权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Development and an application of a PCR-baaed method for the identification of cosumed Japanese flounder, Paralichthys olivaceus, juveniles form the gut contents of crustacean predators

基于 PCR 的方法的开发和应用，用于鉴定食用的日本牙鲆、牙鲆、甲壳类捕食者肠道内容物的幼鱼

基本信息

批准号：
14560160
负责人：
ASAHIDA Takashi
金额：
$ 2.18万
依托单位：
Kitasato University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2004
项目状态：
已结题

项目摘要

It is recognized that predation by benthic crustaceans is one of the major causes of mortality of flatfishes. However, it is difficult to identify ingested larval fish species from the stomach contents of crustacean predators, because crustacean predators finely macerate their prey. We have developed a method for mass screening to detect consumed Japanese flounder-specific DNA from the stomach contents of sand shrimps. The PCR primers used in the present study are designed to amplify a 191 bp region of the mitochondrial control region and are Japanese flounder specific. We applied Southern blot hybridization and an immunological detection method for mass screening. The PCR products were blotted onto a nylon membrane and hybridize with appropriate probes labeled with digoxigenin. The predation experiment was performed in the laboratory under natural light conditions and the temperature was maintained at about 20℃ through the rearing and experiment period. In a laboratory experiment, successful amplification and detection of Japanese flounder-specific DNA were possible up to 15 hrs, with 100% detection within 8 hrs, after the termination of predation. Microscopic examination of the samples revealed the presence of debris, vertebrae and other bones. However, as digestion progressed, we could not observe any debris and bones from the stomach contents sampled after 13 hrs after the termination of predation, and the percentage of successful amplification decreased with time. Since the predator is nocturnal, shrimps should be sampled within 12 hours after sunset for practical use of the method on field-collected shrimp samples. Also, we have got some new knowledge about predator-prey relationship between flounder juveniles and crustaceans in the field. We suggest that this method could be applied to a range of flatfish and crustacean predators.

人们认识到，底栖甲壳类动物的捕食是比目鱼死亡的主要原因之一。然而，很难从甲壳类捕食者的胃内容物中识别摄入的仔鱼种类，因为甲壳类捕食者会精细地浸渍猎物。我们已经开发了一种方法，用于大规模筛选，以检测消费日本比目鱼特异性DNA从胃内容物的沙虾。本研究中使用的PCR引物被设计为扩增线粒体控制区的191 bp区域，并且是日本比目鱼特异性的。我们应用Southern印迹杂交和免疫学检测方法进行大规模筛选。将PCR产物印迹到尼龙膜上，并与地高辛标记的适当探针杂交。捕食试验在室内自然光照条件下进行，饲养期和试验期温度保持在20℃左右。在实验室实验中，成功的扩增和检测的日本比目鱼特异性DNA是可能的长达15小时，与100%的检测在8小时内，捕食结束后。对样本的显微镜检查显示存在碎片、椎骨和其他骨头。然而，随着消化的进展，我们不能观察到任何碎片和骨骼从胃内容物采样后13小时后，捕食终止，成功的扩增的百分比随时间而下降。由于捕食者是夜间活动的，虾应在日落后12小时内取样，以实际使用的方法对现场收集的虾样品。同时，我们也对现场比目鱼幼鱼与甲壳类之间的捕食关系有了新的认识。我们认为，这种方法可以适用于一系列的比目鱼和甲壳类捕食者。