The genetic basis for resistance to bioinsecticides in diamondback moth Plutella xylostella

近年来出现了几种生物杀虫剂的发展，它们的作用方式往往非常具体，因此可能比传统的化学杀虫剂更环保。甚至有人提出，由于它们的生物学起源，这些杀虫剂可能避免了目标害虫物种的抗性进化。不出所料，事实并非如此，而且农业上对其中两种杀虫剂（Bt和Spinosad）的使用增加，导致了小菜蛾（diamondback moth）的抗药性进化，小菜蛾是十字花科作物中最具破坏性的全球害虫。了解耐药性的作用方式和机制对于这些化合物的长期可持续使用至关重要。此外，尽管转基因Bt作物在英国还不能用于商业用途，但了解对现有Bt作物喷剂的抗性基础对于评估未来对转基因作物的抗性可能性至关重要。令人惊讶的是，尽管Bt毒素在叶面喷药和转基因作物中具有全球重要性，但其目标还远未明确。在这里，我们的目标是描述在田间获得对这些杀虫剂抗性的小菜蛾种群的抗性遗传基础。已知Bt （Cry1Ac毒素）和spinosad抗性都涉及一个主要基因，在这两种情况下，我们已经确定了相关的分子标记。我们将用匿名分子标记瞄准这些抗性基因周围的基因组区域。然后，通过大插入BAC文库，我们将对两个抗性基因周围的区域进行完整测序。为了鉴定抗性基因，我们将测试从该区域鉴定的候选基因，看看它们是否在幼虫和适当的组织（如中肠）中表达，以及抗性种群和易感种群之间的表达是否存在差异。此外，我们将使用RNAi敲低实验来确定是否存在降低候选基因表达对易感品系抗性水平的影响。我们还将描述候选位点基因转录的抗性和易感系之间的序列差异（如果有的话）。这将有助于开发基于pcr的快速检测田间捕获样本中抗性等位基因的方法，从而促进田间监测抗性等位基因的传播。鉴定具有新作用模式的杀虫剂在防止农业害虫种群产生交叉抗性方面发挥着重要作用，Bt和spinosad等新型生物农药在补充和/或替代旧的化学杀虫剂方面发挥着重要作用。然而，除非采取措施与之斗争，否则目标害虫的抗药性演变是不可避免的。更好地了解耐药性机制，从而了解这些化合物的作用机制，对于开发具有不同作用模式的新毒素和制定在该领域对抗耐药性的战略至关重要。

Recent years have seen the development of several bio-insecticides, which are often very specific in their mode of action and therefore potentially more environmentally friendly than traditional chemical insecticides. It was even suggested that, due to their biological origin, these insecticides might avoid the evolution of resistance in targeted pest species. Unsurprisingly this has not proven to be the case, and increased agricultural use of two of these insecticides, Bt and Spinosad, has led to the evolution of resistance in the diamondback moth, the most destructive global pest of cruciferous crops. Understanding the mode of action and mechanisms of resistance is crucial for long term, sustainable use of these compounds. Furthermore, although transgenic Bt crops are not available for commercial use in the UK, an understanding of the basis for resistance to existing Bt crop sprays will be vital in assessing the likelihood of resistance to transgenic crops in the future. Surprisingly, despite its global importance in both foliar sprays and transgenic crops, the targets for Bt toxin are far from clear. Here, we aim to characterise the genetic basis of resistance in populations of diamondback moth that have acquired resistance to these insecticides in the field. Both Bt (Cry1Ac toxin) and spinosad resistance are known to involve a single major gene and in both cases we have already identified linked molecular markers. We will target the regions of the genome surrounding these resistance genes with anonymous molecular markers. Then, by means of a large-insert BAC library, we will fully sequence the region surrounding the two resistance genes. In order to identify the resistance gene we will then test candidate genes identified from this region to see whether they are expressed in the larvae and in appropriate tissues such as the midgut, and also whether there are differences in expression between resistant and susceptible populations. Additionally, we will use RNAi knockdown experiments to determine whether there is an effect of reducing the expression of the candidate gene on levels of resistance in susceptible lines. We will also characterise sequence differences, if any, between the resistant and susceptible lines in the gene transcript of the candidate locus. This will allow development of rapid PCR-based methods for detecting resistant alleles in field caught samples that will facilitate field monitoring of the spread of resistance alleles. Identification of insecticides with novel modes of action can play an important role in preventing the evolution of cross resistance in populations of agricultural pests, and novel biopesticides such as Bt and spinosad have played an important role in complementing and/or replacing older chemical insecticides. However the evolution of resistance is inevitable in targeted pests unless measures are taken to combat it. A better understanding of the mechanisms of resistance, and hence the mechanism of action of these compounds will be crucially important in developing novel toxins with distinct modes of action, and in developing strategies for combating resistance in the field.