Progress report for GNE20-228
Bees are essential to the global food system and perform valuable ecosystem services that contribute between 235 to 577 billion dollars to the economy each year 1,2. Within the last decade, Europe and North America have begun to experience high overwintering losses of honeybee colonies and declines in populations of other wild bee species 3. Part of this decline is due to exposure to pesticides which are acutely toxic to pollinators, can reduce brood survival, foraging success and lead to Colony Collapse disorder 4–6. Non-agricultural areas and flowers are increasingly recognized as a significant additional source of exposure yet this is poorly understood outside of agricultural crops 4,7–9. Filling these knowledge gaps are essential to verify the credibility of pollinator conservation techniques. Therefore, my research will investigate the role pollinator friendly flowers play in exposing pollinator species to pesticides. I will use field surveys and greenhouse experiments to 1) test if pollinator friendly flowers translocate pesticides from the soil into floral rewards, and 2) determine the exposure risk posed by each floral species based on their pesticide expression rates and floral traits. Results will inform pollinator conservation practices and agricultural management recommendations to decrease pollinator exposure to pesticides. that aim to increase floral resources to pollinators.
The main goal of my research is to investigate the role pollinator friendly flowers play in exposing pollinators to pesticides and to inform practices used by farmers, researchers and citizens to increase benefits to pollinators. Using greenhouse experiments and observational field surveys, my research will address three objectives:
Objective 1. Determine if different flowers express different quantities of pesticides in pollen and nectar.
Objective 2. Determine which species are those most heavily used by bees.
Objective 3. By combining results obtained under objective 1 and 2, I will build a quantitative model that will predict which species provide the greatest benefit and pose the least risk to pollinators.
My work will benefit management practices of pollinator conservation (farmers, academics, citizens, etc.) by providing crucial information for beneficial flower species and decrease the risk of pesticide exposure to bees. My results will inform policy regarding pollinator conservation techniques and identify floral species that pose the least risk.
Bees pollinate 80-90% of flowering plants, including 75% of agricultural crops, and contribute 235 - 577 billion dollars to the economy each year 10,111 There are an estimated 25,000 bee species worldwide, with many of the species in decline 12. Out of concern for our food supply and ecosystem health, efforts have increased to aid pollinators. Despite the increasing research in this area, nothing has been definitively identified as the cause of the decline bee populations. However the increasing use of pesticides has been strongly implicated as the cause of pollinator declines6.
Pesticides have been implicated in numerous studies as harmful to pollinators4,5,16–19. They are acutely toxic to pollinators, reduce brood survival and foraging success and lead to Colony Collapse disorder 4,5. Although most pesticide applications occur in agricultural habitats, non-agricultural areas and flowers are increasingly recognized as a significant additional source of exposure19–21. As little as 1-10 % of aerosol pesticides, and 1.6-20 % of systemic pesticides, reach target plants directly, with the rest ending up in the soil, water and air 5,22. These pesticides can remain in soils for years after the initial application9,19,23–25, where they can be absorbed by non-target plants and expressed in plant tissue, nectar and pollen3,26–28.
These non-target plants can act as a potential route of exposure of pollinators to pesticides 4,19,30. Despite this knowledge, a common conservation tactic for pollinators is restoring and conserving land and creating pollinator gardens to provide more floral resources to bees1,32. This habitat restoration strategy has received less scientific attention despite the increasing number of guides for pollinator friendly plantings 33. In order to maximize the conservation potential of planting pollinator friendly plants, the beneficial or detrimental parameters of these tactics must be identified 33. Identifying which flowers represent the best choice to aid pollinators in a contaminated landscape is necessary because they will minimize further exposure to pesticides. The risk that non-agricultural soil contamination poses to bees depends critically on the extent to which pesticides are expressed by plants in pollen and nectar, yet this is poorly understood outside of agricultural crops 7,25. We know the lethal and sub-lethal effects that pesticides have on pollinators5. However, there is a gap of knowledge regarding the environmental fate of pesticides and the extent to which flowers pose as risks to pollinators4,8,19,24,34.
The purpose my PhD work and the project proposed here is to examine the role of flowering plants in non-agricultural areas as potential routes of exposure of pollinators to pesticides and quantify the variation among species in absorption of pesticides from soil and expression of pesticides in nectar and pollen.
Objective 1: Determine if flowers vary in their expression of pesticides in floral resources.
Questions: Q1. Do flowers express varying levels of pesticide between different species? Q2. Does this amount differ based on dosage of pesticide?
To test Objective 1, I will conduct greenhouse experiments with pollinator friendly species commonly and the pesticide Imidacloprid, a water-soluble highly used insecticide 24,36.
Study System: The floral species used in this experiment are: Red Clover (Trifolium pretense), Common Self-Heal (Prunella vulgaris), and Showy Goldenrod (Solidago speciose), and Smooth Aster ( Symphyotrichum laeve ). These species were commonly seen throughout the summer of 2019 and are recommended pollinator friendly plants. These species provide floral resources to a variety of pollinators throughout the summer and fall season, including Bombus spp. and other native pollinators. After growing these floral species and exposing them to known amounts of Imidacloprid, I aim to quantify the specific amount of pesticide expression in these floral species and compare these quantities between the different species of flowers.
To answer Q1: Do flowers express varying levels of pesticide between different species: I will conduct a greenhouse experiment with the previous four floral species common across non-agricultural habitats. I will expose growing wildflower plants to known amounts of Imidacloprid and then quantify the amount of pesticides in the floral rewards. Fifteen plants per species, per treatment will be exposed to pesticide soil applications of five different concentrations in for a total of 300 plants. If grown from seed, multiple seeds of the flower will be sewn in 2.7” x 10” deepots with local organic substrate. Immediately after seeds are sewn, they will be watered with either contaminated water or non-contaminated water. The contaminated water will have standardized dosing of Imidacloprid based on field recommended practices. Other flowers with longer germination times that will be obtained from a certified organic sell in their early germination stages. After receiving these plants, they will be watered with either contaminated water or non-contaminated water based on selected treatment. All other watering will be uncontaminated.
To answer Q1: Greenhouse experiments with Trifolium pratense and Plantago lanceolata began in September 2021 to determine expression rates of pesticides and to determine if pesticide type affects expression rates. 40 replicates per treatment for red clover and 35 replicates per treatment for plantain were grown from seed in organic conditions in four-inch pots. After 3 weeks, plants were randomly assigned to one of three potential treatments or the control group. The treatment groups consisted of : Imidacloprid (insecticide), Difenoconazole (fungicide) or a synergistic dose of two different pesticide types, Imidacloprid and Difenoconazole. All plants were treated once a week with a control solution or a diluted solution of the appropriate pesticide treatment solution.
To answer Q2: Does the amount of pesticides expressed in floral rewards differ based on the dosage of pesticides, I will use multiple dosage levels throughout the experiment as well. Treatments of 0, 0.50, 1.01, 5.04, or 10.08 mg per pot will be used, corresponding to one-twentieth, one-tenth, half, and full field recommended rates, respectively37. The pesticide will be dissolved in 15 ml of water and applied as a soil drench. After the initial soil-drench, all plants will be watered regularly.
The question being asked in Q2 changed for the experiment. The focus shifted from pesticide levels to pesticide type because multiple pesticide types are utilized in agricultural sectors. To determine if pesticide type affects expression rates, treatment groups in the experiment were exposed to the same concentration of different pesticide types. Imidacloprid, an insecticide, and Difenoconazole, a fungicide, were the pesticides used in the experiment. In addition to determining expression rates of for each pesticide alone, one group of plants was exposed to a pesticide solution that contained a 1:1 ratio of Imidacloprid and Difenoconazole to determine if the presence of multiple pesticide types affects expression rate.
Sample Collection: Nectar, pollen and foliage samples will be collected for analysis. 10 grams of leaf tissue will be collected from individual plants. If 10 grams cannot be obtained from each plant, the foliage of several samples will be collected as a composite sample. Pollen samples will be collected by removing the anthers from each plant to obtain enough sample material. Composite nectar samples will be collected for each species within the treatments using the best method based on floral morphology and stored in 1.5 ml micro-centrifuge tubes. All samples will be stored at -80°C until analysis.
Pollen and anther tissue were collected for pesticide analysis. To collect the pollen and anthers for Plantago lanceolata, the entire floral head was clipped and stored in a 50ml falcon tube at -10°C. The treatment, pot number, date and unique ID was recorded on the side of the tube for identification. For Trifolium pratense, the inflorescence was removed and stored in a 50 ml falcon tube with the treatment, pot number, date and unique ID recorded on the tube. To collect the anthers and pollen, individual anthers were removed from each floret and stored in a 1.8vml cryo tube. Once all anthers have been removed from the inflorescence and collected in a 2.0 ml cryo tube, the unique ID was written on the tube and stored in a -10°C freezer. 10 gram of for each treatment were collected at 3 time points during the latter half of the experiment. The soil samples will be used to determine amount of pesticide in the soil.
In addition to pollen and anther tissue, aboveground and belowground biomass were collected to determine if the treatments affected growth of the plant. Wet and dry mass of aboveground and belowground biomass were taken for both plant species. The difference between the wet weight and the dry weight, minus the bag, was recorded as the weight of just the living material of the plant.
Sample extraction: Samples will be analyzed using Liquid Chromatography/Mass Spectrometry to quantify pesticides in pollen and nectar at the Cornell University Chemical Core Facility. The QuECHERS method used for multiple pesticides in plant tissue will be used to extract pesticides.
Data analysis: To test if flowers expression of pesticides varies, I will use pesticide level as the response variable and species type as the predicator variable in an ANOVA analysis. Depending on the shape of the resulting dose-response curve, I will use either linear or logistic regression to test for the effect of plant species on expression of soil residues in pollen and nectar.
Objective 2: . Identify the risk flowers grown in contaminated soil pose to pollinators.
To address Objective 2, I will conduce field surveys and observations that measure floral traits and pollinator preference for the different species.
Field observations will be coupled with the greenhouse experiment results to determine risk associated to pollinators based on the floral species. To answer Q2: How do traits such as phenology, floral density, floral abundance, pollinator preference and pollinator visitation influence the risk associated to pollinators, the previously determined traits will be observed and quantified within the field settings.
To measure floral density and abundance, I will use a belt transect of 50m spanning throughout the site. This transect will be walked for 30 minutes and flowers within 2 meters on either side of the belt transect will be recorded. ten random locations at each of 9 previously established sites will be surveyed twice a month. Floral visitors that visit species within the belt transect will be recorded as well. All species that are in bloom and visitors of these species will be recorded. Images will be taken to identify species that cannot be identified on site. To record plant phenology, 50 meter transects will be walked once per week at 10 sites. The number of plants and flowers per plant species will be recorded to allow for floral counts each week.
To address field component of objective 2, To determine what floral species pollinators prefer, bee floral preference was compared between floral species growing in unmown areas. This was done by conducting an observational study at the UVM Horticultural Farm in 2020, June – October. To identify wildflowers growing in unmown areas and what floral species were utilized by bees within the area, nine 50 x 2-meter belt transects were measured, flagged, and given a patch ID (A, B, C, etc.). Patches were left unmown throughout the summer season to allow for proliferation of wildflowers. Weekly floral abundance and diversity surveys were conducted within each patch. This entailed walking through each patch to identify floral species and simultaneously count the number of inflorescences seen for that species. To determine the floral species most preferred by bees, pollinator surveys were conducted within each patch on a weekly basis. Pollinator visitation surveys were standardized at 20 minutes within each patch. Once a bee was spotted visiting a plant, the species or genus of the bee was noted along with the floral species the bee was visiting at the time. If the bee species was not recognizable by eye, a picture was taken of it for later identification, or a brief description of the bee was noted. Any subsequent plants that the bee visited after the initial plant were noted as well until the bee left the transect. These steps were repeated every time a bee was observed in the patch during the pollinator observation time period. Bees were randomly sampled within the patches at the beginning, middle and end of the study to determine what species of pollinators were visiting the patches throughout the season. Samples were pooled together based on time point. Soil samples were also taken at the beginning, middle and end of the season to identify pesticide residues within the patches.
Data Analysis: To analyze theses data, all observations and records will be compiled into one data file to conduct correlative studies as well as ANOVA tests. I will also analyze my results with Multivariate Analysis of Variance (MANOVA) with species as the predictor variable and the other observed traits as the response variables. I will use linear regressions tests to determine for the effect of the multiple factors on visitation is for each floral species.
To address the objective 2, I will create a predictive model that estimates the risk of a habitat to a bee. This risk will be based on soil contamination amounts and estimated importance of the habitat to bees.
The input variables of the model will be soil contamination levels and importance. Using the input soil contamination levels, the model will estimate the pesticide amounts likely to be found in the floral rewards of the wildflowers in the habitat. This estimate will be determined based on the relationship between soil contamination levels and pesticide amount in pollen. Soil contamination levels will be based on literature values and samples collected from sites around the Champlain Valley area and the greenhouse experiment from 2021. The pesticide amounts in pollen will be collected from the greenhouse experiment plants mentioned in the previous section. These amounts will also be supplemented with literature values from floral species grown in similar experiments. The plants and the average pesticide amounts will be used as representative samples for wildflowers and will allow for estimated concentration of pesticides in wildflowers to be determined based on soil contamination levels.
Importance to bees and the average amount of pollen a bee will contact in the field will be additional predictor parameters fitted into the model. Importance to bees is based on how much time a bee spends foraging for resources in the specific habitat. This number will range from 0% to 100%, with 0 meaning the bee does not forage in the specific area at all and 100% meaning the bee only forages in the specific area. Average amount of pollen will be based on literature values for wild bees. Importance to bees and soil contamination levels will be varied across all possible ranges to estimate risk of habitat to bees.
Combining the estimated relationship value and the additional parameters, the output of the model will calculate the percentage of the LD50 a bee is exposed to in a specific habitat. Using the established LD50 values as well as threshold levels, habitat risk to bees can be estimated. This model should confirm predictions that with increasing soil contamination and/or importance to bees, bees will be exposed to more contaminated pollen. This will result in greater risk to bees for that specific foraging area. These results can be visualized on a heat map showing where a habitat would fall based on the varying parameters.
Results for Objective 1:
Data analysis for objective 1 are ongoing due to the recent conclusion of the experiment in mid- January.
Results for Objective 2:
To determine risk, visitation and expression results will be incorporated into a predictive model to calculate cumulative exposure for foragers across the season given soil contamination levels and time bees spend foraging in the habitat.
Based on analysis of the data from the floral and bee observations, 37 wildflower species were observed from June to October in the unmown patches at the Hort farm. Bees from the genera Apis, Bombus, Agapostemon, Augochlora, Halictus Xylocopa and Ceratina were observed foraging within the patches. Visitation by wild bees was seen within the patches throughout the season. Honeybee visitation was not observed until the middle of June but quickly became more abundant than wild bees. Wild bees were also observed in all patches, however, honeybees were only observed in eight of the patches. Of the 37 floral species observed in the unman patches, bees showed preference for only twelve of the floral species and preference was different between wild bees and honeybees. The top preferred floral species for wild bees were the native plants, common yellow wood sorrel (Oxalis stricta) and goldenrod (Solidago spp.), while honeybees preferred the non-native floral species English plantain (Plantago lanceolata ) and great mullein (Verbascum thapsus).
Conclusions: Results indicate that unmown areas provide multiple floral species, but bees show preference for a limited number of species when encountering multiple species.
Education & Outreach Activities and Participation Summary
During my research and after the completion of my experiments, I would like to distribute and discuss my results to academics, farmers and citizens in the Northeast region and other applicable areas. I will host workshops, collaborate with non-profit pollinator organizations, initiate floral plantings with the community as a form of extension work.
Workshops: I would like to host workshops with farmers, conservation biologists, non-profit pollinator organizations and anyone one else interested in pollinator conservation. During these workshops, I will explain the benefits and results of my research. I will be able to make recommendations for bee friendly plantings.
Workshops: I have not hosted a workshop my self, however, I recently became acquainted with the UVM Pollinator Extension coordinator. I have volunteered at the events this person has hosted, including a pollinator day where farmers from around the Chittenden County area come to learn about how they can monitor bee populations on their own farms.
Collaboration: If possible, I would like to collaborate with non-profit organizations that focus on pollinator conservation such as the Xerces Society and the Pollinator Partnership. My goal is to create a more informed flyer for pollinator friendly floral recommendations. This flyer would address the risk each species could have on pollinators by exposing them to pesticides. The flyer could give recommendations to those that want to plant flowers for pollinators but have no knowledge of the condition or history of their habitat.
Extension Work: A goal of my outreach is to disseminate information that is accessible to everyone. I will organize and lead a project throughout the state of Vermont to increase plantings of extra floral resources for pollinators that pose less of a risk based on the findings of my research. I will also host a Pollinator Weekend during the summer open to anyone that wants to learn about pollinators. This weekend would be targeted towards a younger audience to increase awareness of pollinator decline and how people of all ages can get involved and aid pollinators.
Currently, I have not had the opportunity to conduct outreach and/or education opportunities regarding my research. I have been in contact with members of the 4H program to discuss possibly hosting a workshop in the spring/summer that would be focused on my research. This opportunity would likely be a public event on a research farm that focuses on getting people out onto farms while also teaching them about pollinators. They will get the chance to see bees in action and how our plant-pollinator system is connected to the agricultural sector.
Wildflower planting to promote pollinator populations is a popular conservation technique that can involve the many people with varying skillsets in pollinator conservation. I aim to increase public participation in these conservation tactics in ways that still aid pollinators.
I hope that my project will address the many ways bees can be exposed to pesticide within the agricultural sector. The aim is to provide additional knowledge that can be used by people in the agricultural sector to make more sustainable decisions when trying to aid pollinators. My research can facilitate a conversation about how other non-managed areas and plants can be influenced by agricultural practices and how this may directly, or indirectly, affect pollinators.
My view of sustainable agriculture has increased when reviewing the work that others have done through SARE. Sustainable agriculture is more than just one decision but an ongoing process and responsibility. We have a duty to protect and serve our communities and ecosystems to preserve them or the next generation.
My future career path will focus on building the bridge between academia and the public. I envision a career as an outreach/extension coordinator that targets under-represented youth and communities. My goal is to increase STEM awareness in those communities by facilitating science based experiences that the public can attend on a voluntary basis to learn more about specific fields of STEM. I hope to be able to begin some of this work by collaborating with he 4H department at UVM.