Results
ITS2 region naturally differs in size between Anopheles species
of French Guiana (Figure 1A) , ranging from 400 bp to 680 bp forAnopheles eiseni and Anopheles minor respectively[31] . As ITS2 size generally differs for more than 7 bp
among species, we hypothesized this length polymorphism may allow
species identification after PCR amplification and electrophoresis, so
that any additional manipulation or sequencing is not required. Some
species however share identical or similar ITS2 amplicon size
(< 3 bp). For instance, An. braziliensis and An.
oswaldoi both have a 488 bp-long ITS2 sequence, and the species
identification would be impossible using exclusively the size
information. We decided to collect a simple morphological observation
upon mosquito sampling in the field, the color of their three last hind
tarsi (Ta-III3,4,5), in particular the fifth hind tarsus
(Ta-III5, at the tip of the hindleg) (Figure
1A) . This morphological data does not require advanced skills in
taxonomy but is enough to discriminate some species with similar ITS2
length. Anopheles braziliensis and An. darlingi have their
three last hind tarsi totally white while the other species likeAn. oswaldoi have a dark basal band on their fifth hind tarsus.
Additionally, Anopheles outside of the Nyssorhynchussubgenus have a mix of white and black on their three last hind tarsi.
While differences in amplicon size are visible after PCR amplification
and migration on an agarose gel, the analysis of gel images remains
approximative and does not allow precise determination of fragment sizes(Figure 1B) . With an agarose gel, fragment sizes are determined
using the size marker as a reference, yet gel homogeneity and migration
speed are not strictly controlled and slight differences in migration
between wells cannot be corrected in the absence of internal controls.
Capillary electrophoresis migration is another technique that allows
automatization of the process and standardization of the fragment size
detection, using internal controls of defined sizes as references(Figure 1C) . The precision is up to 3 bp for the most precise
migration settings on fragments shorter than 500 bp. Additionally, the
electropherogram allows to verify the quality of the fragment
amplification (Figure 1D) . We decided to set up a method to
identify our Anopheles samples based on capillary electrophoresis
analysis combined with observation of fifth hind tarsus color.