DOI: 10.5061/dryad.x0k6djhv7
title: Species-rich old grasslands have beneficial effects on the health and gut microbiome of bumblebees

description: Recent studies have shown that old, traditionally managed semi-natural
 grasslands (SNGs) harbor specific plant assemblages characterized by high
 species richness, diversity, evenness, and a great abundance and diversity
 of floral resources. As nectar and pollen from many of these plants
 contain a multitude of favorable phytochemical compounds, we hypothesized
 that pollinator health and survival would improve in old, species-rich hay
 SNGs compared to younger SNGs. We monitored experimental colonies of
 bumblebees placed in hay SNGs of different ages. The survival of
 bumblebees increased with grassland age, thus they lived longer and
 produced more workers in the old SNGs. The abundance of actinomorphic
 flowers (with radial symmetry) increased with the age of the SNGs and was
 positively correlated with the body size, body mass, and lipid reserves of
 the bumblebees. The taxonomic composition of gut microbiota changed during
 the experiment, with a significant decrease of core taxa Bombiscardovia
 and Bifidobacterium in young SNGs, while the abundance of
 Bombilactobacillus increased in old SNGs. At the end of the experiment,
 the bumblebees of young SNGs hosted lower abundances of Gilliamella than
 those of old SNGs. In old SNGs, there was a positive relationship between
 floral richness and the abundance of six taxa, including the three core
 taxa Bombiscardovia, Bifidobacterium, and Snodgrassella.
 Microbiota-diversity decreased and microbiota-diversity increased over
 time in young SNGs, while both remained stable in old SNGs. Both
 deterministic and stochastic processes acting simultaneously in bumblebee
 microbiomes within young SNGs seemed to explain these changes. Compared to
 old forests, very little attention has been paid to old, species-rich
 grasslands. Considering their importance for pollinator health, as well as
 their high ecological and cultural values, it is crucial that the rare and
 endangered old, species-rich hay SNGs are protected. 
description: # Species-rich old grasslands have beneficial effects on the health and
 gut microbiome of bumblebees
 [https://doi.org/10.5061/dryad.x0k6djhv7](https://doi.org/10.5061/dryad.x0k6djhv7) ## Description of the data and file structure In this study, we monitored 16 experimental colonies of bumblebees (*Bombus terrestris*) placed in hay grasslands of different ages. We examined colony demographic parameters as well as survival, body size, body mass and lipid reserves of each individual bumblebee in relation to grassland age and floral characteristics. In addition, we sampled the gut microbiota of the bumblebees at the start and end of the experiment. ### Files and variables #### File: microbiome\_R1R2\_good\_demultiplexed\_derepl\_basicfilt\_cl\_agg.fasta **Description:** This fasta file corresponds to the bumblebee microbiome sequencing data after bioinformatic processing - i.e. paired-end assembly, sequence quality filtering, demultiplexing, dereplication and clustering. #### File: cr\_v2.docx **Description:** This information file summarises bioinformatic analyses performed and give summary statistics on the number of sequences kept at each step. microbiome R1, microbiome R2 and microbiome R3 correspond to the 3 PCR replicates carried out on each gut sample. #### File: Data on the number of flowers v2.xlsx **Description:** This file indicates the abundance of the different species of flowers (lines) recorded in 2 quadrates of each monitored grassland (columns) at different dates (sheets). Cells containing an 'X' indicate species detected in the cages but outside the quadrates. #### File: microbiome\_physeq.Rdata **Description:** Phyloseq object integrating an OTU file (OTU abundance in each sample), the sample data (sample metadata) and the Tax table (Taxonomic affiliation of each OTU). This phyloseq object was used for Alpha-diversity, Beta-diversity and Taxonomic composition analyses. Requires the Phyloseq package in R.  #### File: quality\_v2.zip **Description:** Folder containing the files and scripts used to curate the molecular artefacts after bioinformatic processing of the sequences using the R package metabaR. Subfolder "quality/in" contains the input files for this process. Subfolder "quality/analysis" contains the script (in html and Rmarkdown) of the analysis. Subfolder "quality/out" contains the final output files. See below for detailed content (folders in bold): analysis     microbiome_metabar.Rmd *= script of data curation using the R package metabaR in html format*     microbiome_metabar.html *= script of data curation using the R package metabaR in Rmarkdown format * in     microbiome.sampstat *= number of reads and motus in each sample replicate/control after dereplication, filtering and clustering*     ngsfilter*allv2.tab = ngsfilter file which gives the unique set of tag combination for each sample - **Variables** Column A: project name; Column B: sample id; Column C: unique tag combination for each sample; Column D: Forward primer; Column E: reverse primer; Column F: Sample metadata (see samples.tsv for detail)     samples.tsv* = samples description file -**Variables** id: sample id; type: sample type (sample, control, etc.); Controltype: empty column; Codeindividu; individual id number; Datedissection: date of dissection; Dateextraction: date of DNA extraction; Sexe_individu: sex of individual; *Observations: Comment*     tax_slv_ssu_138.1.txt *= silva v.138.1 taxonomy file*     results         ssu             exports                 microbiome2---ssu---otus.csv *= taxonomic annotation produced by SILVAngs*             stats                 sequence_cluster_map                     data                         microbiome2---ssu---sequence_cluster_map---microbiome_R1R2_good_demultiplexed_derepl_basicfilt_cl_agg.clstr *= clustering produced by SILVAngs (the minimum similarity was set to 100% in SILVAngs but as it uses a local alignement algorithm, sequences might be clustered together)* out     microbiome_mblist_clean_agg.rds *= final R object after curation using the metabaR package containing 3 dataframes: motus, pcrs, reads (see .tsv files below for detail)*     microbiome_motus.tsv *= taxonomic affiliation of each OTU using the silva v.138.1 taxonomy database*     microbiome_pcrs.tsv *= metadata of each sample after merging information from the 3 PCR replicates of the sample -  **Variables**: Column A: sample ID; experiment: project name; primer_fwd: forward primer; primer_rev; reverse primer; tag_fwd: forward tag; tag_rev: reverse tage; plate_no; PCR plate number of the 3 PCR replicates of the sample; plate_col; PCR plate column position; plate_row: PCR plate row position; id: id of the 3 PCR replicates of the sample; sample_id: sample id; type: sample type; control_type: control type; Code_individu: individual sample code; Date_dissection: date of bumblebee dissection; Date_extraction: date of DNA extraction; Sexe_individus: sex of bumblebee; Observations: comment; pos_tag_F.rows.: row position of forward tag on 386 plate; pos_tag_R.col.: column position of reverse tag on 386 plate; library: library name of the 3 PCR replicates; nb_reads: number of reads in the 3 PCR replicates; nb_motus: number of OTUs in the 3 PCR replicates;  low_contamination_level: samples showing low contamination levels (TRUE or FALSE); pcrbias: samples showing PCR bias; not_too_degraded: samples showing not too degraded sequences (TRUE or FALSE); seqdepth_ok: samples with sufficient sequencing depth (TRUE or FALSE); functional_pcr: sample for which PCR were functional (TRUE or FALSE); derepl.otus: number of OTUs after dereplication in each PCR replicate; derepl.reads: number of reads after dereplication in each PCR replicate; basicfilt.otus: number of OTUs after basic filtering in each PCR replicate; basicfilt.reads: number of reads after basic filtering in each PCR replicate;  clust97.otus: number of OTUs after clustering at 97% in each PCR replicate;  clust97.reads: number of reads after clustering at 97% in each PCR replicate; solitary_pcr: samples for which only one PCR worked; metabar.reads: number of reads after metabar curation in each PCR replicate; metabar.otus: number of OTUs after metabar curation in each PCR replicate.*     microbiome_reads.tsv *= numbers of reads for each MOTU (columns) in each sample (rows)* #### File: Bumblebee\_Microbiome\_Raw\_Sequencing\_data.zip **Description:** This compressed folder contains 6 compressed fastq files. These fastq files correspond to the raw pre-processed files after Illumina sequencing. The prefixes microbiome-R1, microbiome-R2 and microbiome-R3 correspond to the 3 PCR replicates of each sample performed in different libraries. The suffixes *R1.fastq *and *R2.fastq* correspond the 2 paired-end readings of each sequences. #### File: grassland\_bumblebee\_data.txt **Description:** File compiling data relating to grassland characteristics (first 11 variables) and to individual bumblebee characteristics (following 10 variables) ##### Variables * id_grassland: Grassland identification number * Grassland age: Grassland age in years * Age class: Grassland in class: &gt;50=Old, &lt;50=Young * Plant richness: Number of plant species recorded in each grassland * Total flower abundance: total flower abunance in each grassland * Shannon H': Flower species diversity (shannon index) per grassland * Pielou equitability: Flower species evenness (Pielou index) per grassland * Abundance of zygomorphic flowers: per grassland * Abundance of actinomorphic flowers : per grassland * Latitude: latitude of each grassland * Longitude: longitude of each grassland * Individual bumblebee: Bumblebee identification number * Femur length: Femur length of each bumblebee (mm) * Fat mass: lipid mass (mg) * id_No._colony: Bumblebee colony identification number * No. marked bumblebees at D0: number of marked bumblebee before the start of experiment - day 0 * No. marked bumblebees at D15: number of marked bumblebee at the end of experiment - day 15 * No. unmarked bumblebees at D15: number of newly emerged bumblebees at the end of experiment - day 15 * Weight (g) of the nest at D0: Total weight of colony (g) before the start of experiment - day 0 * Weight (g) of the nest at D15: Total weight of colony (g) at the end of experiment - day 15 * Total dry mass of unmarked bumblebees: Total dry mass of newly emerged bumblebees (g) #### File: Bumblebee\_Microbiome\_Data.txt **Description:** File containing the metadata for each gut microbiome sample including grassland, colony, bumblebee and microbiome characteristics. ##### Variables * id: gut sample id number * id_prairie: grassland id number * Code_individu: bumblebee id number * Date_dissection: date of gut sample dissection (ddmmyyyy) * Date_extraction: date of DNA extraction of gut sample * Sexe_individus: sex of bumblebee * Tarse: Tarsus length (mm) * Comment: * Type: bumblebee sampled before experiment (T0) or after experiment (T1) * id_num_ruchett: colony id number * nbre_ind_marq: number of marked individuals before the experiment * nbre_ind_non_marq: number non-marked individuals (newly emerged during the course of the experiment) * nbre_larves: number of larvae recorded at the end of the experiment * total_ind: Total number of individuals in the colony at the end of the experiment * reine: Presence of a queen in the colony at the end of the experiment * masse_ruchett_T0_ac_reine: Total mass of the hive including the queen at the start of the experiment (g) * masse_ruchett_T1_sans_reine: Total mass of the hive at the end of the experiment excluding the queen (g) * masse_reine: mass of the queen (g) * masse_ruchett_T1_ac_reine: Total mass of the hive at the end of the experiment including the queen (g) * masse_T1moinsT0: Difference in hive mass between the start and the end of the experiment (g) * masse_totale_individu: Total mass of individuals (g) * mesure_patte_moy: mean tarsus length of individuals in each colony (mm) * masse_seche_ac_lipides_moy: mean dry mass of lipids per individual in each colony (mg) * difference_moy: difference in mean * age: age of the grassland (years) * classe_age: age class of grassland (ancienne: old &gt;50 years; jeune: young &lt;50 years) * RichTot: Total number of  flower species recorded in grassland * AbondanceTot: Total abundance of flowers recorded in grassland * Hmoy_Shannon: Mean flower species diversity (Shannon index) in grassland  * Jmoy_EquitabilitePielou: Mean flower species evenness (Pielou index) in grassland * somme_abondance_zygomorphe_prairie: Abundance of zygomorphic flowers per grassland * somme_abondance_actinomorphe_prairie: Abundance of actinomorphic flowers per grassland * latitude: latitude of grassland * longitude: longitude of grassland * Observed: OTU (Operational Taxonomic Unit) richness of bacteria in gut sample * Chao1: Chao1 species richness index of bacteria in gut sample * se.chao1: Standard error of the Chao1 index * Shannon: OTU diversity (Shannon index) of bacteria in gut sample * InvSimpson: OTU diversity (Inverse Simpson index) of bacteria in gut sample * ExpShannon: Exponential of the Shannon index (see Shannon above) * Abundance: Total number of reads in gut sample * RareObserved: OTU richness of bacteria in gut sample after rarefaction to 1000 reads * RareChao1: Chao1 species richness index of bacteria in gut sample after rarefaction to 1000 reads * Rarese.chao1: Standard error of the Chao1 index after rarefaction to 1000 reads * RareShannon: OTU diversity (Shannon index) of bacteria in gut sample after rarefaction to 1000 reads * RareInvSimpson: OTU diversity (Inverse Simpson index) of bacteria in gut sample after rarefaction to 1000 reads * RareExpShannon: Exponential of the Shannon index (see Shannon above) after rarefaction to 1000 reads