Supplementary code and data for: Symbiotic bacteria and fungi proliferate during diapause and may enhance overwintering survival in a solitary bee

Host-microbe interactions underlie the development and fitness of many macroorganisms, including bees. Whereas many social bees benefit from vertically transmitted gut bacteria, current data suggests that solitary bees, which comprise the vast majority of species diversity within bees, lack a specialized gut microbiome. Here we examine the composition and abundance of bacteria and fungi throughout the complete life cycle of the ground-nesting solitary bee Anthophora bomboides standfordiana. In contrast to expectations, immature bee stages maintain a distinct core microbiome consisting of Actinobacterial genera (Streptomyces, Nocardiodes) and the fungus Moniliella spathulata. Dormant (diapausing) larval bees hosted the most abundant and distinctive bacteria and fungi, attaining 33 and 52 times their initial copy number, respectively. We tested two adaptive hypotheses regarding microbial functions for diapausing bees. First, using isolated bacteria and fungi, we found that Streptomyces from brood cells inhibited the growth of multiple pathogenic filamentous fungi, suggesting a role in pathogen protection during overwintering, when bees face high pathogen pressure. Second, sugar alcohol composition changed in tandem with major changes in fungal abundance, suggesting links with bee cold tolerance or overwintering biology. We find that Anthophora bomboides hosts a conserved core microbiome that may provide key fitness advantages through larval development and diapause, which raises the question of how this microbiome is maintained and faithfully transmitted between generations. Our results suggest that focus on microbiomes of mature or active insect developmental stages may overlook stage-specific symbionts and microbial fitness contributions during host dormancy.

Methods
This dataset includes data and code for the following: amplicon sequencing (1A), qPCR (1B), plate inhibition (2), and sugar/sugar alcohol analysis (3). Software and major package versions: R (4.1.1), DADA2 (1.22.0), phyloseq (1.38.0), vegan (2.6.4), microbiome (1.23.1), and ggplot2 (3.4.2). For furhter method details see manuscript.

Amplicon data and qPCR are from stages throughout the lifecycle of a solitary bee, as well as environmental samples. All samples were added whole to DNA extraction, following preprocessing. Extraction for all samples was done per manufacturer’s instructions with the DNeasy PowerSoil Pro kit. Four blanks were included in DNA extractions. Extracted DNA was stored in the included extraction buffer at -80oC for amplicon sequencing and qPCR.

 Amplicon sequencing of extracted DNA was done to assess bacterial and fungal community composition using the 16S rRNA (V5/6) gene and ITS gene at the Integrated Microbiome Resource (IMR) at Dalhousie University in Halifax, Nova Scotia. Phusion Plus high-fidelity polymerase was used with fusion primers, which include the sequences below with Illumina adaptors + indices for multiplexing; sequencing was then performed on Illumina MiSeq. Samples were de-multiplexed at IMR. For bacteria, primers 799F/1115R amplifying V5/V6 region of the 16S gene were used to limit mitochondria and chloroplast amplification (799F= 5'-AACMGGATTAGATACCCKG-3'/ 1115R= 5'-AGGGTTGCGCTCGTTG-3'). These primers amplify a ~300bp length target sequence. For fungi, primers ITS1F/ITS2 were used (ITS1F= 5'-CTTGGTCATTTAGAGGAAGTAA-3'/ ITS2=5'-GCTGCGTTCTTCATCGATGC-3'). These primers amplify the variable length ITS1/2 region.

qPCR: Bacterial copy number was quantified with standard DNA intercalating dye (SYBR) based qPCR. The same extracted samples that were sent for amplicon sequencing were run through this procedure. Identical primers (799F= 5'-AACMGGATTAGATACCCKG-3' /1115R= 5'-AGGGTTGCGCTCGTTG-3') were used so that compositional and quantification could be directly compared and merged. A 1:10 dilution of extracted DNA was determined after dilution testing was done with a representative subset of samples; 1:10 dilution gave in-range Cq values. Master mix, per reaction, was composed of 5ul SSO Advanced Universal SYBR Supermix (Catalog# 1725271), 0.3ul of each primer (10uM), 3.4ul Molecular grade water, and 1ul of extracted DNA (diluted 1:10 in Molecular grade water). Reactions were performed in triplicate for each sample, and arranged semi-randomly across plates to avoid possible correlations of plate and developmental stage. Blanks and standards were included in each plate, and a Cq cutoff for blanks was established at 31. Fungal quantification was done with FungiQuant, using the 18S rRNA gene primers FungiQuant-F= 5′-GGRAAACTCACCAGGTCCAG-3′ and FungiQuant-R = 5′-GSWCTATCCCCAKCACGA-3′, along with the fluorescent probe FungiQuant-Prb = (6FAM) 5′-TGGTGCATGGCCGTT-3′ (MGBNFQ). As with bacteria, dilution testing of samples was done to bring Cq values into the optimal range, and a 1:20 dilution was picked. Master mix, per reaction, was composed of 5ul PCR Biosystems qPCRBIO Probe Mix (No-ROX) (Catalog# 17-512B), 0.3ul of each primer (10uM), 0.3ul fluorescent probe (10uM), 3.1ul molecular grade water, and 1ul of extracted DNA (diluted 1:20 in molecular grade water). Reactions were performed in triplicate for each sample, and arranged semi-randomly across plates to avoid possible correlations of plate and developmental stage. Blanks and standards were included in each plate.

Plate Inhibition: For all inhibition trials we used TSA media without any antimicrobials. For consistency, we used a template to mark the underside of all of the plates, it included in the center a cross “+” then two parallel lines, 30mm long and each 20mm from the center point. These served as guides for inoculations. Streptomyces strains were inoculated with 1ul hoops from stock plates of the TSA without antifungals onto the 30mm parallel lines. Five replicate plates were made for each comparison (25 per trial, including 5 control plates). These were allowed to grow for 10 days, then, from stock plates of each fungus (also TSA, no antimicrobials), plugs were inserted into the center “+” of each plate. Care was taken to ensure that plugs were all taken from just inside the leading edge of the fungal hyphae on the stock plates. These were allowed to grow for seven days. Measurements were taken on the backs of the plate, and measured the distance from the leading edge of the growing fungi to the center “+”, directly perpendicular to the parallel lines, on both sides. A tabletop light pad was used for imaging to qualitatively assess the density of the fungal hyphae, ensuring even back-lighting for the plates. Radius measurements (two per plate, each side of the ‘+’) were averaged for each replicate plate.

Sugar/ Sugar Alcohol Profiles: Samples of whole larvae, prepupae and pupae, as well as one pollen provision from a 4th instar larva were extracted for sugar and sugar alcohol analysis. Whole samples were placed in tubes with metal beads and 1mL of 100% ethanol and run on a bead beater for 8 minutes at full speed with 20s breaks every minute. These were then centrifuged for 30 seconds at 10k rcf. For each sample, the top 700ul of ethanol was moved to a new tube, 700ul 100% hexane was added, and then vortexed for 30 seconds. To this, 100ul MilliQ water was added, and vortexed for another 30 seconds. Once hexane had separated from the aqueous phase, it was removed (800ul). The remaining 1mL of aqueous phase was centrifuged for 2 minutes at 16k rcf, and the bottom 500ul was filtered through a 0.2 micron syringe filter and placed in a new tube in a lyophilizer for 6 hours, without heat. The dried samples were kept in a -20C freezer until analysis, at which time they were re-suspended in 300ul 1:1 water: acetonitrile. Standards of erythritol, sorbitol, fructose, glucose, sucrose, xylose and maltose were made at 0.5 mg/mL, standards of glycerol and trehalose were made at 5mg/mL and 1mg/mL respectively, all in 1:1 water: acetonitrile. Separation of sugars was performed on Thermo UltiMate 3000 HPLC system according to the Waters Application Note: WA60110, except for the following: column was Phenomenex Luna Omega 3um SUGAR (50x2.1mm, Part#: 00B-4775-AN), and flow rate was 0.2mL/min; detection was by CAD (Corona Veo; Dionex). Each sample was run twice, standards were run 2-5 times. Analysis of peaks was performed with Thermo Fisher Chromeleon software. Peak identities were assigned based on retention times of standards, and unassigned peaks were then named by their retention times. Peak area was calculated by the software.