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

开发新的基因组工具和快速、自动化的蚊子幼虫耐药性检测

• 批准号: 1981052
• 金额: --
• 依托单位: Liverpool School of Tropical Medicine
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2017
• 资助国家: 英国
• 起止时间: 2017 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-1981052
• 关键词: Developing; new; genomic; tools; rapid

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.

杀虫剂耐药性对疟疾和针对蚊媒的虫媒病毒控制方案构成了迫在眉睫的威胁。幼虫抗性是一个需要评估的重要表型，然而，现有的WHO推荐的基于死亡率的幼虫抗性分析方法是低通量的，且容易受到调查者的偏见。为了解决这些问题，利用无脊椎动物自动表型分析平台(INVAPP)和分析算法(Vectorgon)开发了一种新的检测方法，可以自动定量测定幼虫在接触杀虫剂后的活动。在对常见杀虫剂表现出抗药性的蚊子中观察到了几个分子变化。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还没有被探索。了解每个机制对蚊子控制方案的潜在影响至关重要。在敏感的实验室蚊子中，代谢酶的上调是使用两部分GAL4-UAS系统来模拟的。用INVAPP法检测了转基因幼虫对杀虫剂的敏感性。转基因幼虫高表达GSTe2、CYP6M2、CYP6P3和SAP2这四种与成虫抗性有关的酶。表达CCEae3a(一种在伊蚊中发现的一种对有机磷(OP)抗性的羧酸酯酶)的转基因品系，对幼虫的三种常见OP、成虫的四种杀虫剂类别表现出抗性，成虫寿命、产卵和幼虫孵化率显著降低，尽管程度不大。利用CRISPR-Cas9，在乙酰胆碱酯酶1(ACE1)基因中引入了单核苷酸多态性(G119S)，ACE1是易感蚊子的OP和氨基甲酸酯类杀虫剂的靶标，并对由此产生的表型进行了评估。这项工作的结果突出了防腐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。

项目成果

Automated phenotyping of mosquito larvae enables high-throughput screening for novel larvicides and offers potential for smartphone-based detection of larval insecticide resistance.

• DOI: 10.1371/journal.pntd.0008639
• 发表时间: 2021-06
• 期刊: PLoS neglected tropical diseases
• 影响因子: 3.8
• 作者: Buckingham SD;Partridge FA;Poulton BC;Miller BS;McKendry RA;Lycett GJ;Sattelle DB
• 通讯作者: Sattelle DB

Genetic modification tools in mosquitoes revolutionize our ability to functionally analyze insecticide resistance Genetic modification tools in mosquitoes revolutionize our ability to functionally analyze insecticide resistance

蚊子基因改造工具彻底改变了我们功能分析杀虫剂抗性的能力 蚊子基因改造工具彻底改变了我们功能分析杀虫剂抗性的能力

• DOI: 10.1127/entomologia/2023/1897
• 发表时间: 2023
• 期刊: Entomologia Generalis
• 影响因子: 6.9
• 作者: Grigoraki L

Functional genetic validation of key genes conferring insecticide resistance in the major African malaria vector, Anopheles gambiae

对非洲主要疟疾媒介冈比亚按蚊赋予杀虫剂抗性的关键基因进行功能遗传验证

• DOI: 10.1101/749457
• 发表时间: 2019
• 作者: Adolfi A