Assigning Culicoides larvae to species using DNA barcoding of adult females and phylogenetic associations

Abstract

Background Orbivirus-induced hemorrhagic diseases cause high mortality in wild and captive white-tailed deer in North America. The role of different Culicoides species in Orbivirus transmission outside of areas of intensive animal production has not been established. At our study location, bluetongue virus (BTV) RNA-positive female Culicoides debilipalpis pools have been detected annually since 2012 when BTV transmission was noted in a captive deer herd. Identifying specific larval habitats of suspected vectors at active transmission sites is crucial both for identifying the source of the vectors and for subsequently planning intervention actions. Since C. debilipalpis larvae are known to develop in tree holes, this study was designed to use DNA barcoding to identify larvae collected from tree holes. Methods Adult female Culicoides were collected using light or emergence traps and morphologically identified to 11 species. Culicoides sonorensis were also obtained from a laboratory colony. Substrate was collected from tree holes and flooded with water to harvest floating larvae. Total DNA from three to seven adult females per species and 19 larvae was extracted. Two loci of the nuclear 18S ribosomal RNA (rRNA) gene, one locus each of the mitochondrial cytochrome oxidase subunit I (COI) gene and the nuclear 28S rRNA gene were amplified using loci-specific primers. Results All 61 adults were sequenced at each of the four loci under study. Since no single locus delineated all putative species and the COI locus yielded unreliable pseudogenes for two individuals of C. arboricola, sequences of all four loci were concatenated to maximize species separation and allow for larval association with identified adults. Sixteen larvae were clearly assigned to species based on DNA barcoding and phylogenetic results. Multiple larvae were assigned to each of the C. debilipalpis clade, the C. villosipennis clade, the C. arboricola clade and the C. nanus clade. Conclusions Of the approximately 62 species described in the southeast USA, 21 have now been barcoded and sequences are publicly available. In this study, we constructed a database composed of species-specific sequences of adult Culicoides and then identified larvae to species by matching their corresponding sequences with adults. Since Culicoides larvae are difficult to identify, using DNA barcoding to facilitate larval habitat surveys can be a valuable tool. Graphical Abstract

Domestic Triatoma spp. Infections with Trypanosoma cruzi, Household Infestations, and Molecular Identification in Oaxaca, México

“…In our entomological surveys, we found that Triatoma spp. nymphs, which are difficult to identify morphologically, could be identified by DNA barcoding, similar to what has been utilized for the molecular identification of immature Culicoides biting midges [34]. Past entomological surveys of the four primary domestic triatomines of this region, T. dimidiata, T. mazzotti, T. pallidipennis, and T. phyllosoma, in 1980 documented their occurrence in 24 municipalities [17]; however, Ramsey et al (2000) [12] conducted a study between 1996-1998 and reported eight species of triatomines in 64 municipalities [12].…”

Development of Conventional Multiplex PCR Assays for the Identification of 21 West Palaearctic Biting Midge Taxa (Diptera: Ceratopogonidae) Belonging to the Culicoides Subgenus Culicoides, including Recently Discovered Species and Genetic Variants

“…[32,33]. This method proves to be difficult for the identification to the species level of females of closely related species that have very similar or identical (isomorphic) features and is even more challenging for juvenile specimens (larvae, pupae) [34][35][36], for which either identification keys have not yet been developed or distinguishing features are not yet even established for corresponding adults. Furthermore, morphological species identification cannot be used for the determination of phylogenetic distances [37] and may require time-consuming analyses of slide-mounted microscopical insect preparations to visualize fine structures [38], a lot of practical experience and fresh material with distinct coloration.…”

Overcoming life stage‐centric biases illuminates arthropod diversity, systematics and biology

“…The potential for molecular identification methods to help address these issues, allowing non‐typical semaphoronts to contribute to studies in biodiversity inventory and systematics, has been recognized since DNA sequences first became readily accessible (Caterino et al, 2000; Stern et al, 1997). Associations of larvae through DNA sequencing have become routine (Ahrens et al, 2007; Caterino & Tishechkin, 2006; Curiel & Morrone, 2012; Jin et al, 2022; Miller et al, 2005; Sinclair et al, 2022; Zhou et al, 2007). A number of authors have also demonstrated successful associations of sexes in dimorphic species (Corley & Ferreira, 2017; Ekrem et al, 2010; Ferreira & Ivie, 2022; Gariepy et al, 2022).…”