title: The prevalence of endosymbiotic bacteria in Culicoides biting midges and the distribution of Torix group Rickettsia in arthropod hosts
Creators: name: Pilgrim, Jack
Publisher: name: University of Liverpool Repository
Year: 2020

description: Culicoides biting midges (Diptera: Ceratopogonidae), are the vectors of several viruses affecting livestock including bluetongue and Schmallenberg viruses. There are no effective control methods of the vectors, and disease control therefore relies on vaccines which, given the rapid emergence/spread of the viruses, are often not available. Thus, there is increasing interest in the heritable bacteria (endosymbionts) of invertebrates as they present novel targets for control initiatives. For example, the endosymbiont Wolbachia is capable of inducing a “virus blocking” phenotype in mosquito hosts. Previous studies on biting midges, have revealed infections with the endosymbiotic bacteria, Wolbachia and Cardinium. However, other common symbionts, such as Rickettsia, are underexplored. I first clarify which Culicoides vector species are appropriate for further study of Cardinium-midge interactions. Reinvestigation of a previous UK screening study indicates spurious identification of Cardinium infection in the vector species C. pulicaris, as a result of inappropriate methodology and interpretation. In addition, this chapter establishes associations between mitochondrial haplotypes (mitotypes), used as a phylogeographic marker, and Cardinium infection in the globally important vector C. imicola. The concordance of mitotypes and Cardinium infection in populations of C. imicola from different geographic regions suggests a potential confounding of future biodiversity studies which fail to consider the presence of Cardinium. I then describe the results of a targeted screening of Culicoides populations for Rickettsia symbionts. Through conventional PCR, I demonstrated that Rickettsia represent a widespread but previously overlooked association, reaching high frequencies in midge populations and present in over a third of the species tested. Sequence typing clusters the Rickettsia within the Limoniae group of the genus, a group known to infect several aquatic and haematophagous taxa. Considering the presence of Rickettsia in several vector species, this result highlights the need to establish the impact of this newly-found association on vector competence. Leading from this, I describe the tropism of a Rickettsia endosymbiont present in the Scottish Highland midge, Culicoides impunctatus. Fluorescence in-situ hybridisation (FISH) and transmission electron microscopy (TEM) analysis indicated the presence of Rickettsia bacteria in ovarian tissue and the ovarian suspensory ligament suggesting a novel germline targeting strategy. In addition, Rickettsia presence in the fat body of larvae indicates potential host fitness effects to be investigated in the future. In the final study, I investigate inadvertent amplification of Rickettsia DNA through the common taxonomic identification technique of DNA barcoding. Through collaboration with the Barcoding of Life Data System (BOLD) curators, I undertook a systematic survey to determine the scale of this phenomenon, as well as investigate its potential to unveil new Rickettsia-infected host species. My results determine that unintended Rickettsia amplification is common and should be considered when designing future barcoding studies. In addition, a new wealth of host information was uncovered which can inform future directions of investigation pertaining to Rickettsia.