New Results

A bacterial symbiont protects honey bees from fungal disease

Delaney L. Miller, Eric A. Smith, Irene L. G. Newton
doi: https://doi.org/10.1101/2020.01.21.914325

Abstract

Fungi are the leading cause of insect disease, contributing to the decline of wild and managed populations1,2. For ecologically and economically critical species, such as the European honey bee (Apis mellifera), the presence and prevalence of fungal pathogens can have far reaching consequences, endangering other species and threatening food security3,4,5. Our ability to address fungal epidemics and opportunistic infections is currently hampered by the limited number of antifungal therapies6,7. Novel antifungal treatments are frequently of bacterial origin and produced by defensive symbionts (bacteria that associate with an animal/plant host and protect against natural enemies 89. Here we examined the capacity of a honey bee-associated bacterium, Bombella apis, to suppress the growth of fungal pathogens and ultimately protect bee brood (larvae and pupae) from infection. Our results showed that strains of B. apis inhibit the growth of two insect fungal pathogens, Beauveria bassiana and Aspergillus flavus, in vitro. This phenotype was recapitulated in vivo; bee brood supplemented with B. apis were significantly less likely to be infected by A. flavus. Additionally, the presence of B. apis reduced sporulation of A. flavus in the few bees that were infected. Analyses of biosynthetic gene clusters across B. apis strains suggest antifungal production via a Type I polyketide synthase. Secreted metabolites from B. apis alone were sufficient to suppress fungal growth, supporting this hypothesis. Together, these data suggest that B. apis protects bee brood from fungal infection by the secretion of an antifungal metabolite. On the basis of this discovery, new antifungal treatments could be developed to mitigate honey bee colony losses, and, in the future, could address fungal epidemics in other species.

Emerging fungal pathogens pose major threats to animal and plant populations2. Among insects, fungal pathogens are currently the most common causal agents of disease, and historically have plagued insect hosts for over 300 million years1,10. In recent years, fungal pathogens have contributed to the unprecedented population decline of honey bees, causing opportunistic infections in already stressed colonies 3,4. Within the colony, the most susceptible individuals are arguably the bee brood (larvae and pupae), which are exposed to fungal pathogens, notably chalkbrood (Ascophaera apis) and stonebrood (Aspergillus flavus) 11,12. Although the spread of fungal disease among the brood can be limited by the hygienic behavior of honey bee nurses13, this behavior does not prevent infection. However, brood fungal infections in other insects are sometimes inhibited by the presence of bacterial symbionts14,15,8. Given that honey bee brood are reared in the presence of a handful of bacterial taxa16,17, it is possible these microbes play similar defensive roles. Indeed, worker honey bee pathogen susceptibility correlates with changes in their microbiome composition and abundance 18,19,20,21. Furthermore, the presence of key microbiome members in worker bees can alter the prevalence of bacterial diseases 22,23,24,25. In aggregate, this evidence suggests that honey bee-associated bacteria can defend against bacterial pathogens and may similarly protect the host from fungal disease.

One of the most prevalent brood-associated bacteria is Bombella apis (formerly Parasaccharibacter apium), an acetic-acid bacterium found in association with nectar and royal jelly. Within the colony it is distributed across niches including larvae, the queen’s gut, worker hypopharyngeal glands, and nectar stores. Many of the niches it colonizes, particularly the larvae, are susceptible to fungal infection and/or contamination, and its localization to these niches may be indicative of a protective role. Furthermore, increased B. apis load is negatively correlated with Nosema (a fungal pathogen) in honey bee adults, suggesting interactive effects. However, since B. apis is rarely found in adult guts, this interaction may be the result of B. apis-fungal interactions in the diet and where brood are reared. Additionally, the mechanism by which B. apis might interact with and/or suppress fungal pathogens is unknown.

Here we examined the potential of B. apis to prevent fungal infection in brood and the bacterial genes underlying pathogen defense. To determine the impact of B. apis on fungal colonization, we used two different insect pathogens in our assays: Beauveria bassiana, a generalist pathogen that infects 70% of insect species, and A. flavus, an opportunistic pathogen of honey bee brood. To determine the ability of B. apis to inhibit fungal growth in vitro, we competed each fungal pathogen with one of five B. apis strains, isolated from apiaries in the US (Fig 1a). In the presence of B. apis strains, fungal growth was either suppressed or completely inhibited, (Fig 1b). To quantify fungal inhibition, we counted spores of B. bassiana or A. flavus co-cultured with B. apis. The number of spores produced by both B. bassiana and A. flavus, was reduced by an order of magnitude on average (Fig 1c), showing that B. apis can suppress growth of both pathogens.

Figure 1:
Figure 1: B. apis outcompetes fungal pathogens in vitro.

a, The ability of each fungal isolate to grow on a B. apis lawns was qualitatively assayed. b, Compared to fungal controls, the presence of B. apis either suppressed or completely inhibited fungal growth, depending on strain identity. c, When co-cultured in liquid media, the presence of B. apis strongly reduced the number of spores produced by B. bassiana (A29: t = 13.114, df = 2, p = 0.19; B8: t = 11.147, df = 3, p = 0.006; C6: t = 10.121, df = 2.7, p = 0.011; SME1: t = 12.352, df = 2, p = 0.025) and A. flavus (A29: t = 2.8807, df = 2, p = 0.40; B8: t = 2.9033, df =2, p = 0.39; C6: t = 3.0137, df = 2, p = 0.37; SME1: t = 3.1679, df = 2, p = 0.34), depending on B. apis strain identity.

To test if B. apis is capable of preventing fungal infections in vivo, we collected larvae from our apiary and reared them on a diet supplemented with either B. apis or a sterile media control. Once reared to pupae, the cohort was inoculated with A. flavus or a sterile media control and presence of infection was scored until adulthood (Fig 2a). Pupae that were supplemented with B. apis as larvae were significantly more likely to resist fungal infection (χ2 = 14.8, df = 1, p < 0.001), with 66% of the cohort surviving to adulthood with no signs of infection (Fig 2b,c). In sharp contrast, without B. apis, no pupae survived to adulthood (Fig 2b, d). Interestingly, in the 34% of B. apis-supplemented pupae that succumbed to fungal infection, the number of spores produced was 68% on average (Fig 2e; t = 2.9116, df = 8.4595, p = 0.02). Taken together, these results suggest that the presence of B. apis increases the host’s likelihood of survival under fungal challenge, while decreasing the pathogen’s spore load and potential to spread infection to new hosts.

Figure 2:
Figure 2: Bee brood supplemented with B. apis are less susceptible to infection with A. flavus.

a, First instar larvae (n = 45) collected from the apiary were reared on sterile larval diet +/- B. apis (AJP2). Five days after pupation, each pupa was inoculated with 103 spores of A. flavus +/- B. apis or 0.01% Triton X-100 as a control. b, Of the pupae inoculated with flavus, those without B. apis all showed signs of infection by 48 hrs d, whereas 66% of those with B. apis never developed infections(χ2 = 14.8, df = 1, p < 0.001) c. e, Pupae with B. apis that did become infected had lower intensity infections, producing significantly (t = 5.5052, df = 5.5751, p = 0.002) fewer spores than those without B. apis.

To determine if B. apis produces antifungal metabolite(s), we incubated fungi in spent media (SM) from B. apis, filtered to exclude bacterial cells and normalized for final optical density reached (Fig 3a). Growth of both B. bassiana and A. flavus were significantly reduced by spent media alone, indicating that B. apis-induced changes in the media are sufficient to suppress fungal growth. To eliminate the possibility that fungal inhibition was mediated by acidification of the media, A. flavus was cultured in media acidified to pH of 5.0 (the same pH of B. apis SM). pH had no significant effect on fungal growth (Fig S3; t = −0.251, df = 35, p = 0.804). Therefore, it is likely that B. apis inhibits fungi via secretion of an antifungal secondary metabolite(s). We used antiSMASH26 to annotate secondary metabolite gene clusters in the genomes of all B. apis strains used in this study and found that all strains have a conserved type 1 polyketide synthase (T1PKS) region. Type 1 polyketide synthases are common among host-associated microbes and produce macrolides which often have antifungal activity 8,27,28,29. Additionally, all B. apis strains contain an aryl polyene synthesis cluster. The commonly used antifungals amphotericin, nystatin and pimaricin are all polyenes, suggesting that this gene cluster may also contribute to the production of antifungal compound(s). Further functional characterization of these gene clusters will help elucidate whether they play a role in the antifungal phenotype of B. apis. Considering the antifungal activity of B. apis secreted metabolites in vitro and our genomic predictions, it is likely that B. apis synthesizes and secretes a metabolite capable of inhibiting fungi.

Figure 3:
Figure 3: Fungal inhibition is mediated by B. apis secreted metabolites.

a, Spores of fungal isolates were incubated in spent media (SM) from B. apis cultures. b, The growth of both B. bassiana (A29: t = −15.315, df = 119, p < 0.001; B8: t = −13.925, df = 119, p < 0.001; C6: t = −13.202, df = 119, p < 0.001; SME1: t = −11.963, df = 119, p < 0.001) and A. flavus (A29: t = −11 .398, df = 59, p < 0.001; B8: t = −13.022, df = 59, p < 0.001; C6: t = −13.282, df = 59, p < 0.001; SME1: t = −11.261, df = 59, p < 0.001) in SM was strongly reduced compared to the control, suggesting secreted metabolites from B. apis mediate fungal inhibition. c, Genomic architecture of the type 1 polyketide synthase and arylpolyene secondary metabolite gene clusters identified by antiSMASH; gene models are colored based on putative function within the cluster and are oriented to show direction of transcription

Our results provide evidence that a honey bee-associated bacterium, B. apis, is capable of suppressing two prevalent insect fungal pathogens both in vitro and in vivo, likely via the synthesis of an antifungal metabolite. Our in vitro results demonstrate antifungal activity in all sampled strains of B. apis, with some variation between strains. Analysis of biosynthetic gene clusters present across all strains of B. apis revealed two putative regions involved in antifungal production: an aryl polyene synthetase and a T1PKS. Given that a significant proportion of known bacterially-produced antifungals are polyketides8,27,28,29, the T1PKS is a promising candidate region.

On the basis of our in vivo experiments, supplementing honey bee colonies with B. apis may decrease colony losses due to fungal disease. Indeed, in the field, supplementation of B. apis is correlated with a reduction in Nosema load in adult bees22. Beyond decreasing colony losses and fungal load via direct inhibition of fungal infection, the presence of B. apis may limit disease transmission by reducing the number of spores produced per infection. In addition, it may suppress adult-specific pathogens, which could be transiently harbored in the larval diet between adult hosts30.

Altering the prevalence of pathogenic fungi within managed honey bee colonies could have further ecological consequences. Floral resources shared among diverse pollinators act as transmission centers for fungi, both pathogenic and saprophytic31. Species-specific fungal pathogens can be seeded in pollen and nectar sources32, after which diverse pollinators, including native bees, can act as vectors to transmit the fungal pathogens to other floral sources, thereby facilitating heterospecific transmission of fungal agents33. As a result of reduced spore loads within colonies, the load of fungal pathogens deposited in local floral resources by foragers might also decrease, and perhaps reduce heterospecific transmission and spillover events 34.

Methods Summary

Competition assays were carried out with stationary cultures of B. apis normalized to the same OD and 103 spores of either fungal isolate in liquid or solid MRS media. The number of spores produced was counted on a hemocytometer under a light microscope at 40x magnification. Larvae were maintained on UV-sterilized larval diet and supplemented with stationary cultures of B. apis. A total of 103 spores of A. flavus were added to half the brood, five days into the pupal phase. Presence of fungal infection was scored daily until adulthood. Spent media (SM) of B. apis was obtained by spinning down stationary cultures and filtering out remaining bacterial cells using a 0.25 um filter. 103 spores of either fungal isolate were incubated in equal volumes SM and fresh media; OD600 was used as proxy for fungal growth. Genomes for all strains were downloaded from GenBank (see Table 1 for accession numbers) and re-annotated with RAST35,36. The resulting GFF files and corresponding genome files were uploaded to antiSMASH 26 and results were compared across strains to determine conserved secondary metabolite synthesis clusters.

View this table:
Table 1:

Sampling of B. apis strains

Methods

Isolates and culturing

All bacterial strains of B. apis and were obtained by sampling either nectar or larvae (Table 1). Isolates were acquired from our apiary or from Leibniz-Institut DSMZ. All cultures were incubated for 48 hours at 30° C in MRS. Fungal isolates, B. bassiana and A. flavus, were maintained at 25°C with 80% RH or 34° C with ambient humidity respectively on PDA or MRS agar plates. Spore solutions were prepared by flooding fungal plates with 0.01% Triton X-100, agitating with a cell scraper, and suspending the spores in the solution.

Competition plates

B. apis strains were grown to their maximal OD, and all strains were normalized to the lowest OD value by diluting in fresh media. A lawn of B. apis was created by plating 100 µL of normalized culture on MRS agar plates. The plate was then inoculated with 103spores of each fungal isolate and incubated at the appropriate temperature for that isolate. Over the course of three to seven days (depending on isolate) the presence of hyphal/conidia growth was monitored.

Competition assays

B. apis strains were grown to their maximal OD, and all strains were normalized to the lowest OD value by diluting in fresh media. 103 spores of each fungal isolate were incubated in 100 µl of density-normalized B. apis culture or 100 µl of fresh media. Fungal growth was monitored daily and once controls showed sporulation, spore counts were quantified for each well via hemocytometer.

Larval collection and in vivo infections

Late first instars were grafted from our apiary at Indiana University Research and Teaching Preserve into queen cups filled with UV-sterilized worker diet prepared as outlined in Schmel et. al, 201637. B. apis supplemented groups were given diet with a ratio of 1:4 stationary (OD=1.0) B. apis in MRS to worker diet. This bacterial load was between 2 × 106 and 6 × 106 cells/mL. Control groups were given diet with a ratio of 1:4 axenic MRS media to worker diet. After 5 days in larval diet, pre-pupae were transferred to new wells after either MRS or B. apis in MRS was added. Five days into pupal development, individuals were inoculated with 103 spores of A. flavus in 0.01% Triton X-100 or an equal volume of 0.01% Trition X-100 as a control. B. apis-supplemented groups were co-inoculated with one final dose of the bacterium (104 cells); controls received the same volume of MRS. Presence of infections (as evidenced by hyphae penetrating through the cuticle and/or spore production) was scored daily until adulthood.

Analysis of biosynthetic gene clusters (BGCs)

Genomes for all strains were downloaded from GenBank (see Table 1 for accession numbers) and re-annotated with RAST35,36. The resulting GFF files and corresponding genome files were uploaded to antiSMASH 26 and results were compared across strains to determine conserved secondary metabolite synthesis clusters. Gene model figures were visualized and adapted for publication using R38.

In vitro antifungal assay

To obtain spent media, strains were grown to their maximal OD (0.6-0.25), and all strains were normalized to the lowest OD value by diluting in fresh media. Cultures were spun down at 9,000 rpm for 5 min and the supernatant filtered through a 0.2 µm filter to remove bacterial cells. Spent media and fresh media were added to a multi-well plate in equal volumes and 103 spores from spore stock solutions were added. Growth was measured daily by assaying OD600. A positive control included spores in fresh media alone used to compare to treatment groups with spent media. Optical densities of spent media alone were monitored to ensure no bacterial growth occurred. Assay plates were incubated at the appropriate temperature for the fungal isolate used. Since B. apis acidifies the media from a pH of 5.5 to 5.0, controls of MRS media reduced to pH 5.0 with HCl were included.

Statistical analyses

All statistical analyses were performed in R 38. Spore counts of fungal isolates in the presence of B. apis were compared to controls with unequal variance, two sample t tests; p-values were Bonferroni-corrected for multiple comparisons across strains. In vivo infections are displayed as Kaplan-Meier survival curves. B. apis +/- infected treatments were compared with a long-rank test using R package, “survminer”39. Interactive effects of B. apis SM on growth of fungi over time were determined with a generalized linear model of OD, time, and strain identity.

Data and code availability

All genomic data used in this manuscript are publicly available through NCBI and listed in Table 1.

Author contributions

Conception and design of the work, ILGN and DLM, acquisition, analysis, or interpretation of data, EAS and DLM, drafted and revised the manuscript, DLM, EAS, ILGN.

Competing interests

ILGN and DLM are co-founders of VitaliBee, a company based partly on the discovery described herein.

Additional information

Supplementary information is available for this paper at: Correspondence and requests for materials should be addressed ILGN.

Supplemental Data

Supplementary Figure 1:
Supplementary Figure 1:

A. Maximum-likelihood 16S rRNA gene sequence tree for strains used in this study. Saccharibacter floricola and Gluconobacter oxydans were used as outgroups. Sequences were downloaded from GenBank and aligned with the SINA aligner40. The tree was constructed with RAxML 41 and visualized with FigTree42. Numbers at nodes represent bootstrap support from 1000 bootstrap pseudoreplicates. B. Core-ortholog maximum-likelihood phylogeny. All genomes were downloaded from GenBank and core orthologs were identified using OrthoMCL43. Alignments of core orthologs were made using MAFFT 44and concatenated together. As above, the tree was constructed with RAxML 41 and visualized with FigTree 42. Numbers at nodes represent bootstrap support from 1000 bootstrap pseudoreplicates.

Supplementary Figure 2:
Supplementary Figure 2: Bee brood are protected from fungal infection, independent of B. apis strain identity.

a, First instar larvae (n = 20) collected from the apiary were reared on sterile larval diet +/- B. apis (A29). Five days after pupation, each pupa was inoculated with 103 spores of A. flavus +/- B. apis or 0.01% Triton X-100 as a control. Pupae supplemented with A29 were more likely to survive to adulthood (χ2 = 3.4, df = 1, p = 0.07) b, Presence of B. apis (A29) significantly reduced (t = 5.5052, df = 5.5751, p = 0.001914) sporulation in infected pupae.

Supplemental Figure 3:
Supplemental Figure 3: Fungal inhibition by SM is not pH-mediated.

B. apis (A29) reduces MRS media from a pH of 5.5 to 5.0. Spent media from B. apis at pH 5.0 significantly reduced fungal growth (t = −6.111, df = 35, p < 0.001)while MRS media reduced to a pH of 5.0 using HCl did not significantly reduce growth (t = −0.251, df = 35, p = 0.804).

Acknowledgements

This work was funded by a Project Apis m. grant to ILGN and a USDA NIFA to EAS.

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Posted January 23, 2020.
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A bacterial symbiont protects honey bees from fungal disease
Delaney L. Miller, Eric A. Smith, Irene L. G. Newton
bioRxiv 2020.01.21.914325; doi: https://doi.org/10.1101/2020.01.21.914325
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