水利建设对辽东半岛香鱼Plecoglossus altivelis的影响

辽东半岛水利建设造成洄游型香鱼资源下降,并在碧流河水库、转角楼水库形成了陆封型香鱼。陆封型香鱼发生了①个体小型化：体长和体重由洄游型香鱼的２０─２５ｃｍ（平均２２．６ｃｍ）,１５０─２５０ｇ（平均２０４ｇ）,分别下降到１０─１８ｃｍ（平均１３．６ｃｍ）,和１３─７３ｇ（平均３６．５ｇ）。②性状变异：洄游型香鱼体长为体高的３．７─４．７倍,为头长的４．５─５．３倍,而陆封型香鱼体长为体高的４．３─６．５倍,为头长的３．９─４．３倍。③怀卵量减少,产卵期提前：洄游型香鱼怀卵量为１．０─８．５万粒（平均３．１万位）．产卵盛期为９月底至１０月上旬,而陆封型香鱼的怀卵量为０．３２─０．６１万粒（平均０．４０万粒）,产卵盛期为９月上旬至下旬,提前２０ｄ左右。     

香鱼消化道及肝脏的形态结构特征

采用解剖及石蜡切片显微技术观察了香鱼消化道及肝脏的组织学结构。香鱼消化道由口咽腔、食道、胃及肠构成。口咽腔大且狭长,其底壁前部有一对粘膜褶,两颌边缘着生宽扁梳状齿,腭骨及舌骨具齿,犁骨无齿;舌由基舌骨突出部分覆盖粘膜构成,舌粘膜上皮为复层扁平上皮,含有较多的杯状细胞和味蕾。食道、胃及肠均由粘膜层、粘膜下层、肌层及外膜构成。食道粘膜层上皮为复层扁平上皮,杯状细胞发达。胃呈V形,由贲门部、胃体部及幽门部组成,胃壁粘膜上皮为单层柱状上皮,贲门部与胃体部的固有层中有胃腺。肠较短,由前、中、后肠构成,肠壁粘膜上皮为单层柱状上皮,其游离面具微绒毛;上皮细胞间有杯状细胞。幽门盲囊有350～400条,其组织学结构与肠相同。肝脏单叶,外被浆膜;肝细胞形态不规则,肝小叶界限不明显。     

香鱼基因组DNA提取方法的优化

为获得高质量的基因组DNA,分别采用传统酚-氯仿法、高盐法、试剂盒法和改进酚氯仿法提取香鱼肌肉基因组DNA。琼脂糖凝胶电泳检测结果表明,改进酚氯仿法提取的基因组DNA电泳条带整齐明亮且无降解。紫外分光度计测定DNA浓度和纯度,结果表明,改进酚氯仿法提取的鱼类基因组DNA浓度约为300μg/mL,A260/A280为1.80-1.86。用改进的酚氯仿法提取的DNA进行AFLP分析,扩增结果稳定,电泳条带清晰。综上所述,改进酚氯仿法能够获得高质量DNA,且可以用于进一步的分子生物学研究。     

关于中国香鱼分类地位的线粒体DNA基因序列分析

测定了辽宁和广东香鱼的Cytb基因序列,与GenBank中日本和韩国香鱼分子数据合并比较。结果表明11尾香鱼402个碱基序列中变异位点8个,信息简约位点有2个,A+T与G+C含量分别为51.4%和48.6%,转换/颠换比为4.9。运用Kimura-2parameter模型,采用邻接法构建的分子系统树,发现中国香鱼与日本和韩国香鱼聚类在一起,不同地理种群的香鱼均未单独分群,表明中国和日、韩香鱼亲缘关系相当近。根据日、韩香鱼的地理分布,推测所分析的辽宁和广东种群可能亦为香鱼指名亚种,但该推论尚需与琉球香鱼亚种的序列进行比较后才能确定。研究虽未发现辽宁香鱼和广东香鱼间存在明显的种群结构差异,但由于中国海域辽阔,生境多样,中国沿海是否会有其它种下群体发生,则需要采用进化速度适中的线粒体和核基因标记,对中国香鱼不同的地理种群进行广泛的大样本分析来确定。     

鸭绿江香鱼耳石日轮与生长的研究

１９９２年对鸭绿江香鱼耳石日轮与生长进行了研究。人工受精卵胚胎发育后期和前仔鱼期连续剖察表明，受精后约９６小时胚体听囊内出现一对矢耳石，孵出后第２天耳石上出现第一个日轮，之后每天形成一轮。在光镜和扫描电镜下测定了幼、成鱼矢耳石的形态、直径、日轮数及其间距变化。耳石短径（ｖ， μｍ）与鱼体长（ｘ， ｍｍ）呈线性关系， ７５尾幼、成鱼的关系式为ｙ＝３．２８ｘ＋２４８．３０。以耳石日轮数推算其产卵孵化期与实地调查结果一致。耳石日轮数（Ｄ）可用孵化后日数（Ｎ）减１表示。日轮间距有规律性变化，与鱼体生长发育和生态条件密切有关。依据日龄和相关体长体重资料进行了香鱼生长特性研究，用生长方程描绘的生长速度曲线和生长拐点（位于２８３日龄）等均较客观地反映了其生长特点。     

福建东张水库陆封型香鱼的遗传变异

测定了福建东张水库18尾陆封型香鱼的ND4-tRNA^Ser基因533 bp序列,发现2个可变位点和2个简约信息位点,共检测到4种单倍型。基于Kimura 2-parameter模型的遗传距离在0.001 9～0.003 9之间,在NJ系统树上无明显谱系结构;单倍型多样性(Hd)和核苷酸多样性(Pi)分别为0.627和0.001 37,平均核苷酸差异数为0.732,远低于两侧洄游型日本香鱼指名亚种(Hd为0.793,Pi为0.002 94),与被列为IUCN濒危物种名录的日本琉球香鱼亚种(Hd=0.519,Pi=0.001 11)相当。东张水库香鱼遗传多样性如此贫乏到底是历史原因还是近期陆封的结果,仍需进一步研究。目前亟待采取有效措施保护东张水库陆封香鱼。     

香鱼肌肉与免疫器官6种同工酶的表达

采用聚丙烯酰胺电泳方法,对香鱼(Plecoglossus altivelis)肌肉和肝脏、脾脏及肾脏3种免疫器官的6种与防御机能相关的同工酶的表达进行研究,共分析了SOD、POD、ACP、ALP、CAT、EST等6种同工酶的表达模式。结果表明,香鱼已经具有了完善的机体防御系统,肝脏和肾脏在香鱼的防御系统中发挥着十分重要的作用。本研究能为筛选具有较强抗病力和适应性的香鱼良种提供有价值的理论参考。     

鸭绿江香鱼耳石日轮与生产的研究

１９９２年对鸭绿江香鱼耳石日轮与生长进行了研究。人工受精孵胚胎发育后期和前仔鱼期连续剖察表明,受精后约９６小时胚体听囊内出现一对矢耳石,孵出后第２天耳石上出现第一个日轮,之后每天形成一轮。在当镜和扫描电镜下测定了幼、成鱼矢耳石的形态、直径、日轮数及其间距变化。耳石矩径与鱼体长呈线性关系,７５尾幼、成鱼的关系式为ｙ＝３＼２８ｘ＋２４８．３０     

宜昌鳅蛇仔稚鱼的形态特征

宜昌鳅蛇系底栖性小型鱼类,在长江宜昌江段的渔获物中时有出现。繁殖期为5—6月,产漂流性卵,膜径在3．1—4．0毫米之间,水温21．2--25℃时,受精卵约经40小时孵出。作者对其仔稚鱼的形态进行了观察。     

宁海地区香鱼弧菌病病原菌鉴定

摘要：【目的】香鱼弧菌病对中国沿海地区的香鱼养殖业造成了巨大的危害,然而,病原不明导致了防治上的许多问题。本文鉴定了引起宁海地区香鱼爆发性弧菌病的病原。【方法】采用TCBS平板分离优势菌;采用回归感染试验确认病原菌,采用改进的寇氏法计算LD50;采用形态学观察、生理生化特征测定、细菌特异性引物PCR扩增检测及细菌16S rRNA和金属蛋白酶（MP）基因序列分析鉴定细菌;采用药敏实验测定它对部分抗生素的敏感性。【结果】分离并鉴定优势菌株ayu-H080701为宁海地区香鱼弧菌病的病原菌,它对香鱼的半致死量为1.2×104 CFU。形态学观察和生理生化特征测定表明,ayu-H080701与鳗利斯顿氏菌最为接近。PCR扩增检测表明,细菌16S rRNA 基因通用引物和鳗利斯顿氏菌MP基因特异引物均能扩增到预期大小的特异性条带。ayu-H080701与鳗利斯顿氏菌16S rRNA基因核苷酸序列同源性最高,为99.4%～99.5%,与同属的海弧菌和美人鱼发光杆菌分别为94.3%和91.9%;ayu-H080701与鳗利斯顿氏菌MP氨基酸序列同源性高达97.6%～98.8 %,与其它弧菌科成员则低于75.6 %,系统进化树分析也揭示ayu-H080701与鳗利斯顿氏菌进化相关性最高。【结论】引起宁海地区香鱼弧菌病的菌株ayu-H080701被鉴定为鳗利斯顿氏菌。     

Development and multiplex PCR amplification of new microsatellite markers for the freshwater fish,ayu (Plecoglossus altivelis)

Multiplex polymerase chain reaction (PCR) technique has recently been applied to gain many advantages in molecular genetics. The present study focused on the development of 15 new microsatellite markers with multiplex PCR systems in ayu, Plecoglossus altivelis, an important freshwater fish in Japan. All loci were followed Mendelian inheritance in 27 F1 progeny except for the one locus. The number of alleles per locus ranged from nine to 44 and the observed heterozygosities ranged from 0.680 to 0.980 in 50 unrelated individuals. The results indicate that these new microsatellite markers are useful for studies of linkage mapping and population genetics for the species.     

Isolation and characterization of 49 polymorphic microsatellite loci in the ayu,Plecoglossus altivelis

A total of 49 novel polymorphic microsatellite markers in the ayu fish, Plecoglossus altivelis, were successfully isolated and characterized. The number of alleles per locus, observed heterozygosity, and expected heterozygosity ranged from two to 25 (11.3 on average), from 0.125 to 1.000 (0.648 on average), and from 0.123 to 0.957 (0.751 on average), respectively. These microsatellite markers will be useful for both studying population structure and identifying quantitative trait loci that control evolutionarily and economically important traits of ayu.     

Development of immersion vaccine for bacterial cold-water disease in ayu Plecoglossus altivelis

Flavobacterium psychrophilum (F. psychrophilum) is the causative agent of bacterial cold-water disease (BCWD) that occurs in ayu Plecoglossus altivelis. Formalin-killed cell of F. psychrophilum has long been studied as an immersion vaccine for BCWD. In this study, we explored the possibility of F. psychrophilum collagenase (fpcol) for use as the immersion vaccine. BCWD convalescent ayu sera contained specific IgM antibodies against somatic F. psychrophilum and fpcol, meaning that fpcol is a promising antigen for the vaccine development. The recombinant fpcol was successfully expressed in Escherichia coli and Brevibacillus chosinensis (B. chosinensis). The culture supernatant of the B. chosinensis was used as an immersion vaccine solution. The vaccinated ayu were then challenged by soaking into F. psychrophilum culture. In two experimental groups, the relative percentages of survivals were 63 and 38%, respectively, suggesting that fpcol is promising as the immersion vaccine for ayu-BCWD.     

Inheritance of amplified fragment length polymorphism markers and their utility in population genetic analysis of Plecoglossus altivelis

The reproducibility, mode of inheritance and polymorphism of amplified fragment length polymorphism (AFLP) markers were examined in ayu Plecoglossus altivelis (Salmoniformes: Plecoglossidae). The AFLP markers were highly reproducible, their inheritance following Mendelian expectations. The number of fragments amplified (34–134), polymorphic ratio (0·15–0·78) and average heterozygosity (0·02–0·25) of the AFLP markers showed significant variation among six primer pairs and among ayu populations, including a landlocked Lake‐Biwa population, two amphidromous populations ( P. a. altivelis ) and two Ryukyu‐ayu populations ( P. a. ryukyuensis ). Although AFLP analysis provided similar results in intra‐population diversity and relationships among populations to those found by analyses of allozymes, microsatellites and mitochondrial DNA sequences, AFLPs showed higher polymorphisms and hence greater distinction between genetically close populations.     

Ultrastructural study of skin and eye of UV‐B irradiated ayu Plecoglossus altivelis

A scanning electron microscopic study of the skin and eye of UV‐B radiated ayu Plecoglossus altivelis (age 30 days, mean ±  s . e . total length: 16·25 ± 0·11 mm) under laboratory condition showed marked changes when compared with the control fish without UV‐B radiation. The exposure of the fish to the radiation resulted in the destruction of microridges in the epidermis and exposed neuromast cells of the skin. Domed protrusions were also more common in the skin of UV‐B radiated fish than in the control fish. The appearance of mucus in both groups was different. In the control skin the mucus was spread over a wide area whereas in the treated fish the mucus was concentrated in a small area. The anastomozing structures of the microridges of the eyes were lost in UV‐B radiated fish and the microridges themselves were fewer in number, fragmented, and aggregated. Mucus cells, prominently visible in the control fish, were distorted in the treated fish. Cell contours were irregular in UV‐B radiated fish and cell to cell contacts had been lost in this group.     

长江荆江段的渔业生态环境评价

1997—2002年,对长江荆江段青、草、鲢、鳙产卵场的水环境质量和浮游生物状况进行了调查,并用河流水质综合评价方法和Kolkwitz&Marsson体系法进行了评价。结果表明:在每年5—6月份的青、草、鲢、鳙繁殖季节,仅个别采样点铜或石油类超过渔业水质标准,该江段水质基本符合渔业水质标准和地表水环境质量Ⅱ类标准,能满足青、草、鲢、鳙繁殖对水质质量的要求。浮游生物的指示性表明其水体属于β中污带。       

Examination of reproductive success of transplanted stocks in an amphidromous fish, Plecoglossus altivelis (Temmink et Schlegel) using mitochondrial DNA and isozyme markers.

Isozymes and mitochondrial DNA (mtDNA) were used as genetic markers to evaluate the reproductive success of transplanted stocks of ayu Plecoglossus ultivelis in a highly stocked Japanese river, the Shinano. Two major alleles at the MPP and GPI-A * loci and 26 mtDNA clonal genotypes were demonstrated and surveyed in two non-native source populations and five collections from the Shinano River. Contributing populations were clearly discriminated in their isozymes and mtDNA composition. Despite the stocking of large numbers of non-native fish, they have had little effect on the genetic characteristics of the native population. Maturation and migration of the stocking and native populations was traced by genetic markers to clear the mechanism of this event. All the contributing populations join together in the migration toward the spawning ground throughout the spawning season but these contributing populations occur independently of each other in other sites of the river.