Progress report for GNE20-226
Beekeepers are experiencing over $1 million in annual losses when pollinating NJ blueberries. Fungicide use over the last decade has increased during bloom which correlates with colony declines. Therefore, investigations on the effects of fungicides are needed.
Initial work has shown that bees may prefer fungicide laced food sources, while other research has shown bee attractiveness to some fungicides. As part of my dissertation, I found 36 pesticides returned to the hive in pollen, including numerous fungicides. New papers point to the negative impact of fungicides on different bee species, including honey bees. My initial tests showed that some high fungicide rates caused larval mortality. Therefore, we need to determine the influence of blueberry fungicide use on foraging behavior as well as impacts on brood development.
My objectives include: 1) determining the influence of commonly used blueberry fungicides on honey bee foraging behavior, 2) determining if this behavior influences residues being introduced to the colony, and 3) determining the impacts of in-hive residues on brood development.
Research components include: Field studies that record honey bee forager visitations in number and consumption rate as influenced by plain syrup vs syrup with various fungicides; pollen and nectar analyses for pesticide residues in hives, and in vitro feeding tests with fungicide amended diet to examine resulting effects on brood and maturation.
Outreach will include extension newsletters, web-based blogs, and grower winter and spring update meetings. I will work with extension faculty to update the pollinator protection chapter in the NJ Blueberry Production Guide.
This project extends the work being done on an existing SARE grant, now coming to a conclusion in its 3rd and final season, but having opened additional questions relative to bee behavior and fungicide toxicity. The proposed work is designed to address those questions not addressed in the original SARE project.
Objective 1. Determine the influence of commonly used blueberry fungicides on honey bee foraging behavior.
Objective 2. Determine fungicide levels in honey bee hives, and when they are most likely to enter the hive in relation to fungicide spray programs.
Objective 3. Determine if there is any impact on honey bee brood development and resulting colony health by fungicide levels found in the hive as influenced by objectives 1 and 2.
The purpose of this project is to identify the behavioral and developmental effects of fungicide use on honey bee colonies used for blueberry pollination. I will be examining the impacts of fungicide use during bloom on bee forager behavior, resulting hive contamination, and effects on brood development. Declines in brood development during and shortly after pollination services have left beekeepers with severe colony losses resulting in up to 90% hive mortality. Thus, threatening the sustainability of commercialized blueberry pollination. The NJ blueberry industry is valued between $65-70 million annually, and is produced on just over 9,000 acres in the southern NJ Pinelands. NJ blueberry growers contract with NJ and regional beekeepers to bring in about 18,000 colonies each year for pollination.
Hives with greater pesticide exposure show higher rates of death. In some cases multiple pesticides may show additive or even synergistic mortality effects to bees. Most older literature has focused on pesticide effects on adult bees, and most of this was done with insecticides. Only recently have researchers started to look at larval responses to pesticides, with fungicides being the newest frontier. Since fungicides are heavily used in blueberries during the pollination period, several areas need to be investigated.
I will be examining the behavior of adult foragers to commonly used fungicides, and the resulting residue levels brought back to the hive. Additionally, I will be looking at the developmental effects of fungicide contaminants.
By conducting larval bioassays with fungicide and miticide laced diets, I will identify pesticide combinations that pose risks to bee development. The identification of any negative impacts of fungicides can be used to support mitigation strategies and improve recommendations by Rutgers research and extension faculty. My findings will be presented at apicultural and agricultural meetings across the Eastern states. Such findings will improve honey bee health, impact pest management practices, and reduce beekeeper losses and reduce production costs for fruit growers.
Behavioral tests for honey bee forager response to fungicides
The formulated products, will all be mixed in sucrose syrup, and compared to blank sucrose syrup alone. I will use tunnel enclosures in a manner similar to that of Liao et al., 2017. I will train 5-framed hives with sister queens, consisting of approximately 3,000 honey bees (one per tunnel) to feed on 4 separate but identical sugar syrup feeders. Feeders will consist of red (non-see-through) inverted plastic containers with approximately 50 holes (1 mm in diameter) evenly dispersed just below the lid of each container. They will be placed upon 2 separate cinder blocks oriented in a straight line on one side of the enclosure (opposite side of the hive). After three days of successful foraging at each feeder, I will begin the experiment. Each feeder will be filled with appropriate diet and weighed before and after each run to determine consumption rate. An experimental run will consist of 30-minute visual observations where the number of foragers visiting each feeder will be recorded. A minimum of 2 runs will be conducted each day per hive and will continue for a total of 3 days. Appropriate diet will be replaced and use measured as needed. On the 4th day, 4th and 5th instar larvae will be visually inspected for adverse effects of treatment and used for comparison in behavior response analysis. Each experiment will be repeated 4 times (over 4 weeks); to ensure there is no biased to feeder location, the feeders will be placed in a new location each time. Active ingredient fungicides to be tested include ziram, azoxystrobin (Abound), difenoconazole (Quash), cyprodinil (in Inspire Super), and boscalid (in Pristine). Pristine and Inspire Super are premixes of azoxystrobin + boscalid and cyprodinil + difenoconazole, respectively. These tests will include the 5 most commonly used FRAC groups: 3-DMIs, 7-SDHIs, 11-Qols, 9-Anilino-Pyrimidines, and M03-dithiocarbamates. Data on consumption rate and larval mortality will be analyzed using analysis of variance between the 4 treatments.
Determination of fungicide levels as reflected by honey bee foraging behavior.
Based on the response of foragers to fungicides, nucleus hives will be placed in the centers of commercial fields and monitored for fungicide residues in pollen, bee bread, and nectar. I will coordinate residue sampling with commercial grower participants so that a total residue load in the colony’s food stores can be calculated. I have previously determined various residues that are present during the middle of bloom. However, this ‘1 shot’ sampling does not give us information over time, or enable a correlation with fresh fungicide applications with the developmental stage of the bee. I will collaborate with 4 blueberry growers who each have individual farm sites of over 200 acres. Four nucleus hives will be placed in the centers of each of these 4 farms so that the foraging ability of the bees is mostly restricted to those fields, and any fungicide residues found will be reflective of that growers’ spray programs. At each farm site, 2 hives will be fitted with pollen traps to prevent the entry of foraged pollen. Each of these 2 hives (treatment hives) on each farm will be provided substitute pollen patties (Mann Lake) as a protein source. The other 2 hives (standard) on each farm will not have pollen traps and will be freely exposed to all pollen and nectar being returned to the hive. This gives an experimental design with 2 treatments and 4 replicates. After each fungicide application (8 to 24 hours, depending on application method), pollen will be sampled along with an internal hive sampling of nectar from 1 of the 2 pollen trapped hives. This will be done every 48 hours for 7 days, or until the next application, upon which time the process will start over. The other pollen trapped hive will not be sampled, but all 4 hives will be examined for parameters of hive health, and pollen+ bee bread samples taken every 7 days. These measurements include hive weight, number of frames with brood, percent brood coverage, egg and queen presence, and disease incidence. At the end of pollination (~3.5-4 weeks), hives will get a final sampling for hive health and residue samples. Data on hive parameters and residues will be analyzed using analysis of variance.
Fungicide impact on honey bee brood development
Using the fungicides found to be attractive, if any in objective 1, and those fungicides found at the highest residue levels in objective 2, I will examine those fungicides alone and in combination with miticides used by beekeepers to treat varroa mites, in in-vitro feeding tests. Each residue will be tested in its pure active ingredient form and by formulated product. The concentrations of each will be tested on a gradient starting with the highest concentration found from objective 2 in both pollen and nectar. Additional tested rates will be cut in half in serial divisions, resulting in a minimum of 5 tested dietary concentrations. A control of pure diet, and an acetone control (solvent) will be included. Laboratory procedures will be adapted from Schmehl et al., 2016. I will establish eight 5-framed honey bee hives (nucleus hives) with newly mated sister queens. Inside each nucleus hive there will be a caged frame on empty drawn comb, this frame will be swapped out every week (four days prior to larvae extraction) to ensure all larvae will be of the exact age. 1st instar larvae will be grafted to 48 well-plates and fed fungicide and miticide laced diets with concentrations corresponding to pollen residue samples with 12 larvae per treatment and each treatment will be replicated 4 times. Larvae will be fed daily until the fifth day where they will be then transported to a pupation incubator (which serves as capped cells) until adult emergence. Dosing of fungicide active ingredients will occur only on day 1-3. Mortality will be recorded at 24, 48, 72, 96, and 120 experimental hours and on the 21st day after adult emergence. All grafting, feeding, and data collection will be conducted inside a positive laminar flow hood which will be sterilized for 30-minutes before and after each procedure via UV light and by wiping down with 10% bleach. Subjects which complete pupation and achieve adult stage will be measured (head dimensions, length and width and weight of body) and compared across treatments for additional sublethal effects. Data on larval mortality and adult measurements will be analyzed using analysis of variance between rate treatments and probit analysis for mortality rates vs pesticide concentration.
Experiments have yet to begin. So far, all materials have been acquired to begin research in April 2021.
Education & Outreach Activities and Participation Summary
Outreach activities will be divided into 1) Extension based to blueberry growers and to commercial and hobbyist beekeepers; and 2) To professional journals and the commercial beekeeping industry.
Extension based information to growers and beekeepers: I will present my work at 2 annual blueberry grower meeting reaching at least 70 growers and 1 statewide NJ Beekeepers Association (NJBA) meeting each year reaching about 150 beekeepers (total 3 per year), and will contribute 1 article annually to the online NJBA newsletter. This online newsletter reaches over 1,000 NJ beekeepers, many of whom have already donated to my research efforts. These meetings will include the blueberry session at the Atlantic City NJ Agricultural Convention each January, the annual Blueberry Open House in Hammonton, NJ in late February, and twilight fruit grower update meetings in Hammonton during the spring season. I will work with commercial beekeepers throughout the year, communicate by email and phone, make annual reports available to them and hold 1 informal meeting per year, likely during the winter. I will contribute efforts to the Rutgers Blueberry Production Guide, so that bee safety information is updated.
Professional journals and commercial beekeeping industry: I will target articles for the American Bee Journal, and on a more scientific level, The Journal of Apiculture Research. I will present my work at the Eastern Branch of the Entomological Society of America (ESA), as well as the National ESA meetings, and at the regional Cumberland-Shenandoah Fruit Workers Meeting (95-100 extension, researchers, industry and graduate students from the mid-Atlantic to New England states).