Development of a test design for a semi-field, colony feeding study for the common eastern bumble bee (Bombus impatiens [Hymenoptera:Apidae])
Data files
Feb 05, 2025 version files 31.70 KB
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BB_Colony_Feeding.zip
20.35 KB
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README.md
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Abstract
Ecological risk assessment is a key component of the regulatory process required for registration of crop protection products around the world. The western honey bee (Apis mellifera) is the model organism for pesticide risk assessments for bees, but there are uncertainties over whether it is predictive of risks to other bees. Consequently, efforts are underway to develop test methodologies for other non-Apis bees. We conducted a semi-field colony feeding study with Bombus impatiens colonies to develop a colony-level methodology for bumble bees. We exposed commercially available bumble bee colonies to diets consisting of four concentration treatments of dimethoate insecticide (0.05, 0.19, 0.75, and 3.0 mg a.i./L) via supplemental sugar solution for six weeks and compared exposed colonies to untreated controls. Each treatment group had 10 replicate colonies, with one replicate per treatment group represented at each of the 10 study rural locations. We collected data on various colony-level endpoints including production of female reproductive (gyne) offspring, colony weight, foraging activity, and consumption of provisioned sugar solution. Our results indicated that the test design could be used to derive concentration-response relationships for several endpoints including the most sensitive, colony mass (No Observed Adverse Effect Concentration = 0.05 mg a.i./L). Overall, our study provides the foundation for a semi-field, colony feeding study test design for bumble bees, thus adding to the growing body of studies that may be used to assess the protectiveness of the honey bee risk assessment framework for non-Apis bees exposed to pesticides.
README: Development of a test design for a semi-field, colony feeding study for the common eastern bumble bee (Bombus impatiens [Hymenoptera:Apidae])
https://doi.org/10.5061/dryad.wwpzgmssv
We have submitted our raw data (cfs.arrival.csv, Bayer.CFS.basic.information.csv, bayer.cfs.cca.2022.csv, bayer.cfs.2020.foraging.csv), treatment groups (bayer.cfs.trts.csv), locations/sites coordinates (Bayer_CFS_sites.csv), codes for land use from the National Land Cover Dataset (NLCD.codes.csv), and R script (bayer.CFS.2020.R).
Descriptions
cfs.arrival.csv - Contains the observations of 80 bumble bee (Bombus impatiens) commercial colonies at the time of receipt in the field facility and from which 60 colonies were selected for the study. The variables recorded were:
- Hive.no: Assigned number from 1 to 80, to identify the colony.
- Date: Date of observations
- Hive.mass: The mass (weight) of the commercial colony including the bumble bee nest, brood, adults, and plastic foundation (box) containing the colony. Units are grams (g).
- Notes: Observations made by personnel.
- Excluded: X denotes colonies that were excluded from the study. Sixty study colonies were selected from 80 initially aquired. The excluded colonies were due to the presence of queen pupae, and by colony mass on both ends of the distribution tail.
- CCA.no: Colony Condition Assessment number. This assessment was done at arrival of the colonies to the field facility.
- Hive.dead: To note if any colony was dead or alive at the time of receipt at the field facility.
Bayer.CFS.basic.information – Lists the basic information regarding the experimental design including the location, site number, site name, position of the study colony within each site, treatment assignment, and hive number (identification assigned at time of arrival into the field facility).
- Location: Farm or location identified by name.
- Site.no: Site number. Identification of the site within locations. There were four sites within the Codling location, three sites within the Stoney location, and three sites within the Pullman location.
- Site: Site identified by a combination of the location name and a number, from 1-4 in the Codling location, from 1-3 in the Stoney and Pullman locations, respectively.
- Position: Position of the colony within a site. The colonies were placed in a linear manner within a site, separated by 10 m. The position of the colonies and treatments was randomly allocated.
- trt: Treatment. There were six treatment: unfed control, fed control, and dimethoate concentrations of 0.05, 0.19, 0.75 and 3.0 ppm via sucrose solution.
- Hive.no: Hive number. Identification made at time of arrival in the field facility.
bayer.cfs.cca.2022.csv - Contains the compiled data and observations at each Colony Condition Assessment (CCA). The variables recorded were:
- Sort: Position of the colony within one site (randomly designated). The position was maintained in the site, but was identified with new numbers as CCAs progressed, thus, for CCA 1, the positions were identified from 1-60, for CCA2, positions were identified from 61-120, and so on.
- Loc.sort: Location of the colony. Colonies were distributed within 10 sites, an dthese sites were clustered into three farms or locations.
- Date: Date of observation.
- CCA.no: Colony Condition Assessment number.
- Location: Farm or location identified by name.
- Site.no: Site number. Identification of the site within locations. There were four sites within the Codling location, three sites within the Stoney location, and three sites within the Pullman location.
- Site: Site identified by a combination of the location name and a number, from 1-4 in the Codling location, from 1-3 in the Stoney and Pullman locations, respectively.
- Position: Position of the colony within a site. The colonies were placed in a linear manner within a site, separated by 10 m. The position of the colonies and treatments was randomly allocated.
- trt: Treatment. There were six treatment: unfed control, fed control, and dimethoate concentrations of 0.05, 0.19, 0.75 and 3.0 ppm via sucrose solution.
- Gynes.total.09.11.2020: Number of gynes (new bumble bee queens) per surviving colony. During the last CCA the colonies were dissected, alloweing for the quantification of all adult new queens.
- Notes.field: Observations made in the field such as presence of other insects, or dead queens, colonies killed by bears, etc.
- Notes.09.11.2020: Observations made on the last day of the experiment on 9 September 2020.
- Dead queens from margin of the hive not collected 09.11.2020: Number of dead queens found at the margin of the hives that were not collected, but their presence was noted. Only two colonies had this scenario, one with one queen and the other with three queens.
- New.bag.no: Identification of the new bag containing the provisioned sucrose solution to each colony upon replacement in the field.
- New.nectar.g: Weight of the new bag containing fresh provisioned sucrose solution. Units are grams (g).
- Old.mass.g: Weight of the old bag containing provisioned sucrose solution upon replacement. Units are grams (g).
- Nectar.consumed: Amount of sucrose solution consumed weekly during the six weeks of exposure. Units are grams (g).
- nectar.notes.2022: Observations made in regard to not being able to measure the old bag weight due to ants chewing the bag (1 colony) and due to plexiglass in the hive being broken (1 colony).
- Hive.no: Hive number. Identification made at time of arrival in the field facility.
- Hive.mass.g: Mass (weight) of the colony including the nest, brood, adults, and plastic foundation (box) containing the colony. Units are grams (g).
- Hive.dead: Survival observation. Noted if the colony was dead.
- No.queens: Number of queens observed.
- Queen.pupae: Number of queen pupae observed. Queen pupae cells are notably bigger than worker/male pupae cells.
- Gynes.present: Presence or absence of gynes (new queens).
- Queen.dead: Foundress (mother) queen survival observation.
- Gyne.or.pupae: Noted the presence or absence of gynes or gyne pupae.
- Males.present: Noted the presence of males in the colony.
- Date.last.CCA: Date of CCA.
- Days.since.last.CCA: While the initial plan was to conduct CCAs in a weekly basis, due to scheduling and other factors, the intervals ranged from 7 to 13 days.
- hrs.since.last.CCA: Hours since last CCA. This was the results of multiplying the days since the last CCA by 24 hours/day.
- Notes.field.Q.verbatim: Informal records of observations made in the field in regard to the foundress queen or new queens.
- Notes.data.entry: Any notes that were taken into consideration for data entry.
- Gyne.count.exclude: Identification of colonies that were excluded from the analysis.
- Gynes.collected.09.11.2020: Number of gynes (new queens) collected at the end of the experiment.
- unopened.gyne.pupae.09.11.2020: Number of unopened gyne pupae.
bayer.cfs.2020.foraging.csv - Contains the compiled data and observations corresponding to the foraging activity assessment. The variables recorded were:
- Location: Farm or location identified by name.
- Site: Site identified by a combination of the location name and a number, from 1-4 in the Codling location, from 1-3 in the Stoney and Pullman locations, respectively.
- Position: Position of the colony within a site. The colonies were placed in a linear manner within a site, separated by 10 m. The position of the colonies and treatments was randomly allocated.
- Hive.no: Hive number. Identification made at time of arrival in the field facility.
- Treatment: Treatment. There were six treatment: unfed control, fed control, and dimethoate concentrations of 0.05, 0.19, 0.75 and 3.0 ppm via sucrose solution.
- Date: Date of observation for purposes of identifying observation events.
- Date.actual: Actual date of observation where bees were recorded entering and exiting their colonies. In some instances this happend -1 day of the date identified for the observation event.
- Start.time: Time of the day when the foraging observation intiated.
- 8am: 8:00
- Noon: 12:00
- Mins.to.noon: The number of minutes before or after 12:00pm.
- Mins.since.8am: The number of minutes after 8:00 am.
- Observer: Initials of personnel.
- Bee.in: Number of bees entering a colony within the 10 minute observation period allocated to each colony.
- Bee.out: Number of bees exiting a colony within the 10 minute observation period allocated to each colony.
- Ratio.in.out: Ration between the number of bees entering and the number of bees exiting a colony within the 10 minute observation period allocated to each colony.
- Bees.in.per.hr: Number of bees entering the colony per hour. This value was obtained by multiplying the number of Bee.in by 6.
- Exclude: Identified colonies excluded from analysis. The reasons are noted in column Notes.1.
- Notes.1: Observations made in regards to the colonies.
- Notes.2: Observation made in one Site (Codling 2).
bayer.cfs.trts.csv - Lists the treatments and a sorting number from 1-6 to enable the random allocation of colonies to positions within sites.
- trt: Treatment. There were six treatment: unfed control, fed control, and dimethoate concentrations of 0.05, 0.19, 0.75 and 3.0 ppm via sucrose solution.
- sort: Numbers 1 to 6, to enable random allocation of the study colonies to a position within a site.
Bayer_CFS_sites.csv - Identifies the site, locations, nearest town, latitude, and longitude of the sites.
- Site: Site identified by a combination of the location name and a number, from 1-4 in the Codling location, from 1-3 in the Stoney and Pullman locations, respectively.
- Location: Farm or location identified by name.
- Town: Nearest town to the farm (location).
- latitude: Coordinate.
- longitude: Coordinate.
NLCD.codes.csv - Contains the code and corresponding description of the land use according to National Land Cover Dataset.
- NLCD.code: Numerical code associated with a category of land use.
- NLCD.desc: Short and general description of the land use category.
- NLCD.desc.verbose: Additional details included in the general description of the land use category.
- NLCD.desc.verbose: Accounting for the 'Perennial' nature of the category Ice/snow.
- NLCD.full.desc: Detailed description of the land use category.
bayer.CFS.2020.R - Contains the R code to analyze the data. The script was created using version 4.3.1. Notes included for clarification of certain steps.
Methods
Test Material
We used pure PestanalTM dimethoate (Sigma Aldrich, St. Louis, MO; Lot # BCBS9338, purity ≥98%) as the test material.
Bumble Bee Colonies
On 16 June 2020, we obtained 80 Excel-type B. impatiens colonies from Koppert Biological Systems Inc. (Michigan, USA). Excel-type commercial colonies as described by the vendor contained a minimum of 70 worker-caste females and a queen nesting on a plastic foundation within a cardboard hive box. The plastic foundation has a hive entry of limited size that acts as a queen excluder, allowing the traffic of worker and drones, but not queens. Each hive was fitted a plexiglass observation lid and without any cover to the nest by request to the supplier. Bees were fed ad libitum with a sugar syrup solution and honey bee-collected pollen for one week. Upon receipt, the weight of each colony was measured. Sixty queenright study colonies were selected for field deployment by removing the most extreme by weight colonies, i.e., the lightest and heaviest colonies. A quick visual assessment of the brood area was also conducted and those that had queen pupae were removed from consideration.
Experimental Design
The colonies were moved to the field on 26 June 2020. Colonies were randomly assigned to treatment group and site. We conducted the study at 10 sites established along the margins of hayfields (organic-certified or managed without pesticides) on working dairy farms in central Vermont. The sites were clustered at three farms (hereafter, “locations”) that were separated by > 500 m. In May 2020, an area that was approximately 3 × 65 meters in size was mowed at each site in preparation for the relocation of colonies from the test facility to the field. Each mowed area was surrounded by a solar-powered electric fence for protection from bears. We performed maintenance on fencing weekly and mowed fence lines as necessary during the season. At each site, six colonies were deployed in one row with a 10 m spacing.
Each bumble bee colony was placed in an open-sided plastic box to prevent rain damage. Each hive was balanced on a stand in a second plastic box filled with 5 to 10 cm of water to prevent ants from entering the hive. Each hive was stabilized with a pair of grade stakes pounded into the ground.
Each of the six treatment groups, two controls (fed and unfed sucrose solution) and four dimethoate concentrations, were represented at each site by one experimental unit (colony). The “Fed” control colonies received 50% (w/v) supplemental sucrose solution whereas the “Unfed” control colonies did not receive supplemental sucrose solution. The four dimethoate treatment groups involved exposure to 0.05, 0.19, 0.75, or 3.0 mg dimethoate/L sucrose solution. In the dimethoate treatments, pure PestanalTM dimethoate powder was dissolved in measured volumes of 50% sucrose solution. One L of solution per week per colony was then provisioned to bees via supplemental nectar feeders. The feeding solutions were poured into the plastic bags, which acted as nectar feeders, with calibrated cylinders. The exposure period lasted six weeks starting on 2 July 2020 and ending 14 August 2020. This exposure period was selected to harmonize the test design to the honey bee colony feeding study design, and to represent a worst-case scenario exposure to an indeterminate blooming crop variety. Following the exposure period, colonies remained at the same locations for post-exposure assessments as described below.
During the field phase encompassing the exposure and post-exposure periods, colony condition assessments (hereafter “CCA”) were performed weekly starting at dusk after the forager bees had returned to the colony for the night. Headlamps provided enough light for the conduct of CCAs. Each hive was weighed (including the colony and the plastic hive insert box), and observations were made on queen survival and production of reproductive offspring (gyne pupae, new gynes, males). New gynes were differentiated from the foundress queen by the presence of intact wings, intact setae especially in the abdomen, and coloration (newly emerged queens have a grey complexion before sclerotization is complete). Presence of wax moths (adult and larva stages), ants, snails, beetles, wasps, mold, and bumble bees from a different species was recorded. At each CCA, the previous sugar solution provisions were removed and replaced with a fresh 1 L bag. We calculated the amount of solution consumed by each colony by subtracting the initial fresh mass of the bag from the mass of the bag remaining after one week in the field.
Foraging activity at each colony was determined on four dates during the exposure period. Hive entrances were observed for 10 minutes between the hours of 0900 to 1600, and the number of bees leaving and returning to the hive recorded. We also made notes on bee behavior and the presence of insects and other invertebrates in/on the colonies.
After the six-week exposure period, bees in the colonies continued to forage freely. Weekly CCAs continued until the experiment was terminated on 9 September 2020. At that time, approximately 75% of the surviving colonies had produced female reproductive offspring (hereafter “gynes,” those individuals who will found new colonies the following spring). Most other colonies had either died or were incapable of reproduction. After the field phase, we placed colonies in a freezer (≤-15 ºC) until dissection in the final CCA.
Statistical Analysis
Statistical analyses were conducted in the R statistical computing environment (Version 4.3.1) using tidyverse, DescTools, lme4, lmerTest, vegan, FedData, and other R libraries (Bates et al. 2015; Kuznetsova et al. 2017; Wickham et al. 2019, R Core Team 2021, Signorell 2023). To evaluate access of the colonies to floral resources across the study, we compared patterns of land use/land cover in a 1 km radius around sites. This radius is within the reported bumble bee foraging ranges of 250 m to 3000 m. The foraging range is affected by species differences in colony size and body size (Goulson 2010). Furthermore, workers from commercial B. impatiens colonies in lowbush blueberry fields foraged up to 400 m from the colony (Desjardins and De Oliveira, 2006). Using the R library, FedData (Bocinsky et al. 2023), the 2016 National Land Cover Dataset was downloaded for an area around the study site. The R library, ‘Raster’, was used to extract pixels within 1 km of each site. Proportional representations of 12 land cover classes at each site were then determined. We also explored land use patterns among sites using non-metric multi-dimensional scaling (NMDS) implemented with the R library, vegan.
Dimethoate treatment groups were compared to the Fed control for sucrose solution consumption, colony mass, numbers of gyne offspring produced, colony survival, and foraging activity. To determine the effect of supplementing hives with 50% sucrose solution, we compared responses between the two controls (Fed and Unfed). Foraging behavior was expressed as the number of bees returning to the hive per hour after normalizing from 10-minute counts. Data from one site were excluded because all six colonies were destroyed by bears between CCAs 2 and 3. In addition, dead colonies were excluded from CCA-specific analyses. For some response variables, such as colony mass, analyses were precluded at CCA 7 because the treatment colonies exposed to 0.75 and 3.0 mg/L dimethoate had poor survival. For final counts of gynes, we excluded colonies that were lost to bears, but included colonies that died before the end of the study. We excluded five hives from the analysis for gyne production because their plexiglass lids had failed, possibly affecting response variables. The Shapiro-Wilk Test was used to test for normality of residuals and Levene’s Test to test for homogeneity of variance among treatment groups. Dunnett’s multiple comparison procedure was used to determine the NOAEC and LOAEC for response variables that met the assumptions of parametric statistical tests. Otherwise, the non-parametric Kruskal-Wallis test was used.