Ratsch Rikki, Kingsbury Bruce A, Jordan Mark A
Department of Biology, Purdue University Fort Wayne, Fort Wayne, IN 46805, USA.
Animals (Basel). 2020 Jun 19;10(6):1057. doi: 10.3390/ani10061057.
Environmental DNA (eDNA) surveys utilize DNA shed by organisms into their environment in order to detect their presence. This technique has proven effective in many systems for detecting rare or cryptic species that require high survey effort. One potential candidate for eDNA surveying is Kirtland's Snake (), a small natricine endemic to the midwestern USA and threatened throughout its range. Due to its cryptic and fossorial lifestyle, it is also a notoriously difficult snake to survey, which has limited efforts to understand its ecology. Our goal was to utilize eDNA surveys for this species to increase detection probability and improve survey efficiency to assist future conservation efforts. We conducted coverboard surveys and habitat analyses to determine the spatial and temporal activity of snakes, and used this information to collect environmental samples in areas of high and low snake activity. In addition, we spiked artificial crayfish burrows with Kirtland's Snake feces to assess the persistence of eDNA under semi-natural conditions. A quantitative PCR (qPCR) assay using a hydrolysis probe was developed to screen the environmental samples for Kirtland's Snake eDNA that excluded closely related and co-occurring species. Our field surveys showed that snakes were found in the spring during the first of two seasons, and in areas with abundant grass, herbaceous vegetation, and shrubs. We found that eDNA declines within a week under field conditions in artificial crayfish burrows. In environmental samples of crayfish burrow water and sediment, soil, and open water, a single detection was found out of 380 samples. While there may be physicochemical and biological explanations for the low detection observed, characteristics of assay performance and sampling methodology may have also increased the potential for false negatives. We explored these outcomes in an effort to refine and advance the successful application of eDNA surveying in snakes and groundwater microhabitats.
环境DNA（eDNA）调查利用生物体释放到环境中的DNA来检测它们的存在。这项技术已在许多系统中被证明对检测需要大量调查工作的稀有或隐秘物种有效。eDNA调查的一个潜在候选对象是基尔特兰蛇（Kirtland's Snake），它是一种小型游蛇科蛇类，特产于美国中西部，在其整个分布范围内都受到威胁。由于其隐秘的穴居生活方式，它也是一种出了名的难以调查的蛇类，这限制了人们对其生态的了解。我们的目标是利用针对该物种的eDNA调查来提高检测概率并提高调查效率，以协助未来的保护工作。我们进行了覆盖板调查和栖息地分析，以确定蛇的时空活动，并利用这些信息在蛇活动频繁和不频繁的区域采集环境样本。此外，我们在人工小龙虾洞穴中添加了基尔特兰蛇的粪便，以评估eDNA在半自然条件下的持久性。我们开发了一种使用水解探针的定量PCR（qPCR）检测方法，以筛选环境样本中基尔特兰蛇的eDNA，同时排除密切相关和共存的物种。我们的野外调查表明，在两个季节中的第一个季节的春季，以及有丰富草丛、草本植被和灌木的区域发现了蛇。我们发现，在野外条件下，人工小龙虾洞穴中的eDNA在一周内就会下降。在小龙虾洞穴水和沉积物、土壤以及开阔水域的环境样本中，380个样本中仅检测到一例。虽然对于观察到的低检测率可能存在物理化学和生物学方面的解释，但检测方法性能和采样方法的特点也可能增加了假阴性的可能性。我们探讨了这些结果，以期完善和推进eDNA调查在蛇类和地下水微生境中的成功应用。
