DOI: 10.25338/b8c05s
title: Comparing bacterial microbiome composition of Xylocopa species across populations using PacBio 16S rRNA gene sequencing

description: The gut microbiota of bees affect nutrition, immunity, and host fitness,
 yet the role of diet, sociality, and geographic variation in determining
 microbiome structure, including variant-level diversity and relatedness,
 remain poorly understood. Here, we use full-length 16S rRNA amplicon
 sequencing to compare the crop and gut microbiomes of two incipiently
 social carpenter bee species, Xylocopa sonorina and Xylocopa tabaniformis,
 from multiple geographic sites within each species’ range. We found that
 Xylocopa species share a set of core taxa consisting of
 Bombilactobacillus, Bombiscardovia, and Lactobacillus, found in
 &gt;95% of all individual bees sampled, and Gilliamella and Apibacter
 were also detected in the gut of both species with high frequency. The
 crop bacterial community of X. sonorina was comprised nearly entirely of
 Apilactobacillus with occasionally abundant nectar bacteria. Despite
 sharing core taxa, Xylocopa species’ microbiomes were distinguished by
 multiple bacterial lineages, including species-specific variants of core
 taxa. The use of long-read amplicons revealed otherwise cryptic species
 and population-level differentiation in core microbiome members which was
 masked when a shorter fragment of the 16S rRNA (V4) was considered. Of the
 core taxa, Bombilactobacillus and Bombiscardovia exhibited differentiation
 in ASVs among bee populations, but this was lacking in Lactobacillus,
 suggesting that bacterial genera in the gut may be structured by different
 processes. We conclude that these Xylocopa species host a distinctive
 microbiome, similar to that of previously characterized social corbiculate
 apids, which suggests that further investigation to understand the
 evolution of bee microbiome and its drivers is warranted.
description: Between 2019 and 2020, 33 <em>X. sonorina</em> and 22
 <em>X. tabaniformis</em> adults were collected. Bees were
 captured in one of three ways: netted while foraging, caught using traps
 over the nest entrance (Supplementary Figure S1), or through the
 excavation of logs to sample entire nests. The type of capture was
 recorded, with bees caught foraging in flight denoted as ‘foraging’ and
 those captured within or exiting a log denoted as ‘nest-caught’. Samples
 were obtained from Davis, CA (21 <em>X. sonorina</em>; 14
 <em>X. tabaniformis</em>), Anza-Borrego Desert State Park in
 Southern California (1 <em>X. sonorina</em>, 8 <em>X.
 tabaniformis</em>), and Tempe, Arizona (11 <em>X.
 sonorina</em>). Samples from Davis and Anza-Borrego were captured
 during late summer and early fall while samples from Tempe were collected
 in early summer (see data file for collection date). Bee species can be
 distinguished morphologically, and identifications were verified using
 voucher specimens at the Bohart Museum of Entomology. Captured carpenter
 bees were photographed, then killed by placing them in a -20
 <sup>o</sup>C freezer where they were stored until
 dissection.  <strong>(B) Sample processing and
 DNA extraction</strong>: Before dissection, carpenter bee samples
 were rinsed in 70% ethanol for fifteen seconds. They were then air-dried
 and placed in a sterile petri plate for dissection. Dissecting tools were
 flame sterilized before dissection and before each organ removal. The crop
 and the rest of the gut (combined midgut and hindgut) were separated and
 stored at -20<sup>o</sup>C until DNA extraction.
 Microbial DNA was extracted from individual crop and gut samples
 separately and kit reagents only as a blank extraction control, using the
 Qiagen DNeasy PowerSoil Kit with slight modifications (Rubin et al. 2014).
 Modifications include adding 4 magnetic beads per PowerBead Tube after
 tissue samples had been added and beating tubes in a BeadBlaster 24
 Homogenizer for 3 cycles of speed 7 for 20 seconds per cycle. Then 60 μL
 of Solution C1 and 2 μL Proteinase K solution (600mAU/ML – from Qiagen
 Tissue and Blood) were then added to each tube and tubes were incubated
 overnight at 56<sup>o</sup>C. The following day tubes were
 beaten once more using the same cycle settings and the rest of the
 protocol followed the manufacturer’s protocol beginning at step 6.
 Extracted DNA was sent for library prep and sequencing at Dalhousie
 University Integrated Microbiome Resource Facility. Briefly, full-length
 16S rRNA region was amplified in duplicate using 27F AGRGTTYGATYMTGGCTCAG
 (Paliy et al. 2009) and 1492R RGYTACCTTGTTACGACTT (Lane 1991) using
 full-length fusion primers (PacBio adapters + barcodes + specific
 regions). PCR products were visualized using a Hamilton Nimbus Select
 robot using Coastal Genomics Analytical Gels. PCR products were pooled
 within a sample, cleaned and normalized using Charm Biotech Just-a-Plate
 96 Well Normalization kit and quantified fluorometrically before
 sequencing. PacBio Sequel 2 chemistry was used in sequencing, performed by
 the Dalhousie University Integrated Microbiome Resource facility (Halifax,
 Canada). <strong>(C)
 Bioinformatics</strong>: Preliminary processing and filtering of raw
 full-length 16S rRNA reads into Amplicon Sequence Variants (ASVs) was
 performed in R v4.1.0 (R Core Team, 2021) using DADA2 (v1.20.0) (Callahan
 et al. 2019). Primer sequences were removed, and reads were filtered by
 size and quality to yield sequences ranging from 1000 – 1600 bp with no
 ambiguous bases, 2 maximum expected errors, and a minimum quality score of
 3. Filtered reads were then dereplicated, and sequencing errors were
 inferred using the PacBioErrfun function and removed. Chimeras were
 inferred with a minFoldParentOverAbundance value of 3.5 and removed using
 sequence consensus as a method. Finally, taxonomy was assigned using the
 BEExact database (Daisley and Reid 2021) and SILVA v 138.1 (Quast et al.
 2012); resulting taxonomy was nearly identical so we present assignments
 from BEExact below. The ASV and taxonomy tables
 generated from the DADA2 pipeline outlined above were merged with metadata
 using phyloseq (McMurdie and Holmes 2013). ASVs classified as chloroplast
 or mitochondria were removed, any ASVs found in the control sample were
 removed, and only samples with greater than 500 total sequences, were
 retained (see sampling curves in Supplementary Figure S2). 
description: R, Dada2