Modification of the Folmer primers for the cytochrome c oxidase gene facilitates identification of mosquitoes

Abstract

Background Accurate identification of mosquito species is essential for the development and optimization of strategies to control mosquitoes and mosquito-borne diseases. Problems with the morphological identification of mosquito species have led to the use of molecular identification techniques, in particular the Folmer cytochrome c oxidase subunit I (COI) PCR system (FCOS), originally designed to identify a range of other invertebrates. Methods As there can be difficulties identifying mosquitoes using FCOS, we re-evaluated the FCOS primers and developed a new COI-based SYBR PCR (the Auburn COI system—AUCOS) to improve the molecular identification of mosquitoes. Sequence data in GenBank for 33 species from 10 genera of mosquitoes were used to develop our AUCOS primers. Two molecular assays (AUCOS, FCOS) and morphological identification were carried out on mosquitoes collected from the field in Auburn, Alabama (USA) and on Saint Kitts. Results With a convenience sample of individual mosquitoes comprising 19 species from six genera in Saint Kitts (n = 77) and Auburn (n = 48), our AUCOS provided higher-quality sequence data than FCOS. It also proved more sensitive than FCOS, successfully amplifying 67.5% (85/126) as opposed to 16.7% (21/126) of the samples. The species determined by morphology, or genus with damaged samples, matched that as determined by AUCOS for 84.9% (62/73) of the samples. Morphological classification was confirmed by FCOS with 81.0% (17/21) of samples producing utilizable sequences. While both FCOS and AUCOS correctly identified all the Aedes, Anopheles, Deinocerites, and Uranotaenia species in the study, identification of Culex species was less successful with both methods: 50.0% (3/6) by FCOS and 35.7% (5/14) by AUCOS. Conclusions The AUCOS DNA barcoding system for mosquito species described in this study is superior to the existing FCOS for the identification of mosquito species. As AUCOS and FCOS amplify the same variable region of the COI, the large amount of existing data on GenBank can be used to identify mosquito species with sequences produced by either PCR. Graphical Abstract

Aedes Mosquito Virome in Southwestern Cameroon: Lack of Core Virome, but a Very Rich and Diverse Virome in Ae. Africanus Compared to Other Aedes Species

“…Mounting evidence show that ISVs interact with other components of the mosquito microbiota and influence mosquito susceptibility to arboviral infection [27,43,44]. Unfortunately, very little is known about the virome of the Aedes mosquitoes circulating in Cameroon [26,41,42] In this study, we employed viral metagenomics to characterize the viral composition of Aedes mosquito pools from four regions in the southwestern part of Cameroon representing three subclimatic zones (Table S1). In this part of the country, sampling showed that the dominant Aedes species was Ae.…”

“…The eluted volume (60 μL) was diluted to 120 μL and divided into 10 aliquots of 12 μL each and stored to avoid multiple freeze thawing cycles of large volumes. The first aliquot was used for the amplification of the cytochrome c oxidase subunit I region of Aedes mosquitoes employing AUCOS primers (Table S2) with the QIAGEN One-step RT-PCR kit, following the manufacturer's protocol [42]. Briefly, the reaction mixture consisted of 10 μL RNase-free water, 5 μL of 5x QIAGEN OneStep RT-PCR Buffer, 1μL of dNTP Mix, 1.5 μL of each Primer (10 μM), 1μL of QIAGEN OneStep RT-PCR Enzyme Mix, and 5 μL of sample, in a total volume of 25 μL [41].…”

Comparison of a multiplex PCR with DNA barcoding for identification of container breeding mosquito species

“…Identification of mosquito species has become increasingly important in the last decades, due to the spread of (potentially) invasive species [ 1 ]. Container breeding mosquito species, which use natural or artificial containers for oviposition [ 2 ], are of particular concern due to their potential to be competent vectors for a variety of pathogens.…”