A novel mosquito species identification method based on PCR and
capillary electrophoresis
Abstract
In the Anopheles genus, various mosquito species are able to transmit
Plasmodium parasites responsible for malaria, while others are
non-vectors. In an effort to better understand the biology of Anopheles
species and to quantify transmission risk in an area, the identification
of mosquito species collected on the field is an essential but
problematic task. Morphological identification requires expertise,
well-preserved specimens and high-quality equipment, and it does not
allow any subsequent verification when samples are later used in a
destructive treatment. Moreover, it involves physical manipulations that
are not compatible with experiments requiring fast sampling and
processing of specimens, hence species identification is often based on
DNA sequencing of reference genes or region such as the Internal
Transcribed Spacer 2 (ITS2) region of nuclear ribosomal DNA. Sequencing
ITS2 for numerous samples is costly, but the design of species-specific
PCR primers is not always possible when local species diversity is high.
Here, we introduce a molecular technique of species identification based
on precise determination of ITS2 length combined with a simple visual
observation, the color of mosquito hindleg tip. DNA extracted from
field-collected Anopheles mosquitoes was amplified with universal
Anopheles ITS2 primers and analyzed with a capillary electrophoresis
device, which precisely determines the size of the fragments. We defined
windows of amplicon sizes combined with fifth hind tarsus color, which
allow to discriminate the major Anopheles species found in our
collections. We validated our parameters via Sanger sequencing of the
ITS2 amplicons. This method can be particularly useful in situations
with a moderate species diversity, i.e. when the number of local species
is too high to define species-specific primers but low enough to avoid
individual ITS2 sequencing. This tool will be of interest to evaluate
local malaria transmission risk and this approach may further be
implemented for other mosquito genera.