DOI: 10.5061/dryad.qbzkh18t1
title: Core symbionts, age at inoculation, and diet affect colonization of the bumble bee gut by a common bacterial pathogen

description: Microbes shape the health of bumble bees, an important group of
 pollinators including species of conservation concern. Most microbial
 research on bumble bees has focused on eukaryotic and viral pathogens or
 the core gut microbiome, a community of host-specialized bacterial
 symbionts that helps protect hosts against eukaryotic pathogens. Bumble
 bees also harbor a third class of microbes: non-core gut bacteria, which
 are non-host-specific and vary among individuals. Understanding their
 functional role and how they interact with core symbionts is important for
 bumble bee ecology and management. We surveyed non-core bacteria in wild
 bumble bee workers (Bombus impatiens) and conducted lab experiments with
 gnotobiotic B. impatiens to examine factors shaping colonization by a
 focal non-core bacterium (Serratia marcescens) and its consequences for
 bee health. Non-core bacteria, including Serratia, frequently
 occur at high abundance in wild bumble bees, with roughly half of
 individuals harboring at least 10% non-core gut bacteria. Experiments
 showed that Serratia marcescens better colonizes the gut when bees are
 inoculated early (within one day of adult emergence) and the core gut
 microbiome is disrupted. A mixed wildflower pollen diet facilitated the
 highest level of infection compared to two monofloral pollen treatments.
 We also provide evidence that Serratia is pathogenic: exposing bees with
 disrupted gut microbiomes to Serratia strongly reduced lifespan and, as a
 result, also reduced total reproduction. These results have three
 important implications: first, non-core bacteria are widespread in wild
 bumble bees, and some species are opportunistic pathogens. Second, the
 core gut microbiome plays a crucial role in protecting against these
 pathogens. Third, the timing of inoculation relative to bee age, as well
 as diet, are key factors controlling bacterial pathogen colonization of
 the gut. Overall, these findings suggest that gut bacterial health could
 be an important target for monitoring and managing bumble bee health.
description: # Core symbionts, host age, and diet affect colonization of the bumble bee
 gut by a common bacterial pathogen
 [https://doi.org/10.5061/dryad.qbzkh18t1](https://doi.org/10.5061/dryad.qbzkh18t1) ## Description of the data and file structure We measured non-core bacterial prevalence in wild bumble bee workers (*Bombus impatiens*) and conducted lab experiments with gnotobiotic *B. impatiens* workers to examine factors shaping colonization by a focal non-core species (*Serratia marcescens*) and its consequences for bee health. ### Files and variables #### File: final\_ASV\_table.txt **Description:** The final amplicon sequence variant table obtained after filtering and removing contaminants. ##### Variables * sample_name: The name of the sample. Field-collected bees are identified with unique IDs following the format ####_####. Bees from lab experiments are identified with unique IDs following the format MC##_B##. * All other columns: Headings correspond to a unique identifier (combination of letters and numbers) of an individual ASV. The numbers in each row correspond to the number of reads of the ASV within each sample. #### File: final\_taxonomy\_table.txt **Description:** The final taxonomy table obtained after filtering and removing contaminants. ##### Variables * OTU_ID: The unique identifier (combination of letters and numbers) of an individual ASV. * Domain: The domain of the ASV. * Phylum: The phylum of the ASV. * Class: The class of the ASV. * Order: The order of the ASV (if known). Unidentified orders are listed as "NA." * Family: The family of the ASV (if known). Unidentified families are listed as "NA." * Genus: The genus of the ASV (if known). Unidentified genera are listed as "NA." * Species: The species of the ASV (if known). Unidentified species are listed as "NA." #### File: metadata.csv **Description:** The metadata associated with the ASV and taxonomy tables. ##### Variables * sample_name: The name of the sample. Field-collected bees are identified with unique IDs following the format ####_####. Bees from lab experiments are identified with unique IDs following the format MC##_B##. * plate_number: The PCR plate on which the sample was placed. * plate_well: The well in the PCR plate where the sample was placed. * project: The project (field collection versus lab experiment) that the sample was in. * sample_or_control: Whether the "sample" was a sample (i.e., bee gut) or control (i.e., no-template control or extraction blank). * dissection_date: The date when the bee was dissected. * sampling_location: The location where the bee was collected from the field, if applicable. * species: The bee species. * sampling_site: The site where the bee was collected from the field, if applicable. * latitude: The latitude where the bee was collected from the field, if applicable. * longitude: The longitude where the bee was collected from the field, if applicable. * colony_id: The colony ID from which the bee was sourced for lab experiments, if applicable. * microcolony_id: The microcolony ID which the bee was placed in for lab experiments, if applicable. * core_treatment: The core gut microbiome transplant treatment for bees in lab experiments. * core_inoc_id: The unique ID of the core gut microbiome treatment inoculum, if applicable. * age_treatment: The age treatment of bees in lab experiments. * serratia_treatment: The *Serratia* treatment of bees in lab experiments. * date_microcolony_established: The date when the microcolony was established for lab experiments. * date_prepped_extraction: The date when homogenized guts were prepared for DNA extraction. * date_extracted: The date when DNA was extracted. * date_pcr_1: The date when the first PCR was run. * date_pcr_2: The date when the second PCR was run. #### File: initial\_feature\_table.txt **Description:** The initial feature table obtained prior to filtering and removing contaminants. After removing contaminants, this generated the final taxonomy table file. ##### Variables * OTU_ID: The unique identifier (combination of letters and numbers) of an individual ASV. * taxonomy: The taxonomic identification of the ASV. * All other columns: Headings correspond to the names of each sample. Field-collected bees are identified with unique IDs following the format ####_####. Bees from lab experiments are identified with unique IDs following the format MC##_B##. Mock community standards are indicated by "Mock comm," PCR no-template controls are "NTC," and extraction blanks are "Extr blank." #### File: serratia\_cfu\_data.csv **Description:** The data table from lab experiments testing whether *Serratia* colonization of *B. impatiens* workers depended on core microbiome and the timing of inoculation with respect to be age. ##### Variables * sample_name: The name of the sample. Bees are identified with unique IDs following the format MC##_B##. * source_colony: The ID of the commercial colony from which the worker was sourced. * microcolony_id: The ID of the microcolony in which the worker was placed. * core_treatment: The core microbiome treatment, indicated by "M+" for core microbiome transplant and "M-" for core microbiome depletion treatment. * core_inoc_id: The ID of the gut homogenate used to inoculate bees in the M+ treatment. "NA" indicates that bees were not inoculated with homogenate. * age_treatment: The timing of *Serratia* inoculation relative to bee age. In "early" treatments, bees were inoculated at &lt;1 day after eclosion. In "late" treatments, bees were inoculated 4-5 days after eclosion. * serratia_treatment: The *Serratia* treatment, with "S+" for *Serratia* inoculation and "S-" for *Serratia*-free control. * date_microcolony_established: The date when the microcolony was first established. * date_core_inoculation: The date when the bee was given the core gut microbiome (M+ or M-) treatment. * date_serratia_inoculation: The date when the bee was given the *Serratia* (S+ or S-) treatment. * date_dissected: The date when the bee was dissected. * dilution_cfus_counted: The dilution at which *Serratia* CFUs were counted from the dissected bee gut. * mean_cfus_per_spot: The mean number of *Serratia* CFUs per 10-uL spot at the indicated dilution. * cfus_per_gut: The mean number of *Serratia* CFUs per gut, calculated based on values reported in the previous two columns. * notes: If a bee died before the end of the end of the experiment, it is indicated as "bee died" in this column. These bees were not dissected. #### File: pollen\_treatment\_data.csv **Description:** The data table from lab experiments testing whether *Serratia* colonization of *B. impatiens* workers depended on pollen diet. ##### Variables * sample_name: The name of the sample. Bees are identified with unique numbers. * source_colony: The ID of the commercial colony from which the worker was sourced. * microcolony_id: The ID of the microcolony in which the worker was placed. * pollen_treatment: The pollen diet treatment. * date_sterilized: The date that pupae were removed from the commercial colony and surface sterilized. * date_microcolony_established: The date when the microcolony was first established. * date_dissected: The date when the bee was dissected. * dilution_cfus_counted: The dilution at which *Serratia* CFUs were counted from the dissected bee gut. * mean_cfus_per_spot: The mean number of *Serratia* CFUs per 10-uL spot at the indicated dilution. * cfus_per_gut: The mean number of *Serratia* CFUs per gut, calculated based on values reported in the previous two columns. * pollen_grains_per_gut: The mean number of pollen grains per gut, estimated by pipetting 10 uL gut homogenate into a Neubauer improved hemocytometer and counting all pollen grains in two separate technical replicates under 100X magnification. * notes: If a bee died before the end of the end of the experiment, it is indicated as "bee died" in this column. These bees were not dissected. #### File: surv\_repro\_data.csv **Description:** The data table from lab experiments testing whether *Serratia* and core gut microbiome colonization affected bee survival and reproduction. ##### Variables * sample_name: The name of the sample. Bees are identified with a unique combination of letters and numbers. * source_colony: The ID of the commercial colony from which the worker was sourced. * microcolony_id: The ID of the microcolony in which the worker was placed. * core_treatment: The core microbiome treatment, indicated by "M+" for core microbiome transplant and "M-" for core microbiome depletion treatment. * core_inoc_id: The ID of the gut homogenate used to inoculate bees in the M+ treatment. "NA" indicates that bees were not inoculated with homogenate. * serratia_treatment: The *Serratia* treatment, with "S+" for *Serratia* inoculation and "S-" for *Serratia*-free control. * date_microcolony_established: The date when the microcolony was first established. * date_died: The date when the bee died. * day: The day of the experiment on which the bee died. * status: The final status of the bee, with "1" indicating that the bee was dead and "0" indicating that it was alive at the end of the experiment. * no_eggs: The total number of eggs present in the microcolony at the end of the experiment. * no_larvae: The total number of larvae present in the microcolony at the end of the experiment. * no_pupae: The total number of pupae present in the microcolony at the end of the experiment. * no_adult_males: The total number of adult males present in the microcolony at the end of the experiment. * no_bee_days: The total number of days all bees were alive in the microcolony. ## Code/software All analyses were conducted in R v4.1.1. Annotated R scripts are attached. ## Access information Other publicly accessible locations of the data: * Raw sequence reads are available in the European Nucleotide Archive (PRJEB79856).