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Heritable microbes that exhibit reproductive parasitism are common in insects. One class of these are the male-killing bacteria, which are found in a broad range of insect hosts. Commonly, our knowledge of the incidence of these microbes is based on one or a few sampling sites, and the degree and causes of spatial variation are unclear. In this paper, we examine the incidence of the son-killer microbe Arsenophonus nasoniae across European populations of its wasp host, Nasonia vitripennis. In preliminary work, we noticed two female N. vitripennis producing highly female biased sex ratios in a field study from the Netherlands and Germany. When tested, the brood from Germany was revealed to be infected with A. nasoniae. We then completed a broad survey in 2012, in which fly pupal hosts of N. vitripennis were collected from vacated birds' nests from four European populations, N. vitripennis wasps allowed to emerge and then tested for A. nasoniae presence through PCR assay. We then developed a new screening methodology based on direct PCR assays of fly pupae and applied this to ethanol-preserved material collected from great tit (Parus major) nests in Portugal. These data show A. nasoniae is found widely in European N. vitripennis, being present in Germany, the UK, Finland, Switzerland and Portugal. Samples varied in the frequency with which they carry A. nasoniae, from being rare to being present in 50% of the pupae parasitised by N. vitripennis. Direct screening of ethanol-preserved fly pupae was an effective method for revealing both wasp and A. nasoniae infection, and will facilitate sample transport across national boundaries. Future research should examine the causes of variation in frequency, in particular testing the hypothesis that N. vitripennis superparasitism rates drive the variation in A. nasoniae frequency through providing opportunities for infectious transmission., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Gregory Hurst reports financial support was provided by Biotechnology and Biological Sciences Research Council. Gregory Hurst reports financial support was provided by Natural Environment Research Council. Eeva Tapio reports financial support was provided by Academy of Finland. Indirikis Krams reports financial support was provided by Latvian Council of Sciences. Ana Norte reports financial support was provided by Fundação para a Ciência e a Tecnologia. Gregorio Moreno-Rueda reports financial support was provided by Spanish Ministry of Education., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

A dynamic continuum exists from free-living environmental microbes to strict host-associated symbionts that are vertically inherited. However, knowledge of the forces that drive transitions in symbiotic lifestyle and transmission mode is lacking. Arsenophonus is a diverse clade of bacterial symbionts, comprising reproductive parasites to coevolving obligate mutualists, in which the predominant mode of transmission is vertical. We describe a symbiosis between a member of the genus Arsenophonus and the Western honey bee. The symbiont shares common genomic and predicted metabolic properties with the male-killing symbiont Arsenophonus nasoniae, however we present multiple lines of evidence that the bee Arsenophonus deviates from a heritable model of transmission. Field sampling uncovered spatial and seasonal dynamics in symbiont prevalence, and rapid infection loss events were observed in field colonies and laboratory individuals. Fluorescent in situ hybridisation showed Arsenophonus localised in the gut, and detection was rare in screens of early honey bee life stages. We directly show horizontal transmission of Arsenophonus between bees under varying social conditions. We conclude that honey bees acquire Arsenophonus through a combination of environmental exposure and social contacts. These findings uncover a key link in the Arsenophonus clades trajectory from free-living ancestral life to obligate mutualism, and provide a foundation for studying transitions in symbiotic lifestyle., (© 2021. The Author(s).)

Mobile elements-plasmids and phages-are important components of microbial function and evolution via traits that they encode and their capacity to shuttle genetic material between species. We here report the unusually rich array of mobile elements within the genome of Arsenophonus nasoniae , the son-killer symbiont of the parasitic wasp Nasonia vitripennis This microbe's genome has the highest prophage complement reported to date, with over 50 genomic regions that represent either intact or degraded phage material. Moreover, the genome is predicted to include 17 extrachromosomal genetic elements, which carry many genes predicted to be important at the microbe-host interface, derived from a diverse assemblage of insect-associated gammaproteobacteria. In our system, this diversity was previously masked by repetitive mobile elements that broke the assembly derived from short reads. These findings suggest that other complex bacterial genomes will be revealed in the era of long-read sequencing. IMPORTANCE The biology of many bacteria is critically dependent on genes carried on plasmid and phage mobile elements. These elements shuttle between microbial species, thus providing an important source of biological innovation across taxa. It has recently been recognized that mobile elements are also important in symbiotic bacteria, which form long-lasting interactions with their host. In this study, we report a bacterial symbiont genome that carries a highly complex array of these elements. Arsenophonus nasoniae is the son-killer microbe of the parasitic wasp Nasonia vitripennis and exists with the wasp throughout its life cycle. We completed its genome with the aid of recently developed long-read technology. This assembly contained over 50 chromosomal regions of phage origin and 17 extrachromosomal elements within the genome, encoding many important traits at the host-microbe interface. Thus, the biology of this symbiont is enabled by a complex array of mobile elements., (Copyright © 2020 Frost et al.)

Maternally heritable symbionts are common in arthropods and represent important partners and antagonists. A major impediment to understanding the mechanistic basis of these symbioses has been lack of genetic manipulation tools, for instance, those enabling transgenic GFP expression systems for in vivo visualization. Here, we transform the 'son-killer' reproductive parasite Arsenophonus nasoniae that infects the parasitic wasp Nasonia vitripennis with the plasmid pOM1-gfp, re-introduce this strain to N. vitripennis and then used this system to track symbiont life history in vivo. These data revealed transfer of the symbiont into the fly pupa by N. vitripennis during oviposition and N. vitripennis larvae developing infection over time through feeding. A strong tropism of A. nasoniae to the N. vitripennis ovipositor developed during wasp pupation, which aids onward transmission. The symbiont was also visualized in diapause larvae. Occasional necrotic diapause larvae were observed which displayed intense systemic infection alongside widespread melanotic nodules indicative of an active but failed immune response. Our results provide the foundation for the study of this symbiosis through in vivo tracking of the fate of symbionts through host development, which is rarely achieved in heritable microbe/insect interactions., (© 2019 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.)