Crayfish of North American origin are amongst the most prominent high-impact invasive invertebrates in European freshwaters. They contribute to the decline of European native crayfish species by spreading the pathogen causing crayfish plague, the oomycete Aphanomyces astaci. In this study we validated the specificity of four quantitative PCR (qPCR) assays, either published or newly developed, usable for environmental DNA (eDNA) screening for widely distributed native and non-native crayfish present in Central Europe: Astacus astacus, Pacifastacus leniusculus, Faxonius limosus and Procambarus virginalis. We then conducted an eDNA monitoring survey of these crayfish as well as the crayfish plague pathogen in a wide variety of habitat types representative for Central and Western Europe. The specificity of qPCR assays was validated against an extensive collection of crayfish DNA isolates, containing most crayfish species documented from European waters. The three assays developed in this study were sufficiently species-specific, but the published assay for F. limosus displayed a weak cross-reaction with multiple other crayfish species of the family Cambaridae. In the field study, we infrequently detected eDNA of A. astaci together with the three non-native crayfish species under examination. We never detected eDNA from A. astaci together with native crayfish, but in a few locations eDNA from both native and non-native crayfish was captured, due either to passive transport of eDNA from upstream populations or co-existence in the absence of infected crayfish carriers of A. astaci. In the study, we evaluated a robust, easy-to-use and low-cost version of the eDNA sampling equipment, based mostly on items readily available in garden stores and hobby markets, for filtering relatively large (~5 l) water samples. It performed just as well as the far more expensive equipment industrially designed for eDNA water sampling, thus opening the possibility of collecting suitable eDNA samples to a wide range of stakeholders. Overall, our study confirms that eDNA-based screening for crayfish and their associated pathogen is a feasible alternative to traditional monitoring.

中文翻译：

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同时在中欧各种生境中从环境DNA样本中同时检测本地和侵入性小龙虾和Aphanomyces astaci

北美起源的小龙虾是欧洲淡水中最著名的高影响力无脊椎动物。它们通过传播引起小龙虾瘟疫的病原体，即卵菌的Aphanomyces astaci，而导致欧洲本地小龙虾种类的减少。在这项研究中，我们验证了四种定量PCR（qPCR）检测方法的特异性，无论是已发表还是新开发的，均可用于环境DNA（eDNA）筛查，以筛查中欧广泛分布的本地和非本地小龙虾：Astacus astacus，Pacifastacus leniusculus， Faxonius limosus和Procambarus virginalis。然后，我们在代表中欧和西欧的各种栖息地类型中对这些小龙虾以及小龙虾鼠疫病原体进行了eDNA监测。针对大量小龙虾D​​NA分离物（包括从欧洲水域记录的大多数小龙虾物种）验证了qPCR分析的特异性。在这项研究中开发的三种测定法具有充分的物种特异性，但是已公开的金线莲的测定法显示出与Cambaridae家族的其他多种小龙虾物种的交叉反应较弱。在现场研究中，我们很少检测到A. astaci的eDNA以及三种非本地小龙虾物种。我们从未检测到A. astaci与天然小龙虾的eDNA，但是在少数位置，由于上游种群的eDNA被动运输或在没有感染小龙虾的情况下共存，因此捕获了来自本地和非本地小龙虾的eDNA。 astaci的载体。在这项研究中，我们评估了一种健壮的，易于使用且价格低廉的eDNA采样设备，主要基于花园商店和业余市场中容易获得的物品，用于过滤相对较大（约5升）的水样。它的性能与工业上为eDNA水采样设计的更昂贵的设备一样好，从而为广泛的利益相关者打开了收集合适eDNA样品的可能性。总体而言，我们的研究证实，基于eDNA的小龙虾及其相关病原体筛查是传统监测的可行替代方案。因此，为广泛的利益相关者提供了收集合适的eDNA样本的可能性。总体而言，我们的研究证实，基于eDNA的小龙虾及其相关病原体筛查是传统监测的可行替代方案。因此，为广泛的利益相关者提供了收集合适的eDNA样本的可能性。总体而言，我们的研究证实，基于eDNA的小龙虾及其相关病原体筛查是传统监测的可行替代方案。