Developing an eDNA toolkit to quantify broadcast spawning events of the sea scallop Placopecten magellanicus: moving beyond fertilization assays
Developing an eDNA toolkit to quantify broadcast spawning events of the sea scallop Placopecten magellanicus: moving beyond fertilization assays
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DOI：
10.3354/meps12991
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发表时间：
2019-07-04
影响因子：
2.5
通讯作者：
Wahle, Richard A.
中科院分区：
文献类型：
--
作者：
Bayer, Skylar R.;Countway, Peter D.;Wahle, Richard A.
广播产卵器释放它们的配子到水柱中进行“机会”受精事件。然而，近实时检测此类事件极其困难，但需要改进渔业和养护管理做法。这是几乎不可能区分许多物种的配子显微镜，因此，DNA为基础的技术是最好的检测和定量配子从现场收集的浮游生物样品。我们开发了一种定量PCR（qPCR）的方法来检测和量化海洋环境DNA（eDNA）样品中的广播产卵事件。我们将这种方法应用于具有商业价值的广播产卵双壳贝类--扇贝Placopecten magellanicus。我们的方法包括（1）测序的内部转录间隔区（ITS）区域，（2）开发一种新的物种特异性探针和引物组，（3）测试的探针和引物组上的一系列稀释的海扇贝精子，以量化配子丰度和DNA拷贝数之间的关系，和（4）进行码头现场测试，我们的方法对浮游生物样品附近的自然产卵海扇贝。定量PCR检测结果表明，DNA拷贝数与麦哲伦栉孔扇贝精子丰度之间存在明显的相关性，表明该方法可用于检测雄性栉孔扇贝在产卵过程中的精子释放量。在扇贝产卵季节收集的浮游生物样本显示，在20亩（可能的鸡蛋）颗粒大小的部分扇贝eDNA的尖峰。这种方法具有很大的潜力，提供更有效的估计的时间，幅度和空间尺度的生殖事件比传统的方法为广泛的广播产卵。
Broadcast spawners release their gametes into the water column for 'chance' fertilization events. However, detection of such events in near real-time is extremely difficult, but needed to improve fisheries and conservation management practices. It is practically impossible to distinguish the gametes of many species by microscopy; therefore, DNA-based techniques are preferable to detect and quantify gametes from field-collected plankton samples. We developed a quantitative PCR (qPCR) approach to detect and quantify broadcast spawning events in marine environmental DNA (eDNA) samples. We applied this approach to a commercially valuable broadcast spawning bivalve species, the sea scallop Placopecten magellanicus. Our approach includes (1) sequencing the internal transcribed spacer (ITS) region, (2) developing a novel species-specific probe and primer set, (3) testing the probe and primer set on a dilution series of sea scallop sperm to quantify the relationship between gamete abundance and DNA copy number, and (4) conducting dockside field tests of our method on plankton samples adjacent to naturally spawning sea scallops. Quantitative PCR revealed a clear relationship between DNA copy number and P. magellanicus sperm cell abundance, indicating that this method is reliable for detecting sperm release by male scallops during spawning events. Plankton samples collected during the scallop spawning season revealed spikes of scallop eDNA in both the 20 mu m (possible eggs) particle size-fractions. This method holds great potential to provide more efficient estimates of the timing, magnitude, and spatial scale of reproductive events than conventional methods for a wide range of broadcast spawners.