Rapid and high throughput molecular identification of diverse mosquito species by high resolution melting analysis

Mosquitoes are a diverse group of invertebrates, with members that are among the most important vectors of diseases. The correct identification of mosquitoes is paramount to the control of the diseases that they transmit. However, morphological techniques depend on the quality of the specimen and often unavailable taxonomic expertise, which may still not be able to distinguish mosquitoes among species complexes (sibling and cryptic species). High resolution melting (HRM) analyses, a closed-tube, post-polymerase chain reaction (PCR) method used to identify variations in nucleic acid sequences, has been used to differentiate species within the Anopheles gambiae and Culex pipiens complexes. We validated the use of PCR-HRM analyses to differentiate species within Anopheles and within each of six genera of culicine mosquitoes, comparing primers targeting cytochrome b ( cyt b ), NADH dehydrogenase subunit 1 (ND1), intergenic spacer region (IGS) and cytochrome c oxidase subunit 1 ( COI ) gene regions. HRM analyses of amplicons from all the six primer pairs successfully differentiated two or more mosquito species within one or more genera ( Aedes ( Ae. vittatus from Ae. metallicus ), Culex ( Cx. tenagius from Cx. antennatus , Cx. neavei from Cx. duttoni , cryptic Cx. pipiens species), Anopheles ( An. gambiae s.s. from An. arabiensis ) and Mansonia ( Ma. africana from Ma. uniformis )) based on their HRM profiles. However, PCR-HRM could not distinguish between species within Aedeomyia ( Ad. africana and Ad. furfurea ), Mimomyia ( Mi. hispida and Mi. splendens ) and Coquillettidia ( Cq. aurites , Cq. chrysosoma , Cq. fuscopennata , Cq. metallica , Cq. microannulatus , Cq. pseudoconopas and Cq. versicolor ) genera using any of the primers. The IGS and COI barcode region primers gave the best and most definitive separation of mosquito species among anopheline and culicine mosquito genera, respectively, while the other markers may serve to confirm identifications of closely related sub-species. This approach can be employed for rapid identification of mosquitoes.