The effects of dietary imidacloprid on bumblebee health in lowbush blueberry fields in Maine

The effects of dietary imidacloprid on bumblebee health in lowbush blueberry fields in Maine

Summary

Declines of native bumblebee species have been recorded for over six decades in Europe and, more recently, are also being reported in North America. Bumblebees are a temperate species and are well-adapted to Maine’s cool spring climate, making them an important pollinator for the May to June bloom of lowbush blueberry. Bumblebee pollination is especially imperative in contemporary times as we see honeybee colony losses and the rising costs of honeybee hive rentals causing a burden on Maine’s growers. Although there is currently little consensus about the exact cause of bumblebee losses, there has been recent interest in the use of systemic neonicotinoid pesticides in North America’s crop systems and possible sublethal effects on bumblebee colonies.

This project seeks to investigate such effects using managed colonies of Bombus impatiens by feeding them a range of field-realistic doses of the neonicotinoid imidacloprid and then following the colonies’ progress during the lab dosing period and then through the season as the colonies are placed out into blueberry fields. Colonies that consumed more imidacloprid during the dosing period were found to have fewer bees at the end of the season (P = 0.10), gain significantly less weight in during the dosing period (P <0.0004*), and have a significantly smaller brood area at the end of the season (P = 0.0132*). Index (consumed) dose was found to have a marginally significant effect on brood weight at the end of the season (P =0.073), although field management type had a significant effect where colonies placed in fields with low-medium input had higher brood mass as compared to the organic-low input fields (P =0.036*).

Finally, another component will be to measure the immunocompetence of individuals in the differentially dosed colonies to investigate if imidacloprid exposure affects the immune system. Levels of conopid fly parasitism will also be investigated to identify possible enhanced susceptibility of dosed colonies to parasites.

Objectives/Performance Targets

Objective 1. To dose colonies of commercial B. impatiens with a range of field-realistic doses of imidacloprid and track their progress through the season:

24 small (~30 individuals per colony) colonies were ordered from Koppert Biological Systems (Romulus, MI) and delivered to Maine on 15 May 2014. The colonies were divided into four groups of six and each group was given a range of imidacloprid treatments as added into their only food source (a bag of Koppert “Bee Happy” food). The doses ranged from the control (0 ppb) up to 125ppb of added imidacloprid in the form of AdmirePro®. The bees were allowed to feed on the dosed food ad libitum for two weeks in the lab and colonies and their food bags were weighed daily to monitor growth and track food consumption. Bees were kept at growth chamber conditions (15:9 LD, 20°C, 55% rH) during the dosing period.

After this two-week period, samples from each food bag and five individuals from each colony were collected and frozen at -20°C for chemical analysis to analyze actual dosage. Each group was placed into one of six blueberry fields around Waldo and Hancock Counties (Figure 1), the same sites where wild bumblebee collections took place. These fields had management practices that ranged from small and organic-low input (three fields) to low-medium input (three fields). Colonies were placed > 10m apart to mitigate bees switching between colonies and were weighed once a week to monitor their growth.

Colonies were picked up from the fields on 29 July and 30 July 2014 and placed into a 5.5°C cold room overnight. Final colony weights were taken and then colonies were moved to a -20°C freezer to freeze-kill. Final counts of workers, drones, and queens along with estimated of brood area were made in the following weeks. The intertegular widths of all individuals from each colony were measured to estimate average worker size of each colony.

Worker abdomens will be dissected to assess macroparasite presence or absence (conopid fly larvae) and their ages will be estimated using a four-point scale (0-3) based on wing wear and their intertegular spans will be measured as a proxy for individual size. Gut contents will be removed for examination under a phase contrast microscope and remaining body parts (head and thorax) will stored in the -80ºC freezer. 5 minutes will be spent on each slide of gut tissue to determine presence or absence of any pathogenic organism. Specimens will be considered to be positive if two or more pathogenic spores are seen of Nosema bombi.

Immune analysis will be performed this winter and spring to estimate immune strength of each colony.

Objective 2. To monitor levels of conopid fly parasitism in the commercially-raised dosed colonies as well as in wild caught native bumblebees collected from blueberry fields around the state:

From 14 July 2014 to 24 September 2014, the six field sites where the colonies were placed were visited approximately every two weeks in the Midcoast and Downeast regions of Maine (Figure 1). Two to three researchers spent either an hour or collected 20 wild bumblebees at each site (whichever came first) as a measure of sample effort. Obvious queens were not captured. Researchers split up at field sites to minimize the possibility that collected bumblebees were all from the same colony. Specimens were marked with the date, field site, and the common name of the flower on which they were collected (if known) then brought back to the lab and placed in a -20º C freezer to freeze-kill. Each bee will be identified to the species level and dissected using the method described above.

Objective 3. To establish an effective rearing method for the native bumblebee species B. ternarius in order to use true native colonies in similar experiments as in Objective 1.

Approximately 20 B. ternarius and B. vagans queens were captured during the month of May in blueberry fields in Downeast Maine and placed into plastic “rearing boxes” with a pollen ball and fed Koppert Bee Happy food ad libitum and kept in the growth chamber with the experimental Koppert colonies. Because no queens demonstrated nesting behavior and no eggs were laid within a 21 day period, all queens were released.

An attempt to use radio telemetry of captured and then released queens to locate nest sites for possible experimental use was not successful. One B. impatiens, one B. vagans, and two B. bimaculatus queens were fitted with 200 mg radio transmitters (Advanced Telemetry Systems, Isanti, MN) and released. We were able to locate three of the bees –all of which had died and were found on the ground.

Objective 4. To measure the immunocompetence of the imidacloprid-challenged bees using measures of the enzyme phenoloxidase (PO) in bees collected from each colony before dosing, during dosing, directly after dosing, and at regular intervals throughout the season.

This work will be done this winter and spring using the bee thoraces from the dissected bees, but I have prepared for this by learning the technique of how to measure PO levels using the frozen thorax of bumblebees.

• Figure 1. Locations of field sites of bumblebee collections and imidacloprid experiment in 2014, separated by management type

Accomplishments/Milestones

Due to the fact that feeding aversion was seen to occur wherein the colonies given the highest dose of imidacloprid were observed to eat significantly less (Figure 2), it was necessary to calculate an “index dose” of imidacloprid consumed per colony using the amount of food consumed and the dose to get a more accurate representation of the amount of imidacloprid consumed per colony (Figure 3). From this calculation, we replaced the treatment (ppb) with this index dose for each individual colony.

A significant effect of index dose on the weight difference of each colony from the start to the end of the two-week dosing period was observed (Figure 4).

Measurements of the ITS of workers of each colony taken at the end of the season showed no significant effect of either field management or index dose on final worker size. Numbers of bees (drones and queens included) counted at the end of the season in each colony also display a downward linear trend with increasing index dose with a nearly significant effect of index dose on bee number (P = 0.10) (Figure 5).

Index dose had a marginally significant effect (P = 0.073) on brood weight taken at the end of the season, but interestingly field management had a significant effect (P = 0.036*; Figure 6) where the low-med input fields produced colonies with more brood mass than the org-low input fields.

Calculated index dose was also found to have a significant effect on brood area at the end of the season (P = 0.0132; Figure 7).

Objectives that have not yet been completed include dissections on the dosed bees to investigate possible differential susceptibility to parasites and pathogens and the immune analysis on dosed bees.

The project did progress as expected on the timeline, although weather became an issue when several severe windstorms managed to overturn colonies resulting in loss of mass. Ants were also a problem at a couple of fields, which most likely resulted in smaller colonies not due to the dosing itself. We also discovered honeybees in the boxes at one site indicating there was possibly an antagonistic relationship between the bumblebees and the honeybees left behind in the blueberry field once the hives were picked up for the season. Finally, one of the objectives was to count number of queens produced by each colony by the end of the season but there were very few queens found in the colonies. The colonies were in the field with the queen excluders until late July meaning the colonies have been approximately 18 weeks old when picked up. Because this was enough time for the queens to have been produced and impatiens drones were observed at the field sites, it would appear that the queen excluders did not and produced queens were able to disperse from the colonies.

• Figure 3. Calculated “index dose” based on initial imidacloprid concentration in the food and food consumption over the two week dosing period.
• Figure 4. The significant effect of index dose on the weight change of each colony in the lab (P < 0.0004*).
• Figure 5. The releationship between index dose and number of bees in each colony at the end of the season. There was no significant effect of management type on the number of bees (P = 0.43), but there was a near significant effect of index dose (P = 0.10).
• Figure 6. The effect of index dose (P = 0.073) and field management type (P = 0.036*) on brood mass at the end of the season.
• Figure 7. The effect of index dose (P = 0.0132) on brood area at the end of the season. Field management type was not found to be significant (P = 0.2618), although colonies in low-med input fields had larger average brood area.
• Figure 2. Average amount of food consumed by colonies during the dosing period. There was a significant effect of treatment (ppb) on the amount of food consumed by each colony (P < 0.0001*).

Impacts and Contributions/Outcomes

I expect that this project will be a unique contribution to the study of the sublethal effects of neonicotonoids on bumblebees. Currently, similar work has been done but in a lab setting only and using the European managed bumblebee, B. terrestris. Therefore, this work will be the first using North American bee species that not only follows the colonies through the lab-dosing period, but also their progress throughout the season when the dosing period ends and the colonies are placed out in field sites. This will be key in determining susceptibility of exposed bees to parasites in pathogens in a natural setting.

It is expected that these findings will generate a publication or publications in a peer-reviewed journal for the focus on North American bumblebees. Although predicting possible effects on agricultural management practices is difficult, the results of this research will be informative in determining sublethal effects of imidacloprid on bumblebees, which will be useful in the future for making suggestions for best growing strategies in the lowbush blueberry system. Beyond this, the results will help shape future investigations into the effects of neonicotinoids on native bees and potentially lend clues as to why some bumblebee species are becoming more prevalent while others are in decline.

In addition, my cooperation with blueberry growers in Maine will enable me to not only relate my research findings to them directly, but also through an annual presentation at the Maine Wild Blueberry Field Day at the University of Maine’s research farm in Jonesboro, ME and an annual report submitted to the University on research in blueberry fields. As I am also on Specialty Crop Research Initiative (SCRI) grant, I will continue to work toward the goals outlined there in which I contribute to a “Pollinator Toolbox” to aid stakeholder growers in assessing pollinator efficacy and monitor the native bee populations in their fields.

Electronic access to information about the SCRI pollinator security project will be available to stakeholders, as well as information specific to my project in the form of a video interview (attached).

The preliminary results of this project were presented at the Annual Meeting of the Entomological Society of America on November 17 2014 in Portland, OR.

• Research Topics in Lowbush Blueberry Pollination – eXtension.org/bee_health

Collaborators: