Figure 1: The method is based on natural variation of ITS2
sequence size between Anopheles species coupled with visual
observation of mosquito fifth hind tarsus color (A) .
Species-specific amplicon sizes are visible on an agarose gel(B) but more precisely measured with capillary electrophoresis
including internal reference size controls (C) . Additionally,
capillary electrophoresis allows quality check of fragment amplification
and migration (D) .
We collected Anopheles mosquitoes from five different locations
in French Guiana (Figure S1) over a four-year period from 2018
to 2021. The color of the fifth hind tarsus was noted for each mosquito
sample at the time of capture. Back in the laboratory, we extracted the
DNA of each individual, amplified the ITS2 sequence and ran capillary
electrophoresis (Figure 2) .
Our method development was divided in three phases based on different
mosquito collections, with a first phase of establishment of parameters
(step 1) and two phases of adjustment of these parameters (steps 2 and
3) (Figure 2) . During step 1, we sequenced ITS2 in 167Anopheles samples, identified species by BLAST and ran capillary
electrophoresis to link mosquito species with observed ITS2 amplicon
size. During steps 2 and 3, we collected 163 and 73 mosquitoes
respectively, we tested the method identification process, coupling ITS2
size and fifth hind tarsus color to determine the Anophelesspecies, and we verified the result by sequencing again 100 % of our
ITS2 amplicons. This enabled us to identify errors, adjust size
intervals and add new species to the method. Finally, we applied this
method routinely in our laboratory on a total of 372 samples and more
than 99 % could be identified without the need for sequencing(Figure 2) . Only 2/372 samples needed to be sequenced because
of uncertain identification.