The ISME Journal (2021) 15:2956–2968 https://doi.org/10.1038/s41396-021-00977-z

ARTICLE

Transitions in : evidence for environmental acquisition and social within a of heritable symbionts

1,2 3 2 4 2 Georgia C. Drew ● Giles E. Budge ● Crystal L. Frost ● Peter Neumann ● Stefanos Siozios ● 4 2 Orlando Yañez ● Gregory D. D. Hurst

Received: 5 August 2020 / Revised: 17 March 2021 / Accepted: 6 April 2021 / Published online: 3 May 2021 © The Author(s) 2021. This article is published with open access

Abstract A dynamic continuum exists from free-living environmental microbes to strict -associated symbionts that are vertically inherited. However, knowledge of the forces that drive transitions in symbiotic lifestyle and transmission mode is lacking. 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 . The symbiont shares common genomic and predicted metabolic properties with the male-killing symbiont , however we present multiple lines of evidence that the bee

1234567890();,: 1234567890();,: 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 life stages. We directly show horizontal transmission of Arsenophonus between 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 trajectory from free-living ancestral life to obligate mutualism, and provide a foundation for studying transitions in symbiotic lifestyle.

Introduction horizontal transmission (HT) during host development, by acquisition from environmental reservoirs or via infected Microbes that associate with hosts span a continuum from conspecifics or other host taxa [3, 4]. In some cases, a mutualism to and employ disparate transmission combination of these routes operate, creating a complex strategies [1]. Heritable bacterial symbionts, such as Wol- transmission landscape [5, 6]. Across this transmission axis bachia and Arsenophonus, transmit vertically (VT) from lies the impact of the symbiosis on each partner—whether parent to offspring [2]. Other microbial symbioses form via the host and microbe benefit from the interaction, and whether one party requires the other to complete their life cycle. Symbioses vary from being facultative (where one partner does not require the other) to obligate (where there Supplementary information The online version contains supplementary material available at https://doi.org/10.1038/s41396- is dependence). Obligacy may be a component of symbiont 021-00977-z. or host biology, or in some cases both parties are mutually dependent [2, 7, 8]. * Georgia C. Drew Clades of heritable symbionts commonly include strains [email protected] that are both facultative and obligate from the host per- 1 Department of Zoology, University of Oxford, Oxford, UK spective. In contrast, for the microbe, life is commonly 2 Institute of Infection, Veterinary and Ecological Sciences, obligately symbiotic, generally lacking replicative or dor- University of Liverpool, Liverpool, UK mant phases outside of host organisms [7]. Despite this 3 School of Natural and Environmental Sciences, Newcastle dependence, many of these symbionts can also transmit University, Newcastle upon Tyne, UK horizontally (inter and intra-specifically) over evolutionary – 4 Institute of Bee Health, Vetsuisse Faculty, University of Bern, timescales [9 11]. However, it is generally accepted that Bern, Switzerland VT predominantly drives population dynamics [12], but see Transitions in symbiosis: evidence for environmental acquisition and social transmission within a clade. . . 2957

[13]. In contrast, exclusively horizontally acquired sym- those in solitary species. This difference arises from a bionts must be transmitted to new hosts via infected con-/ higher density of hosts, greater host relatedness and a hetero-specifics or environmental reservoirs [14, 15]. As a homoeostatic nest environment, as well as reproductive result, symbiotic life for these microbes is commonly division of labour, overlapping generations and cooperative facultative [3]. The absence of VT can promote higher rates brood care [46, 47]. Specialised social behaviours [48] of partner switching [2, 16] and a weaker association additionally foster the transmission of microbes by direct between host and symbiont fitness. Consequently, selection contact, such as via proctodeal (anus—mouth feeding) and for higher and trajectories toward parasitism are stomodeal trophallaxis (mouth—mouth feeding) [49]. Thus, commonly assumed to be favoured more readily in HT host sociality is an important driver of symbiont phenotypes symbionts [1, 17, 18], but see [19, 20]. [47, 49, 50], and indeed interesting heterogeneities in Symbioses thus exist on an evolutionary landscape in symbiont infections are emerging based on caste, sex which transitions between transmission mode and lifestyles [51–55] and degree of sociality [50]. Despite this, the occur. However, study of the drivers and impact of transi- potential effects of host sociality on symbiont ecology and tions in transmission mode is inhibited by a lack of clades in , including important phenotypes such as trans- which different lifestyles co-occur among members. Clades mission, remain largely unexplored. encapsulating diverse transmission biology enable under- This study focuses on characterising the ecology and standing of both the ecological and evolutionary mechan- transmission of Arsenophonus from a eusocial host. We use isms driving transitions, and the consequences for processes a phylogenomic approach to show robust placement of the such as genome evolution [21–24]. For instance, the strain within the Arsenophonus clade and genomic analysis emergence of a horizontally transmitted vertebrate to report on its predicted metabolic properties and genome (Coxiella burnetti) from a clade of maternally inherited tick features. We then present data tracking the prevalence of the [22] provides insight into how infectious symbiont in honey bee colonies over space and time, to transmission can emerge from a heritable, obligate clade. search for indicators of stable maintenance (implying VT) Likewise, the presence of an opportunistic human infective or changes in prevalence (implying infectious transmission Sodalis provides an important comparator for understanding epidemics). These data are complemented by fluorescence the evolution of symbiosis, through comparison to - in situ hybridisation (FISH) analysis highlighting tissue associated mutualistic lineages of Sodalis [21]. interactions with honey bees. Finally, the capacity for ver- As a monophyletic clade of heritable Enterobacteriaceae, tical transmission is assessed directly by screening for Arsenophonus provides a valuable base for exploring the Arsenophonus across host life history, and the ability to evolution of a heritable lifestyle [25], due to its wide host horizontally transmit intra-specifically is investigated under distribution (est. 5% of species) [26] and diversity differing social conditions. These data demonstrate the in symbiotic lifestyle [27]. Members of the clade include presence of an infectiously transmitting Arsenophonus reproductive parasites [28], facultative mutualists [29], and without , highlighting transitions in life highly coevolved obligate endosymbionts undergoing history within this important genus of insect-associated genomic decay [30–33]. Despite this diversity, all strains microbes. characterised to date are VT transmitted. While frequent HT over ecological timescales has been shown for a number of strains, including the male-killer Arsenophonus nasoniae Methods [13, 34, 35] and two species of insect-vectored phyto- (Arsenophonus phytopathogenicus and Phlomo- Phylogenomic position of Arsenophonus from honey bacter fragariae)[36–38], this route occurs alongside bees and comparative genomic analysis vertical transmission. As a result, Arsenophonus is com- monly reported from arthropod screening efforts and pre- To assess the relatedness of the Arsenophonus strain asso- sumed heritable without further characterisation [26]. ciated with honey bees to other Arsenophonus, a phyloge- An economically important eusocial host, the Western nomic approach was adopted. To this end, a draft genome honey bee (Apis mellifera), has previously been associated was assembled (bioproject accession: PRJEB39047) using with Arsenophonus [39–43] and infection has been linked Illumina paired end shotgun library reads obtained in a to poor health outcomes [44] including colony collapse previous study derived from haemolymph template pur- disorder [45]. Whilst this interaction has attracted interest ified through a Nycodenz gradient (see Gauthier et al. from the community, basic information on the epidemiol- [56]). The assembled contigs were annotated using prokka ogy and transmission of Arsenophonus in honey bee v1.14.0 [57] and completeness was assessed using BUSCO populations is lacking. Symbionts in eusocial hosts are v4.1.4 based on the presence of 124 universal bacterial exposed to markedly different selection pressures from marker genes [58]. The phylogenetic position of the 2958 G. C. Drew et al.

Arsenophonus strain was estimated in relation to other bees (heads removed prior to extraction to minimise Arsenophonus, with the free-living species mir- PCR inhibition [61]) by Promega Wizard purification, with abilis and Providencia stuartii used as outgroups, using PCR detection of Arsenophonus (see Supplementary first an alignment of 53 ribosomal proteins and then a information). concatenated set of 155 single copy core orthologus pro- teins. In addition, we compared the predicted metabolic Localisation of Arsenophonus within the gut potential of Arsenophonus from bees to other strains, and in greater detail examined functional differences and the To visualise Arsenophonus using FISH, whole guts were degree of synteny to A. nasoniae (see Supplementary dissected from live worker bees and placed into Carnoy’s information for details). fixative for 24–48 h, washed with 100% EtOH (×3) and incubated with hybridisation buffer at room temperature in Spatial and seasonal dynamics of Arsenophonus in the dark (~15 h, see Supplementary information for detail). honey bee colonies The symbiont was targeted using an Arsenophonus specific probe (TCATGACCACAACCTCCA) [62]witha5′ Alexa To monitor Arsenophonus prevalence over time and space, Fluor 647 fluorochrome. Tissues were washed with pre- adult workers were collected from the outer frames of 159 heated buffer (∼48 °C, ×3 for 10 min) and mounted on colonies stemming from 45 apiaries in ten counties across glass slides with DAPI counter-staining. Tissues cured for England. Colonies were sampled from April to November 24 h before visualisation by confocal microscopy (ZEISS during 2014–2018, and repeated screening of some colonies LSM 88, ×40 objective lens), after which multiple optical resulted in a total of 230 sampling events. Apiaries are sections were assembled into Z-stacks under maximum defined here as a collection of colonies that are colocalised intensity settings using ImageJ [63]. Gut tissue of bees within a small area. Bees were preserved in 70% EtOH at from colonies not associated with Arsenophonus underwent −20 °C until DNA extraction. For each colony, posterior the same process to function as negative controls (see legs were pulled from workers (n = 12), pooled in groups of Supplementary information). In addition to gut imaging, four and exposed to UV light for 10 min to cross link DNA faecal samples were collected from workers (n = 22) iso- from surface microbes. Legs were used as this tissue is a lated in sterile petri dishes and extracted by Promega reliable marker of Arsenophonus association in bees and did Wizard purification (see Supplementary information). not inhibit downstream PCR assays when DNA was Material was then tested for Arsenophonus DNA through extracted using a high throughput Chelex protocol [59]. PCR assay. Extraction quality was verified by amplification of host DNA (EF1-α)[60] and the presence of Arsenophonus spp. Assessing the heritability of Arsenophonus and established by PCR assays targeting fbaA (adapted from acquisition from infected conspecifics Duron et al. [34], see Supplementary information) and Sanger sequencing of the product to determine broad To determine heritability of Arsenophonus in honey bees, identity of the Arsenophonus strains. Sensitivity of PCR infection was assessed across life history. Frames of worker assays was established through serial dilution, and was brood were removed from managed field colonies (N = 8, robust over two orders of magnitude (see Supplementary A+=6, A− = 2) where Arsenophonus status had previously information). been determined with high confidence. Eggs (n = 29), larvae To determine if Arsenophonus is lost from honey bee (n = 67), pupae (n = 49), newly emerged workers (NEWs) colonies during overwintering, the status of 25 colonies (n = 36), adult workers (n = 45) and where possible drones (from seven apiaries) was tracked from autumn to spring at (n = 22) were collected concurrently. To asses if Arseno- a greater depth than described above. In the autumn, 15 phonus association is heritable but only becomes detectable worker bees were screened from each colony (three indi- later in the life history, a further 64 NEWs were removed viduals pooled per extraction) to determine Arsenophonus from colonies and left to develop to forager age (25 ± 2 days prevalence. If 80% of extractions (i.e. 4 of 5) were positive post-emergence) in the company of other NEWs only. DNA for Arsenophonus the colony was included in the infected was extracted individually from eggs and early stage larvae cohort (group A, n = 19). The uninfected cohort (group B, using a Qiagen DNeasy Blood & Tissue Kit, for all other life n = 6) comprised of colonies where all extractions returned stages Promega Wizard purification was used. Molecular negative. Colonies were left to overwinter, and the sampling detection was completed as previously. process was repeated in the spring for colonies that over- To assess if infections were maintained in the absence of wintered successfully. To determine infection status in the colony and foraging environment, infected individuals spring a total of 24 bees were screened per colony (eight were detained in the laboratory and their Arsenophonus pools of three bees). DNA was extracted from whole status tracked over 15 days. Adult workers were collected Transitions in symbiosis: evidence for environmental acquisition and social transmission within a clade. . . 2959 from two colonies (total N = 76, Colony A = 36, Colony B = 40) with a high prevalence of Arsenophonus in 15 workers (>90%) or one control colony where none of 15 workers tested positive (N = 32) (based on Arsenophonus detection of individual bees, see Supplementary informa- tion). On entering the laboratory (day 1) the Arsenophonus status of all individuals was established by removal of posterior tarsus tissue (‘leg snip’) and PCR assays for symbiont presence (see Supplementary information). Additional bees (‘no leg snip control’, N = 40) from the infected source colonies were not subjected to leg snips to establish if this method biased results. Individuals were Fig. 1 Transmission mode and lifestyle diversity in the Arseno- marked and maintained in groups of four. To track the phonus clade. Phylogenomic position of the Arsenophonus associated maintenance of Arsenophonus over time, individuals were with honey bees (this study) among Arsenophonus spp. with available genomes. Bacterial species names are given, if formally recognised, randomly selected from each group and culled at days 4, 8 else the associated insect host is given. Symbols indicate known and 15. Individual Arsenophonus status was determined bacteriocyte associations and transmission modes, vertical transmis- using the same methodology as day 1, but using opposing sion (green), horizontal transmission (blue), if an environmental tarsus tissue. transmission route is additionally inferred this is indicated in pink. Analysis based on 53 ribosomal proteins support for Bayesian infer- To assess the capacity for horizontal acquisition of the ence (posterior probabilities) is shown at nodes (Color figure online). symbiont, adult workers (donors) were taken from colonies (N = 6) with a high prevalence of Arsenophonus in 15 adult bees (>85% infected, see Supplementary information) and Results mixed in 340 ml pots with uninfected NEWs (recipients). Each pot contained ten donors and five recipients. Two Phylogenomic position of the honey bee transmission treatments were established, each with ten Arsenophonus in the wider clade and metabolic replicates. In the ‘general contact’ treatment, bees were competence allowed to freely contact one another within the pot and food (50% sucrose solution) was openly available. In the Phylogenomic analysis based on 53 ribosomal protein ‘trophallaxis’ treatment, recipients with no direct access to sequences unambiguously placed the honey bee associated food were separated from donors (with food) by fine mesh, strain within the Arsenophonus clade (Fig. 1) alongside forcing infected bees to feed uninfected individuals by known heritable symbionts. The strain was established as a trophallaxis, but preventing other social contact. Contacts sister strain, with strong support, to Arsenophonus strains were allowed for 5 days, after which the fate (dead or alive) infecting other hymenopteran hosts ( ), of all individuals was recorded, i.e. those that had died including the male-killing reproductive parasite A. naso- during the course of the experiment (prior to final cull) were niae. These results were mirrored in analysis of a wider set labelled dead and analysed separately. In each case, of core genes, in which the Arsenophonus from honey bees experiments were compared to controls in which uninfected again clustered as sister to A. nasoniae (Supplementary bees were mixed with recipient bees. DNA was extracted information Fig. 1). individually from whole bees (test; n = 300, control; n = We assembled a draft genome for Arsenophonus from 165) by Promega Wizard purification. bees and examined synteny and compared gene content to A. nasoniae (Fig. 2), which is a closed completed genome Statistical methods [69]. At 3.3 Mb, the draft genome was larger than most other sequenced Arsenophonus with the exception of A. For statistical analyses, generalised linear models (GLM) nasoniae and Candidatus Arsenophonus triatominarum and mixed models (GLMM) were fitted in R version 3.3.1 (Supplementary information Table 1). BUSCO complete- [64] with binomial error distributions and logit link func- ness score for the bee Arsenophonus strain mirrored that of tion, using the packages glm and lme4 [65]. Minimum A. nasoniae (C:99.2% [S:99.2%, D:0.0%], F:0.8%, adequate models were selected by Akaike information cri- M:0.0%, n:124) (Supplementary information Fig. 2) sug- teria [66, 67] and likelihood ratio tests (LRT), with the latter gesting a near complete genome. Synteny analysis sup- being used to assess the significance of fixed and random ported the close relationship of the two strains, with effects [68]. Overdispersion was assessed using the Blemco evidence of some structural differences including at least package [64]. See Supplementary information tables one inversion event and several indels (Fig. 2). As expec- detailing selection of statistical models. ted, accessory elements () were less well 2960 G. C. Drew et al.

Fig. 2 Overview of the genome assembly of Arsenophonus from searches. B Syntenic comparison between ArsBeeCH and ArsFIN honey bees (ArsBeeCH) and comparison with close relative genomes as determined by D-Geneis and minimap2. C COG func- Arsenophonus nasoniae (ArsFIN). A GC% vs coverage plot of the tional annotation of strain-specific genes. The inset Venn diagram ArsBeeCH draft assembly. Contigs with putative phage origin were shows the number of shared and unique ortholog groups between the identified using PHASTER web server based on sequence similarities two Arsenophous strains. conserved between the two genomes. Overall, 2451 Spatial and seasonal dynamics of Arsenophonus in orthologous groups of proteins were shared between the honey bee colonies two symbionts, with 319 found solely in Arsenophonus from bees, and 909 solely in A. nasoniae. This higher Arsenophonus was found associated with 38.9% (95% CI: number of unique orthologous genes in A. nasoniae may 32.5–45.6%, N = 159 colonies) of honey bee colonies reflect the difference between draft and polished genomes, across the UK. Infections were spatially widespread and particularly in accessory genes carried on plasmids and identifiable in all ten county regions tested (Fig. 3A). prophage elements. County region was not a significant predictor of Arseno- Arsenophonus from bees also clustered with A. nasoniae phonus infection (GLMM, LRT: X2 = 0, df = 1, p = 1), but in analysis of predicted metabolic potential (Supplementary spatial patterns were evident at a local scale, with apiary information Figs. 3 and 4). These strains shared higher emerging as an important covariate, with colonies within an predicted competency for the glyoxylate cycle, polyamine apiary having correlated infection status (GLMM, LRT: biosynthesis compared to other sequenced Arsenophonus, X2 = 13.8, df = 1, p = 0.002**). All Arsenophonus detected and reduced capacity for cysteine biosynthesis. Predicted had identical sequence at the fbaA locus (N = 159). differences in metabolic capacity between Arsenophonus Strong seasonal dynamics in Arsenophonus infections from bees and A. nasoniae were modest, making similar (Fig. 3B) were evident, with prevalence of the bacterium metabolic potential and culture requirements likely. changing in a non-linear fashion during the main foraging Transitions in symbiosis: evidence for environmental acquisition and social transmission within a clade. . . 2961

Fig. 3 Spatial and seasonal dynamics in the Arsenophonus–honey spring to autumn was modelled using 229 binomial observations of bee association. A Circles show the approximate locations of 45 Arsenophonus status from 159 colonies (2014–2018). An overall honey bee apiaries (colonies colocalised within a small area) sampled prediction for all apiaries (black) and individual predictions for each for Arsenophonus across England (2014–2018). Circle size reflects the apiary (grey, n = 45) are shown. Green circles represent the mean number of colonies sampled within an apiary location and colour prevalence of Arsenophonus by month at the mean monthly sampling indicates the proportion of colonies associated with Arsenophonus. date, size is proportional to the square root of the number of colony B The proportion of colonies testing positive for Arsenophonus from samplings. Error bars represent binomial CI (Color figure online). period of the host (March to October). Arsenophonus pre- valence is lowest during the spring (2.63% of colonies, 95% CI: 0.0670–13.8%, N = 38) with the first infected colony detected in May. Prevalence continues to rise, reaching a peak in late summer (August: 71% of colonies, 95% CI: 55.7–83.6, N = 45), before dropping into Autumn (43.5% of colonies, 95% CI: 34.3–53.0, N = 115). Significant temporal variation by year was observed for Arsenophonus prevalence (GLMM, LRT: X2 = 3.87, df = 1, p = 0.049*). See Supplementary information Table 2 for a full summary. In temperate environments, overwintering conditions represent a distinct state for honey bees, with foraging ceasing temporarily and survival dependent on stored col- ony resources. The Arsenophonus infection status of 25 Fig. 4 Overwintering loss of Arsenophonus at a colony level. Group honey bee colonies was tracked from autumn to spring A colonies (n = 19) were infected with Arsenophonus in autumn but (Fig. 4). Of the 23 colonies that survived winter, all of those the bacterium was undetectable by the following spring. Group B = colonies (n = 6) were uninfected with Arsenophonus in autumn, one infected with Arsenophonus in the autumn (Group A, n colony gained Arsenophonus association by spring. Colonies that did 17) had lost the bacterium by spring. Of the colonies where not survive the winter (n = 2) are denoted by an X. The proportion of Arsenophonus was not detected in the autumn (Group B, bees testing positive for Arsenophonus in each colony is shown. Note, n = 6), five remained uninfected in spring, and one case fewer bees were sampled in Autumn (n = 15 bees per colony, pooled fi in groups of 3) than spring (n = 25 bees per colony, pooled in groups gained Arsenophonus. Con dence in this newly positive of 3). Pink dots (0 = Arsenophonus−) and green dots (1 = Arseno- colony is high, as all pools tested (N = 8 pools of three phonus+) show the raw binomial data jittered. Error bars indicate bees) were positive for Arsenophonus. Notably, this colony binomial SE (Color figure online). was sampled latest in the spring season. [39] and in haemolymph [56]. To obtain confirmation of Localisation of Arsenophonus within the gut symbiosis, targeted imaging of gut tissue from infected honey bees was conducted. This analysis showed aggrega- Previous work using metagenomic analysis has reported tions of Arsenophonus (coloured red) in the midgut (Fig. 5) Arsenophonus in the gut [40, 41], on the cuticular surface sufficiently large to imply colonisation and replication. 2962 G. C. Drew et al.

Fig. 6 Vertical transmission is largely absent in the Arsenophonus–honey bee association. Samples from across the host life history were taken from colonies (n = 6) identified as Arseno- Arsenophonus phonus positive (based on workers) and screened for Arsenophonus. Fig. 5 Localisation of in the midgut of a Eggs (n = 29), larvae (n = 67), pupae (n = 49) and newly emerged worker honey bee. Confocal microscopic image of a whole honey bee = fi ® workers (NEWs) (n 36) were all of worker caste. Isolated workers gut mount with an Arsenophonus speci c Alexa Fluor 647 labelled (n = 45) were removed from colonies as NEWs and allowed access probe (red fluorescence) and DAPI counter-staining (blue fluores- fi only to other NEWs before screening for Arsenophonus at forager age cence). Hybridisation of an Arsenophonus speci c probe (A) is visible (25 days ± 2). Binomial GLM predictions for the proportion of each within the lumen (L) of the midgut (ventriculus) alongside pollen life-stage infected are shown with 95% CI. Pink dots (0 = Arseno- grains (P). The image is a composite Z-stack comprised of 32 optical phonus−) and green dots (1 = Arsenophonus+) show the raw bino- slices (ZEISS LSM 880) assembled in ImageJ under maximum mial data jittered (Color figure online). intensity, scale bar represents 100 μm. Amplicon based approaches also report Arsenophonus from haemolymph [56]. For confocal images of whole gut mounts from honey bees uninfected with Arsenophonus [43]. In other early life stages Arsenophonus was detected fi see Supplementary information Fig. 5 (Color gure online). only at low frequencies, with 5.97% of larvae (95% CI: 1.65–14.6, p < 0.001***), 4.081% of pupae (95% CI: More sporadic infections were apparent in the crop, and also 0.498–14.0, p < 0.001***) and 2.78% of NEWs (95% the rectum (consistent with low representation of Arseno- CI: 0.07–14.5, p < 0.001***) testing positive for Arseno- phonus in honey bee gut microbiome studies that focus on phonus. In contrast, adult life stages showed high inci- the hindgut). Control images of uninfected bee tissue sug- dences of Arsenophonus, with the bacterium detectable in gested neither autofluorescence nor inadequate probe 54.5% of drones (95% CI: 32.2–75.6, p = 0.0198*) and removal contributed to artefactual Arsenophonus visualisa- 82.2% of workers (95% CI: 67.9–91.0). Workers isolated tion. The absence of a strong signal in the rectum is con- from the colony since emergence also did not develop sistent with specific binding, as there was no widespread Arsenophonus association on reaching forager age (95% probe binding in the rectum of Arsenophonus positive CI: 0.00–0.06, p < 0.001***). individuals (see Supplementary information Fig. 5). The maintenance of Arsenophonus in worker bees in the Arsenophonus was also detected by PCR assay in 59.1% of absence of sources of infection was then tested (Fig. 7). faeces from infected bees (95% CI: 36.4–79.3) suggesting Bees were removed from two infected colonies (colony A the bacterium may be shed from the gut, however viability and colony B) and maintained in the laboratory for 15 days of the symbiont was not confirmed. with access only to sterile food. At day 0 all individuals in the leg snip group were confirmed positive for Arseno- Assessing the heritability of Arsenophonus and phonus. Individuals in the ‘no snip control’ group were acquisition from infected conspecifics presumed, from identical colony origin, to be infected at a similar prevalence. There was no significant effect of To date, all characterised Arsenophonus strains show ver- treatment (leg snip or no snip) on the proportion of bees tical transmission in their arthropod hosts. To assess if the infected with Arsenophonus at each time point (LRT: X2 = honey bee strain conforms to this transmission strategy, the 0.253, df = 1, p = 0.615), suggesting the leg snip did not presence of Arsenophonus was assessed across host life impact our results. Overall, the proportion of bees infected history (Fig. 6). There was no evidence of transovarial with Arsenophonus decreased significantly over time (p < transmission of Arsenophonus with eggs consistently test- 0.001***) indicating loss of the bacterium. However, var- ing negative, corroborating previous findings in honey bees iation was evident at a colony level, and a significant effect Transitions in symbiosis: evidence for environmental acquisition and social transmission within a clade. . . 2963

Fig. 7 Loss of Arsenophonus in individuals removed from the colony and foraging environment. Binomial GLMM predictions (with 95% CI) show the proportion of individual honey bees that remain infected with Arsenophonus over time when removed from colonies and detained under lab conditions. The initial Arsenophonus status of each bee (n = 76) was determined by a leg snip (treatment group). An additional group of bees (n = 40) from the same colonies did not undergo leg snip (control group), and thus Arsenophonus starting prevalence was unknown. Overall predictions are shown for colony A (green) and colony B (orange), and the proportion of honey bees infected at days 0, 4, 8, and 15 is plotted independently for each colony and treatment (Color figure online). of colony was observed (LRT: X2 = 20.2, df = 1, p < 0.001***). Colony B lost Arsenophonus at a significantly faster rate (p < 0.001***), with fewer than 20% of indivi- duals infected by day 15. Control bees taken from unin- fected colonies maintained 0% infection throughout the study. See Supplementary information Table 3 for a full summary. Horizontal acquisition of Arsenophonus was tested in a separate experiment, where uninfected recipient bees were exposed to infected donor bees, either through trophallaxis or general contact. Acquisition of Arsenophonus among recipient bees was observed under both of these social conditions (Fig. 8A). The rate of Arsenophonus gain for Fig. 8 Horizontal transmission and maintenance of Arsenophonus recipients was higher under general contact exposure in honey bees under two social conditions. A Uninfected (recipient) (recipients, 40.0%, 95% CI: 26.4–54.8) compared to tro- bees were mixed with (B) infected (donor) bees and allowed either phallaxis (recipients, 22.0%, 95% CI: 11.5–36.0) and social general contact or contact via trophallaxis only. For control groups, fi recipients were mixed with uninfected bees. Dotted lines indicate context had a signi cant effect in the model (GLMM, LRT: the prevalence of Arsenophonus in recipients and donors at the start X2 = 5.22, df = 1, p = 0.0223*). of the transmission period. Note, all control bees started uninfected Within this experiment, a subset of donors lost the (Arsenophous prevalence = 0%). After 5 days of social interaction infection from the 85% prevalence starting threshold the Arsenophonus status of recipients (A) and donors (B)isshown. Transmission to recipients occurred under general contact and tro- (Fig. 8B), paralleling the previous observation that infection phallaxis, but at varying levels. Recipients mixed with uninfected was unstable under laboratory isolation. Horizontal acqui- (control) bees did not acquire Arsenophonus, with the exception of sition between donor and recipient bees contrasted to con- two individuals in the general contact treatment. Coloured dots show raw binomial data jittered (0 = Arsenophonus−,1= Arseno- trol pots where the same contact was with uninfected + ‘ ’ phonus ) and error bars indicate binomial SE. Each group was donor bees. Here, recipient controls under trophallaxis replicated (n = 10), with five donor bees and ten recipient bees per remained uninfected (control recipients, 0%, 95% CI: replicate. 0–13.7), however two control recipients under general 2964 G. C. Drew et al. contact tested positive for Arsenophonus (control recipients, lies in the same subclade as the male-killing microbe, 6.66%, 95% CI: 0.818–22.1). The fate of individuals (live/ Arsenophonus nasoniae. Whilst predominantly associated dead at end of transmission period) emerged as an important with the parasitic , A. nasoniae is an extra- predictor of infection status (GLMM, LRT: X2 = 7.64, df = cellular symbiont that also has phases outside of the wasp 1, p = 0.00571**), with individuals that had died during the host [71], is culturable [28], and is also maintained by a study more commonly associated with Arsenophonus combination of vertical and infectious transmission [13]. infection (see Supplementary information Fig. 6 and Sup- Arsenophonus nasoniae and the bee Arsenophonus also plementary information Table 4 for a full summary). share the capacity to establish through the gut [71], indi- cating this pathway is preserved in the clade. The genome of Arsenophonus from the honey bee is, like A. nasoniae, Discussion relatively large in size and not subject to the degradation process that occurs in the obligate symbionts in the genus. The diversity of existing symbiotic interactions represents The two strains have markedly similar predicted metabolic the result of historical evolutionary transitions between competence profiles, which further supports the likely cul- lifestyles. Here, we investigate the symbiotic lifestyle of turability of the Arsenophonus from Apis, and its capacity to Arsenophonus associated with honey bees. A significant survive and replicate outside of the host environment. contingent of the Arsenophonus clade is in the latter stages Taken together, the A. nasoniae/honey bee Arsenophonus of an evolutionary trajectory towards obligate intracellular clade is most parsimoniously regarded as a retained free- life [30–32], and until now all characterised members of the living, insect-associated microbe in a clade that has evolved genus demonstrate a heritable lifestyle, at least to some into endosymbiosis, and indeed obligate endosymbiosis, in degree [26, 27]. We here show that Arsenophonus asso- other cases. The alternate model—reacquisition of free- ciated with honey bees deviates significantly from this living capacity—is less probable as this would require the heritable model, instead demonstrating strong seasonal re-establishment of multiple genetic systems allowing patterns and a dynamic association that appears to be driven growth outside of a host environment. This scenario is not, by social transmission with an additional, unidentified, however, impossible, as previous work on Coxiella indi- environmental reservoir. These results shed new light on cated the striking emergence of an infectious vertebrate evolutionary transitions and symbiotic diversity within a pathogen from a clade of heritable, and sometimes obligate, clade of heritable arthropod symbionts. endosymbionts of [22]. We have identified the Arsenophonus–honey bee strain Our data thus indicate that the clade Arsenophonus as the first within the clade to show little evidence of her- contains more transmission diversity than previously con- itability, with evidence for this coming from multiple sidered. Knowledge of the selective forces that drive the sources. First, the absence of the bacterium in eggs of emergence of symbiotic diversity is imperative for under- infected colonies corroborates previous findings that standing important transitions such as the emergence of Arsenophonus is not transovarially transmitted in honey pathogenic agents from commensal partners, and vice versa bees [43], while very low prevalence in early host life stages [21, 22, 72]. Enterobacteriaceae genera appear to be well provides new evidence that no alternative routes of VT are adapted for transitioning between ecological and symbiotic operating in the association. Secondly, the pronounced niches [73, 74]; for example, Pantoea, Sodalis and Serratia seasonal dynamics we observed for Arsenophonus are not include notable symbionts of [75–77] but are largely consistent with the epidemiology of heritable symbionts, comprised of representatives from soil, plants and clinical whose infection prevalence generally remains relatively settings [21, 74, 77]. Likewise, the genus static within a host generation though may vary dynami- includes both defensive symbionts and environmentally cally between generations [70]. Within these dynamics, acquired pathogens of invertebrates [78, 79], but also a colonies appear to lose Arsenophonus over the winter, or primary pathogen of humans with an unidentified trans- the association declines to a level undetectable by our sur- mission route [80, 81]. Among Enterobacteriaceae, the vey. Finally, experiments show first that workers bees with Arsenophonus genus differs, in that all strains to date are Arsenophonus infection lose it under laboratory conditions believed to be arthropod host-restricted and previously under social isolation, and also that social contact can result environmental acquisition has not been shown to occur in acquisition of infection. Overall, Arsenophonus presents routinely [27]. as a horizontally acquired infection of honey bees rather Given that vertical transmission is not operating in the than a persistent VT transmitted one. honey bee–Arsenophonus association, the questions arises: This acquisition-loss cycle occurs for a bacterium that is what are the main drivers of HT? Do honey bees acquire otherwise nested into a clade of heritable microbes, with infection predominantly from infected conspecifics, or are phylogenomic analysis indicating Arsenophonus from bees infections acquired from the wider environment (e.g. Transitions in symbiosis: evidence for environmental acquisition and social transmission within a clade. . . 2965 additional host species or reservoirs)? While the symbiotic the scope for the evolution of virulence [17, 18], although phenotype of the honey bee–Arsenophonus remains this trait alone is insufficient to draw conclusions regarding unknown, we identified horizontal (infectious) transmission symbiont phenotype, as many avirulent or beneficial when general contact between conspecifics was allowed, microbes are transmitted horizontally [15, 95]. The asso- and when contact was via trophallaxis exclusively. For a ciation with dead hosts may implicate Arsenophonus heritable symbiont, maintenance or reversal to the ancestral directly or represent opportunistic proliferation in a com- (horizontal) transmission route has a clear adaptive benefit promised host. Alternatively, saprophytic growth on cada- in a eusocial host, typified by high host density and low vers may be occurring, a capacity demonstrated by A. genetic diversity [82], and social acquisition can prevent nasoniae in fly puparia [96]. Further work is needed to workers being the evolutionary dead ends they are often determine if health outcomes are causal in honey bees and considered for heritable symbionts [83]. Localisation in the to characterise the transmission phenotype of Arsenophonus gut and detection in faeces is consistent with a faecal—oral reported from other bee species [94, 97–99]. route, which could allow effective social transmission To conclude, the honey bee–Arsenophonus contrasts in within a colony and to other hosts via shared floral its phenotype from the rest of the heritable clade, as vertical resources [84–86]. Indeed, Arsenophonus has previously transmission is not the predominant transmission mode. been detected on flowers [87], but until now direct evidence Instead, HT occurs via social interactions, colonisation can for the potential capacity to transmit via this route was occur in the gut and environmental reservoirs appear to be lacking. necessary for maintenance of the association. Our data The marked seasonal incidence of Arsenophonus, peak- establish the honey bee–Arsenophonus as a key link in a ing in autumn, has a number of potential drivers. Shedding symbiont clades trajectory from free-living ancestral life to and accumulation of the bacterium in the foraging envir- obligate mutualism threatened by mutational decay. This onment (e.g. on flowers) may generate increased infection will provide new avenues for research into the emergence of risk as the foraging season proceeds, as observed for some symbiotic diversity. parasite species [88]. However, this would be contingent on the survival of Arsenophonus in the environment, a trait Data availability conventionally considered absent from this class of insect symbionts, despite relatively large symbiont genomes and All data associated with this study can be publicly accessed cell free cultivability found within the clade [89, 90]. at https://doi.org/10.6084/m9.figshare.c.5073479.v1. Alternatively, but not mutually exclusively, Arsenophonus dynamics may reflect the activity period of an environ- Acknowledgements We thank Dr. Katherine Roberts, Dr. Laurent mental reservoir or additional host species that is respon- Gauthier and Dr. Kirsty Stainton for assistance on the project, Dr. Andri Manser for statistical input, and three anonymous reviewers for sible for transmission to honey bees. Comparable improving the manuscript. Jack Wilford, and many other bee keepers, seasonality is observed for Spiroplasma in honey bees, with were pivotal in sampling efforts. This work was financially supported peak prevalence of S. melliferum aligning with peak flow- by a BBSRC iCASE studentship to GCD (BB/L016133/1) and Bee ering periods [91, 92]. This latter hypothesis may explain Disease Insurance Ltd. Microscopy was conducted at the Centre for Cell Imaging (CCI), using equipment under grant BB/M012441/1. our observation that apiary is a significant predictor of a colonies Arsenophonus status, while over broad spatial Author contributions The project was conceived initially by GEB, scales infection prevalence does not vary notably. Here, CLF and GDDH. GCD designed and completed survey, experimental localised spatial pattern reflects shared exposure to reser- and statistical analysis, with input from GDDH and GEB. PN, OY and voirs of infection, with colonies originating from the same SS completed the sequencing and phylogenomic analysis elements. GCD wrote the paper with GDDH and SS, which was edited by OY, apiary often overlapping in foraging area [93]. Relevant PN and GEB with the final draft approved by all authors. here are reports of Arsenophonus associated with pollen and nectaring sites [42, 87], providing further evidence for Compliance with ethical standards environmental transmission [87]. Other processes, such as drifting of infected bees within apiaries, could drive inter- Conflict of interest The authors declare no competing interests. colonial transmission of Arsenophonus and feedback to drive the localised infection prevalence we observed. Publisher’s note Springer Nature remains neutral with regard to The position of the honey bee Arsenophonus on the jurisdictional claims in published maps and institutional affiliations. parasitism-mutualism continuum remains speculative. Our observations of HT, systemic infections and a higher pre- Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, valence among dead hosts strengthens interpretations of adaptation, distribution and reproduction in any medium or format, as previous work correlating Arsenophonus with poor health long as you give appropriate credit to the original author(s) and the outcomes in bees [44, 45, 94]. HT is considered to increase source, provide a link to the Creative Commons license, and indicate if 2966 G. C. Drew et al. changes were made. The images or other third party material in this 18. Frank SA. Host-symbiont conflict over the mixing of symbiotic article are included in the article’s Creative Commons license, unless lineages. Proc Biol Sci. 1996;263:339–44. indicated otherwise in a credit line to the material. If material is not 19. Sachs JL, Essenberg CJ, Turcotte MM. 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