of Macrobrachium rosenbergii Nodavirus（MrNV）.The specific primers were designed according to the high conserved region of RNA2 sequence of MrNV.The 224 bp specific amplification product was obtained in positive sample determined with 3%agarose gel electrophoresis,while no product was generated from shrimp infected with other viruses including DNA viruses（IHHNV,WSSV）and RNA viruses（TSV,IMNV,YHV）.The detecting limit of the assay was 8pg nucleic acid,which is more sensitive than that of PCR method.

Variation Laws and Influencing Factors of pH in Fishery Waters in Quzhou City

[Objectives] This study was conducted to strengthen the understanding of the variation laws of pH in fishery waters. [Methods]Based on the investigation data of pH, dissolved oxygen(Do), water temperature(T) and chlorophyll a(Chl. a) of the fishery waters in Quzhou City from 2017 to 2020, the seasonal variation characteristics and influencing factors of pH in the fishery waters were analyzed and discussed. [Results] The pH of the fishery waters in Quzhou City was in the range of 6.40-11.3, and was characterized by the highest seasonal variation in spring and the lowest in winter. The correlation analysis showed that water temperature and pH were significantly positively correlated in spring and summer(P<0.05), and the photosynthesis of phytoplankton was the main reason that affected the pH of water bodies in spring and summer. There was a negative correlation between water temperature and pH in winter, and the pH in the natural waters had a significant negative correlation with water temperature(P<0.05). The balance of carbonate system in the water bodies was the main reason that affected the pH of water in winter. The level of water eutrophication(Chl. a) affected the pH of fishery waters to a certain extent. When Chl. a<10 μg/L, there was a significant positive correlation between the pH of fishery waters and Chl. a(P<0.05). Various environmental factors had different effects on the pH of different types of fishery waters. The stepwise regression analysis showed that the pH of natural waters was mainly affected by Chl. a, and the pH of aquaculture waters was mainly affected by water temperature and dissolved oxygen. [Conclusions]This study provides certain theoretical support for actual production management.

Community Characteristics Analysis of Eukaryotic Microplankton via ITS Gene Metabarcoding Based on Environmental DNA in Lower Reaches of Qiantang River, China

Eukaryotic microplankton plays an important role in water biotic community and in maintaining the stability of water ecosystems. Environmental DNA metabarcoding provides the opportunity to integrate traditional and emerging approaches to discover more new species, and develop molecular biotic indices that can be more rapidly, frequently, and robustly used in water quality assessments. In order to examine assemblages of eukaryotic microplankton in lower reaches of Qiantang River, ITS gene metabarcoding technology based on environmental DNA was carried out. As a result, various species of phytoplankton, fungi and zooplankton were annotated on. More phylum, classes and specieses of eukaryotic phytoplankton and zooplankton were found after compared communities taxa based on metabarcoding with that obtained from morphological examination. Nevertheless, Chlorophyceae was the most common assemblage both identified by using these two methods, also Mesocyclops leuckarti and Acanthocyclops bicuspidatus were both found to be the dominant species of Cyclopoida in the river. Additionally, the reads proportions of phytoplankton and zooplankton at the three freshwater sampling sites (Tonglu, Fuyang and Wenyan) decreased as temperature drop. Meanwhile, twenty classes of fungi were annotated on, of which the community characteristic was first researched in the river. There were significant spatial differences in values of Chao1 index for eukaryotic microplankton. Cluster analysis and Non-metric multidimensional scaling ordination further confirmed that the community composition of eukaryotic microplankton at class level for Jiashao-September sample had the most dissimilarity with the others.

Characteristics of a Critical Calcium Transportation Regulator: Plasma Membrane Ca²⁺-ATPase Involved in Calcium Homeostasis from <i>Hyriopsis cumingii</i>(Lea)

Plasma Membrane Calcium ATPase (PMCA) plays a critical role in transporting Ca2+ out of the cytosol across the plasma membrane. Here, a full-length cDNA sequence of plasma membrane Ca2+-ATPase gene was isolated from the gill of Hyriopsis cumingii (HcPMCA) by using SMART RACE technique. The entire cDNA was 5230 bp, including a 417-bp 5'-UTR, a 3588-bp ORF and a 1225-bp 3'-UTR, encoding a 1195-amino acid protein, and no putative signal peptide was predicted. Compared with PMCA homologs from seawater mollusks, HcPMCA had high similarity with them in both sequence and structure. Tissue-specific expression analysis revealed that HcPMCA mRNA was detected in all the sampled tissues, but was prominently expressed in the gill and mantle. When exposed to a serie of increasing Ca2+ that lasted for 7 days, the mRNA expression of HcPMCA in the mantle was slightly downregulated, but peaked at 60 mg/L. Moreover, the temporal expression of HcPMCA transcripts in the mantle after 60 mg/L Ca2+ exposure was shown to be bell-shaped, which was slightly downregulated at 24 h, but upregulated from 24 h to 48 h post-treatment, peaking at 48 h. The result of present study provides useful information for further studies on function and regulation mechanism of HcPMCA gene.

Distribution Characteristics of Methylmercury in Eriocheir sinensis Produced in Zhejiang Province and the Risk of Eating It

Based the detection of content of methylmercury in the meat and butter of Eriocheir sinensis cultured in Zhejiang Province by liquid chromatography-atomic fluorescence spectroscopy,the distribution characteristics of methylmercury were analyzed,and the exposure risk of methylmercury to consumers was evaluated. The results showed that the content of methylmercury in the crab meat was higher than that of the crab butter. According to China＇s national standard and industry standard,the content of methylmercury in the crab meat was lower than the limit and accorded with relevant regulations. According to the reference intake of methylmercury recommended by the World Health Organization（ WHO） and the United States Environmental Protection Agency（ US EPA）,the weekly intake of E. sinensis meat should not exceed 3.2 kg,and the daily intake should not exceed 200g.

Isolation and Identification of the Pathogens of Bullfrog Streptoccus Disease

The tested strain from typical clinical cases,and the physio-chemical characteristics,16 S rRNA gene sequence and drug sensitivity challenge property were analyzed.This isolate was determined as Streptococcus agalactiae,16 S rRNA gene fragment of 1 280 bp in length.Blast analysis showed that this fragment shared a 100% homology with the conservative region of Streptococcus agalactiae isolate（AB023574）,Streptococcus dysqalactiae isolate（HM146084.1,AB537922,EU075033）,Streptococcus difficilis isolate（AB112407） accessed in Genbank.The isolate was sensitive to ciprofloxacin,neomycin,spectinomycin,furadantin and enoxacin.The results lay foundation for the effective control,rational medication and vaccine preparation for Streptococcus disease on bullfrogs.

Study on Regulating Effects of "Aquatic Environment Protection Oriented Fishery" on Water Environment of Qiaodun Reservoir

In order to evaluate the regulating effect of ＂ aquatic environment protection oriented fishery＂ on aquatic ecosystem,the water quality,plankton community structure,biomass,diversity index,eutrophication index and ecosystem maturity evaluated with Ecopath model were compared from 2010 to 2012 in Qiaodun Reservoir of Zhejiang Province. It was indicated that the water quality had been improved obviously. The water transparency maximally increased by 130%,while TP,TN and CODCrwere reduced. The density and biomass of the plankton were decreased,so was the ratio of Cynobacteria in phytoplankton community. The Shannon-Weaver index was increased; the trophic level was decreased; the ecosystem maturity was improved. By all accounts,the ＂ aquatic environment protection oriented fishery＂ had positive effects on maintaining the balance of reservoir ecosystem and ensuring the safety of drinking water.

Effects of flow velocity on water quality and ammonia excretion in recirculating aquaculture system culturing juvenile largemouth bass(Micropterus salmoides)

Flow velocity plays an important role in recirculating aquaculture systems(RAS)and the growing practice of culturing juvenile largemouth bass(Micropterus salmoides).In this study,the effects of flow velocity on the water quality as well as the ammonia excretion were discussed from the perspective of actual production,and a polynomial model of ammonia nitrogen excretion was established,using the juvenile largemouth bass.Results showed that the range of ammonia nitrogen and nitrite nitrogen decreased with flow velocity increasing,while the number and volume share of large particles increased.According to the polynomial model,compared with the medium flow velocity(11 cm/s,2.45 body length(bl)/s),the ammonia excretion of juvenile largemouth bass at high(18 cm/s,4.00 bl/s),and low(4 cm/s,0.90 bl/s)flow velocity changed faster with time,and the excretion rate peaked at the 6th hour after feeding,earlier than that under medium flow velocity.Therefore,it is suggested to increase the flow velocity at the 5th hour after feeding and then decreased it at the 10th hour,to ensure better water quality in RAS culturing juvenile largemouth bass.

Effects of lighting mode on the growth performance,cortisol level and oxidative stress of juvenile Chinese three-keeled pond turtle(Chinemys Reevesii)

To improve the light environment and welfare of the turtle cultured indoors,the effects of lighting mode on growth performance,cortisol level,and oxidative stress of juvenile Chinese three-keeled pond turtle,Chinemys reevesii,were investigated in this study.The experimental turtles with an initial weight of 5.61±0.09 g were reared in tanks under four different lighting modes:three groups with light(lighting the basking area and water area,LBW;lighting the water area only,LW;lighting the basking area only,LB)and control group(no light,NL).The experiment was conducted for more than six months,with each group having three replicates.After 203 d of the experiment,the turtle in the LW group exhibited higher weight gain rate(WGR)and a specific growth rate(SGR,%/d)compared to other treatments.Also,results showed that the final body weight of the turtle exposed to LW was higher than that exposed to other treatments.On the physiological level,serum cortisol level in turtles exposed to LW was significantly lower than that in other treatments.Regarding oxidative stress,the level of catalase(CAT)in turtles exposed to LW and LB was significantly lower than that exposed to LBW and NL.The malonaldehyde(MDA)activity in turtles exposed to LW was significantly lower than other treatments.Based on the growth performance and health status,it is suggested that lighting the water area only is the optimal lighting mode for the juvenile threekeeled pond turtle cultured indoors.

Isolation and characterization of a novel strain(YH01) of Micropterus salmoides rhabdovirus and expression of its glycoprotein by the baculovirus expression system

As one of the most important aquatic fish,Micropterus salmoides suffers lethal and epidemic disease caused by rhabdovirus at the juvenile stage.In this study,a new strain of M.salmoides rhabdovirus(MSRV)was isolated from Yuhang,Zhejiang Province,China,and named MSRV-YH01.The virus infected the grass carp ovary(GCO)cell line and displayed virion particles with atypical bullet shape,300–500 nm in length and 100–200 nm in diameter under transmission electron microscopy.The complete genome sequence of this isolate was determined to include 11 526 nucleotides and to encode five classical structural proteins.The construction of the phylogenetic tree indicated that this new isolate is clustered into the Vesiculovirus genus and most closely related to the Siniperca chuatsi rhabdovirus.To explore the potential for a vaccine against MSRV,a glycoprotein(1–458 amino acid residues)of MSRV-YH01 was successfully amplified and cloned into the plasmid pFastBac1.The high-purity recombinant bacmid-glycoprotein was obtained from DH10Bac through screening and identification.Based on polymerase chain reaction(PCR),western blot,and immunofluorescence assay,recombinant virus,including the MSRV-YH01 glycoprotein gene,was produced by transfection of SF9 cells using the pFastBac1-gE2,and then repeatedly amplified to express the glycoprotein protein.We anticipate that this recombinant bacmid system could be used to challenge the silkworm and develop a corresponding oral vaccine for fish.

Application of a recombinant replicase to localize the Trionyx sinensis hemorrhagic syndrome virus and evaluate its effects on antiviral genes of T.sinensis

Trionyx sinensis Hemorrhagic Syndrome Virus(TSHSV)is an arterivirus newly discovered in Chinese softshell turtles.Little is known about the effect of antibodies against the virus or the distribution of the virus in different organs of infected turtles.In this study,a partial protein of TSHSV-HP4 was produced using a prokaryotic expression system,and its polyclonal antibody was generated.The polyclonal antibody was confirmed by western blot and dot enzyme-linked immunosorbent assay(dot-ELISA).The distribution of TSHSV in different organs of T.sinensis was examined by immunohistochemistry(IHC)and the expression of immune-related genes was analyzed using quantitative real-time polymerase chain reaction(qRT-PCR).The results indicated that the recombinant TSHSV-HP4 protein was successfully expressed,and the generated polyclonal antibody showed specific binding to viral particles in the lung tissues of infected turtles.The IHC assay indicated that the virus was highly localized in various cells,including intestinal lymphocytes,enterocytes,kidney epithelial cells,spleen cells,lung macrophages,and cardiomyocytes.The qRT-PCR analysis revealed that TSHSV was detected in all organs tested,including the lungs,liver,kidneys,spleen,and heart.The numbers of viral mRNA copies in lung and heart tissues were significantly higher in the virus-antibody group than in the virus group.The interferonstimulated genes(ISGs),myxovirus resistance protein 2(MX2)and radical S-adenosyl methionine domain containing 2(RSAD2)were highly upregulated in all groups of infected turtles.Antibody-dependent enhancement(ADE)seemed to occur after stimulation by the polyclonal antibody,because significantly greater expression of the two genes was detected in the virus-antibody group than in the virus group.Overall,these results are important in understanding the cell localization of TSHSV and the immune response of infected turtles.

Optimizing protein and lipid levels in practical diet for juvenile northern snakehead fish(Channa argus)

A 3×3 factorial feeding trial was conducted to evaluate the production response of juvenile northern snakehead fish(Channa argus). Nine diets containing 3 protein levels(45%, 48% and 51%) and 3 lipid levels(9%,12% and 15%) were formulated and fed to triplicate groups of juvenile northern snakehead(15.78 ± 0.09 g/fish) for 8 weeks. The formulated diets were named as P45 L9, P45 L12, P45 L15, P48 L9,P48 L12, P48 L15, P51 L9, P51 L12 and P51 L15(P-Protein, L-Lipid), respectively. Fish fed diets with the lowest protein and lipid combination(P45 L9) had the lowest growth performance. Weight gains(WG) of fish fed the 4 diets P48 L12, P48 L15, P51 L9, and P51 L12 were not significantly different(P> 0.05), but significantly higher(P < 0.05) than those of fish fed the other diets. Fish fed diets P48 L12 and P48 L15 had significantly lower(P < 0.05) feed conversion ratios(FCR) than the rest of the treatments. Protein retentions(PR) among fish fed the diets P45 L12, P45 L15, P48 L12, P48 L15, P51 L9, and P51 L12 were similar and significantly higher(P < 0.05) than those of fish fed the remaining diets. Protein sparing effect was observed in the treatments when fish was fed diets containing 45% or 48% dietary protein levels with dietary lipid increased from 9% to12%. Fish fed diets with 9% lipid tended to have lower viscerosomatic index(VSI), hepatosomatic index(HSI),and whole-body lipid. Increasing dietary protein level significantly increased(P < 0.05) liver moisture and lipid while dietary lipid level increased liver lipid. Intestinal lipase activity increased significantly(P < 0.05)with increasing dietary lipid and protein levels while intestinal a-amylase and protease activities were not significantly influenced(P > 0.05) by dietary treatments. Based on these results, the diet containing 48%protein with either 12% or 15% lipid is the optimal for supporting growth and feed utilization of juvenile northern snakehead under the current testing conditions.