Genome-wide characterization of mapk gene family in black rockfish Sebastes schlegelii and their expression patterns against Edwardsiella piscicida infection

Mitogen-activated protein kinases(MAPKs)play pivotal roles in response to environmental stresses and bacterial infections.Compared with those in the higher vertebrates,studies of mapk gene family are still limited in teleost.Identification,characterization,classification,and expression profiling of totally 15 mapk genes in black rockfish(Sebastes schlegelii)were conducted.Phylogenetic relationships show that these mapk genes could be divided into extracellular signal-regulated kinase(ERK),c-Jun N-terminal kinase(JNK),and p38 sub-families.In addition,gene structures,syntenic analysis,and selective pressure analysis are performed to confirm their annotations.Results of selective pressure analysis indicate that mapk1,mapk3,mapk7,mapk10,mapk11,and mapk12 underwent significantly-positive selections,while the others genes such as mapk4,mapk6,mapk15,mapk8a,mapk8b,mapk9,mapk13,mapk14a,and mapk14b were under purifying selections.Moreover,results of qRT-PCR indicate that mapk genes in 8 healthy tissues displayed different expression patterns.The expression patterns of several mapk genes including mapk12,mapk13,mapk14a,mapk14b,and mapk15 were significantly changed in mucosal tissues after Edwardsiella piscicida infection.This study demonstrates that mapk genes in black rockfish play vital prevention roles against bacterial infection,which not only helps us understand the structure and function of mapk genes in black rockfish,but also provides a reference to understand the role of mapk genes in teleost immune responses.

Characterization of a CD209-Like Lectin in Black Rockfish(Sebastes schlegelii):Structure Features and Binding Activities as a Pathogen Recognition Receptor

CD209,a transmembrane lectin belonging to the C-type lectin family,can recognize carbohydrates on the surface of host cells and invading pathogens,and play an important role in cell adhesion and migration,pathogen recognition and immune activation.Although well characterized in mammals,CD209 is still under-researched in fish.Here,we report a CD209-like gene,which was named SsCD209like,in black rockfish Sebastes schlegelii,and analyzed its structure features,expression patterns and ligand-binding activities.SsCD209like displays structural similarities to mammalian CD209s,with a cytosolic tail at N-terminus,a transmembrane region and an extracellular part containing a neck region and a CRD at C-terminus.The extracellular region and the neck region of SsCD-209like can both form dimers,which is different with the tetramer in human homologue.This result demonstrates the multimerization of CD209 homologue in fish for the first time.The EPN motif,a functional motif participating in sugar binding and affinity determination,is conserved in the CRD of SsCD209like,which is consistent with the higher binding strength of this lectin to L-fucose,D-GlcNAc and D-mannose.The binding of SsCD209like to different bacteria strains and bacteria-derived pathogen associated molecular patterns(PAMPs)are also observed in a dose-dependent manner.Results in this study show the sequence and structure features of SsCD209like and demonstrate its binding properties as a pathogen recognition receptor,which promotes our understanding of CD209 homologues in fish and provides basis for more in-depth studies of this molecule in the future.

Expression profile of long non-coding RNAs in the intestine of black rockfish Sebastes schlegelii in response to Edwardsiella tarda infection

Long non-coding RNAs(lncRNAs)are a class of transcripts longer than 200 bp,which have been emerged as essential regulators in numerous biological processes.Black rockfish(Sebastes schlegelii)is an economic fish that widely cultured in the coastal areas of China,Japan,and South Korea.With the expansion of aquacultural scale,various pathogens have threatened its industry and reduced its economic values.It has been reported that lncRNA were involved in the immune response and metabolic pathway in teleost,while no study is available on identification and functional analysis of lncRNAs in black rockfish so far.Herein,this study was performed to identify lncRNAs in the intestine of black rockfish after Edwardsiella tarda infection.In our results,a total of 9311 lncRNAs were identified through highthroughput sequencing,and 102 lncRNAs were significantly regulated following challenge,which were predicted to target 3348 mRNAs.Results of Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analyses of the se target genes showed they were function in catalytic activity,hydrolase activity,defense response and peptidase activity,which involved in metabolic pathways and immune related pathways.In addition,47 lncRNAs and 8 differentially expressed mRNAs(DEmRNAs)showed co-expression at two or more infection time points with metabolism and immunity functions.Moreover,real-time quantitative PCR(qRT-PCR)was performed to verify the reliability of sequencing gene expression analysis results.This research laid the foundation for further investigation of the regulatory roles of lncRNAs in the intestinal immune response of black rockfish.

Use of calcein and alizarin red S for immersion marking of black rockfish Sebastes schlegelii juveniles

Black rockfish Sebastes schlegelii juveniles （30-40 mm total length） were immersed in a range of calcein （CAL） solutions at concentrations ranging from 50 to 250 mg/L and alizarin red S （ARS） solutions at concentrations ranging from 100 to 500 mg/L in filtered seawater （salinity 30） for 24 h. Fluorescent marks were detected in otoliths （sagittae, asteriscus）, scales, fin rays （dorsal, pectoral, ventral, anal, and caudal fin rays）, and fin spines （dorsal, ventral, and anal fin spines） after a 60-d growth experiment. With the exception of 50-100 rng/L CAL, acceptable marks were produced in the otoliths and fin spines by all concentrations of CAL and ARS. In particular, marks were clearly visible under normal light in the sagittae, asteriscus, and fin spines offish immersed in 200 500 mg/L, 300-500 rag/L, and 200-500 mg/LARS, respectively. Scales and fin rays had acceptable marks at much higher concentrations （≥50 mg/L CAL, ≥300 mg/L ARS for scales and ≥50 mg/L CAL,≥200 mg/L ARS for fin rays）. The mark quality was highest （i.e., acceptable marks were observed in all sampled structures after immersion marking） in fish immersed in 150-250 mg/L CAL or 300-500 mg/LARS. In addition, there was no significant difference in survival and growth of marked fish compared with controls 60 d post-marking （P〉0.05）.

Use of tetracycline hydrochloride and alizarin complexone for immersion marking black rockfish Sebastes schlegelii

We tested the utility of chemical marking techniques in the juvenile black rockfish Sebastes schlegelii. Juveniles （30-40 mm total length） were immersed in a range of tetracycline hydrochloride （TC） solutions at concentrations ranging from 300 to 500 mg/L, and alizarin complexone （ALC） solutions at concentrations ranging from 200 to 400 mg/L in filtered sea water （salinity of 30） for 24 h, respectively. Otoliths （sagittae, asteriscus）, scales, fin rays （dorsal, pectoral, ventral, anal, and caudal fin rays）, and fin spines （dorsal, ventral, and anal fin spines） were sampled and used to detect fluorescent marks after a 60-day growth experiment. With the exception of 300 mg/L TC, acceptable marks were produced in the otoliths and fin spines by all concentrations of TC and ALC. In particular, we observed clearly visible marks in the sagittae, asteriscus, and fin spines under normal light at concentrations of200~00 mg/L, 250-400 mg/L, and 250-400 mg/L ALC, respectively. Scales and fin rays had acceptable marks at much higher concentrations （_〉350 mg/L TC, 〉250 mg/L ALC for scales and _〉350 mg/L TC, 〉300 mg/L ALC for fin rays）. The best mark quality （i.e., acceptable marks were observed in all sampled structures after immersion marking） were obtained following immersion in TC at between 350-500 rag/L, and ALC between 300-400 mg/L. In addition, there was no significant difference in survival and growth of TC and ALC marked fish compared to their controls up to 60 days post-marking （P〉0.05）.

Isolation and Expression Analysis of FTZ-F1 Encoding Gene of Black Rock Fish (Sebastes schlegelii)

Sex related FTZ-F1 is a transcriptional factor regulating the expression of fushi tarazu (a member of the orphan nuclear receptors) gene. In this study, FTZ-F1 gene (FTZ-F1) was isolated from the testis of black rockfish (Sebastes schlegeli) by homology cloning. The full-length cDNA of S. schlegeli FTZ-F1 (ssFTZ-F1) contained a 232bp 5′UTR, a 1449bp ORF encoding FTZ-F1 (482 amino acid residules in length) with an estimated molecular weight of 5.4kD and a 105bp 3′UTR. Sequence, tissue distribution and phylogenic analysis showed that ssFTZ-F1 belonged to FTZ group, holding highly conserved regions including Ⅰ, Ⅱ and Ⅲ FTZ-F1 boxes and an AF-2 hexamer. Relatively high expression was observed at different larva stages. In juveniles (105 days old), the transcript of ssFTZ-F1 can be detected in all tissues and the abuncance of the gene transcript in testis, ovary, spleen and brain was higher than that in other tissues. In mature fish, the abundance of gene transcript was higher in testis, ovary, spleen and brain than that in liver (trace amount), and the gene was not transcribed in other tissues. The highest abundance of gene transcript was always observed in gonads of both juvenile and mature fish. In addition, the abundance of gene transcript in male tissues were higher than that in female tissue counterparts (P<0.05).

Growth Characteristics and Resource Evaluation of Sebastes schlegelii in Zhangzidao Artificial Reef Area

[Objective]The paper was to formulate catchable size and total allowable catch of Sebastes schlegelii in Zhangzidao artificial reef area.[Method]Based on analysis of length-weight formula,body length and weight growth equations,and instantaneous mortality rate,the inflection age and critical age of weight growth were calculated,and the biomass of S.schlegelii in Zhangzidao artificial reef area was estimated.[Result]The growth equation of body length was Lt=412.5×[1-e^-0.21（t＋0.65）]and the growth equation of body weight was Wt=1 734.2×[1-e^-0.21（t＋0.65）]^2.92.The inflection age and critical age for weight growth of S.schlegelii were 4.45 and 4.82 a,respectively.The biomass in Zhangzidao artificial reef area was about 456.8 t.[Conclusion]For S.schlegelii flock in Zhangzidao artificial reef area,the catchable length was about 271.2-281.7 mm,the catchable weight as about 509.4-569.5 g,and the total allowable catch was about 60.43 t.

Successful transplantation of cryopreserved spermatogonia in Sebastes schlegelii:A simple and suitable alternative approach for conservation of viviparous fish

Black rockfish(Sebastes schlegelii)is one of the most important marine economic viviparous fishes.Recently,germplasm degradation and genetic diversity reduction have occurred due to overfishing and long-term artificial breeding.Germ cell transplantation combined with cryopreservation may be an alternative way to protect genetic resources.However,in viviparous fish that undertake fertilization and embryo development in vivo,transplantation is more difficult than in oviparous fish,including selection of transplantation stage,isolation of germ stem cells,and preparation of sterile recipients.This seriously restricts the development of viviparous transplantation.Therefore,in this study,we aimed to explore a transplantation method suitable for these species.Donor cells were isolated from cryopreserved whole testes of 300–400​g male Sebastes schlegelii in May,labeled by PKH26,and intra-peritoneally transplanted into allogeneic larvae at 5–10 days post-birth.Subsequently,the development of donor-derived cells in recipients were continuously detected by fluorescence labeling,histology,microsatellite markers,and fecundity tests.The results showed that donors were rich in spermatogonia(75%)and recipients maintained a high survival rate after transplantation,with a rate of>20%at sexual maturity.Further,donor-derived cells successfully migrated(100%),colonized,and incorporated into the developing recipient gonad(93.33%).Finally,transplanted recipients could normally develop and differentiate into male and female individuals,with donor-derived gametes found in 65.38%of mature recipients.In the present study,we first establish a simple and suitable transplantation method for Sebastes schlegelii using immature males and specific larvae,which will serve as a promising tool in the protection of germplasm resources for this transplantation-restricted marine viviparous species.

Genome-Wide Characterization of the Hyaluronidase Gene Family and Their Potential Roles in Viviparous Black Rockfish(Sebastes schlegeli)

Black rockfish(Sebastes schlegelii)has evolved viviparous reproduction mode.Different from the oviparous fishes,the spermatozoa of black rockfish are stored on the surface of ovarian follicle for over four months until the egg mature in ovary,while the adaptive evolution of fertilization-related genes remains to be studied.In the present study,based on the genome and transcriptome information of black rockfish,a total of 10 hyaluronidase genes were identified by phylogenetic and sequence analyses,including hyal1,hyal2,hyal3,hyal4,hyal6 and spam1a,spam1b,spam1c and spam1d.The spam1 subfamilies,including spam1a,spam1b,spam1c and spam1d,were significantly expanded compared to other species.The adaptive evolution of hyaluronidase was further investigated by selection pressure analysis of branch model and branch site model.The results showed that only spam1 subfamily was positively selected with a large number of positive selection sites,and the evolution rate was significantly higher thanthose of other teleosts.Two positively selected sites,LYS-171 and GLY-164,were located in the neutral hyaluronidase activity domain,which was pivotal for SPAM1 to participate in enzymatic hydrolysis of the extracellular hyaluronic acid matrix and mediate acrosome reaction in mammals.Furthermore,spam1 was mainly expressed in spermatocytes,secondary spermatocytes,and spermatids of testis,whereas it was not found in metamorphic sperm and mature sperm based on the spatiotemporal expression analysis.All results indicated that spam1 might originate from the testis and might be an adaptation of viviparous trait in black rockfish.

Molecular characterization and expression patterns of nuclear androgen receptors in the ovoviviparous black rockfish Sebastes schlegelii

nARs are ligand-activated transcription factors associated with gonadal development and reproductive regulation.In this study,two nuclear androgen receptor genes(ara and arb)were cloned from an ovoviviparous teleost,black rockfish(Sebastes schlegelii).The phylogenetic analysis of nARs showed that nARαand nARβclustered into teleost nARαand nARβ,respectively,but differed from the nARs of tetrapods.Four module domains of the nuclear receptor superfamily are present in the black rockfish nARs,an N-terminal domain(NTD),a DNA-binding domain(DBD),a hinge region(HR)and a ligand-binding domain(LBD).Among the four domains,only the DBD and LBD of nARαand nARβare relatively conserved.Tissue distribution analysis revealed that ara was mainly expressed in the gonad,muscle,intestine and kidney in males and in female,while arb was mainly detected in the gonad,followed by the intestine,kidney and head kidney.During the gonadal development process,the expression levels of ara and arb were significantly decreased from the regenerating to late stages of spermatogenesis and significantly increased in the degeneration stage in the testis(P<0.05).In the ovary,the expression levels of ara and arb were not significantly different in different stages.In situ hybridization revealed that in black rockfish,both ara and arb transcripts were localized to the Sertoli cells of the testis in black rockfish.In general,the present study is the first to characterize and analyse the expression of nuclear androgen receptors,including ara and arb,in black rockfish.

复方中草药对许氏平鲉幼鱼生长和非特异性免疫酶活性的影响

为探究复方中草药对许氏平鲉(Sebastes schlegelii)幼鱼生长与免疫酶活性的影响,选取144尾重量相近的许氏平鲉幼鱼分为4组,每组设3个平行,每个平行12尾鱼。将黄芪、当归、红枣、干姜、甘草、白术六种中草药等比例混合后超微粉碎,分别按质量百分数0(对照组)、1%、2%、3%加入到许氏平鲉基础饲料中进行投喂,试验为期42 d。结果表明:与对照组相比,各试验组许氏平鲉的过氧化氢酶(CAT)、总超氧化物歧化酶(T-SOD)活性显著提高(P<0.05),1%组的增重率、特定生长率和摄食率显著高于其他组(P<0.05),总抗氧化能力(T-AOC)无明显差异(P>0.05),而饲料系数低于对照组及3%组(P<0.05)。综上所述,在饲料中添加复方中草药对许氏平鲉生长和免疫功能有显著提升作用,建议的最适添加量为1%。

许氏平鮋源嗜水气单胞菌分离鉴定与药敏试验

为了解青岛地区人工养殖许氏平鮋(Sebastes schlegelii)病原菌嗜水气单胞菌(Aeromonas hydrophila)的流行情况和耐药性情况。对2020~2021年采集青岛地区人工养殖场中病死许氏平鮋的肝脏、肠道等病料组织127份,采用常规的细菌分离鉴定方法和PCR方法进行嗜水气单胞菌分离鉴定,采用人工感染动物试验和K-B药敏纸片法分别检测嗜水气单胞菌分离菌株的致病性和耐药性。结果显示:从127份病料样品中分离到68株嗜水气单胞菌,分离率为53.5%(68/127),50株嗜水气单胞菌可以引起许氏平鮋死亡为致病性菌株;50株许氏平鮋对新霉素、氟苯尼考、大观霉素等9种药物耐药率在52.0%以上,对其他的药物耐药率为4.0%~30%。该研究为许氏平鮋源嗜水气单胞菌合理用药及防控提供了参考。

基于体长标准的大长山人工鱼礁区许氏平鮋资源评估

为了评价大长山人工鱼礁区许氏平鮋(Sebastes schlegelii)资源利用状态,为当地许氏平鮋资源的合理开发与管理提供科学依据,采用基于体长的贝叶斯评估方法(LBB)对2011-2015年大长山人工鱼礁区许氏平鮋资源进行了评估。研究结果表明大长山人工鱼礁区许氏平鮋最优体长Lopt为31cm,最佳开捕体长Lc-opt为28cm,平均M/K=1.5、F/K=4.82、Z/K=6.34、F/M=3.24,许氏平鮋最优现存资源量与原始资源量之比B/B0(F=M,Lc=Lc-opt)=0.366、实际年平均B/B0=0.038 6,许氏平鮋最优的单位补充量渔获量Y/R’(F=M,Lc=Lc-opt)=0.045 9kg,实际年平均Y/R’=0.003 44kg。

三种海水鱼类肌肉基本营养成分及感官品质分析

为指导消费者合理选择喜爱的海水鱼类,对大西洋鲑(Salmo salar)、白卜鲔(Euthynnus affinis)和许氏平鲉(Sebastes schlegelii)三种海水鱼类肌肉的基本营养成分、物理特性及肌肉质构特性进行比较分析,结果发现:在营养方面,大西洋鲑蛋白质和脂肪含量最高,白卜鲔次之,许氏平鲉最低;白卜鲔肌肉钙质丰富,优于大西洋鲑和许氏平鲉(P<0.05)。在肌肉颜色方面,大西洋鲑和白卜鲔肌肉均呈偏红色,其中大西洋鲑肌肉更偏橘红色,但许氏平鲉肌肉肉色呈现少红。在系水力方面,许氏平鲉肌肉蒸煮损失最大,达到了29.12%,显著高于其他两种鱼类(P<0.05)。在肌肉质构方面,白卜鲔和大西洋鲑肌肉硬度显著低于许氏平鲉(P<0.05),入口即化。