Monitoring of noble, signal and narrow-clawed crayfish using environmental DNA from freshwater samples

For several hundred years freshwater crayfish (Crustacea—Decapoda—Astacidea) have played an important ecological, cultural and culinary role in Scandinavia. However, many native populations of noble crayfish Astacus astacus have faced major declines during the last century, largely resulting from human assisted expansion of non-indigenous signal crayfish Pacifastacus leniusculus that carry and transmit the crayfish plague pathogen. In Denmark, also the non-indigenous narrow-clawed crayfish Astacus leptodactylus has expanded due to anthropogenic activities. Knowledge about crayfish distribution and early detection of non-indigenous and invasive species are crucial elements in successful conservation of indigenous crayfish. The use of environmental DNA (eDNA) extracted from water samples is a promising new tool for early and non-invasive detection of species in aquatic environments. In the present study, we have developed and tested quantitative PCR (qPCR) assays for species-specific detection and quantification of the three above mentioned crayfish species on the basis of mitochondrial cytochrome oxidase 1 (mtDNA-CO1), including separate assays for two clades of A. leptodactylus. The limit of detection (LOD) was experimentally established as 5 copies/PCR with two different approaches, and the limit of quantification (LOQ) were determined to 5 and 10 copies/PCR, respectively, depending on chosen approach. The assays detected crayfish in natural freshwater ecosystems with known populations of all three species, and show promising potentials for future monitoring of A. astacus, P. leniusculus and A. leptodactylus. However, the assays need further validation with data 1) comparing traditional and eDNA based estimates of abundance, and 2) representing a broader geographical range for the involved crayfish species.数百年来，淡水螯虾（甲壳纲—十足目—螯虾下目，Crustacea—Decapoda—Astacidea）在斯堪的纳维亚地区发挥着重要的生态、文化及食用层面的作用。近一个世纪以来，诸多本土高贵螯虾（Astacus astacus）种群遭遇严重衰退，其主要诱因是人类活动辅助扩散了携带有螯虾瘟疫病原菌的外来信号螯虾（Pacifastacus leniusculus）。在丹麦，外来的狭螯螯虾（Astacus leptodactylus）也因人为活动出现种群扩张。掌握螯虾的分布情况，并对外来入侵物种开展早期检测，是成功保护本土螯虾的核心要素。从水体样本中提取的环境DNA（environmental DNA, eDNA）技术，是一种极具应用前景的新型早期非侵入式水生生物物种检测手段。本研究基于线粒体细胞色素氧化酶亚基1（mtDNA-CO1）基因，针对上述三种螯虾开发并验证了物种特异性检测与定量的实时荧光定量PCR（quantitative PCR, qPCR）检测方法，其中还包含针对狭螯螯虾两个演化支的专属检测体系。本研究通过两种不同实验方法确定检测限（LOD）为每PCR反应体系5个拷贝，定量限（LOQ）则根据所选方法不同分别为每PCR反应体系5个和10个拷贝。该检测方法可在三种螯虾均有已知种群分布的自然淡水生态系统中检出螯虾，展现出用于后续监测高贵螯虾、信号螯虾与狭螯螯虾的良好应用潜力。不过，该检测方法仍需进一步验证：一是需对比传统种群丰度估算方法与eDNA技术的丰度估算结果，二是需在涵盖目标螯虾物种更广泛地理分布范围的区域开展验证。