Egg collection from hatchery-reared broodstock of spotted halibut Verasper variegatus treated with LHRH analog

The objective of this study is to establish a method for acquiring large quantities of high-quality eggs for artificial fertilization from hatchery-reared broodstock of spotted halibut Verasper variegatus. We estimated the optimum conditions for implantation of luteinizing hormone-releasing hormone analog (LHRHa) cholesterol pellet (LHRHa-CP) to induce ovulation from two experiments which focused on the dose and the timing combined with monitoring of oocyte development by ovarian cannulation. From the results of cannulation, vitellogenesis of hatchery-reared broodstock was suggested to occur normally, although final oocyte maturation was not initiated in the first clutch of oocytes. In late December, oocytes in the most advanced clutch still underwent vitellogenesis, having diameter of about 0.82?mm. LHRHa-CP implantation during this period had no remarkable effects, except for administration at high dose (100???g/kg). In contrast, in mid January, when oocyte diameter reached about 0.95?mm, ovulation occurred in most individuals, even at low dose (20???g/kg). In mid February atretic oocytes became remarkable and LHRHa-CP implantation showed much lower performance in terms of egg quality. The diameter of growing oocytes converged to about 0.95?mm, which was that of fully grown postvitellogenic oocytes. Thus, oocyte diameter is suggested to be an effective indicator to estimate the timing of LHRHa-CP implantation.     

圆斑星鲽线粒体基因组全序列结构及其进化

利用圆斑星鲽(Verasper variegatus Temminck et Schlegel)和相关鱼类的部分线粒体基因序列,设计出6对扩增引物,通过PCR扩增产物直接测序和引物行走(Primer walking)法测定圆斑星鲽线粒体基因组全序列,并对其进行结构与进化分析。圆斑星鲽线粒体基因组序列长17273 bp,其基因序列及构成都与其他硬骨鱼基本相同,包括37个基因(2个rRNA基因、22个tRNA基因和13个蛋白质编码基因)和2个非编码区(控制区和WANCY区)。在22个tRNA基因中,tRNAGln、tRNAAla、tRNAAsn、tRNACys、tRNATyr、tRNASer(第2个)、tRNAGlu和tRNAPro的编码基因位于L链上,其余则位于H链上。在13个蛋白质编码基因中,除了COⅠ基因的起始密码子为GTG外,其余均以ATG为起始密码子。蛋白质编码基因包括具有完整TAA终止密码子的ND1,COⅠ,ATP8,ND4L,ND5,而其他蛋白编码基因则具有不完全终止密码子。在L链上,ND6是仅有的一个蛋白质编码基因。圆斑星蝶的控制区包含1个终止相关序列区(ETAS)、6个中央保守序列区(CSB-A、B、C、D、E、F)和3个保守序列区(CSB-1、2、3)以及长串联重复区(Tandemly repeat sequence)。用NJ法和MP法对5个目22种鱼mtDNA的13个蛋白质编码基因的氨基酸序列进行系统分析,结果显示,圆斑星蝶与石鲽关系最近,鲽形目的鲆、鲽类与鲈形目的科(Carangidae)、鲷科(Sparidae)鱼类亲缘关系较近,而同属于鲽形目的塞内加尔鳎(Solea senegalensis)没有和任何目聚在一起,成为一个独立的分支。圆斑星蝶的基因组全序列序列已提交到GenBank,登录号为DQ403797,控制区单元型序列的GenBank登录号为DQ834444~7。     

圆斑星鲽促性腺激素释放激素基因克隆及表达特性

采用RACE技术,在圆斑星鲽(Verasper variegatus)脑中克隆到3种GnRH基因的cDNA序列: cGnRH-Ⅱ、sGnRH和sbGnRH.每种GnRH都包括1个信号肽、1个Gly-Lys-Arg连接序列和1个GnRH相关肽.其中, cGnRH-Ⅱ的cDNA全长为568 bp,编码85个氨基酸, ORF为255 bp,5′UTR为141 bp,3′UTR为169 bp.sGnRH的cDNA全长为457 bp,编码90个氨基酸, ORF为270 bp,5′UTR为41 bp,3′UTR为143 bp.sbGnRH的cDNA全长为381 bp,编码98个氨基酸, ORF为294 bp,5′UTR为48 bp,3′UTR为36 bp.分析了3种GnRH基因的编码氨基酸序列与其他脊椎动物的同源性,圆斑星鲽 GnRH 与鲽形目鱼类氨基酸同源性最高,其次为鲈形目鱼类.对3种 GnRH 基因的系统进化分析表明,圆斑星鲽 GnRH 基因与其他鲽形目鱼类亲缘关系最近,其次为鲈形目、鲑形目和鳗鲡目鱼类.荧光定量PCR分析表明,3种GnRH基因都在脑中表现出最高表达水平且具有性别特异性表达模式:雌性中不同组织的 GnRH mRNA 表达水平都相应高于雄性.组织表达分析表明, cGnRH-Ⅱ的 mRNA 仅在脑中表达,而sbGnRH在各个组织都有表达, sGnRH仅在脑、垂体和性腺中表达.脑中sbGnRH mRNA的表达水平在卵巢成熟过程中变化显著(P<0.05),而其他两种GnRH的mRNA表达水平变化不显著(P>0.05).本研究首次在圆斑星鲽脑中克隆到3种GnRH基因,其组织和季节表达水平变化表明sbGnRH可能是圆斑星鲽生殖调控的关键GnRH类型,本结果可为圆斑星鲽生殖调控机制和人工繁育技术研究提供理论支撑.     

水温对圆斑星蝶摄食和生长的影响

采用实验生态学的方法研究了水温时圆斑星鲽(Verasper variegatus)摄食和生长的影响.试验结果表明:水温对圆斑星鲽生长发育的影响明显,在适宜的水温条件下,圆斑星鲽的生长发育速度是不适宜水温条件下的5倍.圆斑星鲽的适宜摄食温度为15～24℃,最佳摄食温度为18～21℃,每天的平均摄食率为鱼体重的1%左右.水温<18℃或＞21℃时,摄食量开始迅速下降,到了12℃时,摄食率比最高时下降了80%左右,6℃时几乎不摄食.2～26℃水温是圆斑星鲽养殖生产的安全上下限.     

Pedigree analysis of recaptured fish in the stock enhancement program of spotted halibut Verasper variegatus

ABSTRACT:   Spotted halibut Verasper variegatus hatchery juveniles produced in 2002 were genotyped using three microsatellite DNA markers (msDNA) and then released into natural waters. Subsequently, recaptured individuals were examined using msDNA. In order to evaluate the effectiveness of the stock enhancement program, from the genetic point of view, a pairwise F _ST test was implemented to estimate the genetic divergence between the wild captive broodstock, the hatchery offspring and the recaptured samples. The analysis showed significant differentiation between the broodstock and recaptured samples. Pedigree determination using msDNA was used to calculate the effective population size of the recaptured stock, which was found to be very low ( N _e ≈ 8). Equal family survivability was observed between the two recaptured stocks, but not between the released and recaptured stock. The number of identified families was higher and more equalized in the hatchery offspring compared to the recaptured samples, where the number of families declined. This fact was caused by an unequal family survivability just before or just after release. Separately, the number of contributing parents to the hatchery offspring was lower than the broodstock census number. Consequently, these two facts caused the genetic divergence of the recaptured stock from the broodstock.     

圆斑星鲽卵巢发育的组织学和数量形态特征研究

研究了圆斑星鲽卵巢年周期发育的组织学规律及其数量形态特征变化.结果表明,圆斑星鲽产卵属1年1次成熟分批产卵类型,卵巢发育属非同步分批成熟类型.性成熟亲鱼在年周期发育中经历5个发育时期,同时可见5个发育时相的卵母细胞,不同发育时期的卵巢中卵母细胞类型和组成比例不同.性腺指数(GSD、肝脏指数(HSI)和肥满度(CF)在亲鱼的年周期发育中呈现规律性的变化,与卵巢发育呈现密切的关联.亲鱼卵巢发育的有效积温为1050日度.     

发酵豆粕对圆斑星鲽生长及表观消化率的影响

为探讨圆斑星鲽(Verasper variegatus)饲料中发酵豆粕的适宜添加量,本试验以0%、10%、20%、30%、40%的发酵豆粕添加在五组等蛋等脂的饲料中,分别替代鱼粉蛋白的0%,9.81%,19.63%,29.44%,39.26%,标记为A、B、C、D、E共5个试验组,每组设3个重复,每池15尾圆斑星鲽(49.0±1.0 g),经过10周的饲养试验,结果表明:随饲料中发酵豆粕水平的升高,特定生长率逐渐降低,A、B组显著高于其他各组(P<0.05),饲料系数则逐渐升高,A、B和C组显著低于D、E组(P<0.05)。营养物质表观消化率以B组最高,显著高于其他各组。综上所述,饲料中添加10%的发酵豆粕在一定程度上能提高鱼体对营养物质的表观消化率,且不会对鱼体生长造成不利影响,过量则抑制鱼体生长。     

不同生长期圆斑星鲽形态性状对体重影响的通径分析

圆斑星鲽(Verasper variegatus)作为中国鲽形目(Pleuronectiformes)养殖鱼类的重要代表种类,具有生长速度快,可食用部分多,肉质细嫩等特点。为了探究圆斑星鲽形态性状与体重的关系,本研究分别测定11月龄、14月龄和17月龄3个不同生长期圆斑星鲽的体重及全长(FL)、体长(BL)、体高(BH)、头长(HL)、头高(HH)、尾柄长(CL)、尾柄高(CH)、吻长(SL)共计8个形态性状,分别进行相关分析和通径分析,剖析形态性状对于体重的直接作用和间接作用;利用回归分析的方法,建立各个时期以形态指标为自变量,体重为因变量的最优线性回归方程。结果显示,1)不同生长阶段,影响体重的重点形态指标存在差异,影响11月龄圆斑星鲽体重的主要形态性状为体高和体长,14月龄阶段为全长、体高和头长,17月龄阶段为全长和体高;2)11月龄、14月龄和17月龄形态性状对体重(Y)的多元回归方程分别为:Y=-186.412+17.381XBH+10.517XBL,Y=-362.484+13.145XFL+20.172XBH+21.199XHL,Y=-816.147+24.121XFL+40.575XBH;3)除本研究所包括的形态指标外,体厚可能为另外一个影响圆斑星鲽体重的重要指标。本研究阐明了体重与形态指标间的相关关系,为圆斑星鲽的良种选育提供了生物学依据。     

A low body-color regulating ability of spotted halibut Verasper variegatus: Evaluation of the roles of melanin-concentrating hormone and proopiomelanocortin systems

In teleosts, melanin-concentrating hormone (MCH) induces body brightening. On the other hand, α-melanophore-stimulating hormone (α-MSH), derived from the precursor proopiomelanocortin (POMC), leads to body darkening. In many teleosts, the expression level of pmch is high in light backgrounds, while that of pomc is low in dark backgrounds. In the present study, we investigated the ability of spotted halibut Verasper variegatus to regulate body coloration. Intraperitoneal administration of MCH resulted in a dose-dependent brightening of body color. The body color and brain contents of pmch1 and pmch2 in spotted halibut acclimated to the black or white background were not almost affected by background color. In addition, pituitary mRNA levels of pomc-a, pomc-b, and pomc-c genes in fish acclimated to the white background often exceed those levels of fish kept under the black background. These results suggest that the weak response of expression levels of pmch genes in response to background color is a factor in the blunt ability of spotted halibut to regulate body color. The different profiles of pomc gene expression to background color compared to that reported in other teleosts may be another factor for the weak ability of body color change in the spotted halibut.

     

Growth and habitat of spotted halibut Verasper variegatus in the shallow coastal nursery area, Shimabara Peninsula in Ariake Bay, Japan

ABSTRACT:   To determine the growth performances and habitat characteristics of spotted halibut Verasper variegatus , successive samplings were conducted in the south-eastern coastal area of Shimabara Peninsula in Ariake Bay. The push-net samplings were performed at Kamaga beach from May 2003 to April 2004 to determine the horizontal distribution of larvae and juveniles, while the gill net samplings were done in the coastal waters of Tatsuishi beach from July 2003 to July 2004 to collect the larger juveniles and adults. A total of 478 individuals from metamorphosing larvae to 2-year-old fish (15.2–447.0 mm total length [TL]) were sampled. In particular, the 2003-year class fish were successively collected from May 2003 to June 2004 (15.2–350.0 mm TL; n  = 418) and the mean total length in March, June, September, December 2003 and May 2004 was 22.4, 82.5, 172.5, 203.9 and 296.4 mm, respectively. The present study found that metamorphosing larvae migrated into the estuarine tidal flats during March 2003; newly settled juveniles inhabited the shallow intertidal areas; and juveniles had a remarkably high growth in shallow coastal areas. It is suggested that young fish around 300 mm TL migrated from the nursery grounds in Ariake Bay to deeper waters around Tachibana Bay during May–June 2004.     

圆斑星蝶MHC ⅡB基因结构、多态性及组织表达分析

通过表达序列标签法和cDNA末端快速扩增(RACE)技术,分离和克隆了圆斑星鲽(Verasper variegatus)主要组织相容性复合体(MHC)IIB的全长cDNA序列,该cDNA全长为1 144 bp,5'UTR(untranslated region)为7 bp,3'UTR为450 bp,开放阅读框(ORF)长度为687 bp,可编码228个氨基酸,包含信号肽、抗原结合域(β1),IGC区(β2),跨膜区和胞质区5个结构域.同源分析表明,圆斑星鲽MHC IIB氨基酸序列与其他硬骨鱼具有49%-79%的同源性,与鼠、人、红原鸡和护士鳖的相似性较低,分别为34%,33%,31%和30%.圆斑星鲽MHC IIB基因含有5个内含子,与其他硬骨鱼不同,其β2结构域编码区内存在I个109 bp的内含子.根据获得的MHC IIB基因组序列设计特异性引物,在10尾野生圆斑星鲽中扩增了包括完整内含子1和外显子2的长度约388 bp的DNA片段,PCR产物直接测序后发现在270 bp的抗原结合域中共有23个位点发生变异,密码子第1位和第2位的变异明显高于第3位.利用荧光定量PCR分析组织表达发现,MHC IIB基因在健康圆斑星鲽9种组织中均有表达,但表达量存在差异,肾的表达量最高,肌肉的表达量最低,肾、心、脾脏和鳃的表达量显著高于肝、皮肤、脑、血和肌肉.本研究旨在为MHC基因家族的遗传进化分析、结构与功能的解析提供基础,同时为圆斑星鲽的分子免疫学和标记辅助育种研究提供参考依据.     

Occurrence and distribution of settling and newly settled spotted halibut Verasper variegatus and Japanese flounder Paralichthys olivaceus in shallow nursery grounds around Shimabara Peninsula, western Japan

The occurrence and distribution of settling and settled spotted halibut Verasper variegatus were compared with those of Japanese flounder Paralichthys olivaceus in shallow habitats around Shimabara Peninsula, western Japan, to elucidate the settlement and recruitment characteristics of spotted halibut, a rare pleuronectid flatfish in Japan. Push-net sampling conducted at nine sites in March and April 2003 collected spotted halibut from four nurseries located at eastern areas of Shimabara Peninsula in Ariake Bay; Japanese flounder were sampled from eight nurseries. Comparison of microhabitat distributions in a tidal-flat nursery during February?CApril in 2003?C2007 revealed that metamorphosing larvae and juveniles of spotted halibut, which showed the drastic body coloration (black to brown) and behavioral changes (frequent swimming to settlement), were distributed mainly in the lower edge of the intertidal area, whereas Japanese flounder inhabited both intertidal and subtidal areas. Metamorphosing larvae of spotted halibut (12.6?C16.5?mm standard length; n?=?24) were collected in March when the highest densities were observed, whereas metamorphosing larvae of Japanese flounder (10.0?C15.9?mm, n?=?55) were observed during March?CApril. Our results suggest that spotted halibut has a narrower time/space window for recruitment to the specific nurseries compared with Japanese flounder.     

Habitat utilization,feeding, and growth of wild spotted halibut <Emphasis Type="Italic">Verasper variegatus</Emphasis> in a shallow brackish lagoon: Matsukawa-ura,northeastern Japan

Habitat utilization, feeding, and growth of a rare pleuronectid flatfish, spotted halibut Verasper variegatus, were examined in a brackish lagoon in northeastern Japan: Matsukawa-ura. The distribution and date–length data of spotted halibut collected mainly from beam-trawl samplings during 1983–2008 indicated that age-0 juveniles [n = 25, 6.0–18.0 cm total length (TL)] and older spotted halibut (n = 71, 13.8–43.0 cm TL) inhabited almost the entire northern part of Matsukawa-ura. Comparative distribution surveys of spotted halibut, stone flounder, and marbled flounder during 1985–1989 revealed similar distribution patterns of these flatfishes; the highest densities were found around the central part of Matsukawa-ura, although significantly lower abundance was detected for spotted halibut. Linear growth equations of age-0 juveniles demonstrated that spotted halibut were able to achieve high growth after June, probably because abundant prey (e.g., mysids and gammarids) and suitable physical conditions (warmer water temperature and lower salinity) were present. The main prey items shifted from various crustaceans including mysids, caridean shrimps, and anomurans to brachyura Hemigrapsus spp. (≥20 cm TL). Our study shows that spotted halibut use the shallow brackish lagoon as an important nursery for juveniles, and also as a feeding ground for young and adults.     

Horizontal movement and emigration of juvenile spotted halibut <Emphasis Type="Italic">Verasper variegatus</Emphasis> released in a shallow brackish lagoon: Matsukawa-ura,northeastern Japan,revealed by acoustic telemetry

To elucidate the post-release movement and emigration of juvenile spotted halibut Verasper variegatus, ten hatchery-reared age-0 individuals (10 months old, 17.0–19.7 cm total length) surgically implanted with acoustic transmitters were released near the center of Matsukawa-ura, a shallow brackish lagoon, on 25 November 2009 (water temperature 13.8 °C). They were monitored by ten acoustic receivers for 5 months. Of ten individuals, eight left the release site during December–March. Three of these emigrated to the outer ocean on 17 and 29 January and 30 March 2010, respectively. Juveniles probably started wintering migration in December and January when water temperatures decreased considerably in the lagoon (mean 9.5 and 6.0 °C, respectively). They stopped their migration in the coldest month, February (mean 5.7 °C), and restarted it in March when water temperatures frequently exceeded a plausible threshold for movement (≥6 °C). Statistical analyses revealed that the fish started migration significantly more frequently at nighttime. The migration track of an individual recorded from 11 to 30 March showed gradual nocturnal movements and a slow migration speed (estimated maximum speed 2.2 m/min). Our results revealed that water temperature primarily governed the seasonal timing of nocturnal migratory movements of juvenile spotted halibut.