Laurentian Forestry Centre, Natural Resources Canada, Quebec City, Quebec, Canada.
Ministère des Forêts, de la Faune et des Parcs du Québec, Direction de la Protection des Forêts, Quebec City, Quebec, Canada.
Pest Manag Sci. 2022 Jan;78(1):336-343. doi: 10.1002/ps.6645. Epub 2021 Oct 21.
In eastern Canada, surveys of overwintering 2 instar spruce budworm (Choristoneura fumiferana) larvae ('L2s') are carried out each fall to guide insecticide application decisions in the following spring. These surveys involve the collection of fir and spruce branches in selected stands, followed by the mechanical/chemical removal of larvae. The latter then are counted manually on filter papers, using a stereomicroscope. Considering the significant effort and difficulties which this manual counting entails, we developed a quantitative (q)PCR-based 'molecular counting' approach designed to make this step less tedious.
Using the C. fumiferana mitochondrial cytochrome c oxidase 1 (COI) gene as a target for qPCR DNA quantification, we show that the amount of DNA in a larval extract is strongly correlated with the number of larvae used to generate that extract, and that molecular estimates of L2 counts are comparable to those generated using the manual approach. In addition, we used the same DNA extracts to monitor the microsporidian pathogen Nosema fumiferanae, and the hymenopteran parasitoids Glypta fumiferanae and Apanteles fumiferanae in overwintering L2s employing a subset of a TaqMan assay developed by Nisole et al. (2020) for the identification of budworm natural enemies. We show that the proportion of individuals affected by each natural enemy in samples containing a known number of larvae can be estimated from presence/absence data through the binomial probability distribution.
The present proof-of-principle study shows that a molecular approach for counting L2s and assessing their natural enemy load is clearly possible and is expected to generate reliable results. © 2021 Her Majesty the Queen in Right of Canada. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry. Reproduced with the permission of the Minister of Natural Resources Canada.
在加拿大东部，每年秋季都会对越冬的 2 龄云杉卷叶蛾幼虫（L2）进行调查，以指导次年春季的杀虫剂应用决策。这些调查涉及在选定的林分中收集冷杉和云杉枝条，然后通过机械/化学方法去除幼虫。然后，在滤纸上使用立体显微镜手动计数幼虫。考虑到这种手动计数所涉及的大量工作和困难，我们开发了一种基于 qPCR 的“分子计数”方法，旨在使这一过程不那么繁琐。
使用云杉卷叶蛾线粒体细胞色素 c 氧化酶 1（COI）基因作为 qPCR DNA 定量的靶标，我们表明幼虫提取物中的 DNA 量与用于生成该提取物的幼虫数量密切相关，并且分子估计的 L2 数量与使用手动方法生成的数量相当。此外，我们使用相同的 DNA 提取物监测越冬 L2 中的微孢子虫病原体 Nosema fumiferanae 以及膜翅目寄生蜂 Glypta fumiferanae 和 Apanteles fumiferanae，采用 Nisole 等人（2020 年）开发的 TaqMan 检测的一部分，用于鉴定卷叶蛾的天然天敌。我们表明，可以根据二项式概率分布从存在/不存在数据中估计含有已知幼虫数量的样本中每个天然敌害个体的比例。
本原理验证研究表明，一种用于计数 L2 并评估其天然敌害负荷的分子方法显然是可行的，并且有望产生可靠的结果。2021 年，加拿大女王陛下以加拿大的名义享有版权。害虫管理科学由 John Wiley & Sons Ltd 代表化学工业协会出版。经加拿大自然资源部许可转载。
