A novel mosquito species identification method based on PCR and
capillary electrophoresis
Estelle Chabanol1,2,3,4, Ottavia
Romoli1, Stanislas Talaga1, Yanouk
Epelboin1, Katy Heu1, Ghislaine
Prévot2,3, Mathilde Gendrin1,5,*
1 Microbiota of Insect Vectors Group, Institut Pasteur de la Guyane,
Cayenne, French Guiana
2 Tropical Biome and Immunophysiopathology Laboratory, Université de
Guyane, Cayenne, French Guiana
3 Université de Lille, CNRS, Inserm, CHU de Lille, Institut Pasteur de
Lille, U1019 – UMR 9017 – CIIL – Center for Infection and Immunity of
Lille, F-59000, Lille, France
4 École Doctorale 587, Université de Guyane, Cayenne, French Guiana
5 Institut Pasteur, Université de Paris, Department of Insect Vectors,
F-75015, Paris, France
* Correspondence: mathilde.gendrin@pasteur.fr
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 majorAnopheles 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.
Keywords: Species Identification; Capillary Electrophoresis;
ITS2; Length Polymorphism; Anopheles ; French Guiana