Background Chromosomal inversion polymorphisms have been associated with adaptive behavioral, physiological,
morphological and life history traits in the two main Afrotropical malaria vectors, Anopheles coluzzii and Anopheles
gambiae. The understanding of the adaptive value of chromosomal inversion systems is constrained by the feasibility
of cytological karyotyping. In recent years in silico and molecular approaches have been developed for the genotyping
of most widespread inversions (2La, 2Rb and 2Rc). The 2Ru inversion, spanning roughly 8% of chromosome 2R,
is commonly polymorphic in West African populations of An. coluzzii and An. gambiae and shows clear increases in frequency
with increasing rainfall seasonally and geographically. The aim of this work was to overcome the constraints
of currently available cytological and high-throughput molecular assays by developing a simple PCR assay for genotyping
the 2Ru inversion in individual specimens of both mosquito species.
Methods We designed tetra-primer amplification refractory mutation system (ARMS)-PCR assays based on five tag
single-nucleotide polymorphisms (SNPs) previously shown to be strongly correlated with 2Ru inversion orientation.
The most promising assay was validated against laboratory and field samples of An. coluzzii and An. gambiae karyotyped
either cytogenetically or molecularly using a genotyping-in-thousands by sequencing (GT-seq) high-throughput
approach that employs targeted sequencing of multiplexed PCR amplicons.
Results A successful assay was designed based on the tag SNP at position 2R, 31710303, which is highly predictive
of the 2Ru genotype. The assay, which requires only one PCR, and no additional post-PCR processing other than electrophoresis,
produced a clear banding pattern for 98.5% of the 454 specimens tested, which is a 96.7% agreement
with established karyotyping methods. Sequences were obtained for nine of the An. coluzzii specimens manifesting
2Ru genotype discrepancies with GT-seq. Possible sources of these discordances are discussed.
Conclusions The tetra-primer ARMS-PCR assay represents an accurate, streamlined and cost-effective method
for the molecular karyotyping of the 2Ru inversion in An. coluzzii and An. gambiae. Together with approaches already

Anopheles gambiae complex, Chromosomal inversion, Inversion genotyping, Malaria vector, Molecular karyotyping