Dataset DOI: 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

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.