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, placing them in one of six field sites in Midcoast Maine, and then taking measurements that serve as proxies for colony strength: the number of adults left in the colony at the end of the season; the area of the brood clump left in the colony; the weight of the brood clump; proportional weight change while out in the field; and average worker size. In 2015, I compared these measurements to field components that included: field region; field management (organic – low input and low – medium input); bumblebee species richness; flower species richness; and the cumulative conopid incidence in each field measured by dissecting wild caught bumblebees.

I found differences in bumblebee and flower diversity between the study fields and included these measures as field components that may affect the experimental Kopperty colonies placed in those fields. The cumulative incidence of conopid infected bees over the season (from mid July through September) across all fields was 15.8%, although there was a peak observed in mid-late August, after the Koppert colonies had been removed from the fields on 7/29/14. In late July, cumulative incidence across all fields was only 10.4%.

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/conventional 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 region 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 was 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 2014 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 has not yet been completed, but I have prepared for this by learning the technique of how to measure PO levels using the frozen thorax of bumblebees. We have also compared the PO levels of different species of bumblebees (Koppert B. impatiens, B. ternarius, and B. vagans) and this data can be used in future comparisons.

• Objective/Performance Targets Figure

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, 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 2). From this calculation, we replaced the treatment (ppb) with this index dose for each individual colony and these numbers were used in analyses of the Koppert data.

In 2014 from 7/14/14 to 9/22/14, 304 wild bumblebees were collected from the six fields and all were identified to species and dissected to look for conopid larvae. 48 (15.8%) of those bees were found to contain a conopid larva. Using the wing wear as a proxy for age on an assigned 0-3 scale, we found that older bees were significantly more likely to contain a conopid larva (Wald’s X² = 11.51, df = 1, P = 0.0008**) (Figure 3). However, it should be noted that conopid infection may cause a bee to act “clumsily”, which may also result in excessive wing wear. There was no significant relationship between conopid infection and size (ITS) (Wald’s X² = 0.77, df = 1, P = 0.38).

There was also a difference in wild bumblebee diversity between fields. For instance, only three unique bumblebee species were caught at Bucksport low-organic input field in Region 3, but seven unique species were captured in Stockton Springs 2 low-organic in Region 1. A Shannon’s Diversity Index for each field was performed (Table 1).

Plant diversity was also calculated at each field by using the recorded plants the bees were foraging on when captured (Table 2).

Four field characteristics were used as predictors of macroparasite (conopid) rates at each field. Bumblebee species richness + diversity and Flower species richness + diversity were both found to be highly correlated (bumblebee: 0.8326, plant: 0.8780), and so only richness was used in analysis.

• • Bumblebee species richness (total number of species found at each field)

• • Region (1, 2, or 3)

• • Field management

• • Flower species richness

Stepwise linear regression was used to select models to examine relationships between the predictor variables and macroparasite rates. We used a mixed-procedure with the probability to leave and enter at a = 0.250. We found that there was a significant difference between regions, with region 3 having a significantly lower rate of macroparasite infection as compared to regions 1 and 2 (Figure 4).

Cumulative incidence through the 2014 season for each field (Table 3).

Koppert Boxes:

I used the data from the wild caught bees for field variables and then used these in analyses to ascertain if any of these variables might have had effects on the Koppert boxes. Here, we look for significance of field effects on the health measurements of the Koppert boxes that go beyond the possible effects of the dietary imidacloprid.

AVERAGE WORKER SIZE:

Conopid incidence in each field and field region were found to be signifincant effects on average worker size in each of the Koppert boxes at the end of the season.

Conopid incidence: F(1, 20) = 8.1608; P = 0.0098**, the higher the conopid incidence at the end of the season, the larger the bees (Figure 5).

Field Region {3&2 -1}: F(1, 20) = 4.9518; P = 0.0377*, region 1 had a higher average worker ITS than regions 3 & 2 (Figure 6).

AVERAGE PROPORTIONAL WEIGHT CHANGE OF THE COLONIES FROM END OF LAB TO END OF FIELD:

Here, there was only a possible correlation with Field region{1 vs. 3&2} (Figure 7). Colonies placed in region 1 lost significantly more weight while out in the field as compared to regions 2 and 3 where the colonies gained weight.

• Figures for Accomplishments/Milestones section

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.

2015 presentations that contained data acquired from my SARE project:

Bickerman-Martens, K., F. Drummond, and B. Swartz. The Status of Native Bumblebees in Maine. Maine Bumble Bee Atlas Volunteer Training Workshop. Gorham, ME, July 18^th, 2015. Oral presentation.

Bickerman-Martens, K.. Birds, sea turtles, and bumblebees – the life of a biologist. Summer Camp at the Challenger Learning Center of Maine. Bangor, ME, July 16^th, 2015. Oral presentation.

Bickerman, K.E. and F. Drummond. The Status of Native Bumblebees in Maine. Maine Bumble Bee Atlas Volunteer Training Workshop. Orono, ME, May 16^th, 2015. Oral presentation.

Bickerman, K.E. and F. Drummond. The Status of Native Bumblebees in Maine’s Wild Blueberry Fields. Androscoggin Beekeepers Club Meeting. Lewiston, ME, May 13^th, 2015. Oral presentation.

Bickerman, K.E.. The Health of Maine’s Native Bees. Maine Arborist Association Spring Workshop. Portland, ME, April 8^th, 2015. Oral presentation.

Bickerman, K.E. and F. Drummond. Bumblebee Health in Maine’s Lowbush Blueberry Fields. Spring Growers’ Meeting. Bangor, ME, March 20, 2015. Poster presentation.

Collaborators: