鱼类杂交育种及其在鲌属鱼类中的应用

对中国鲌鱼杂交研究的现状及其问题进行了介绍.重点阐述了鲌属鱼类杂交育种的研究现状、细胞遗传学、生化遗传特征及分子标记技术的应用和进展.     

“石斑鱼杂交育种技术”首次批量培育出石斑鱼新品种

2009年12月29日,由广东省大亚湾水产试验中心承担的广东省科技计划项目“石斑鱼杂交育种技术”项目顺利通过由广东省海洋与渔业局组织的专家验收。     

石斑鱼类的人工繁育技术

石斑鱼（Epinephalus）属鲈形目能科,是广泛分布于印度洋和太平洋的暖水性鱼类,在中国近海有31种,其中,南海有30种,东海8～9种,黄渤海仅1种。目前我国大陆沿海养殖的种类主要有：赤点石斑鱼、青石斑鱼、点带石斑鱼、斜带石斑鱼、鲑点石斑鱼、云纹石斑鱼、鞍带石斑鱼等。     

鱼类杂交育种的原理和应用

所谓杂交育种是人们将遗传性不同的生物体相互交配(或结合)使之产生优良杂种的过程。它已作为培育鱼类新品种的基本方法之一，即使在选择育种的许多情况下也往往要应用杂交育种的一些基本原理才能凑效。     

杂交育种技术在海水养殖贝类中的应用及研究进展

我国是世界上最大的水产养殖大国,同时也是海水养殖大国。2001年海水养殖产量达1.13×108t,其中贝类养殖产量占海水养殖总量的80%以上[1],贝类养殖在整个海水养殖业中占有十分重要的地位。但是长期以来,我国贝类只是进行单一的野生品种的养殖,很多养殖品种一直处于野生状态。长期近亲繁育、累代养殖,导致了近交衰退,种质逐年下降,表现为生长缓慢、经济性状下降、抗逆性降低等等。再加上病害的发生、养殖海区环境恶化等因素,致使近些年几种重要的养殖贝类均发生了大规模的死亡事件,造成巨大经济损失,其中仅1997、1998年的扇贝死亡,山东省…     

石斑鱼类养殖技术体系的创建和石斑鱼养殖产业持续发展的思考

石斑鱼是驰名世界的名贵海产鱼类,是中国南方沿海广东、海南、福建、广西等海水养殖业的主要对象,经济价值巨大。中国石斑鱼类养殖兴起于20世纪80、90年代;进入21世纪,中山大学的研究团队围绕石斑鱼苗种繁育和养殖产业化的理论和技术进行了一系列研究,对其生殖调控、生长发育、营养需求、病害防治、种质分析、养殖技术和养殖模式等方面进行集成创新,建立苗种繁育和成鱼养殖各个环节的技术规范和管理措施,实现了石斑鱼苗种规模化繁育和自养自足,从而带动石斑鱼养殖产业发展到相当的规模。石斑鱼类养殖产业化持续健康发展的总体思路是：以建设资源高效利用、改善生态环境、产品优质安全的现代渔业基本内涵为目标,改革和提升传统养殖技术和养殖模式,转变产业发展方式,不断优化和提升产业链上的各项技术,包括：（1）优化苗种人工繁育技术,提高苗种成活率和苗种质量;（2）培育抗逆、抗病、优质、高产的优良品种;（3）配制高效、优质的人工配合饲料;（4）建立病害检测和防控新技术和研制主要疾病的特异性疫苗;（5）建立高效、低碳、节能的养殖技术,因地制宜地采用先进的养殖模式;（6）加强石斑鱼类的生物学基础研究以及生物科学和工程技术的交叉融合、协同发展,为石斑鱼养殖产业的技术创新和集约化与工程化奠定基础。     

人工增殖放流石斑鱼类的标识技术研究

本文介绍了对平均体长5cm～8cm的石斑鱼苗进行背部挂线、尾部挂线、剪背鳍、剪尾鳍和尾部烙印5种标识手术和术后40d对比饲养观察结果。研究表明：①尾部烙印标志法具有对石斑鱼幼鱼的行为习性、生长、存活影响较小,标识脱标率低、保留时间长、标志醒目,操作简单的优点,是五种标识方法中结果最好的标识技术②标识操作的鱼苗规格以6．0cm～8．0cm为宜;③鱼苗在标识后暂养期以14d为宜。     

杂交石斑鱼(斜带石斑鱼♀×赤点石斑鱼♂)与其亲本形态性状比较研究

研究了杂交石斑鱼[斜带石斑鱼(Epinephelus coioides)♀×赤点石斑鱼(E.akaara)♂]与其亲本的生长差异。采用形态学测量方法,分别测定了3种石斑鱼体质量和7个形态性状,对各形态性状对体质量影响进行通径分析,并分析杂交子代杂种优势。结果显示,3种石斑鱼各形态性状与体质量之间均呈极显著相关(P0.01),斜带石斑鱼、赤点石斑鱼体质量与全长的相关性最高,相关系数分别为0.946、0.932,杂交子代体质量与尾柄高的相关性最高(0.955),与全长的相关系数为0.927;形态性状与体质量的多元线性回归方程中,斜带石斑鱼保留体长、体高、尾柄高和体厚,赤点石斑鱼保留全长、体高和尾柄高,杂交子代保留全长、体高、尾柄高和体厚。对杂交子代杂种优势进行分析发现,体质量平均杂种优势为3.43%,体长平均杂种优势为3.02%,杂交子代同时表现出正向超亲优势和负向超亲优势。该研究表明杂交子代(斜带石斑鱼♀×赤点石斑鱼♂)与斜带石斑鱼的差异要小于赤点石斑鱼,杂交子代形态性状更偏向于母本。     

石斑鱼人工繁育技术研究进展

石斑鱼是我国南方海水养殖的主要种类之一,由于天然种苗不足,人工育苗技术日显关键和重要。文章根据国内外石斑鱼人工繁育技术的研究现状,对石斑鱼的繁殖生物学、人工繁殖、早期发育、人工育苗技术及种苗生产所面临的问题等方面进行了综述和展望。     

石斑鱼人工育苗技术研究

通过对点带石斑鱼人工育苗生产试验,从饵料准备、环境条件控制、管理措施等方面进行了探索。共培育出规格5～8cm的石斑鱼苗60多万尾。     

Temporal genetic heterogeneity of juvenile orange-spotted grouper (Epinephelus coioides, Pisces: Serranidae)

Juveniles of orange-spotted grouper ( Epinephelus coioides ), a tropical serranid species, are heavily harvested for aquaculture seeds from nursing grounds in several Southeast Asian countries. Because juveniles of similar sizes are present in a nursery area throughout the year, we aimed to determine whether more than one genetically distinct population contributes to juvenile aggregations. We examined the temporal genetic heterogeneity of juvenile aggregations collected at four different times of the year at a nursery area in coastal waters of the Andaman Sea in Trang province, Thailand. Also, we examined the differences between these temporal samples and an outgroup collected from the Gulf of Thailand (Chantaburi). The genetic variation at six polymorphic microsatellite loci within each sample was moderate, with observed heterozygosities across all loci ranging from 0.551 to 0.629 and number of alleles per locus ranging from 7.0 to 8.33. Results indicated substantial genetic differences between the two geographically distant samples, Trang and Chantaburi (F_st=0.040–0.050, P <0.005), and between the July sample and the remaining samples from Trang (F_st=0.096–0.106, P <0.005). The observed temporal genetic heterogeneity of E. coioides juveniles may reflect high variability in the reproductive success of each spawning event and the existence of spatially isolated groups of spawners.     

云龙石斑鱼与云纹石斑鱼、珍珠龙胆石斑鱼的生长性状及对比分析

为了对云纹石斑鱼与鞍带石斑鱼的杂交种云龙石斑鱼生长特性进行分析,本研究对工厂化养殖条件下云龙石斑鱼与云纹石斑鱼、珍珠龙胆石斑鱼的生长特性进行了对比。通过云龙石斑鱼家系与云纹石斑鱼家系建立和培育,对不同家系的受精率、正常发育率和畸形率进行统计;分别对45~245日龄的云龙石斑鱼与云纹石斑鱼的生长性状进行了测量;分别对云龙石斑鱼与珍珠龙胆石斑鱼在生长时间跨度为2~13月龄的生长性状进行测量,并于13月龄时统计成活率(苗种为2016年繁育);利用单因素方差分析法和多重比较分析对测定的生长性状进行比较分析,同时利用Excel软件拟合云龙石斑鱼、云纹石斑鱼的生长曲线。结果显示,杂交种云龙石斑鱼受精率平均为55.5%±26.7%,正常发育率平均为33.9%±23.6%,畸形率平均为8.3%±0.9%。至245日龄时云龙石斑鱼体长、体质量的平均值分别为(22.5±1.7) cm、(316.7±57.3) g,云纹石斑鱼的体长与体质量的平均值分别为(16.8±1.3) cm、(123.2±30.2) g,云龙石斑鱼比云纹石斑鱼生长快,云龙石斑鱼的体长为云纹石斑鱼的1.3倍,体质量为云纹石斑鱼的2.6倍。云龙石斑鱼在45~245日龄阶段的生长曲线为W=0.039 2L^{2.891 2}(R²=0.986 9),云纹石斑鱼生长曲线为W=0.025 5L^{3.021 6} (R²=0.990 8),在本阶段云龙石斑鱼为异速生长型,云纹石斑鱼为等速生长型。经过12个月的对比养殖,云龙石斑鱼成活率为97.2%,珍珠龙胆石斑鱼的成活率为93.2%~94.5%,云龙石斑鱼全长与体质量平均值分别为(35.3±4.1) cm、(700.0±247.2) g,珍珠龙胆石斑鱼全长与体质量平均值分别为(28.6±3.5) cm、(550.0±224.8) g,云龙石斑鱼体质量是珍珠龙胆石斑鱼的1.3倍,全长为1.2倍。研究表明,云纹石斑鱼(♀)×鞍带石斑鱼(♂)是一个比较理想的杂交组合,杂交后代云龙石斑鱼具有生长快、畸形率低、成活率高的杂交优势,为杂种优势的研究提供了基础材料,同时也为新品种云龙石斑鱼的推广提供了科学依据。     

Establishment and characterization of a new cell line derived from kidney of grouper, Epinephelus akaara (Temminck & Schlegel), susceptible to Singapore grouper iridovirus (SGIV)

A marine fish cell line derived from the kidney of red-spotted grouper, Epinephelus akaara, designated as EAGK was established and characterized. The EAGK cells multiplied well in Leibovitz's L-15 medium containing 10% foetal bovine serum at 25 °C and have been subcultured for more than 90 passages. Karyotyping, chromosomal typing and ribosomal RNA (rRNA) genotyping analysis revealed that EAGK had a modal diploid chromosome number of 82 and was a fibroblast cell line originated from grouper. A severe cytopathic effect was observed in EAGK cells incubated with Singapore grouper iridovirus (SGIV), but not with soft-shelled turtle iridovirus, viral nervous necrosis virus or spring viraemia of carp virus. SGIV replication was further confirmed by immunofluorescence, electron microscopy and virus titre determination. Bright fluorescence was observed after transfection with fluorescent protein reporter plasmids, indicating that EAGK cells can be used to identify gene functions in vitro. In addition, the cell organelles including mitochondria and endoplasm reticulum changed and aggregated around virus factories after SGIV infection, suggested that the EAGK cell line could be an important tool for investigation of iridovirus-host interactions.     

Possible application of mibolerone for induced sex inversion of grouper Epinephelus coioides

SUMMARY: Thirty immature juvenile grouper Epinephelus coioides (19–168 g bodyweight, BW) were randomly stocked in four units 6 t tanks to determine if mibolerone can be used to induce sex inversion in groupers. After acclimatization and weaning to artificial feed, the feed given daily (4% BW/day) was supplemented with 0, 50, 100, and 200 μg mibolerone/kg feed for about 18 weeks. Thereafter, the hormone treatment was withdrawn and the experiment was terminated at Week 24. Ten fish were killed for gonad histology at stocking to serve as an initial control while about three to five fish were killed every 8 weeks. In general, ovaries of initial controls showed the presence of moderate stromal cells and gonia and few primary oocytes. At Weeks 8 and 16, ovaries of the control fish (0 μg/kg) were similar to that of the initial control except that primary oocytes increased at Week 24. Gonads of fish fed diets containing 100 and 200 μg/kg had none to moderate spermatocytes and few spermatids at Week 8 and 16, although spermatozoa were not observed, indicating that the fish were undergoing spermatogenesis. Spermatogenesis at 50 μg/kg was not as advanced since only few spermatocytes occurred at Weeks 8 followed by moderate gonia and no spermatocytes and spermatids at Week 16. However, the presence of few primary oocytes was observed when mibolerone was withdrawn suggesting that sex-inversed fish reverted back to a female condition. These results show that sex inversion in juvenile grouper can be induced by oral administration of mibolerone and may have possible application on mature females to produce functional males.     

Characterization of grouper nervous necrosis virus (GNNV)

Grouper nervous necrosis virus (GNNV) was isolated from moribund grouper larvae, Epinephelus sp., using a fish cell line GF-1. The present study describes the biochemical and biophysical properties of GNNV and the expression of GNNV in diseased grouper larvae. Viral protein was detectable in most of the GNNV-infected GF-1 cells by the fluorescent antibody technique (FAT) after 12 h post-infection (p.i.), although no cytopathic effect (CPE) appeared at that time. Clear CPE developed on the third day, and complete disintegration of the monolayer occurred over the subsequent two days. The infectivity of GNNV can be blocked following treatment at 60 °C for 1 h. GNNV was sensitive to pH 3 and pH 10–12 with a 4 log₁₀ drop in infectivity. Purified GNNV was analysed by SDS–PAGE, and then stained with periodic acid silver. The positive staining indicated that its two capsid proteins were glycoproteins. Genomic RNAs of GNNV were extracted from purified virions and analysed. The molecular weights of genomic RNAs were 1.02 × 10⁶ and 0.50 × 10⁶ Da. The T2 region of the coat protein gene of GNNV was amplified by polymerase chain reaction (PCR), and the multiple alignment of the T2 sequence of two GNNV isolates with four genotypes of fish nodaviruses revealed that these two isolates (GNNV9410 and GNNV9508) belong to the red-spotted grouper nervous necrosis virus (RGNNV) genotype. The tissue distribution of GNNV in naturally infected grouper larvae was investigated by in situ hybridization using a dig-labelled probe, which showed that GNNV was not only detected in the brain and retina, but also in the gill, skeletal muscle, liver, pyloric gland, intestine and blood cells in the heart.     

Increasing survival and growth in larval leopard coral grouper (Plectropomus leopardus) using intensively cultured Parvocalanus crassirostris nauplii

Leopard coral grouper, Plectropomus leopardus are a heavily exploited, high-value fish commonly found in the Asian live reef food fish trade. In past decades, many attempts at the mass culture of various grouper species have been undertaken; however, their small mouth gape at first feed has resulted in very low survival when using traditional live feeds such as rotifers. The use of wild caught or extensively cultured copepods has yielded potentially promising increases in survival and growth, but overall survival to the juvenile stage remains low, making mass culture currently impractical. The current study sought to build on past developments in grouper culture and recent advancements in copepod culture technology by observing how growth and survival were influenced by the addition of intensively cultured copepods to the early diet of P. leopardus larvae. Six tanks of larvae, three replicates per treatment, were fed either eggs and nauplii of the calanoid copepod Parvocalanus crassirostris, at a starting density of 5 mL⁻¹, and the rotifer Brachionus rotundiformis, at a starting density of 10 mL⁻¹, or were fed only B. rotundiformis, at a density of 15 mL⁻¹, starting on the evening of 2 days post-hatch (dph) and continuing until 9 dph. After this initial period, all larvae were fed the same diet of rotifers, Artemia, and dry feed until the cessation of the trial at 21 dph. Larvae fed P. crassirostris in addition to rotifers had a significantly higher survival, 9.9 versus 0.5%, than those fed only rotifers. Growth was also significantly enhanced in larvae offered copepods. Larvae only fed rotifers were, on average, 1.5 mm shorter at 21 dph than those that had been fed copepods. More rapid development and the earlier onset of flexion were also noted in the larvae that were offered copepods. The use of intensively cultured copepods, in this study, increased survival tenfold over previous studies, with P. leopardus larvae fed wild-caught copepods. The application of intensively cultured copepods to the early diet of P. leopardus, along with future research to evaluate late-stage mortality issues, may facilitate commercial production of this species.     

石斑鱼循环水养殖系统及水源热泵应用研究

通过构建石斑鱼的循环水养殖系统及水源热泵加温系统,达到在北方大规模养殖石斑鱼的目的。养殖系统由养殖池、弧型筛、循环泵、蛋白分离器、浸没式生物滤池、脱气池、溶氧池、紫外线灭菌器、液氧站组成。采用养殖废水收集及过滤装置处理后的养殖废水作为水源热泵的水源,通过2个冬季的运行,其冬季制热的平均制热能效比(COP)为2.66。在系统中养殖的第1批青斑鱼11个月内由24.41 g生长到480.66 g,存活率超过97%。养殖的第2批青斑12个月内由23.36 g生长到400.46 g,存活率达到84.5%。养殖的珍珠龙胆石斑7个月内由48.46 g生长到511.36 g,存活率达到71.24%。养殖的东星斑12个月内由41.13 g生长到223.56 g,存活率达到65.52%。本系统可实现青斑、珍珠龙胆石斑、东星斑等品种的常年均衡生长,并可降低石斑鱼的养殖成本。     

Utilization of glucose and starch by the grouper Epinephelus malabaricus at 23°C

ABSTRACT : The aim of the present study was to investigate carbohydrate utilization by the grouper Epinephelus malabaricus reared at 23°C. Two isoenergetic semipurified diets were prepared with two carbohydrate sources (glucose and starch). Each diet was fed to triplicate groups of grouper in a recirculating rearing system for 8 weeks. Water temperature was held constant by a thermal controller at 23 ± 1°C. Weight gain, feed efficiency (FE) and protein efficiency ratio (PER) of fish fed the starch diet were significantly higher ( P  < 0.05) than those of fish fed the glucose diet. Body lipid content of the starch-fed group of fish was higher than that of glucose-fed group of fish. Hepatic hexokinase and glucose-6-phosphate dehydrogenase activities were higher in fish fed the starch diet than fish fed the glucose diet. Fish fed the glucose diet had higher hepatic glucose-6-phosphatase activity than fish fed the starch diet. These results suggest that starch is better utilized by grouper than glucose when the water temperature is 23°C.     

Identification and characterization of a late gene encoded by grouper iridovirus 2L (GIV‐2L)

Grouper iridovirus (GIV) belongs to the Ranavirus genus and is one of the most important viral pathogens in grouper, particularly at the fry and fingerling stages. In this study, we identified and characterized the GIV‐2L gene, which encodes a protein of unknown function. GIV‐2L is 1242 bp in length, with a predicted protein mass of 46.2 kDa. It displayed significant identity only with members of the Ranavirus and Iridovirus genera. We produced mouse monoclonal antibodies against the GIV‐2L protein by immunizing mice with GIV‐2L‐His‐tag recombinant protein. By inhibiting de novo protein and DNA synthesis in GIV‐infected cells, we showed that GIV‐2L was a late gene during the viral replication. Finally, immunofluorescence microscopy revealed that GIV‐2L protein accumulated in both the nucleus and cytoplasm of infected cells. These results offer important insights into the pathogenesis of GIV.