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 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.
Highlights
Mosquitoes are among the most important disease vectors, known to transmit and maintain the circulation of pathogens that cause both global and neglected tropical diseases in humans and animals[1]
Despite the fact that the COI-AnophF/HCO2198R primers were originally designed based on Anopheles mitochondria genome alignments, they were most efficient in differentiating among Mansonia (Ma. africana and Ma. uniformis (Figure 1A)), Culex (Cx. neavei and Cx. duttoni, Cx. tenagius and Cx. antennatus, and two genetic variants of Cx. pipiens (Figure 2A)), and Aedes (Ae. vittatus and Ae. metallicus (Figure 3)) mosquitoes (Table 4)
There are single nucleotide polymorphisms (SNPs) within species DNA that resulted to the slight changes observed in their high resolution melting (HRM) profiles, the SNPs across species were enough to distinguish between them
Summary
Mosquitoes are among the most important disease vectors, known to transmit and maintain the circulation of pathogens that cause both global and neglected tropical diseases in humans and animals[1]. Molecular approaches that efficiently differentiate conspecific mosquitoes such as the barcode region[5] improve identification accuracy considerably[6], but are time consuming, expensive in terms of post-polymerase chain reaction (post-PCR) processing and depend heavily on DNA sequencing. Recent approaches have taken advantage of the unique melting profiles generated by homologous PCR products with small sequence differences during high resolution melting (HRM) analysis[7,8]. HRM analysis has proven to offer higher resolution of PCR product based species identification on sequence variants than electrophoretic methods by revealing even single nucleotide polymorphisms (SNPs) in the simple sequence repeats (SSRs) among products of similar sizes[16,17]. Combining HRM analysis of barcode region sequences (Bar-HRM) has been successfully used to rapidly and accurately distinguish between closely related antelope species[19] and medicinal plants[20,21] and to authenticate the source of vegetable oils[22]
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