High-throughput sorting of mosquito larvae for laboratory studies and for future vector control interventions

Eric Marois,Christine Kappler,Christina Scali,Flaminia Catteruccia,Julien Soichot,Elena A Levashina

doi:10.1186/1475-2875-11-302

Abstract

BackgroundMosquito transgenesis offers new promises for the genetic control of vector-borne infectious diseases such as malaria and dengue fever. Genetic control strategies require the release of large number of male mosquitoes into field populations, whether they are based on the use of sterile males (sterile insect technique, SIT) or on introducing genetic traits conferring refractoriness to disease transmission (population replacement). However, the current absence of high-throughput techniques for sorting different mosquito populations impairs the application of these control measures.MethodsA method was developed to generate large mosquito populations of the desired sex and genotype. This method combines flow cytometry and the use of Anopheles gambiae transgenic lines that differentially express fluorescent markers in males and females.ResultsFluorescence-assisted sorting allowed single-step isolation of homozygous transgenic mosquitoes from a mixed population. This method was also used to select wild-type males only with high efficiency and accuracy, a highly desirable tool for genetic control strategies where the release of transgenic individuals may be problematic. Importantly, sorted males showed normal mating ability compared to their unsorted brothers.ConclusionsThe developed method will greatly facilitate both laboratory studies of mosquito vectorial capacity requiring high-throughput approaches and future field interventions in the fight against infectious disease vectors.

Highlights

Mosquito transgenesis offers new promises for the genetic control of vector-borne infectious diseases such as malaria and dengue fever
Rapid and precise establishment of homozygous transgenic Anopheles gambiae lines by COPAS sorting The goal of this work was to evaluate the possibility of performing accurate, fast and high-throughput larval screening and sorting using a flow cytometry machine, the Complex Object Parametric Analyzer and Sorter (COPASW, Union Biometrica)
Non-transgenic mosquitoes were crossed to DSX mosquitoes and the resulting F2 larvae were analysed. Such progeny was expected to segregate into five fluorescence classes according to single gene Mendelian inheritance and sex-specific expression of the GFP marker: 12.5% of homozygous transgenic females, 12.5% of homozygous transgenic males, 25% of heterozygous females, 25% of heterozygous males, and 25% of homozygous nontransgenic males and females

Summary

Introduction

Mosquito transgenesis offers new promises for the genetic control of vector-borne infectious diseases such as malaria and dengue fever. Past attempts at curbing the disease by the massive use of insecticides and of insecticideimpregnated bed nets have promoted the spread of genetic resistance to a wide range of insecticides across mosquito populations This is reducing the impact of Vector control methods that target the desired species represent a valid and environmentally friendly alternative to insecticides. The SIT approach is well suited for Anopheles mosquitoes, as the majority of females mate once in their lifetime and use the sperm stored in their spermatheca to fertilize egg batches produced every time they take a blood meal [6]. Labourintensive procedures for selection of male-only populations and a large variability in the efficiency of sterilization have to date posed vast blocks for a massive application of this approach

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