Environmental DNA (eDNA) metabarcoding assays to detect invasive invertebrate species in the Great Lakes

Describing and monitoring biodiversity comprise integral parts of ecosystem management. Recent research coupling metabarcoding and environmental DNA (eDNA) demonstrate that these methods can serve as important tools for surveying biodiversity, while significantly decreasing the time, expense and resources spent on traditional survey methods. The literature emphasizes the importance of genetic marker development, as the markers dictate the applicability, sensitivity and resolution ability of an eDNA assay. The present study developed two metabarcoding eDNA assays using the mtDNA 16S RNA gene with Illumina MiSeq platform to detect invertebrate fauna in the Laurentian Great Lakes and surrounding waterways, with a focus for use on invasive bivalve and gastropod species monitoring. We employed careful primer design and in vitro testing with mock communities to assess ability of the markers to amplify and sequence targeted species DNA, while retaining rank abundance information. In our mock communities, read abundances reflected the initial input abundance, with regressions having significant slopes (p<0.05) and high coefficients of determination (R2) for all comparisons. Tests on field environmental samples revealed similar ability of our markers to measure relative abundance. Due to the limited reference sequence data available for these invertebrate species, care must be taken when analyzing results and identifying sequence reads to species level. These markers extend eDNA metabarcoding research for molluscs and appear relevant to other invertebrate taxa, such as rotifers and bryozoans. Furthermore, the sphaeriid mussel assay is group-specific, exclusively amplifying bivalves in the Sphaeridae family and providing species-level identification. Our assays provide useful tools for managers and conservation scientists, facilitating early detection of invasive species as well as improving resolution of mollusc diversity.

生物多样性的描述与监测是生态系统管理的核心组成部分。近年来，结合元条形码（metabarcoding）与环境DNA（environmental DNA, eDNA）的相关研究表明，此类方法可作为生物多样性调查的重要工具，同时大幅降低传统调查方法所需的时间、经费与资源投入。现有研究均强调了遗传标记开发的重要性，因为标记决定了eDNA检测体系的适用性、灵敏度与分辨能力。

本研究以线粒体DNA（mtDNA）16S核糖体RNA基因为靶标，依托Illumina MiSeq测序平台，开发了两套元条形码eDNA检测体系，用于检测劳伦琴五大湖及周边水域的无脊椎动物群落，重点适配入侵性双壳类与腹足类物种的监测需求。我们通过严谨的引物设计与模拟群落体外验证实验，评估了该标记扩增并测序靶标物种DNA的能力，同时保留了丰度等级信息。在模拟群落实验中，测序读段的丰度与初始投放的物种丰度高度吻合，所有对比组的回归斜率均具有统计学显著性（p<0.05），且决定系数（R²）较高。对野外环境样本的检测结果显示，该标记同样能够准确测定物种的相对丰度。

由于此类无脊椎动物的参考序列数据较为匮乏，在分析实验结果并将测序读段鉴定至物种水平时需格外谨慎。该标记体系拓展了软体动物的eDNA元条形码研究范围，同时也适用于轮虫、苔藓虫等其他无脊椎动物类群。此外，球蚬科蚌类检测体系具有类群特异性，仅能扩增球蚬科（Sphaeriidae）的双壳类物种，并可实现物种水平的鉴定。本研究开发的检测体系可为管理者与保护科学家提供实用工具，助力入侵物种的早期检测，并提升软体动物多样性的分辨能力。