Developing new genomic tools and rapid, automated resistance detection in mosquito larvae

杀虫剂耐药性对针对蚊子媒介的疟疾和虫媒病毒控制规划构成迫在眉睫的威胁。幼虫耐药是一种重要的表型评估，然而，现有的世卫组织推荐的基于死亡率的幼虫耐药测定是低通量的，并且容易受到研究者的偏见。为了解决这些问题，使用无脊椎动物自动表型平台（INVAPP）和分析算法（Vectorgon）开发了一种新的检测方法，该方法可以自动量化杀虫剂暴露后的幼虫运动。在对常见杀幼虫剂产生抗性的蚊子中观察到一些分子变化。然而，在缺乏其他潜在抗性机制的情况下，它们的表型特征在很大程度上未被探索。了解每种机制对蚊虫控制规划的潜在影响至关重要。在易感的实验室蚊子中，使用双侧GAL4-UAS系统模拟代谢酶的上调。利用INVAPP检测转基因幼虫对杀虫剂的敏感性。转基因幼虫过表达GSTe2、CYP6M2、CYP6P3和SAP2酶，这些酶与杀虫剂抗性有关。一个表达CCEae3a（一种羧酸酯酶，已在具有有机磷抗性的伊蚊中发现）的转基因系，在幼虫中显示出对三种常见有机磷的抗性，在成蚊中显示出对四种杀虫剂的抗性，并且在成蚊寿命、产卵量和幼虫孵化率方面显着（尽管适度）减少。利用CRISPR-Cas9技术，在易感蚊虫对OP和氨基甲酸酯类杀虫剂的靶基因乙酰胆碱酯酶1 （ACE1）中引入了单核苷酸多态性（G119S），并对其表型进行了评价。这项工作的结果突出了杀虫剂和杀幼虫剂抗性机制之间的重要联系，以及单独评估机制的必要性。

Insecticide resistance is a looming threat to malaria and arbovirus control programmes targeting the mosquito vector. Larval resistance is an important phenotype to assess, however, the existing WHO recommended mortality-based larval resistance assay is low-throughput and subject to investigator bias. To address these issues, a novel assay was developed using the Invertebrate Automated Phenotyping Platform (INVAPP) and analysis algorithm (Vectorgon) which provides automated quantification of larval motility after insecticide exposure. Several molecular changes have been observed in mosquitoes displaying resistance to common larvicides. However, their phenotypic characterisation in the absence of other potential resistance mechanisms is largely unexplored. Understanding the potential impact on mosquito control programmes of each mechanism is vital. In susceptible laboratory mosquitoes, upregulation of metabolic enzymes was mimicked using a bipartite GAL4-UAS system. The INVAPP assay was used to assess insecticide susceptibility of transgenic larvae overexpressing individual enzymes - GSTe2, CYP6M2, CYP6P3 and SAP2 - which are implicated in adulticide resistance. A transgenic line expressing CCEae3a (a carboxylesterase which has been identified in Aedes mosquitoes displaying organophosphate (OP) resistance), was found to display resistance to three common OPs in larvae, four-insecticide classes in adults and significant, though moderate, reductions in adult longevity, egg laying, and larval hatch-rate. Using CRISPR-Cas9, a single nucleotide polymorphism (G119S) was introduced in the Acetylcholine esterase 1 (ACE1) gene - the target for OP and carbamate insecticides of susceptible mosquitoes and the resulting phenotypes assessed. The results of this work, highlight important links between adulticide and larvicide resistance mechanisms and the necessity to assess mechanisms individually.