DOI: 10.5061/dryad.d51c5b0h8
title: Heather nectar extracts reduce within-colony epidemics of the bumblebee parasite <em>Crithidia bombi</em>

description: Disease and habitat loss are significant causes of pollinator declines,
 and evidence indicates the two are linked. In Britain, lowland heath is an
 important bee habitat, but over 80% has been lost since the 1800s. This
 deprives wild bees of a rich food resource, but also the health benefits
 of an antimicrobial metabolite from Calluna vulgaris nectar, callunene,
 which reduces infection by the gut parasite Crithidia bombi in individual
 bumblebees. Here, we test whether these individual impacts can control
 epidemics in social colonies. We inoculated workers in early-stage Bombus
 terrestris colonies with C. bombi, and randomly assigned colonies to the
 following treatments: a) heather extract, b) callunene and c) a
 sugar-water control. Colony infection rates were measured weekly. Colonies
 that were exposed to heather-metabolites or callunene had lower prevalence
 of C. bombi, especially the flagellated, infectious form, when compared to
 controls. Consequently, we conclude that these antimicrobial metabolites
 can control epidemics in social bumblebees. This suggests that lowland
 heaths may act, not just as an important food source, but as an essential
 natural pharmacy for pollinators.
description: # Heather nectar extracts reduce within-colony epidemics of the bumblebee
 parasite *Crithidia bombi* Dataset DOI:
 [10.5061/dryad.d51c5b0h8](https://doi.org/10.5061/dryad.d51c5b0h8) ##
 Description of the data and file structure This experiment tested the
 effectiveness of diets containing heather nectar extract (referred to as
 "heather extract") and a semi-purified solution containing
 callunene and related compounds (referred to as "callunene") on
 the spread of a gut parasite (*Crithidia bombi*) in bumblebee (*Bombus
 terrestris*) colonies. The treatment diets were prepared through a
 chemical extraction of heather honey using methanol as a solvent to
 produce heather extract, and a semi-purified callunene was produced by
 fractioning the heather extract using LC-MS and separating fractions
 containing callunene and a related compound called callunol. In the
 bumblebee experiment, colonies of Bombus terrestris were reared to 10-15
 workers in size in the laboratory and inoculated with *Crithidia bombi* by
 feeding 10 workers sugar water mixed with 25,000 *Crithidia bombi* cells.
 The colonies were randomly assigned to heather extract, callunene, or
 sugar water control as diet, and infection rates were screened weekly for
 10 weeks by examining the faeces of 10 worker bees under a phase-contrast
 microscope for Crithidia cells. We also counted the number of
 reproductives produced by the colonies (males or queens - in this case, it
 was just males), and recorded the survival rate of the colony queens in
 each treatment group. Males, which were frozen after removal from
 colonies, were later dissected and gut contents examined for infection
 with *Crithidia bombi*.  ### Files and variables #### File:
 Worker_screening_data.csv **Description:** This contains the results of
 presence/absence faecal screening of worker bumblebees in each colony,
 with each line being the results of a weekly screening of 10 worker bees
 (if this many were available) from a given, numbered colony. There are 27
 colonies in total, 9 in each treatment. **Variables** * **Treatment:**
 colonies assigned at random to one of 3 treatments: **A Control** (sugar
 water diet), **Heather** (crude heather extract mixed with sugar water)
 and **Callunene** (semi-purified fraction of heather extract containing
 callunene and callunol) * **Colony:** numbers were assigned to queens when
 captured, and for those that successfully founded colonies the same number
 was used to refer to the colony * **Capture date:** day (DD/MM/YYYY) the
 queen was collected by net in the winter heather garden of Windsor Great
 Park * **10-worker date:** the date (DD/MM/YYYY) at which a colony was
 first observed to have 10 or more workers, the minimum number for
 inclusion in an experiment * **Time to 10-workers:** number of days
 between when a queen was captured and when her colony reached 10 workers
 in size * **Time to 10-workers rescaled:** Time to 10-workers divided by
 10 (rescale for inclusion in models) * **Capture date (days since 1st
 February):** capture date expressed as days after the 1st February *
 **Initial population:** number of workers in the colony on the day it was
 experimentally inoculated (10 workers fed 25000 *Crithidia bombi* cells in
 sugar water) * **Screen date:** date (DD/MM/YYYY) of weekly screening of
 10 workers'  faeces for Crithidia. This began 2 weeks after
 inoculation and 1 week after commencing treatment diets. Screening 10
 workers was not always possible if there was fewer than 10 unmarked (i.e.,
 not inoculated) bees in the colony, or because some were unwilling to
 defecate. * **Population:** number of workers on the given screen date
 (counted by visually eyeballing workers in real time for smaller colonies,
 for large colonies by taking a photo and counting from that - we note that
 this was not a perfect method and in comparison to exact counts at colony
 termination may have underestimated true populations by up to 20%) * **Max
 population:** highest weekly population count for the colony * **Week:**
 week of data collection, with week 1 being 2 weeks after inoculation and 1
 week after commencing treatment diets * **No. inoculated:** number of bees
 out of the 10 we attempted to inoculate with *Crithidia bombi* that
 actually drank the droplet of infected faeces mixed with sugar:water we
 gave them. A fractional number (e.g., 9.5) means nine drank the full
 droplet, and the tenth bee drank only part of it (we didn't subdivide
 any further than 0.5) * **No. recovered:** number of living marked bees we
 were able to remove from the colony a week after inoculation. Reasons for
 this being less than 10 could include deaths, escapes, or the mark flaking
 off the bee's back. We cannot rule out the possibility that some
 bees' marks did get erased, although the fact that we could still see
 marks on bees even after 10 weeks suggests they were, in general, quite
 robust to wear and tear. * **No. defecated:** number of recovered bees
 that we could extract faeces from. * **No. infected:** number of bees that
 defecated that had live *Crithidia bombi* cells in their faeces *
 **Initial infection %:** percent of recovered marked bees that defecated
 that had *Crithidia bombi* in faeces * **Inoculation score:** % initial
 infection x Number of bees inoculated * **Flagellated Crithidia:** number
 of workers in weekly screenings that had at least one flagellated
 *Crithidia bombi* cell in their faeces when observing a line down the
 length of a coverslip under the microscope * **Unflagellated Crithidia:**
 number of workers in weekly screenings that had no flagellated *Crithidia
 bombi* but at least one apparantly alive amastigote stage *Crithidia
 bombi* cell in faeces. This designation was used for faecal samples in
 which most of the Crithidia cells appeared alive. * **Unflagellated or
 dead Crithidia:** number of workers in weekly screenings that had no
 flagellated *Crithidia bombi*, at least one apparently alive amastigote
 *Crithidia bombi* cell; however, the live cells were outnumbered by dead
 ones. * **Dead Crithidia:** number of workers in weekly screenings in
 which all *Crithidia bombi* cells in faeces appeared to be dead, as
 evidenced by signs of decay, rupturing, and/or motionless flagella *
 **Clean:** number of workers in weekly screenings in which no *Crithidia
 bombi* cells of any description were seen in faeces * **Total:** total
 number of workers screened from a colony in a particular week (usually 10,
 unless fewer than this were available) **File:
 Worker_screening_summary.csv** **Description:** This contains the overall
 proportion of workers screened in each colony that were infected with
 flagellated *Crithidia bombi* and with any live *Crithidia bombi*
 (combining flagellated, unflagellated and unflagellated or dead
 categories). **Variables** * **Colony:** numbers were assigned to queens
 when captured, and for those that successfully founded colonies the same
 number was used to refer to the colony * **Treatment:** colonies assigned
 at random to one of 3, A Control (sugar water diet), Heather (crude
 heather extract mixed with sugar water diet) and Callunene (semi-purified
 fraction of heather extract containing callunene and callunol mixed with
 sugar water diet) * **All live Crithidia:** percentage of screened workers
 in the colony (pooled across all screening dates) that had any live
 *Crithidia bombi* in their faeces (combining the categories of
 'Flagellated Crithidia', 'Unflagellated Crithidia',
 and 'Unflagellated or dead Crithidia' from previous file) *
 **Flagellated Crithidia:** percentage of screened workers in the colony
 (pooled across all screening dates) that had flagellated *Crithidia bombi*
 in their faeces **File: Male_production.csv** **Description:** This file
 lists the number of males produced by each colony in the experiment,
 sorted by treatment group. **Variables** * **Colony:** numbers assigned to
 queens when captured, and for those that successfully founded colonies,
 the same number was used to refer to the colony * **Treatment:** colonies
 assigned at random to one of 3, A Control (sugar water diet), Heather
 (crude heather extract mixed with sugar water), and Callunene
 (semi-purified fraction of heather extract containing callunene and
 callunol) * **Males:** total number of males produced by a given colony
 **File: Male_gut_dissections.csv** **Description:** This file contains the
 results of screening dissected midguts from males removed from
 experimental colonies for *Crithidia bombi*, sorted by colony, treatment,
 and date removed from the colony. Infection intensity was measured for
 samples positively identified as being infected with *Crithidia bombi*. 
 **Variables** * **Treatment:** colonies assigned at random to one of 3, A
 Control (sugar water diet), Heather (crude heather extract mixed with
 sugar water) and Callunene (semi-purified fraction of heather extract
 containing callunene and callunol) * **Date:** day (DD/MM/YYYY) on which
 the male was removed from its colony and frozen * **Colony:** numbers
 assigned to queens when captured, and for those that successfully founded
 colonies the same number was used to refer to the colony * **Status:**
 result of screening gut contents under a phase contrast microscope,
 Clean=no Crithidia seen, Dead Crithidia=dead Crithidia cells seen,
 Crithidia (unflagellated or dead)=more than half the Crithidia cells dead,
 those alive did not possess flagella, Crithidia (unflagellated) = live
 Crithidia cell(s) without flagella, Crithidia=at least one live Crithidia
 cell with flagellum * **Count (cells/µl):** number of Crithidia cells per
 microlitre measured using a Neubaer improved haemocytometer. This was only
 done with males that had Crithidia in faeces; consequently, most cells in
 this column are blank **File: Queen_survival.csv**  This file contains the
 date of death for the queen of each colony, with a blank cell indicating
 she survived until the end of the experiment (day 70). The
 'Span' is calculated by the number of days between the start
 date and the end date.  **Variables** * **Treatment:** colonies assigned
 at random to one of 3, A Control (sugar water diet), Heather (crude
 heather extract mixed with sugar water) and Callunene (semi-purified
 fraction of heather extract containing callunene and callunol) *
 **Colony:** numbers assigned to queens when captured, and for those that
 successfully founded colonies the same number was used to refer to the
 colony * **Start date:** date (DD/MM/YYYY) on which treatment diet was
 applied to the colony * **Queen death date (blank if she survived the
 entire 10 weeks):** date (DD/MM/YYYY) at which the queen of an
 experimental colony was found to have died. A blank cell indicates the
 queen survived until the colony was terminated 70 days after commencing
 treatment * **Span**: number of days from the start date until either the
 queen's death or termination of the colony * **Event:** 1=queen death
 occurred at the end of the period indicated in "Span", 0=queen
 remained alive for the full 70 days. #### File:
 LCMS_data_for_quantification_3.7z **Description:** **LC–MS Data
 Description** This dataset was generated using a **Thermo Fisher
 Scientific Orbitrap Exploris 480 Mass Spectrometer** coupled to a liquid
 chromatography (LC) system. Raw data files were collected in Thermo
 **.raw** format, using Thermo Xcalibur™ Software. The .sld file is a
 sequence list document file used by Thermo Scientific Xcalibur for batch
 data acquisition. It specifies the sample order, file names, vial
 positions, and the instrument method used during LC–MS/MS runs. Load the
 .sld file in Xcalibur’s Sequence view to reproduce the sample acquisition
 settings. GPUCache is an automatically generated cache directory created
 by Xcalibur to store temporary GPU-accelerated rendering data. It does not
 contain experimental results and is not required for data processing or
 reproducibility. It can be safely deleted and is not included as part of
 the dataset.  **Technical Terms and Definitions** | **Term** |
 **Definition** | | :-------------------------------- |
 :------------------------------------------------------------------------------------------------------------------------------------------------- | | **LC–MS** | Liquid Chromatography–Mass Spectrometry. Analytical technique combining chromatographic separation with mass-based detection of analytes. | | **Orbitrap Exploris 480** | A high-resolution, accurate-mass Orbitrap-based mass spectrometer by Thermo Fisher Scientific, designed for small-molecule and proteomic analysis. | | **.raw file** | Proprietary Thermo Fisher binary file containing all LC–MS acquisition data, including spectra, chromatograms, and metadata. | | **Molecular Ion** | The ion corresponding to the intact molecule with minimal fragmentation, often used to confirm compound identity. | | **MS¹ (Full Scan)** | Primary scan detecting all ions within a defined m/z range, used to identify precursor ions (e.g., callunene or callunol). | | **MS² (Tandem MS / MS/MS)** | Secondary scan where a selected ion is fragmented to yield structural information (fragment ions). | | **m/z (Mass-to-Charge Ratio)** | Ratio of the mass (m) of an ion to its charge (z), the primary measurable quantity in mass spectrometry. | | **Retention Time (RT)** | The elution time of a compound from the LC column, used to distinguish between analytes of similar m/z. | | **Resolution** | Ability of the mass spectrometer to differentiate between ions with close m/z values. | | **Electrospray Ionization (ESI)** | A soft ionization technique that produces ions from liquid samples under atmospheric pressure for MS analysis. | ## Code/software #### File: R_code_for_bumblebee_colony_Crithidia_experiment.R **Description:** R code (we used version 4.3.0) used to perform statistical tests on the data contained in the **Worker_screening_data.csv, Male_production.csv** and **Queen_survival.csv** files. R outputs of statistical tests are included. Plain English notes on the test being performed and reasons for them are given on lines following a hash (#) which are integrated into the lines of code.