PDF(Pubmed)
Abstract：
BACKGROUND: Environmental DNA (eDNA) monitoring is growing increasingly popular in aquatic systems as a valuable complementary method to conventional monitoring. However, such tools have not yet been extensively applied for metazoan fish parasite monitoring. The fish ectoparasite Gyrodactylus salaris, introduced into Norway in 1975, has caused severe damage to Atlantic salmon populations and fisheries. Successful eradication of the parasite has been carried out in several river systems in Norway, and Atlantic salmon remain infected in only seven rivers, including three in the Drammen region. In this particular infection region, a prerequisite for treatment is to establish whether G. salaris is also present on rainbow trout upstream of the salmon migration barrier. Here, we developed and tested eDNA approaches to complement conventional surveillance methods.
METHODS: Water samples (2 × 5 l) were filtered on-site through glass fibre filters from nine locations in the Drammen watercourse, and DNA was extracted with a CTAB protocol. We developed a qPCR assay for G. salaris targeting the nuclear ribosomal ITS1 region, and we implemented published assays targeting the mitochondrial cytochrome-b and NADH-regions for Atlantic salmon and rainbow trout, respectively. All assays were transferred successfully to droplet digital PCR (ddPCR).
RESULTS: All qPCR/ddPCR assays performed well both on tissue samples and on field samples, demonstrating the applicability of eDNA detection for G. salaris, rainbow trout and Atlantic salmon in natural water systems. With ddPCR we eliminated a low cross-amplification of Gyrodactylus derjavinoides observed using qPCR, thus increasing specificity and sensitivity substantially. Duplex ddPCR for G. salaris and Atlantic salmon was successfully implemented and can be used as a method in future surveillance programs. The presence of G. salaris eDNA in the infected River Lierelva was documented, while not elsewhere. Rainbow trout eDNA was only detected at localities where the positives could be attributed to eDNA release from upstream land-based rainbow trout farms. Electrofishing supported the absence of rainbow trout in all of the localities.
CONCLUSIONS: We provide a reliable field and laboratory protocol for eDNA detection of G. salaris, Atlantic salmon and rainbow trout, that can complement conventional surveillance programs and substantially reduce the sacrifice of live fish. We also show that ddPCR outperforms qPCR with respect to the specific detection of G. salaris.
METHODS: Water samples (2 × 5 l) were filtered on-site through glass fibre filters from nine locations in the Drammen watercourse, and DNA was extracted with a CTAB protocol. We developed a qPCR assay for G. salaris targeting the nuclear ribosomal ITS1 region, and we implemented published assays targeting the mitochondrial cytochrome-b and NADH-regions for Atlantic salmon and rainbow trout, respectively. All assays were transferred successfully to droplet digital PCR (ddPCR).
RESULTS: All qPCR/ddPCR assays performed well both on tissue samples and on field samples, demonstrating the applicability of eDNA detection for G. salaris, rainbow trout and Atlantic salmon in natural water systems. With ddPCR we eliminated a low cross-amplification of Gyrodactylus derjavinoides observed using qPCR, thus increasing specificity and sensitivity substantially. Duplex ddPCR for G. salaris and Atlantic salmon was successfully implemented and can be used as a method in future surveillance programs. The presence of G. salaris eDNA in the infected River Lierelva was documented, while not elsewhere. Rainbow trout eDNA was only detected at localities where the positives could be attributed to eDNA release from upstream land-based rainbow trout farms. Electrofishing supported the absence of rainbow trout in all of the localities.
CONCLUSIONS: We provide a reliable field and laboratory protocol for eDNA detection of G. salaris, Atlantic salmon and rainbow trout, that can complement conventional surveillance programs and substantially reduce the sacrifice of live fish. We also show that ddPCR outperforms qPCR with respect to the specific detection of G. salaris.
摘要：
背景：环境DNA（eDNA）监测在水生系统中越来越受欢迎，作为常规监测的一种有价值的补充方法。然而,这些工具尚未广泛用于后生动物鱼类寄生虫监测。鱼外寄生虫，1975年引入挪威，对大西洋鲑鱼种群和渔业造成了严重破坏。在挪威的几个河流系统中成功根除了寄生虫，大西洋鲑鱼只在7条河流中被感染，包括三个在Drammen地区.在这个特殊的感染区域，治疗的先决条件是确定鲑鱼迁徙屏障上游的虹鳟鱼上是否也存在G.salaris。这里,我们开发并测试了eDNA方法来补充常规监测方法.
方法：通过Drammen水道9个地点的玻璃纤维过滤器现场过滤水样（2×5升），用CTAB方案提取DNA。我们开发了针对针对核核糖体ITS1区域的G.salaris的qPCR检测方法，我们实施了针对大西洋鲑鱼和虹鳟鱼的线粒体细胞色素b和NADH区域的公开试验，分别。将所有测定成功转移至液滴数字PCR(ddPCR)。
结果：所有qPCR/ddPCR检测在组织样本和现场样本中均表现良好，证明eDNA检测对S.salaris的适用性，虹鳟鱼和大西洋鲑鱼在自然的水系统。使用ddPCR，我们消除了使用qPCR观察到的Gyrodactylusderjavinoides的低交叉扩增，从而大大提高了特异性和敏感性。已成功实施了针对G.salaris和大西洋鲑鱼的DuplexddPCR，可在未来的监测计划中用作方法。记录了受感染的Lierelva河中存在G.salariseDNA，而不是其他地方。虹鳟鱼eDNA仅在某些地方被检测到，这些地方的阳性可归因于上游陆基虹鳟鱼养殖场的eDNA释放。电捕捞支持所有地区都没有虹鳟鱼。
结论：我们提供了可靠的现场和实验室方案，用于G.salaris的eDNA检测，大西洋鲑鱼和虹鳟鱼,这可以补充传统的监测计划，并大大减少活鱼的牺牲。我们还表明，ddPCR优于qPCR的特异性检测。
方法：通过Drammen水道9个地点的玻璃纤维过滤器现场过滤水样（2×5升），用CTAB方案提取DNA。我们开发了针对针对核核糖体ITS1区域的G.salaris的qPCR检测方法，我们实施了针对大西洋鲑鱼和虹鳟鱼的线粒体细胞色素b和NADH区域的公开试验，分别。将所有测定成功转移至液滴数字PCR(ddPCR)。
结果：所有qPCR/ddPCR检测在组织样本和现场样本中均表现良好，证明eDNA检测对S.salaris的适用性，虹鳟鱼和大西洋鲑鱼在自然的水系统。使用ddPCR，我们消除了使用qPCR观察到的Gyrodactylusderjavinoides的低交叉扩增，从而大大提高了特异性和敏感性。已成功实施了针对G.salaris和大西洋鲑鱼的DuplexddPCR，可在未来的监测计划中用作方法。记录了受感染的Lierelva河中存在G.salariseDNA，而不是其他地方。虹鳟鱼eDNA仅在某些地方被检测到，这些地方的阳性可归因于上游陆基虹鳟鱼养殖场的eDNA释放。电捕捞支持所有地区都没有虹鳟鱼。
结论：我们提供了可靠的现场和实验室方案，用于G.salaris的eDNA检测，大西洋鲑鱼和虹鳟鱼,这可以补充传统的监测计划，并大大减少活鱼的牺牲。我们还表明，ddPCR优于qPCR的特异性检测。
