The study of microbial symbioses reveals a spectrum ranging from mutualistic to parasitic relationships, with diverse transmission strategies. Heritable symbionts like *Wolbachia* and *Arsenophonus* typically exhibit vertical transmission (VT), passing from parent to offspring. Conversely, horizontal transmission (HT) involves acquisition from environmental reservoirs or infected conspecifics. Some symbioses utilize a combination of both routes, creating complex transmission patterns. Symbiotic relationships vary in obligacy, from facultative (where one partner doesn't require the other) to obligate (where dependence exists). Clades of heritable symbionts often encompass both facultative and obligate strains from the host's perspective, while the symbiont's lifestyle is usually obligately symbiotic. Despite this dependence, many heritable symbionts can also transmit horizontally over evolutionary timescales, although VT usually drives population dynamics. Exclusively horizontally transmitted symbionts rely on infected individuals or environmental reservoirs for transmission, typically resulting in facultative symbiotic lifestyles. HT often leads to increased virulence and parasitic tendencies. The study of transitions between transmission modes and symbiotic lifestyles is hampered by a lack of clades showing co-occurrence of different lifestyles. The *Arsenophonus* clade, a monophyletic group of heritable Enterobacteriaceae, provides a valuable model for studying the evolution of a heritable lifestyle due to its diverse host range and varied symbiotic relationships, including reproductive parasites, facultative mutualists, and obligate endosymbionts. While HT has been observed in some *Arsenophonus* strains, VT is considered predominant. The Western honey bee (*Apis mellifera*) hosts *Arsenophonus*, and this association has been linked to negative health outcomes. However, the epidemiology and transmission dynamics of *Arsenophonus* in honey bee populations remain poorly understood. Eusocial hosts like honey bees present unique selective pressures due to high host density, relatedness, and social behaviors, influencing symbiont transmission and evolution. This study aims to characterize the transmission ecology of *Arsenophonus* in honey bees, combining phylogenomic analysis with field and laboratory investigations to determine the transmission mode and impact of host sociality.

The literature review section is implicitly woven into the introduction, referencing key studies on symbiosis transmission modes, the *Arsenophonus* clade, and the impact of sociality on symbiont evolution. Specifically, the authors cite numerous studies on *Wolbachia*, *Arsenophonus*, and other symbionts to establish the context of their research. They highlight the existing knowledge gaps concerning transitions in symbiotic lifestyles and the need for model systems to study these dynamics. The *Arsenophonus* clade is presented as a suitable model due to its diversity in transmission and symbiotic relationships. Existing work on honey bee symbionts and the influence of sociality on symbiont interactions are also discussed to justify their focus on this system. The review implicitly positions the current study within the broader field of symbiosis research and highlights the novelty of investigating *Arsenophonus* transmission in a eusocial host.

The study employed a multi-faceted approach. First, a phylogenomic analysis was conducted to determine the phylogenetic position of the honey bee *Arsenophonus* strain within the *Arsenophonus* clade. A draft genome was assembled using Illumina sequencing data, annotated using Prokka, and its completeness assessed using BUSCO. Phylogenetic relationships were inferred using alignments of ribosomal proteins and concatenated single-copy orthologous proteins, with outgroups included. Comparative genomic analysis explored the predicted metabolic potential and compared the honey bee *Arsenophonus* to other strains, especially *A. nasoniae*. Second, the spatial and seasonal dynamics of *Arsenophonus* in honey bee colonies were investigated. Adult worker bees from numerous colonies across England were sampled over several years. DNA was extracted from pooled leg samples, and *Arsenophonus* presence was determined using PCR targeting *rpoB*. The sensitivity of the PCR assays was validated through serial dilutions. To study overwintering effects, *Arsenophonus* prevalence in colonies was monitored from autumn to spring. Third, the localization of *Arsenophonus* within the honey bee was determined using fluorescent in situ hybridization (FISH). Whole guts were dissected from bees, hybridized with an *Arsenophonus*-specific probe, and visualized using confocal microscopy. Fourth, the heritability of *Arsenophonus* and potential acquisition from infected colonies were investigated. Brood samples (eggs, larvae, pupae, newly emerged workers, adult workers, drones) were collected from field colonies to assess infection across life stages. Newly emerged workers were also reared separately to observe infection development. A laboratory experiment tracked *Arsenophonus* maintenance in infected bees over time. Lastly, the capacity for horizontal acquisition was tested by mixing infected adult bees (donors) with uninfected newly emerged bees (recipients) in two treatments: allowing social interactions and only allowing trophallaxis. Generalized linear models (GLM) and generalized linear mixed models (GLMM) were used for statistical analysis.

Phylogenomic analysis confirmed the honey bee *Arsenophonus* strain's placement within the *Arsenophonus* clade, sharing genomic and metabolic similarities with *A. nasoniae*. Spatial and seasonal dynamics revealed fluctuations in *Arsenophonus* prevalence in honey bee colonies, indicating non-vertical transmission. Rapid infection loss events were observed both in the field and in laboratory settings. FISH revealed *Arsenophonus* localized in the gut, with rare detection in early life stages. Horizontal transmission of *Arsenophonus* was directly demonstrated in laboratory experiments, both through social contact and trophallaxis. The study found no evidence of vertical transmission of *Arsenophonus* in honey bees.

The findings demonstrate that the honey bee *Arsenophonus* strain deviates from the typical vertical transmission model observed in other *Arsenophonus* members. The observed spatial and seasonal variations in prevalence, coupled with the demonstration of horizontal transmission, strongly suggest environmental acquisition and social transmission as primary routes of infection. The lack of detectable *Arsenophonus* in early life stages further supports the absence of vertical transmission. These results reveal a key transitional stage in the *Arsenophonus* clade's evolutionary trajectory, from a free-living ancestor towards obligate mutualism. The study highlights the significant influence of host sociality on symbiont transmission and evolution, particularly in eusocial species. The findings provide a strong foundation for future studies investigating the evolutionary dynamics and ecological consequences of transitions in symbiotic lifestyles and transmission modes.

This study provides compelling evidence for the horizontal transmission of *Arsenophonus* in Western honey bees, contrasting with the primarily vertical transmission observed in other members of this clade. The absence of vertical transmission and the demonstration of horizontal transmission through both social contact and trophallaxis highlight a critical evolutionary transition within the *Arsenophonus* lineage. Future research should focus on identifying the environmental reservoirs of *Arsenophonus*, characterizing the specific mechanisms of horizontal transmission, and exploring the broader ecological and evolutionary implications of this transitional symbiosis.

The study primarily focused on a specific geographic region (England) and a limited time frame, which might affect the generalizability of the findings to other honey bee populations and environments. The laboratory experiments, while demonstrating horizontal transmission, may not perfectly replicate the complexity of natural transmission dynamics. Furthermore, the study did not explore the potential impact of environmental factors other than social contact on the transmission dynamics of *Arsenophonus*. Future research should address these limitations by expanding the geographical scope and duration of the study, incorporating more sophisticated laboratory models, and investigating the roles of various environmental factors.