Immunization with recombinant baculovirus expressing the VP6 protein of grass carp reovirus induces immunity in grass carp

Grass carp reovirus（GCRV）,a member of the genus Aquareovirus in the family Reoviridae（Attoui H,et al,2002）,is one of the most virulent aquareoviruses iden-tified so far,leading to high fatality rates（up to 85%）of fingerlings and yearlings of grass carp（Fang Q,et al,2007）.No commercial vaccines against GCRV infection have been developed as yet.

Morphological changes and allometric growth in hatcheryreared Chinese loach Paramisgurnus dabryanus(Dabry de Thiersant, 1872)

The length-weight relationship and allometric growth patterns of hatchery-reared Chinese loach, Paramisgurnus dabryanus(Dabry de Thiersant, 1872), were determined from hatching to 60 days after hatching. A best power model was estimated for the length-weight relationship during the early life stages. Positive allometric growth for the head segment, trunk length, tail length and eye diameter was also found in the early life stages, while body depth, tail depth, tail fi n length, pectoral fi n length and barbel length displayed a negative coeffi cient. During the subsequent early developmental stage, the growth coeffi cients showed a clear and common tendency towards isometry for all measured body ratios. The allometric growth changes in Chinese loach during the early stage are possibly the result of selective organogenesis directed towards survival priorities.

Heterosis and combining ability evaluation for growth traits of blunt snout bream(Megalobrama amblycephala)when crossbreeding three strains

The blunt snout bream(Megalobrama amblycephala)is a major aquaculture species in the Chinese freshwater polyculture system.In this study,complete diallel crossing of three strains(i.e.,Liangzi[LZ],Poyang[PY]and Yuni[YN])was used to evaluate the combining ability and heterosis effect of intraspecific crossbreeding on the growth performance(i.e.,body weight,total length,body length,body height,and Fulton’s coefficient of condition).The offspring produced from the three strains were reared in a communal pond for 20 months,and nine microsatellites were used to assess their pedigree.Of the749 offspring,708(94.53%)could be assigned directly to a single parental pair.Significant differences were observed in body weight(BW)among the different combinations,indicating that the combining ability of BW from each strain was significantly different.The general combining ability(GCA)of BW from the sire was much higher than that from the dam.The GCA for the dam and sire of the LZ and YN were the largest and the smallest,respectively.The special combining ability of YN$9 PY#was the largest,and YN$9 PY#showed significantly positive heterosis effects on all F1growth traits,except for Fulton’s condition coefficient(K)(P\0.05).In addition,significant positive linear correlations were found between the mean BW of corresponding progeny and the H E of the parental generation(y=600.7x-29.472,r=0.8651,P=0.0026),H O(y=1206.9x-518.14,r=0.7436,P=0.0216),and the polymorphism information content(y=1021.3x-331.31,r=0.8245,P=0.0063)as determined by a correlative test.The findings obtained from the study will be important for future considerations of M.amblycephala stock improvement programs.

Effects of transport stress on immune response, physiological state, and WSSV concentration in the red swamp crayfish Procambarus clarkii

Transport is an essential part of the aquaculture and research of the main freshwater aquaculture crayfish Procambarus clarkii in China.However,transport is often accompanied by a low survival rate.Assessing the physiological state of P.clarkii before and after transport may discover the cause of this high mortality rate.In this study,ice-cold and exposed-to-air transport methods were compared using an array of parameters,including relative expression level of heat shock protein 70(HSP70),content of serum glucose and cortisol,immune parameters(enzyme and immune-related genes),and white spot syndrome virus(WSSV)concentration were investigated to understand the physiological state of P.clarkii before and after transport,as well as the cause of dying crayfish on days 5 and 7 after transport stress.Histological sections of hepatopancreas,gills,and intestines reflected pathological changes.The survival rate of crayfish with ice-cold transport was significantly higher than that with exposed-to-air transport,and mortality peaked at 3-9 days after transport stress.A prolonged response to oxidative stress and short-term immunosuppression was present after transport,and the trend of the WSSV concentration in the hepatopancreas was similar to the mortality rate of P.clarkii.The contents of serum glucose and cortisol,antioxidant enzymes and immune-related indexes,and the concentration of WSSV in hepatopancreas of dying crayfish were significantly higher than those of vibrant crayfish on the 5th and 7th days after transport.The hepatopancreas,intestines,and gills of dying crayfish had varying degrees of damage,and the hepatopancreas and intestines were severely damaged.The results suggested that the death of P.clarkii after transport stress is caused by oxidative stress,the imbalance of reactive oxygen species regulation,and decreased WSSV resistance,which eventually led to irreversible tissue damage.The increase of WSSV in the body of crayfish might be the direct cause of crayfish death.

Transcriptional profiles in zebrafish atp7a mutants and responses of atp7a mutants to Cu stress

As a copper(Cu)transport ATPase,ATP7A plays an important role in maintaining Cu homeostasis in the body,but the developmental and physiological roles of atp7a in zebrafish embryogenesis are rarely studied.In this study,normal morphological phenotypes of atp7a^(−/−)homozygous zebrafish were observed at both embryonic and adult stages,however,atp7a^(−/−)larvae exhibited delayed touch response and obvious transcriptome changes.Compared with the WT(wild type),differentially expressed genes(DEGs)in atp7a^(−/−)larvae showed the enrichment in gene ontology(GO)terms related to several processes including ATPase activity,oxidoreductase activity,active transmembrane transporter activity,ion binding,and the citrate cycle.Furthermore,decreases in both ATP content and Na+/K+-ATPase activity in atp7a^(−/−)embryos and larvae were unveiled.57 overlapping DEGs were found both in WT stressed with Cu and in WT mutated with atp7a,and GO term analysis indicated the enrichment in the genes related to neurexin family protein binding and neuronal cell-cell adhesion.Moreover,42 overlapping DEGs in Cu stressed WT and Cu stressed atp7a^(−/−)were identified.GO term analysis showed an enrichment in the genes related to heme binding,implying that Cu was independent of the integral function of atp7a to affect heme binding.In addition,genes involved in the negative regulation of angiogenesis were down-regulated in atp7a^(−/−)mutants with and without Cu stress,which failed to occur in WT,implying that the integral function of atp7a is required for maintaining the normal expression of angiogenesis genes.The integrative data in this study demonstrated that atp7a is required for ion transport and angiogenesis,and for Cu-induced neurexin family protein binding defects,rather than for Cu-induced heme binding defects,during zebrafish embryogenesis.These findings provide possible clues for human diseases with ATP7A dysfunction and imbalanced Cu homeostasis.

Food odors alter behavioral responses and olfactory receptors expression in grass carp(Ctenopharyngodon idellus)

Olfaction,the sense of smell,is crucial for animal survival,especially in localizing prey or food.However,little is known about the fish behavioral responses to specific food odors and which olfactory receptor might be activated by the food odors.Here,we examined the evolutionary relationships and classified the subfamilies of olfactory receptors(ORs),and conducted behavioral and gene expression analysis in grass carp(Ctenopharyngodon idellus).We identified 196 intact ORs in the grass carp genome and classified them into 8 subfamilies:β,δ,ε,η,γ,κ,θ,andζ.12 representative ORs(4β,4δ,and 4ε)were highly expressed in the olfactory epithelium(OE)of 24-month-old grass carp.Behavioral exploration found that grass carp showed different behaviors to different food odors at a series of concentrations.Specifically,0.01μmol/L amino acid mixtures and 0.005×Hydrilla verticillata extracts strongly changed the preference index and swimming trajectories in 6-month-old grass carp.In addition,the two food odors stimulation differentially induced ORs expression in three tissues,especially strong in the olfactory bulb and brain.Altogether,our results suggest that olfaction plays an important role in the regulation of fish feeding behavior and provide a basis for further studies on the function and specific recognition of olfactory receptors in fish.

Genetic basis and biotechnological manipulation of sexual dimorphism and sex determination in fish

Aquaculture has made an enormous contribution to the world food production,especially to the sustainable supply of animal proteins.The utility of diverse reproduction strategies in fish,such as the exploiting use of unisexual gynogenesis,has created a typical case of fish genetic breeding.A number of fish species show substantial sexual dimorphism that is closely linked to multiple economic traits including growth rate and body size,and the efficient development of sex-linked genetic markers and sex control biotechnologies has provided significant approaches to increase the production and value for commercial purposes.Along with the rapid development of genomics and molecular genetic techniques,the genetic basis of sexual dimorphism has been gradually deciphered,and great progress has been made in the mechanisms of fish sex determination and identification of sex-determining genes.This review summarizes the progress to provide some directive and objective thinking for further research in this field.

Essential roles of stat5.1/stat5b in controlling fish somatic growth

Signal transducer and activator of transcription 5b（STAT5b） has been identified as a key downstream mediator of growth hormone（GH） signaling in somatic growth of mammalian. However, the corresponding homologue gene of Stat5b is unknown in fish species. In this study, we generated loss-offunction mutants in stat5.1 and stat5.2, two stat5 homologues existing in zebrafish. In stat5.1-deficient zebrafish, a significant reduction of body length and body weight was detected in the embryos/larvae and adults compared with the wild-type control fish, and sexual size dimorphism in adult zebrafish was also eliminated. However, the stat5.2-deficient zebrafish displayed a normal developmental phenotype during all lifespan. Chromatin immunoprecipitation combined with deep sequencing（Ch IP-seq） method was adopted to further investigate the potential transcriptional targets of Stat5 protein and cast much light upon the biological function of Stat5. We identified more than 800 genes as transcriptional targets of Stat5 during zebrafish embryogenesis. KEGG analysis indicated that the Stat5 target gene network is predominantly linked to the metabolic pathways, neuroactive ligand-receptor interaction and JAK-STAT signaling pathways. Further validation studies suggested that Stat5.1 protein could directly regulate the expression of gh1, and stat5.1-mutated zebrafish showed a reduction of gh1 mRNA level. In the present study, stat5.1 was revealed as the corresponding homologue gene of Stat5b in fish species. Additionally,we found a novel molecular interaction between Stat5.1/Stat5b and GH, and unraveled a positive feedback loop Stat5.1-GH-Stat5.1 which is necessary for somatic growth and body development in zebrafish.

Loss of scleraxis leads to distinct reduction of mineralized intermuscular bone in zebrafish

Intermuscular bones(IBs)are ossified from tendons and only occur in lower teleosts.Positive association between the regulation of scleraxis gene(scx)and tendon development gave us reasons to speculate that the scx gene may play a potential role in regulating the development of IBs.A phylogenetic analysis conducted for this study revealed potential functional differentiation between two scx orthologues,scxa and scxb.The scxa^(-/-) and scxb^(-/-) zebrafish were generated through CRISPR-Cas9 technology to study the role of scx in the IB and rib development.The results showed a significant reduction of the number of IBs in adult scxa-1^(-/-) zebrafish,with almost 70%reduction(15-25 IBs)compared to the wild type scxa-1^(+/+)zebrafish(76-80 IBs).In the scxa-1^(+/+)adults,IBs were observed in both dorsal and tail segments;however,in scxa-1^(-/-)fish IBs were observed only in the tail segment(none in the dorsal segment).Although scxa-1^(-/-) zebrafish had rib defects,the mutants were viable and fertile as adult fish.The scxb^(-/-) zebrafish had the same number of IBs and same skeletal phenotype as the wild-type fish.This suggests that only scxa has a crucial role in the IB development,and confirms functional differentiation of scx orthologues.To further clarify the molecular mechanism by which scxa affects the IB development,we conducted comparative transcriptome analysis of dorsal tissue samples of scxa-1^(+/+)(with IBs)and scxa-1^(-/-) (without IBs),and further verified the expression of key genes via qPCR.This is the first study to identify a gene that controls the amount of IBs in fish,and it provides a new sight into the effects of scxa on the molecular mechanism of IB development in fish.

Sex differences in the expression of GH/IGF axis genes underlie sexual size dimorphism in the yellow catfish(Pelteobagrus fulvidraco)

Dear Editors,Sexual dimorphism is the systematic difference in size,shape,color,physiology,and behavior,between male and female individuals of the same species（Mei and Gui,2015）.Some studies have indicated that the traits of sexual dimorphism in vertebrates are the consequences of sex-biased gene expression and are controlled by multiple critical genes during growth and development（Williams and Carroll,2009）.However,the exact molecular mechanism underlying sexual dimorphism remains unclear.

Loss of stat3 function leads to spine malformation and immune disorder in zebrafish

STAT （Signal Transducers and Activators of Transcription） gene family members have been revealed to be involved in cell growth and differentiation in vertebrates. Despite their physiological importance, their functions are poorly studied at organ and systemic levels. In this study, we performed a genome-wide analysis using data from invertebrates to vertebrates to identify STAT genes and analyze their evolutionary history. Interestingly, the STAT gene family undergoes genome duplications during the evolutionary history with STAT3 homologues firstly appearing in the basal extant vertebrate, sea lamprey, suggesting its possible roles in spine formation. To investigate the functions of stat3 in fish species, TALEN technology was performed to generate mutant zebrafish lines, star3 mutant zebrafish showed no obvious defects at early developmental stage but displayed severe lateral and vertical curvature of the spine （scoliosis）, spine fracture and the incomplete bone joints with narrower junction between vertebrae at early juvenile stage, as indicated by Alizarin red and Alcian blue staining, radiography and micro-computed tomography （MicroCT） analysis. Transcriptome analysis reveals dramatic alterations in a number of genes involved in immune and infection response, skeletal development and somatic growth, especially downregulated expression of collagen gene family, in the juvenile stat3 mutant zebrafish. Moreover, most of the collagen genes were detected to have abnormal expression pattern during the formation of spine deformities in stat3 mutants. Our data reveal that stat3 is specially expressed in vertebrates and required for normal spine development and immune function in zebrafish.

Transcriptional responses of mu-, pi- and omega-class glutathione S-transferase genes in the hepatopancreas of Cipangopaludina cahayensis exposed to microcystin-LR

The glutathione S-transferases(GSTs)play important roles in detoxification of microcystins(MCs),but the responses of GSTs to MC-LR have not been well characterized in freshwater gastropod,Cipangopaludina cahayensis.In the present study,we cloned full-length cDNAs of mu-and pi-class GSTs(GSTM and GSTP)and partial cDNA of omega-class GST(GSTO),and determined the transcriptional responses of the three GST genes to different concentrations of MC-LR(0,1,10 and 100 lg/L)in C.cahayensis.The full-length cDNAs of GSTM and GSTP of C.cahayensis were 813 and 820 bp,containing an open reading frame(ORF)of 648 bp(encoding 215 amino acids)and 624 bp(encoding 207 amino acids),respectively.The mRNA expression of GSTM and GSTO significantly decreased after exposure to 10 lg/L MC-LR,and the mRNA expression of GSTP significantly decreased after100 lg/L MC-LR exposure.This might contribute to the detoxication of MCs in C.cahayensis,which is consistent with its sedentary life and filter-feeder status.The mRNA expression of the three GST isoforms in C.cahayensis could be used as biomarkers for water contamination.

Effects of dietary tert-butylhydroquinone on domoic acid metabolism and transcription of detoxification-related liver genes in red sea bream Pagrus major

Domoic acid (DA) is a neuroexcitatory amino acid that is produced by Pseudo-nitzschia during harmful algal blooms (HAB). Accumulation of DA can be transferred through food chain and cause neuronal damage in marine animal and in human. Like other algal toxins, DA was suggested to increase the oxidative stress and increase the detoxification-related gene expression in fish. The widely used food antioxidant, tert-butylhydroquinone (tBHQ), was known to induce a wide range of antioxidative potentials such as elevation of the glutathione levels and glutathione S-transferases (GSTs), via the activation of antioxidant response elements (AREs). In this study, the influences of dietary tBHQ on domoic acid (DA) metabolism and detoxification-related gene transcription were investigated both in vivo and in vitro. Oral administration of tBHQ resulted in significant decreases of DA accumulation of liver tissues in which red sea bream were fed with a single dose of 10 mg DA and 100 mg tBHQ per kg body weight per fish. Real-time PCR further revealed that the mRNA levels of AHR/ARNT/CYP1A1/GSTA1/GSTR were up-regulated in the above liver tissues at 72 h post tBHQ treatment. In consistence, tBHQ exposure also resulted in increased mRNA transcription of GSTA1, GSTA2 and GSTR in cultured red sea bream hepatocytes. Collectively, our findings in this research suggested that the dietary intake of tBHQ accelerated DA metabolism in fish, through mechanisms involving altered transcription of detoxification- related liver genes.

Effects of Bacillus subtilis on hepatic lipid metabolism and oxidative shess response in grass carp(Ctenopharyngodon idellus)fed a high-fat diet

Bacillus subtilis is widely used in aquaculture as a probiotic.However,few studies have been conducted to examine the effect of B.subtilis on liver lipid metabolism.A total of 135 healthy grass carp(50.24±1.38 g)were randomly divided into three groups:control(Con),high-fat diet(HF),and high-fat diet+B.subtilis(HF4-B.subtilis),and fed for 8 weeks.The results showed that compared with the HF group,the weight gain rate(WGR)significantly increased(Pc0.05)and the hepatic lipid content,serum low-density lipoprotein cholesterol(LDL-C),and aspartate aminotransferase(AST)decreased in the group supplemented with B.subtilis(P<0.05).Moreover,the hepatic mRNA expression of fatty acid synthase(FAS)was significantly down-regulated and the carnitine palmitoyl transferases(CPTlala)were up-regulated in the HF+B.subtilis group compared to the HF group(P<0.05),respectively.Additionally,in the HF+B.subtilis group,glutathione(GSH)significantly increased(Pv 0.()5),while hydrogen peroxide(H2O2)and malondialdehyde(MDA)contents significantly decreased compared to the HF group(P v0.05).B.subtilis may reduce the hepatic lipid content by inhibiting its synthesis and promoting p-oxidation of fatty acids.B.subtilis may also alleviate dyslipidaemia and prevent oxidative damage in the liver caused by the high-fat diet of grass carp.Hence,dietary supplementation with B.subtilis shows promise as a therapeutic or preventive tool against fatty liver disease.

Identification of three members of the Bmp family from yellow catfish Pelteobagrus fulvidraco and their transcriptional responses to a high fat diet

In this study,three members of the Bmp family were cloned and characterized in yellow catfish Pelteobagrus fulvidraco,including Bmp2a,Bmp4 and Bmp9.The predicted amino acid sequences of P.fulvidraco Bmp2a,Bmp4 and Bmp9 showed the characteristic domains of the Bmp family,including an N-terminal signal peptide,Arg-X-XArg site,TGF-β family signature and seven conserved cysteines,indicating that function is likely to have been conserved during evolution.mRNAs of the three Bmp genes had a variable level of expression in tissues.Compared to the control diet,a high fat diet tended to down-regulate the mRNA expression of Bmp2a,Bmp4 and Bmp9 in mesenteric fat,liver and ovary,while it tended to up-regulate their mRNA levels in muscle and kidney.The responses to dietary lipid status and the potential role in lipid metabolism have not previously been reported and reinforces the idea of their multiple functions.Our findings provide the first data about the potential role of the Bmp family in lipid metabolism in teleost.

Zebrafish ELL-associated factors Eaf1/2 modulate erythropoiesis via regulating gata1a expression and WNT signaling to facilitate hypoxia tolerance

EAF1 and EAF2,the eleven-nineteen lysine-rich leukemia(ELL)-associated factors which can assemble to the super elongation complex(AFF1/4,AF9/ENL,ELL,and P-TEFb),are reported to participate in RNA polymeraseⅡto actively regulate a variety of biological processes,including leukemia and embryogenesis,but whether and how EAF1/2 function in hematopoietic system related hypoxia tolerance during embryogenesis remains unclear.Here,we unveiled that deletion of EAF1/2(eaf1^(-/-)and eaf2^(-/-))caused reduction in hypoxia tolerance in zebrafish,leading to reduced erythropoiesis during hematopoietic processes.Meanwhile,eaf1^(-/-)and eaf2^(-/-)mutants showed significant reduc-tion in the expression of key transcriptional regulators scl,lmo2,and gata1a in erythropoiesis at both 24 h post fertilization(hpf)and 72 hpf,with gata1a downregulated while scl and lmo2 upregulated at 14 hpf.Mechanistically,eaf1^(-/-)and eaf2^(-/-)mutants exhibited significant changes in the expression of epigenetic modified histones,with a significant increase in the binding enrichment of modified histone H3K27me3 in gata1a promoter rather than scl and lmo2 promoters.Additionally,eaf1^(-/-)and eaf2^(-/-)mutants exhibited a dynamic expression of canonical WNT/β-catenin signaling during erythropoiesis,with significant reduction in p-β-Catenin level and in the binding enrichment of both scl and lmo2 promoters with the WNT transcriptional factor TCF4 at 24 hpf.These findings demonstrate an important role of Eaf1/2 in erythropoiesis in zebrafish and may have shed some light on regeneration medicine for anemia and related diseases and on molecular basis for fish economic or productive traits,such as growth,disease resistance,hypoxia tolerance,and so on.

Efficient gene editing in a medaka(Oryzias latipes)cell line and embryos by SpCas9/tRNA-gRNA

Generation of mutants with clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9(Cas9)is commonly carried out in fish species by co-injecting a mixture of Cas9 messenger RNA(mRNA)or protein and transcribed guide RNA(gRNA).However,the appropriate expression system to produce functional gRNAs in fish embryos and cells is rarely present.In this study,we employed a poly-transfer RNA(tRNA)-gRNA(PTG)system driven by cytomegalovirus(CMV)promoter to target the medaka(Oryzias latipes)endogenous gene tyrosinase(tyr)or paired box 6.1(pax6.1)and illustrated its function in a medaka cell line and embryos.The PTG system was combined with the CRISPR/Cas9 system under high levels of promoter to successfully induce gene editing in medaka.This is a valuable step forward in potential application of the CRISPR/Cas9 system in medaka and other teleosts.

The dysregulated autophagy signaling is partially responsible fordefective podocyte development in wt1a mutant zebrafish

Podocytes are essential elements of the glomerular filtration barrier and relevant to many kidney diseases.But the molecular mechanism in podocyte development is still unclear.Wilms tumor protein(WT1)is an essential factor for urogenital system development.Zebrafish have two wt1 genes,naming wt1a and wt1b.Here,we examined the roles of both wt1 genes in zebrafish embryonic development employing the CRISPR/Cas9 technology.The homozygous wt1a mutants displayed developmental malformations including pericardial edema,yolk sac edema and failure of glomerulus development.Loss of wt1a function disrupted podocyte differentiation and inhibited expression of podocin and nephrin.In contrast,the homozygous wt1b mutants were phenotypically normal and had no severe phenotypes of nephrogenesis defects that were observed in wt1b morpholino knockdown embryos.Therefore,wt1a plays an essential role in kidney development,whereas wt1b might not be essential for kidney development.Interestingly,from previous studies we knew that autophagy is involved in podocyte differentiation.By detecting the protein level of P62 and LC3A/B,we found the autophagy signal was greatly reduced in wt1a mutants.To better understand the relationship between autophagy signal and wt1a function in podocyte development,rapamycin was added to activate autophagy in wt1a-mutated embryos.As a result,podocyte injury was ameliorated in wt1a homozygous mutants by rapamycin treatment.In conclusion,our findings not only confirmed the essential function of wt1a during kidney development,but also provided a possible way for treatment of diseases involving podocyte injury.