Hurst, Gregory D. D.

AbstractSymbiotic bacteria can alter host biology by providing protection from natural enemies, or alter reproduction or vectoral competence. Symbiont‐linked control of vector‐borne disease in Anopheles has been hampered by a lack of symbioses that can establish stable vertical transmission in the host. Previous screening found the symbiont ‘Candidatus Tisiphia’ in Anopheles plumbeus, an aggressive biter and potential secondary vector of malaria parasites and West Nile virus. We screened samples collected over 10‐years across Germany and used climate databases to assess environmental influence on incidence. We observed a 95% infection rate, and that the frequency of infection did not fluctuate with broad environmental factors. Maternal inheritance is indicated by presence in the ovaries through FISH microscopy. Finally, we assembled a high‐quality 1.6 Mbp draft genome of ‘Ca. Tisiphia’ to explore its phylogeny and potential metabolic competence. The infection is closely related to strains found in Culicoides biting midges and shows similar patterns of metabolism, providing no evidence of the capacity to synthesize B‐vitamins. This infection offers avenues for onward research in anopheline mosquito symbioses. Additionally, it provides future opportunity to study the impact of ‘Ca. Tisiphia’ on natural and transinfected hosts, especially in relation to reproductive fitness and vectorial competence and capacity.

Symbiotic microbes from the genus 'Candidatus Megaira' ( Rickettsiales ) are known to be common associates of algae and ciliates. However, genomic resources for these bacteria are scarce, limiting our understanding of their diversity and biology. We therefore utilize Sequence Read Archive and metagenomic assemblies to explore the diversity of this genus. We successfully extract four draft 'Ca. Megaira' genomes including one complete scaffold for a 'Ca. Megaira' and identify an additional 14 draft genomes from uncategorized environmental metagenome-assembled genomes. We use this information to resolve the phylogeny for the hyper-diverse 'Ca. Megaira', with hosts broadly spanning ciliates, and micro- and macro-algae, and find that the current single genus designation 'Ca. Megaira' significantly underestimates their diversity. We also evaluate the metabolic potential and diversity of ''Ca. Megaira' from this new genomic data and find no clear evidence of nutritional symbiosis. In contrast, we hypothesize a potential for defensive symbiosis in 'Ca. Megaira'. Intriguingly, one symbiont genome revealed a proliferation of ORFs with ankyrin, tetratricopeptide and leucine-rich repeats such as those observed in the genus Wolbachia where they are considered important for host–symbiont protein–protein interactions. Onward research should investigate the phenotypic interactions between 'Ca. Megaira' and their various potential hosts, including the economically important Nemacystus decipiens, and target acquisition of genomic information to reflect the diversity of this massively variable group.

AbstractSymbiotic microbes from the genus ‘Candidatus Megaira’ (Rickettsiales) are known to be common associates of algae and ciliates. However genomic resources for these bacteria are scarce, limiting our understanding of their diversity and biology. We therefore utilized SRA and metagenomic assemblies to explore the diversity of this genus. We successfully extracted four draft ‘Ca. Megaira’ genomes including one complete scaffold for a ‘Ca. Megaira’ and identified an additional 14 draft genomes from uncategorised environmental Metagenome-Assembled Genomes. We use this information to resolve the phylogeny for the hyper-diverse ‘Ca. Megaira’, with hosts broadly spanning ciliates, micro- and macro-algae, and find that the current single genus designation ‘Ca. Megaira’ significantly underestimates their diversity. We also evaluate the metabolic potential and diversity of ‘Ca. Megaira’ from this new genomic data and find no clear evidence of nutritional symbiosis. In contrast, we hypothesize a potential for defensive symbiosis in ‘Ca. Megaira’. Intriguingly, one symbiont genome revealed a proliferation of ORFs with ankyrin, tetratricopeptide and Leucine rich repeats like those observed in the genus Wolbachia where they are considered important for host-symbiont protein-protein interactions. Onward research should investigate the phenotypic interactions between ‘Ca. Megaira’ and their various potential hosts, including the economically important Nemacystus decipiens, and target acquisition of genomic information to reflect the diversity of this massively variable group.Data SummaryGenomes assembled in this project have been deposited in bioproject PRJNA867165Impact statementBacteria that live inside larger organisms commonly form symbiotic relationships that impact the host’s biology in fundamental ways, such as improving defences against natural enemies or altering host reproduction. Certain groups like ciliates and algae are known to host symbiotic bacteria commonly, but our knowledge of their symbiont’s evolution and function is limited. One such bacteria is ‘Candidatus Megaira’, a Rickettsiales that was first identified in ciliates, then later in algae. To improve the available data for this common but understudied group, we searched the genomes of potential hosts on online databases for Rickettsiales and assembled their genomes. We found 4 ‘Ca. Megaira’ this way and then used these to find a further 14 genomes in environmental metagenomic data. Overall, we increased the number of known ‘Ca. Megaira’ draft genomes from 2 to 20. These new genomes show us that ‘Ca. Megaira’ is far more diverse than previously thought and that it is potentially involved in defensive symbioses. In addition, one genome shows striking resemblance to well characterized symbiont, Wolbachia, in encoding many proteins predicted to interact directly with host proteins. The genomes we have identified and examined here provide baseline resources for future work investigating the real-world interactions between the hyper diverse ‘Ca. Megaira’ and its various potential hosts, like the economically important Nemacystus decipiens.

AbstractMembers of the bacterial genusRickettsiawere originally identified as causative agents of vector-borne diseases in mammals. However, manyRickettsiaspecies are arthropod symbionts and close relatives of ‘CandidatusMegaira’, which are symbiotic associates of microeukaryotes. Here, we clarify the evolutionary relationships between these organisms by assembling 26 genomes ofRickettsiaspecies from understudied groups, including the Torix group, and two genomes of ‘Ca. Megaira’ from various insects and microeukaryotes. Our analyses of the new genomes, in comparison with previously described ones, indicate that the accessory genome diversity and broad host range of TorixRickettsiaare comparable to those of all otherRickettsiacombined. Therefore, the Torix clade may play unrecognized roles in invertebrate biology and physiology. We argue this clade should be given its own genus status, for which we propose the name ‘CandidatusTisiphia’.

AbstractCulicoides biting midges (Diptera: Ceratopogonidae) are disease vectors responsible for the transmission of several viruses of economic and animal health importance. The recent deployment of Wolbachia with pathogen-blocking capacity to control viral disease transmission by mosquitoes has led to a focus on the potential use of endosymbionts to control arboviruses transmitted by other vector species. Previous screens of Culicoides have described the presence of Candidatus Cardinium hertigii (Bacteroidetes). However, the biological impact of this symbiont is yet to be uncovered and awaits a suitable system to study Cardinium-midge interactions. To identify candidate species to investigate these interactions, accurate knowledge of the distribution of the symbiont within Culicoides populations is needed. We used a sensitive nested PCR assay to screen Cardinium infection in 337 individuals of 25 Culicoides species from both Palearctic and Afrotropical regions. Infections were observed in several vector species including C. imicola and the pulicaris complex (C. pulicaris, C. bysta, C. newsteadi and C. punctatus) with prevalence ranging from low and intermediate, to fixation. Infection in C. pulicaris was very rare in comparison to a previous study, and there is evidence the prior record of high prevalence represents a laboratory contamination error. Phylogenetic analysis based on the Gyrase B gene sequence grouped all new isolates within “group C” of the genus, a clade which has to date been exclusively described in Culicoides. Through a comparison of our results with previous screens, we evaluate the suitability of Cardinium-infected species for future work pertaining to the symbiont.