Monitoring Anopheles diversity in Madagascar

As part of an ongoing project to monitor Anopheles diversity in Madagascar, we aim to gain a detailed understanding of species composition, population structure and Plasmodium transmission rates through the targeted sequencing of 50,000 individual mosquitoes.
The main aim of the project is to improve our understanding of Anopheles species diversity, population structure and malaria transmission in Africa. In collaboration with partners working in malaria-endemic countries, 500,000 mosquitoes will be sequenced using a targeted amplicon panel known as ANOSPP.

ANOSPP is a multi-locus amplicon sequencing panel targeting 62 short nuclear loci in the generic Anopheles genome and 2 mitochondrial loci in the generic Plasmodium genome. The panel uses a highly multiplexed two-step PCR in which the target sequences are first amplified and then unique barcodes are added so that amplicons from hundreds of mosquitoes can be combined in a single Illumina MiSeq sequencing run (Makunin et al. 2022). The sequences are de-multiplexed using these barcodes and analysed using k-mer approaches to determine a species identity for each individual (Boddé et al. 2022). The sequencing data of each individual mosquito provides information about its species, population structure and whether Plasmodium parasites are present - important information for vector control.

In Madagascar, in collaboration with the National Malaria Control Programme and other partners such as the EVOLVE programme of the President Malaria Initiative, mosquitoes are collected at more than 120 sites across the country, allowing a comparison of species composition, population structure and transmission intensity in space and in different habitats. Samples are collected multiple times at a limited number of sites so that temporal fluctuations and population continuity across seasons can be analysed. All samples collected will be annotated with extensive metadata, e.g. collection method, insecticide resistance status, etc., as described in the ANOSPP manifesto.
This valuable dataset will be used to develop standardised analyses from ANOSPP sequencing data that can be used to inform national and international authorities involved in vector control. In addition, we will investigate how ANOSPP sequencing data can be used for population genetic analyses, such as inversion genotyping, estimation of genetic diversity and estimation of spatial and temporal relationships.
The ANOSPP results will also be used to select individuals of major and minor vector species for short-read whole genome sequencing to further investigate their population structure and emerging and existing insecticide resistance.
This project is led by Diego Ayala from the Institut Pasteur de Madagascar and Mara Lawniczak from the Wellcome Sanger Institute in the UK. Marilou Boddé (LIB and IPM) is responsible for analysing the data. The project is funded by the Bill and Melinda Gates Foundation.