黑鲷×真鲷杂交子代与真鲷的Calmodulin基因克隆与表达分析

采用cDNA末端快速扩增技术(RACE)获得的黑鲷(Acanthopagrus schlegelii,♂)×真鲷(Pagrus major,♀)的杂交子代F_1(AP)与真鲷(Pm)的两组Calmodulin(CaM)基因序列。通过生物软件分析了两者的差异及所表达的蛋白质特性;同时,应用实时荧光定量PCR技术检测了APCaM与PmCaM在仔鱼及2龄鱼的6种不同组织中的表达特征。结果表明:1)克隆得到APCaM与PmCaM的cDNA全长分别为1 230 bp、1 210bp,两基因序列均具有一个450 bp的开放阅读框(Open reading frame,ORF),编码了一个由149个氨基酸组成的多肽,该多肽包含有高度保守的4个EF-hand功能结构域。杂交F_1与真鲷的CaM基因非翻译区差异位点主要在3'端,ORF区域有5个氨基酸差异。2)两基因与其它鱼类的核苷酸序列相似性最高为95%,与氨基酸序列相似性最高为100%;从基因序列结构及蛋白质属性表明APCaM与PmCaM属保守的CaM基因家族。3)定量分析表明CaM在检测组织中均有表达,其中APCaM在脑中表达量最高,PmCaM在性腺中表达最高;APCaM与PmCaM在脑、肌肉、性腺及鳃中的表达存在显著差异(P0.01),在肝、肾组织及仔鱼中的表达无显著差异(P0.05);结合杂交F_1与双亲本在生长及性腺发育上的性状,推测CaM可能与生长及性腺发育调控有关。这些结果为探讨CaM基因在杂交F_1及真鲷亲本中的作用及鲷科育种研究提供基础资料。     

黑鲷和真鲷自交及正反交子代生长性状和肌肉营养成分的分析比较

以黑鲷(Acanthopagrus schlegelii)和真鲷(Pagrus major)为繁育亲本,采用完全杂交配组方法,分别对黑鲷(BB)、真鲷(RR)自交与正反交后代(RB,Pagrus major♀×Acanthopagrus schlegelii♂;BR,Acanthopagrus schlegelii♀×Pagrus major♂)的生长性状和肌肉营养成分进行了分析比较。结果显示:养殖180 d时,黑鲷、真鲷自交及正反交后代的体质量绝对增长率的排序为正交>真鲷自交>反交>黑鲷自交,体质量特定增长率各组间无显著差异(P>0.05);养殖240 d时,正交组的体质量绝对增长率显著高于其他组(P<0.05),真鲷自交组与反交组无显著差异(P>0.05),黑鲷自交组最低(P<0.05)。4个群体鱼的粗蛋白质量分数在19.3%~22.0%,粗脂肪质量分数在0.9%~2.6%,其中真鲷自交组最低(0.9%),正交组最高(2.6%);4个群体鱼的必需氨基酸含量占氨基酸总量的40.16%~41.69%,必需氨基酸指数在82. 17%~87.36%,鲜味氨基酸占氨基酸总量的38.1%~39.0%,杂交组均高于自交组。研究表明:正交组生长速度最快,杂交后代的生长速度均快于黑鲷;4个群体鱼肌肉的蛋白质含量均较高,氨基酸种类齐全,就蛋白质营养水平而言,反交子代与黑鲷、真鲷相当,高于正交子代;就蛋白质的氨基酸水平而言,反交子代优于黑鲷和正交子代;杂交后代的鲜味氨基酸含量均优于黑鲷和真鲷,表现出一定的杂交优势。研究结果证明黑鲷、真鲷杂交能够提高后代的生长性能,改良肌肉品质,增强选育的综合效果。     

真鲷与黑鲷杂交繁育技术研究

2006年3月从海区选购真鲷、黑鲷亲鱼,通过强化培育,性腺促熟,于4月开展真鲷♀×黑鲷♂及黑鲷♀×真鲷♂,结果真鲷♀×黑鲷♂受精率为87.4%,孵化率为85%,出苗率14.28%;黑鲷♀×真鲷♂受精率为92.1%,孵化率为90%,出苗率7.2%,共获得正交反交杂交鱼苗32 000尾。     

黑鲷×真鲷杂交子代回交繁育及子代早期发育观察

为筛选获得类型丰富、具杂交优势的海水养殖新品种,在黑鲷(♀)×真鲷(♂)杂交子代养殖成功的基础上,于2016年4—5月,挑选经强化培育的杂交子代(♂)及黑鲷(♀)为亲鱼(雌雄配比1∶1),养于室内同一水泥池,水温16~20℃,盐度23~25,收集同步发育的自然受精卵进行孵化,采用显微摄像观察,记录回交子代胚胎和早期仔鱼的发育时序与形态特征。试验结果显示,回交子代的受精卵孵化、胚胎及仔鱼发育正常;在水温18~20.2℃、盐度25、微充气的条件下,受精卵历时42.5h完成胚胎发育,仔鱼孵化出膜;初孵仔鱼全长为2.08~2.45mm;在水温18~21.8℃、盐度23~25的条件下,经过约4d时间,仔鱼卵黄囊消失,完成早期仔鱼发育阶段。试验表明,黑鲷(♀)×真鲷(♂)杂交子代的可育性,也为进一步的鲷科优良品种选育提供更多基础资料。     

真鲷与黑鲷杂交繁育技术研究

通过对真鲷黑鲷杂交繁育，培育出一种能结合父母本性状，具有生长速度快、抗逆性强的杂交子一代。2006年3月从海区选购真鲷（Pagrosomusmajor）、黑鲷（Spraus macrocephalus）亲鱼，通过强化培育，性腺促熟，于4月进行干法人工授精。通过对真鲷♀&#215;黑鲷8及黑鲷♀&#215;真鲷♂获得杂交受精卵，结果真鲷♀&#215;黑鲷♂受精率为87．4％，孵化率为85％，出苗率14．28％；黑鲷♀&#215;真鲷♂受精率为92．1％，孵化率为90％，出苗率7．2％，该批次共获得正交反交杂交鱼苗32000尾。从杂交实际结果看，不管正交还是反交都比自交的结果差，说明它们之间存在生殖隔离，杂交繁育难度相当大，要达到规模生产。需要继续研究、试验。     

黑鲷钙调磷酸酶B同源蛋白2(CHP2)基因克隆及表达差异

钙调磷酸酶B同源蛋白2(CHP2)为参与细胞内pH及细胞增殖调控的关键因子。用RACE末端PCR方法得到黑鲷CHP2(AsCHP2)基因全长cDNA,并利用生物信息学方法分析该基因的序列特征与同源性;同时利用实时荧光定量PCR方法检测AsCHP2的mRNA在黑鲷整体仔鱼,90 d、180 d黑鲷,杂交F_2(真鲷♂×黑鲷♀)及回交F_1(黑鲷♂×杂交F_1♀)的脑、心脏、肌肉、肝脏和性腺等组织中的表达。研究结果表明:(1)AsCHP2 cDNA序列全长1722 bp, 5′-非编码区(UTR)长度69 bp, 3′-UTR长度1069 bp,开放阅读框长度为585 bp,编码194个氨基酸;其结构域包含2个EF-hand结构及1个核输出信号;其蛋白分子质量为21.87 ku,理论等电点为4.89;同源建模获得了CHP2的二级与三维结构。AsCHP2基因的核苷酸序列与其他鱼类的相似性为86%～81%,编码的氨基酸序列与其他鱼类的相似性为80%～72%。邻接系统树显示了黑鲷CHP2氨基酸序列与其他鱼类的进化关系。序列比对与结构分析表明,AsCHP2基因具有典型的CHP2基因结构。(2)AsCHP2的mRNA在黑鲷的仔鱼期(5～35 d)及大规格个体的脑、肝脏及肌肉组织中均有表达,在90 d个体各组织中的表达量均高于在180 d个体各组织中的表达量;在180 d黑鲷、杂交F_2及回交F_1的个体中,基因在脑、肝脏、肌肉及性腺等不同组织中均有表达,且均在心脏中高表达、在性腺中低表达。这些结果显示,CHP2基因在海水鱼类的生长、发育过程中发挥功能。     

真鲷与黑鲷杂交繁育技术研究

2006年3月从海区选购真鲷、黑鲷亲鱼,通过强化培育,性腺促熟,于4月开展杂交试验,结果真鲷♀×黑鲷♂受精率为87.4%,孵化率为85%,出苗率14.28%;黑鲷♀×真鲷♂受精率为92.1%,孵化率为90%,出苗率7.2%,共获得正交反交杂交鱼苗32 000尾。     

杂交鲷与黑鲷投喂频率的比较研究

比较了黑鲷(Acanthopagrus schlegelii)♀×真鲷(Pagrus major)♂杂交子二代(杂交鲷或杂交F2)与黑鲷幼鱼在不同投喂频率下生长性能、生理生化指标的差异表现,为新品种选育提供基础依据.选用杂交F2及黑鲷幼鱼为研究对象,设4个投喂频率组(1T:1次·d?1、2T:2次·d?1、3T:3次·...     

真鲷♀×黑鲷♂人工育苗技术研究

通过真鲷♀×黑鲷♂杂交,获得杂交F1.研究发现,杂交鱼苗在培育期间有3个死亡高峰;培育密度以1万～1.8万尾/m<'3>为合适,在此密度下培育至稚仔鱼最高培育成活率可达36%;在水温22～23℃,从初孵仔鱼到1 cm需27 d,水温17～18℃,从初孵仔鱼到1 cm需36 d.     

真鲷与黑鲷及杂交F1耐受温度、生长比较

本文通过对真鲷、黑鲷及真鲷♀×黑鲷♂杂交的F1耐高温、低温的比较，杂交F1具有父母本优良性状，能安全度夏和过冬；通过二三者之间生长测量，真鲷和杂交F1的生长速度明显快于黑鲷，从体重增长比较杂交F．增重速度比黑鲷快30％以上，但低于真鲷。     

Distribution and abundance of the larval black porgy, Acanthopagrus schlegeli (Bleeker), in coastal waters and an estuary of western Taiwan

This study compared the distribution, abundance and body length of larval black porgy, Acanthopagrus schlegeli (Bleeker), to relative environmental conditions in the coastal waters near Wan-kung and the Da-du estuary of western Taiwan to evaluate the utilization of these two water bodies as nursery grounds. Sampling was carried out on spring tides from October 1993 to September 1994. In the coastal waters, salinity (mean = 31.5^0/00) and current speed (39.1 cm s^?1) were significantly higher than those in the estuary (28.3^0/00 and 29.6 cm s^?1). Most of the larvae occurred from late winter to early spring. The period of occurrence was longer and the density was higher in the coastal waters. The mean body length in the coastal waters (9.28 mm) was greater than that in the estuary (7.33 mm), but the coefficient of variation of body length was smaller (coastal waters, 8.15%; estuary, 19.87%). The occurrence of the larvae in the two regions was negatively correlated with water temperature and positively with salinity and dissolved oxygen, and the change of density of larval black porgy was similar to that of all larval species discriminated by principal component analysis. The range and mode of the length-frequency distributions were different between the two regions. It was concluded that coastal waters are overall a better nursery habitat for black porgy.     

Preliminary Results on Rearing of Sparus Aurata × Pagrus Pagrus Hybrids. Performance Comparison With the Parental Species

Hybridization between gilthead sea bream, Sparus aurata, and red porgy, Pagrus pagrus, was undertaken and viable hybrids were produced by an artificial hormonal-induced cross. The hybrids, from the cross of female red porgy and male gilthead sea bream (Pp × Sa), survived through the yolk-sac larval stage. The hybrids produced from the cross of female gilthead sea bream and male red porgy (Sa × Pp) presented higher mortality in the larval stage (98.4%) compared to gilthead sea bream (80.0%) and red porgy (92.5%). Afterwards, the Sa × Pp hybrid was compared to the offspring of its parental species under the same rearing conditions (tanks of 500 l capacity, natural light, ambient temperature, self-feeding with commercial feed) in a 40–week experiment. In this comparative study, the Sa × Pp hybrid had intermediate mortality rate (6%), compared to gilthead sea bream (0%) and red porgy (21%). The growth profile of this hybrid resembled the slow growing phases of its parents (red porgy in warm period and gilthead sea bream in cold period). The feed conversion ratio between fish forms was not significantly different (1.54–1.84 in the Sa × Pp hybrid, 1.53–1.56 in the maternal species and 1.33–1.40 in the paternal one). The body weight variation in the Sa × Pp hybrid was quite high (31.7–37.9%), compared to that in gilthead sea bream (19.7–24.7%) and red porgy (18.0–31.3%). With respect to body coloration, the Sa × Pp hybrid was distinguishable and classified into groups of red porgy-like (55.1%), gilthead sea bream-like (40.6%), and intermediate forms (4.3%). The preliminary results of this study proved that the produced Sa × Pp hybrid had too low a performance for aquaculture use, but the interesting consequent hereditary potential could be a useful tool in fish hybridization practice.     

温度对黑鳍棘鲷耗氧率与排氨率的影响

对体重为240.9±27.1 g的黑鳍棘鲷在不同水温(10℃、15℃、20℃、25℃、30℃)条件下的耗氧率和排氨率进行了测试分析。结果表明:黑鳍棘鲷在一定温度范围内的耗氧率和排氨率均随温度的升高而升高,但当水温超过一定值后,其耗氧率和排氨率均随温度的升高而下降。在实验条件下,黑鳍棘鲷的耗氧率与温度的回归方程为Y=-0.489 7X2+27.39X-147.39;排氨率与温度的回归方程为Y=-0.009 3X2+0.508 2X-1.247;耗氧率的最大值为235.61μg/g.h(温度为28.0℃),排氨率的最大值为5.70μg/g.h(温度为27.3℃)。黑鳍棘鲷快速生长期的最适温度为25~28℃。     

Effect of dietary ascorbate fortification on lipolysis activity of juvenile black sea bream Acanthopagrus schlegeli

ABSTRACT:   Hatchery-reared black sea bream Acanthopagrus schlegeli juveniles averaging 0.05 g in body weight were fed either a control diet (commercial diet) or an experimental diet in which the commercial diet was fortified with 50 mg l -ascorbyl 2-monophosphate Mg (APM)/100 g diet for 50 days. Calcium ascorbate supplemented as a vitamin mixture in the control diet was completely destroyed during storage. Fortification with APM significantly increased the ascorbic acid levels in the muscle, liver, brain and eye. Although APM fortification did not influence growth, survival or fish body composition, adipocyte diameter in the intraperitoneal fat body (IPF) was significantly reduced. After the feeding experiment, the fish were kept for 39 days without feeding. Fortification with APM resulted in high survival, high muscle protein retention and low body weight loss. The results suggested the necessity of fortification with an adequate amount of ascorbate in the diet. While fatty acid compositions of the IPF, muscle and liver were not significantly influenced by APM fortification, characteristic changes in the fatty acid profile were found after starvation. Vitamin C and highly unsaturated fatty acids seemed crossly interactive in relation to lipolysis activity in black sea bream juveniles.     

Ontogeny of tolerance to and avoidance of ultraviolet radiation in red sea bream Pagrus major and black sea bream Acanthopagrus schlegeli

ABSTRACT:   Ontogenetic changes of tolerance to, and avoidance of, ultraviolet-B radiation (UV-B) were examined in red sea bream Pagrus major and black sea bream Acanthopagrus schlegeli . In the tolerance experiment, larvae and juveniles (age 13–46 days) were put in beakers, and were exposed to one of five different levels of UV-B radiation (1.8, 1.1, 0.2, 0.1, and 0 W/m²) for one hour. Their survival rates were calculated either 12 or 24 h later. In the avoidance experiment, fish (age 3–49 days) were put in a long experimental tank, half of which was covered with UV-blocking film and placed under two levels of UV-B radiation (1.1 and 0.2 W/m²), and their avoidance indices were calculated. Black sea bream had significantly better survival compared to red sea bream for most ages. Only black sea bream of ages 37 and 49 days showed significant avoidance of UV radiation under the higher level of UV-B, whereas both species did not show avoidance on any days at the lower level. The present results suggest that black sea bream are significantly better adapted to habitats with high UV-B radiation, than red sea bream, reflecting that back sea bream live in shallower waters through their early life stages.     

Comparative stomach contents and growth of the juvenile black seabream, Acanthopagrus schlegeli, reared in illuminated and unilluminated cages

The present study was designed to determine the positive effects of artificial illumination on the juvenile black seabream, Acanthopagrus schlegeli , by comparing stomach contents and growth between juveniles exposed to light and those maintained in the absence of light. The major prey items for juvenile black seabream reared in illuminated cages were amphipods (IRI%=50.5), copepods (IRI%=44.7) and polychaetes (IRI%=3.0), whereas those for the juveniles maintained in unilluminated cages were copepods (IRI%=96.0), amphipods (IRI%=3.4) and polychaetes (IRI%=0.6). The specific growth rates (SGR) of the juveniles reared in illuminated cages (0.99%) were significantly higher than those of the juveniles maintained in unilluminated cages (0.78%).     

Ploidy polymorphism and morphological variation among reciprocal hybrids of Pseudosciaena crocea (♀) × Miichthys miiuy (♂)

Diploid and triploid hybrids of large yellow croaker (Pseudosciaena crocea, Pc) and Miiuy croaker (Miichthys miiuy, Mm) were produced by interspecific hybridization. The result showed that Pc (♀) and Mm (♂) can be successfully crossed and hybrid offspring of normal hatch rate were produced. In contrast, a lower fertilize and hatch rate were produced for hybrids of Pc (♂) and Mm (♀), and the offspring died day 19 after hatching. To confirm the ploidy of hybrid offspring, we examined the karyotypes of offspring. The result of DNA relative content investigation and genetic relationship analysis demonstrated that the major hybrid offspring were hybrid diploid. In addition, a small number of hybrid were triploid and gynogenesis diploid. Morphological analysis based on the measurable traits data indicated the success of the interspecific crosses. The natural gynogenetic diploid large yellow croaker (PP), diploid hybrids (PM) and triploid hybrids (PPM) were closer to maternal parent (Pc) than paternal parent (Mm). This study provided genetic evidences of the chromosome, DNA content and morphological levels to support the successful establishment of the polyploidy hybrids of Pc (♀) and Mm (♂).     

Natural Spawning and Mass Larviculture of Black Porgy Acanthopagrus schlegeli in Captivity in Taiwan

Abstract.— The black porgy Acanthopagrus schlegeli is one of the most important marine fish cultured in Taiwan. Wild-caught broodstock were spawned naturally in captivity at water temperatures of 19–26 C in 1989 and 1990. Females produced 352,000 to 957,000 eggs per female during the spawning period. The hatched larvae were reared in 45-m³ rectangular cement tanks and fed initially on rotifers, Brachionus plicatilis . followed by Artemia nauplii. and finally weaned onto an artificial diet. Larviculture trials to 40 d produced juveniles of 13.1–14.2 mm average total length, at a survival rate ranging from 18.3 to 74.8%. Lordotic deformation was observed in the juvenile stage. However, when an oil skimmer was used to remove the oil film on the water surface, the incidence of lordosis was reduced from 14.4 to 6.1%. High mortality of the larvae occurred during the initial 10–15 d after hatching and cannibalism was observed when larvae reached 7 mm total length. Juveniles grew to size of 83.7–101.4 mm total length with survival rate of 27.9–28.2% in 100 d. The results indicate that the present technique can be used for mass seed production of black porgy.     

大亚湾黑鲷标志放流及回捕率调查

为评价大亚湾海域黑鲷(Acanthopagrusschlegelii)增殖放流效果,使用塑料椭圆标志牌(plasticovaltags,POTs),开展了黑鲷幼鱼标志保持率与生长实验。结果显示,背鳍鳍棘基部第4～6鳍棘下方5mm左右的背部肌肉为最适标志位置, 90 d后标志保持率为81.76%。POTs标志黑鲷幼鱼45 d内生长与对照组无显著性差异(P0.05),而90 d后标志组生长显著缓于对照组(P0.05)。并于2014—2016年每年1次在大亚湾开展黑鲷幼鱼标志放流试验。采用有奖回收方式进行标志鱼回捕,测量体长、体重,收集回捕日期、回捕地点、回捕方式等信息,整理分析相关数据。3年共放流标志黑鲷幼鱼60115尾,共2758尾回捕标志鱼被定置网、流刺网及垂钓捕获(总回捕率4.59%);大规格放流苗种回捕率高于小规格放流苗种;垂钓回捕鱼数量占总回捕数量的比例为62.08%。通过对反馈信息的分析,认为放流后的标志鱼最初主要沿湾内和湾外两个方向呈近岸辐射状扩散,最远扩散距离约70 km。放流后1～2个月标志黑鲷生长较慢,随后相对加快,放流5～7个月后,黑鲷幼鱼体长、体重分别增长59.51%～127.20%和322.75%～989.83%。结论认为,POT是黑鲷幼鱼体外标志的适宜选择;在研究海域,放流后的标志黑鲷更易于被垂钓方式捕获;丰富的食物和可供躲避敌害的空间是影响其扩散、生长的因素。