SummaryThere is increasing interest in the heritable bacteria of invertebrate vectors of disease as they present novel targets for control initiatives. Previous studies on biting midges (Culicoides spp.), known to transmit several RNA viruses of veterinary importance, have revealed infections with the endosymbiotic bacteria, Wolbachia and Cardinium. However, rickettsial symbionts in these vectors are underexplored. Here, we present the genome of a previously uncharacterized Rickettsia endosymbiont from Culicoides newsteadi (RiCNE). This genome presents unique features potentially associated with host invasion and adaptation, including genes for the complete non‐oxidative phase of the pentose phosphate pathway, and others predicted to mediate lipopolysaccharides and cell wall modification. Screening of 414 Culicoides individuals from 29 Palearctic or Afrotropical species revealed that Rickettsia represent a widespread but previously overlooked association, reaching high frequencies in midge populations and present in 38% of the species tested. Sequence typing clusters the Rickettsia within the Torix group of the genus, a group known to infect several aquatic and hematophagous taxa. FISH analysis indicated the presence of Rickettsia bacteria in ovary tissue, indicating their maternal inheritance. Given the importance of biting midges as vectors, a key area of future research is to establish the impact of this endosymbiont on vector competence.
Biting midges of the genus Culicoides (Diptera: Ceratopogonidae) are insect vectors of economically important veterinary diseases such as African horse sickness virus and bluetongue virus. However, the identification of Culicoides based on morphological features is difficult. The sequencing of mitochondrial cytochrome oxidase subunit I (COI), referred to as DNA barcoding, has been proposed as a tool for rapid identification to species. Hence, a study was undertaken to establish DNA barcodes for all morphologically determined Culicoides species in Swedish collections. In total, 237 specimens of Culicoides representing 37 morphologically distinct species were used. The barcoding generated 37 supported clusters, 31 of which were in agreement with the morphological determination. However, two pairs of closely related species could not be separated using the DNA barcode approach. Moreover, Culicoides obsoletus Meigen and Culicoides newsteadi Austen showed relatively deep intraspecific divergence (more than 10 times the average), which led to the creation of two cryptic species within each of C. obsoletus and C. newsteadi. The use of COI barcodes as a tool for the species identification of biting midges can differentiate 95% of species studied. Identification of some closely related species should employ a less conserved region, such as a ribosomal internal transcribed spacer.
Investigations of host preferences in haematophagous insects, including Culicoides biting midges (Diptera: Ceratopogonidae), are critical in order to assess transmission routes of vector-borne diseases. In this study, we collected and morphologically identified 164 blood-engorged Culicoides females caught in both light traps and permanent 12-m high suction traps during 2008-2010 in Sweden. Molecular analysis of the mitochondrial cytochrome c oxidase subunit I (COI) gene in the biting midges was performed to verify species classification, discern phylogenetic relationships and uncover possible cryptic species. Bloodmeal analysis using universal vertebrate cytochrome b primers revealed a clear distinction in host selection between mammalophilic and ornithophilic Culicoides species. Host sequences found matches in horse (n = 59), sheep (n = 39), cattle (n = 26), Eurasian elk (n = 1) and 10 different bird species (n = 18). We identified 15 Culicoides species previously recorded in Scandinavia and four additional species haplotypes that were distinctly different from the described species. All ornithophilic individuals (n = 23) were caught exclusively in the suction traps, as were, interestingly, almost all mammalophilic species (n = 41), indicating that many biting midge species may be able to cover long distances after completing a bloodmeal. These results add new information on the composition of Culicoides species and their host preferences and their potential long-distance dispersal while blood-engorged.
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