Open Peer Review Any reports and responses or comments on the article can be found at the end of the article. METHOD ARTICLE Rapid and high throughput molecular identification of diverse mosquito species by high resolution melting analysis [version 1; peer review: 2 approved] Yvonne Ukamaka Ajamma , Enock Mararo , David Omondi , Thomas Onchuru , Anne W. T. Muigai , Daniel K Masiga , Jandouwe Villinger 1 Martin Lüscher Emerging Infectious Diseases (ML-EID) Laboratory, International Centre of Insect Physiology and Ecology, Nairobi, Kenya Department of Botany (Genetics), Jomo Kenyatta University of Agriculture and Technology, Juja, Kenya Biochemistry and Molecular Biology Department, Egerton University, Egerton, Kenya Molecular Biology and Virology Laboratory, Department of Medical Biosciences, University of Western Cape, South Africa Insect Symbiosis Research Group, Max Planck Institute for Chemical Ecology (MPI-CE), Jena, Germany Department for Evolutionary Ecology, Institute for Zoology, Johannes Gutenberg University, Mainz, Germany Abstract 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 complexes. We validated the use of Anopheles gambiae Culex pipiens PCR-HRM analyses to differentiate species within and within Anopheles each of six genera of culicine mosquitoes, comparing primers targeting cytochrome b ( ), NADH dehydrogenase subunit 1 (ND1), intergenic cyt b spacer region (IGS) and cytochrome c oxidase subunit 1 ( ) gene COI regions. HRM analyses of amplicons from all the six primer pairs successfully differentiated two or more mosquito species within one or more genera ( ( from ), ( Aedes Ae. vittatus Ae. metallicus Culex Cx. tenagius from , from , cryptic Cx. antennatus Cx. neavei Cx. duttoni Cx. pipiens species), ( from ) and ( Anopheles An. gambiae s.s. An. arabiensis Mansonia from )) based on their HRM profiles. However, Ma. africana Ma. uniformis PCR-HRM could not distinguish between species within ( Aedeomyia Ad. and ), ( and ) and africana Ad. furfurea Mimomyia Mi. hispida Mi. splendens ( , , , Coquillettidia Cq. aurites Cq. chrysosoma Cq. fuscopennata Cq. , , and ) metallica Cq. microannulatus Cq. pseudoconopas 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 1,2 1 1,3,4 1,5,6 2 1 1 1 2 3 4 5 6 Reviewer Status Invited Reviewers version 1 11 Aug 2016 1 2 report report , Thermo Fisher Scientific, Michael Zianni Carlsbad, USA 1 , University of Malaya, Kuala Hwa Chia Chai Lumpur, Malaysia 2 11 Aug 2016, :1949 ( First published: 5 ) https://doi.org/10.12688/f1000research.9224.1 11 Aug 2016, :1949 ( Latest published: 5 ) https://doi.org/10.12688/f1000research.9224.1 v1 Page 1 of 15 F1000Research 2016, 5:1949 Last updated: 03 APR 2020