Rapid and high throughput molecular identification of diverse

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 and Anopheles gambiae complexes. We validated the use of PCR-HRM analyses to Culex pipiens differentiate species within and within each of six genera of culicine Anopheles mosquitoes, comparing primers targeting cytochrome b ( ), NADH cyt b dehydrogenase subunit 1 (ND1), intergenic spacer region (IGS) and cytochrome c oxidase subunit 1 ( ) gene regions. HRM analyses of COI amplicons from all the six primer pairs successfully differentiated two or more mosquito species within one or more genera ( ( from Aedes Ae. vittatus Ae. ), ( from , from metallicus Culex Cx. tenagius Cx. antennatus Cx. neavei Cx. , cryptic species), ( from duttoni Cx. pipiens Anopheles An. gambiae s.s. An. ) and ( from )) based on their arabiensis Mansonia Ma. africana Ma. uniformis HRM profiles. However, PCR-HRM could not distinguish between species within ( and ), ( and Aedeomyia Ad. africana Ad. furfurea Mimomyia Mi. hispida ) and ( , , Mi. splendens Coquillettidia Cq. aurites Cq. chrysosoma Cq. , , , and fuscopennata Cq. metallica Cq. microannulatus Cq. pseudoconopas Cq. ) genera using any of the primers. The IGS and COI barcode region versicolor 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. 1,2 1 1,3,4 1,5,6 2 1