Introduction
Several species of mosquitoes are vectors of viruses or parasites
causing serious diseases in humans. In particular, some species ofAnopheles transmit the Plasmodium parasite, which causes
malaria in humans [1] , and thus strongly impacts human
health with more than 247 million cases over 84 countries in 2021[2] . Despite global effort to reduce the burden of
vector-borne diseases, they remain a sanitary and economic threat in the
intertropical area and beyond [3] . However, among the 3500
species of mosquitoes, only a minority are vectors of pathogens,
therefore the identification of species is an important point in
surveillance and studies.
In French Guiana, 245 mosquito species are known [4,5]including 22 species of Anopheles [6] , nine of which
belong to the Nyssorhynchus subgenus whose elevation to genus
level is under consideration [7–9] . Among them,Anopheles darlingi is known to be the main malaria vector in
South America [10,11] and Anopheles aquasalis ,
although not being incriminated as a vector in French Guiana, is also
considered as one of the principal vectors in other neighboring
countries on the continent [10,12] . Moreover,Anopheles medialis , Anopheles nuneztovari andAnopheles oswaldoi have been found naturally infected byPlasmodium in French Guiana [13] , as well asAnopheles braziliensis and Anopheles triannulatus in
Brazil [14,15] , but the real extent of their involvement in
parasite transmission between humans is currently unknown. Given these
differences in their ability to transmit malaria, it is thus important
to identify the exact species of Anopheles mosquitoes when
studying and monitoring field population distributions and dynamics.
Originally, mosquito species identification was only based on visual
observation of morphological characteristics with the help of
dichotomous taxonomic keys [16–19] . This method is
effective and relatively accessible when morphological differences are
substantial, yet it is arduous and requires advanced skills when
variations are subtle. The use of additional equipment and the
dissection of internal structures are also sometimes mandatory to
distinguish between morphologically close species, as for Culexmosquitoes, in which meticulous dissection of male genitalia is required[20,21] . Moreover, even with properly trained and
experienced professionals the task frequently remains a challenge,
misidentifications are common and results for later processed samples
cannot be checked afterward in case of doubt [22] .
Alternative methods have been developed based on molecular biology
techniques. CO1 (Cytochrome C Oxidase Subunit 1), a mitochondrial
gene, and ITS2 (Internal Transcribed Spacer 2), a non-coding nuclear
sequence located between 5.8S and 28S ribosomal RNA genes,
are often used for molecular identification of animals. CO1 or
ITS2 DNA barcoding are two of the most prevalent techniques for species
identification today, with databases usually already available.
Amplification of DNA regions with species-specific sizes via multiplex
PCR (Polymerase Chain Reaction) is often used as a substitute to
sequencing to discriminate a limited number of species found locally[23–26] , notably Anopheles coluzzii andAnopheles gambiae found in Africa [27,28] and other
species from the Gambiae Complex [29,30] . When a higher
number of species are involved, PCR may be combined with a treatment
with restriction enzymes in the RFLP (Restriction Fragment Length
Polymorphism) technique [31,32] .
Other approaches for mosquito species identification are currently being
considered. Mosquito protein profiling using MALDI-TOF technology is
promising [33] , even though it requires a substantial
investment in terms of equipment and the databases are still under
development [34] . Nowadays, increasingly powerful software
solutions make it possible to perform morphometry of mosquito wings[35] and the use of artificial intelligence may allow for
automatic visual identification of mosquitoes [36] . The
sound and frequency of wing beats is also used to develop identification
tools that are meant to be accessible on a simple smartphone[37] . Yet the implementation of these tools needs more
development and advanced computational knowledge.
In this study, we have developed a new method to distinguish between
nine Anopheles species from French Guiana based on precise
discrimination of their natural ITS2 sequence size polymorphism using
capillary electrophoresis, combined with a simple morphological
observation, the color of the hindleg tip (fifth hind tarsus,
Ta-III5).