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

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，从甲壳类捕食者的胃内容物中识别摄入的幼鱼种类是困难的，因为甲壳类捕食者对猎物进行了精细的浸渍。我们开发了一种从沙虾胃内容物中检测消费日本比目鱼特异性DNA的大规模筛选方法。本研究中使用的PCR引物旨在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。我们采用Southern blot杂交和免疫检测方法进行大量筛选。PCR产物印迹在尼龙膜上，与标记有地高辛的合适探针杂交。捕食实验在室内自然光条件下进行，饲养和实验期间温度保持在20℃左右。在实验室实验中，可以在15小时内成功扩增和检测到日本牙鲆特异性DNA，在停止捕食后8小时内检测到100%。对这些样本的显微镜检查发现了碎片、椎骨和其他骨头。然而，随着消化的进行，在捕食结束13小时后，我们无法从胃内容物中观察到任何碎片和骨头，并且扩增成功的百分比随着时间的推移而下降。由于捕食者是夜间活动的动物，为了在实地收集的虾类样本上实际使用该方法，虾类样本应在日落后12小时内取样。同时，我们对幼体比目鱼与甲壳类动物之间的捕食关系也有了新的认识。我们建议这种方法可以应用于一系列比目鱼和甲壳类食肉动物。

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.