Development and evaluation of a single‐step multiplex PCR to differentiate the aquatic stages of morphologically similar Aedes (subgenus: Stegomyia) species

Abstract

Abstractobjective To develop a single-step multiplex PCR to differentiate the aquatic stages of Aedes aegypti, Aedes albopictus and Aedes vittatus collected from different breeding spots in arbovirus endemic ⁄ epidemic areas and to detect the most abundant species by the multiplex PCR.methods Aquatic stages of different mosquito species were sampled by inspecting artificial and natural breeding sites in domestic and peridomestic areas. DNA was isolated from different stages of the three Aedes species. Using novel primers based on 18S rDNA sequence, a single-step multiplex PCR was developed to clearly distinguish the three Aedes species. It was then evaluated in the aquatic stages of Aedes species collected from different areas.results A total of 1150 aquatic stages were collected from 294 breeding spots, of which 156 contained Aedes species. Discarded tires were the major breeding spots of Aedes species. The aquatic stages were clustered into 230 pools; Ae. albopictus was detected in the largest number of pools, followed by Ae. aegypti and Ae. vittatus.conclusions The Multiplex PCR clearly differentiated the aquatic stages of the three Aedes species and detected that Ae. albopictus was most profuse in different breeding spots surveyed, hence indicating to be the main vector in this region. So control measures can be designed against Ae. albopictus at an early stage to prevent any arboviral outbreak. This method is a convenient tool for precise identification of Aedes vectors during entomological surveys in arbovirus endemic ⁄ epidemic areas where several species coexist.

Genetic Structure and Wolbachia Genotyping in Naturally Occurring Populations of Aedes albopictus across Contiguous Landscapes of Orissa, India

“…aegypti and Ae. vittatus were the main Aedes vectors found here [17] and implicated Ae. albopictus to be the major vector responsible for the arboviral outbreaks [1]–[2], [18].…”

“…1) was assessed by performing species specific PCR using 18S ribosomal DNA [17]. All the specimens were screened initially for Wolbachia infection by PCR using wsp gene, (81F, 5′-TGGTCCAATAAGTGATGAAGAAAC-3′ and 691R, 5′-AAAAATTAAACGCTACTCCA-3′), which amplified 615 bp product.…”

First report of the invasive mosquito species Aedes koreicus in the Swiss-Italian border region

“…It is not possible to distinguish the eggs unambiguously by morphology through a stereo microscope. The eggs need either to be hatched out and reared to late developmental stages for species identification or analysed by technically more sophisticated tools such as DNA sequencing [ 20 ] or the MALDI-TOF MS approach applied here [ 8 ]. In this study, the full cost per MALDI-TOF MS sample was 10 Euros.…”

Anopheles culicifacies sibling species in Odisha, eastern India: First appearance of Anopheles culicifacies E and its vectorial role in malaria transmission

“…Molecular methods of identification, such as multiplex polymerase chain reaction (PCR) assays, have advantages over conventional identification methods (Das et al . ). Cytochrome oxidase II (COII) region from mitochondrial DNA allowed the development of a sibling species‐specific primer, which combined with universal primers, led to a simple and sensitive PCR assay for identification of An.…”

“…Furthermore, identification of sibling species complex is cumbersome as only half-gravid mosquitoes are required for differentiating the polytene chromosome inversions or bandings (Harrison 1980;Subbarao 1998;Van Bortel et al 2001). Molecular methods of identification, such as multiplex polymerase chain reaction (PCR) assays, have advantages over conventional identification methods (Das et al 2012). Cytochrome oxidase II (COII) region from mitochondrial DNA allowed the development of a sibling species-specific primer, which combined with universal primers, led to a simple and sensitive PCR assay for identification of An.…”