Alshari Norli Fauzani Mohd Abu Hassan, Ahmad Siti Zuliana, Azlan Azali, Lee Youn-Ho, Azzam Ghows, Nor Siti Azizah Mohd
School of Biological Sciences, Universiti Sains Malaysia, 11800, Penang, Malaysia. E-mail:
Korean Institute of Ocean Science and Technology, Republic of Korea. E-mail:
Zool Stud. 2021 Dec 22;60:e76. doi: 10.6620/ZS.2021.60-76. eCollection 2021.
The Merbok River (north-west of Peninsular Malaysia) is a mangrove estuary that provides habitat for over 100 species of fish, which are economically and ecologically important. Threats such as habitat loss and overfishing are becoming a great concern for fisheries conservation and management. The identification of larval fish in this estuarine system is important to complement information on the adults. This is because the data could inform the spawning behaviour, reproductive biology, selection of nursery grounds and migration route of fish. Such information is invaluable for fisheries and aquatic environmental monitoring, and thus for their conservation and management. However, identifying fish larvae is a challenging task based only on morphology and even traditional DNA barcoding. To address this, DNA metabarcoding was utilised to detect the diversity of fish in the Merbok River. To complete the study, the fish larvae were collected at six sampling sites of the river. The extracted larval DNA was amplified for the Cytochrome Oxidase subunit 1 () and 12S ribosomal RNA (12S rRNA) genes based on the metabarcoding approach using shotgun sequencing on the next-generation sequencing (NGS) Illumina MiSeq platform. Eighty-nine species from 65 genera and 41 families were detected, with , , and among the most common species. The lower diversity observed from previous morphological studies is suggested to be mainly due to seasonal variation over the sampling period between the two methods and limited 12S rRNA sequences in current databases. The metabarcode data and a validation Sanger sequencing step using 15 species-specific primer pairs detected three species in common: , and . Several discrepancies observed between the two molecular approaches could be attributed to contaminants during sampling and DNA extraction, which could mask the presence of target species, especially when DNA from the contaminants is more abundant than the target organisms. In conclusion, this rapid and cost-effective identification method using DNA metabarcoding allowed the detection of numerous fish species from bulk larval samples in the Merbok River. This method can be applied to other sites and other organisms of interest.
默博克河（位于马来西亚半岛西北部）是一个红树林河口，为100多种鱼类提供了栖息地，这些鱼类具有重要的经济和生态价值。栖息地丧失和过度捕捞等威胁正成为渔业保护和管理的重大关切。识别该河口系统中的幼鱼对于补充成鱼信息很重要。这是因为这些数据可以为鱼类的产卵行为、生殖生物学、育幼场选择和洄游路线提供信息。此类信息对于渔业和水生环境监测以及它们的保护和管理来说非常宝贵。然而，仅基于形态学甚至传统的DNA条形码来识别鱼幼体是一项具有挑战性的任务。为了解决这个问题，利用DNA宏条形码技术来检测默博克河中的鱼类多样性。为完成该研究，在该河的六个采样点收集了鱼幼体。基于宏条形码方法，使用下一代测序（NGS）Illumina MiSeq平台上的鸟枪法测序，对提取的幼体DNA进行细胞色素氧化酶亚基1（COI）和12S核糖体RNA（12S rRNA）基因的扩增。检测到来自65个属和41个科中的89个物种，其中，、、和是最常见的物种。先前形态学研究中观察到的较低多样性主要被认为是由于两种方法在采样期间的季节变化以及当前数据库中12S rRNA序列有限。宏条形码数据和使用15对物种特异性引物对的验证性桑格测序步骤检测到三种共同的物种：、和。两种分子方法之间观察到的一些差异可能归因于采样和DNA提取过程中的污染物，这些污染物可能掩盖目标物种的存在，特别是当污染物的DNA比目标生物的DNA更丰富时。总之，这种使用DNA宏条形码的快速且经济高效的识别方法能够从默博克河的大量幼体样本中检测到众多鱼类物种。该方法可应用于其他地点和其他感兴趣的生物。
