We aim to understand the impacts of neonicotinoid treatments of pumpkin plants on foraging behavior and pathogen transmission in bumble bees by addressing the following specific questions:
1. Do bumble bees forage longer on flowers that have been treated with neonicotinoids? If so, do longer visits impact their survival? We hypothesize that bumble bees will forage longer on neonicotinoid treated plants and will have a shorter lifespan than bumble bees that forage on pesticide free flowers.
2. Do longer foraging visits lead to more pathogens left behind on pumpkin flowers? If so, do longer visits to neonicotinoid treated flowers lead to more pathogens on flowers? We hypothesize that pathogen load increases as bees spend more time foraging on the flowers.
3. Are pathogen loads on pumpkin flowers variable between organic and conventional pumpkin farms? We hypothesize that the pathogen loads will be higher in farms that use neonicotinoids for pest management than farms that use organic approaches.
These questions will be addressed through greenhouse and field experiments. We will submit a report to each of our farmer partners about the results of this research to inform them about the health status of the bumble bee populations pollinating their pumpkin fields.
The importance of pollinators in pumpkin fields is irrefutable. Without them, there would be no pumpkins for human consumption like there are today. The purpose of this project is to assess how insecticide use in agroecosystems impacts bee foraging behavior and pathogen transmission. Recent studies have demonstrated that bumble bees develop a preference toward neonicotinoid treated plants (Arce; et. al. ,2018). In pumpkin fields in Pennsylvania, a similar behavior has been observed where bumble bees showed a tendency to visit neonicotinoid treated plants more frequently (Treanore, E., 2017). Exposure to high doses of neonicotinoids can be lethal for bees but it can have sub-lethal effects, like impaired foraging, reduced fecundity and memory loss at lower doses (Fauser, et. al. 2017). Because neonicotinoid use in agroecosystems has been linked to declines in bee populations (Stoner, et. al., 2010), we are interested in studying how these pesticides may be impacting wild bee populations in the Northeast of the United States.
Pumpkin (Cucurbita pepo) belongs to the family Cucurbitaceae alongside gourds, cucumbers, and squash. Domesticated pumpkins are grown across the United States and harvested annually or semi-annually as food and decoration. In the state of Pennsylvania in 2018, pumpkins occupied 7,300ac of agricultural fields of which 5,700ac were harvested, representing a total production value of $13,852,000 (Pennsylvania Agricultural Statistics USDA, National Agricultural Statistics Service 2017-2018). Pumpkins are monecious plants that require insect pollination to set fruit. In Pennsylvania, pumpkins are pollinated by bumble bees, squash bees, and honey bees (McGrady, C. 2018), which can provide sufficient pollination services on their own but are in decline (Garibaldi, et. al., 2013). Despite this, renting honey bee colonies to increase yield is a common practice amongst farmers.
Pumpkin production is constrained by damage from herbivores including striped cucumber beetles, squash bug, and aphids. Systemic insecticides, like neonicotinoids, are used as seed treatments, foliar applications, and soil drenches that usually represent a one-time application solution for these pests. Nevertheless, neonicotinoids have been linked to pollinator decline because of its presence in plants’ nectar and pollen. Despite evidence of preferential behavior towards neonicotinoid treated plants, insecticide studies have not attempted to link how insecticide use can lead to increased pathogen transmission on flowers. There is ample evidence indicating that gut parasites that are transmitted on flowers, like Crithidia bombi (Durrer, S., & Schmid-Hempel, P., 1994; Graystock, P., Goulson, D., & Hughes, W. O., 2015), can have detrimental effects on bumble bee populations. These can be reduced offspring and shortened lifespan (Fouks, B., & Lattorff, H. M. G., 2014) particularly when exposed to pesticides (Fauser, et. al. 2017). However, the effects of pesticides on pathogen transmission remains unknown.
This project will determine the effects of bumble bees’ preferential behavior towards neonicotinoids and how that preference impacts pathogen transmission. I hypothesize that the widespread application of neonicotinoid insecticides is altering the foraging patterns and pathogen dynamics in bumble bees. Both effects are a threat to bumble bee populations that provide pollination services to pumpkins and economic gain to farmers that depend on their services for a high yield. Planting pumpkin fields that provide appropriate conditions for the long-term stability of wild bumble bee populations can help farmers maintain free pollination services. Growers understanding the impact of insecticide use in pathogen transmission and bee health can lead to better informed management decisions to maintain bee populations in the area. The results obtained by answering our objectives can be used to incorporate more sustainable management practices in pumpkin fields (and other crops that use neonicotinoids for pest control) and will fill a void in the literature by defining the relationship between insecticide use and pathogen transmission.
The greenhouse experiment will be conducted in February. Edited methods from proposal follow. Most significant change is that molecular (qPCR) will not be conducted. After a series of tests for the pathogen using PCR we were unable to detect the pathogen (presumably because of low titers in the samples), however we are able to quantify the pathogen using a compound microscope available in the lab. The relocation of $761 from Materials and Supplies Budget to obtain Colonies needed for the Greenhouse assessments. These colonies are needed because of the death of the colonies we had previously.
METHODS FOR OBJECTIVES 1 AND 2: Do bumble bees forage longer in flowers that have been treated with
neonicotinoids? If so, do longer visits impact their survival? Do longer foraging visits lead to more pathogens left
behind on pumpkin flowers? If so, do longer visits to neonicotinoid treated flowers lead to more pathogens on
I will complete a fully-crossed greenhouse experiment using neonicotinoid treated plants, untreated plants, and
bumble bees infected and uninfected with the gut parasite Crithidia bombi.
Money destined for molecular equipment in the original Budget
Experiment: I will create enclosures to hold Bombus impatiens (bumble bee) microcolonies (see next section) and
two pumpkin plants. There will be eight enclosures in total, four with neonicotinoid treated plants and four with
untreated plants. Bumble bee microcolonies will be infected with C. bombi and four will be left uninfected as
controls. Of the four enclosures with neonicotinoid treated plants, two will be given infected microcolonies and two
will have C. bombi free colonies. The four enclosures with untreated plants will be treated with bumble bee
microcolonies the same way treated plants will (two will have infected microcolonies and two will have C. bombi
free microcolonies). The bees will be allowed to forage from 9:00 to 11:00 am for ten days. Behavioral data will be
collected to capture: flower visitation, duration of visits (time) and sex of visited flowers. The sex of the flowers and
number of flowers open per plant will be recorded each day. The bee’s feces on the surface of the flowers will be
collected using microcapillary tubes at the end of the foraging assay each day. C. bombi amounts will be
quantified by doing Micrsocopy. After each experiment, bumble bees
will be kept in the laboratory for 10 more days to estimate survival curves for the colonies that were foraging on
plants from the 2 different treatments. This experiment will be replicated three times.
Bumble bee microcolonies and pathogens: I will purchase three Bombus impatiens (bumble bees) colonies from
BioBest Sustainable Crop Management and mark the queen upon arrival. I will test a subsample of the bees per colony for
presence of C. bombi in the guts. To do so, I will randomly select ten bees from each colony and keep them in
separate falcon tubes in the fridge. I will then proceed to dissect the bees’ guts and macerate them in 1000ul of
distilled water (five bees per 1000ul). I will leave the solution at room temperature for three to four hours. Then, I
will use a hemocytometer to look for C. bombi in 10ul of solution using a compound microscope. Once I confirm
that the bees are not infected with C. bombi, I will proceed with the creation of microcolonies for the experiments.
To create microcolonies, I will mark newly emerged bees with water-based markers every day on the dorsal side
of the thorax and place them back in the colony. The day after marking the bees, I will remove them from the main
colony and add them to a microcolony container and proceed to mark the newly emerged bees that I see that day.
The bee marking and microcolony creation will continue until enough are created for the experiments (a total of
24, eight per experimental run).
After eight microcolonies are created, I will infect the bees in half of the colonies with C. bombi. I will collect five
bees from an infected colony kept in the lab as a source of C. bombi, dissect the guts, and macerate them in
distilled water. The solution will be at room temperature for three to four hours. I will then use a hemocytometer to
count C. bombi cells and estimate the amounts of cells present in 1000ul of solution. I will calculate the amount of
sucrose solution and C. bombi suspension needed to have 6000 C. bombi cells per 10ul of inoculum . While
the C. bombi suspension is resting at room temperature, I will remove the sugar water source from the
microcolonies that will be infected. After four hours of starvation, I will separate the bees into individual falcon
tubes with holes in the lids. Each bumble bee will be fed 10ul of inoculum. To ensure consumption; I will let a drop
of inoculum in the tip of the pipette touch the antennae of the bee, then release the inoculum slowly near the
bumble bees’ head once I see proboscis extension and observe until the inoculum has been consumed.
To quantify the thiamethoxam in the nectar of insecticide treated plants, I will collect nectar samples from two
male flowers of each plant in the experiment. The nectar will be collected by inserting microcapillary tubes in the
flowers’ nectaries. Nectar from all plants in a treatment will be pooled to obtain the 500ul needed for the chemical
analysis of the nectar. A control sample from four untreated plants will be collected and analyzed as a controlled.
The samples will be stored in a -80 freezer and shipped overnight to Nicolas Baert at Cornell’s Pesticide Residue
Analysis Lab for thiamethoxam concentration analyses.
METHODS FOR OBJECTIVE 3: Are pathogen loads on pumpkin flowers variable between organic and
conventional pumpkin farms? I will visit 20 farms with different management practices and quantify the C. bombi in
a subset of flowers to know how much of it exists in fields.
Interviews to farmers: I will electronically or physically distribute a survey to 14 pumpkin farmers to collect detailed information
about the management practices they use in their pumpkin fields. This information will be correlated with the field
collected data using the statistical analyses outlined below.
Field Experiments: I will visit 14 cucurbit fields with an undergraduate research assistant. Bee visitation data will
be collected during standardized 10 minute sampling periods. Data on bee visitation, visit type, flower sex, and
species visiting the flowers will be collected. Observations will be made from 9:00 am to 11am by walking along
the rows in the field. Flowers with bee visitors will be observed for 90 seconds. Then, flowers of eight plants per
field will be inspected for feces that will be collected using microcapillary tubes. These samples will be analyzed
using a compound Microscope available to use in the lab.
Statistical Analyses for all objectives: I will use a generalized mixed linear model to investigate how bee visitation
rate is impacted by pesticide treatment and infection status while incorporating microcolony and replicate of the
experiment as random effects. I will use a Cox proportional-hazards model to analyze the survival data of
objective 1. All the data will be analyzed in the R environment.
Will not be done:
Molecular quantification of C. bombi: I will extract DNA from bumble bee feces left on flowers using the Zymo
Quick DNA Fecal Microprep kit. I will set up a qPCR reaction using 28S as a reference gene and 3 replicates per
sample to count the C. bombi using the materials and methods described in Fouks and Lattorff (2014).
There are no results yet.
There are no conclusion yet.
Education & Outreach Activities and Participation Summary
I presented a poster describing the proposed experiments at the Pennsylvania State Beekeepers Association (PSBA)Annual Fall Conference and at the Second Pollinator In-Service Meeting at Penn State. I interacted with approximately 30 beekeepers at the PSBA Conference who were exposed to range of information about bumble bee and neonicotinoid impacts on pollinator behavior. At the Pollinator In-Service Meeting I interacted with Master Gardeners, aproximately 20, who had questions about seed coating of neonicotinoids and its impact on pollinators.
I participated of the Great Insect Fair (GIF) on an information table created to educate the general public on topics related to pesticide leaching into the soil and native bees. The table was co-organized with another Graduate Student in the Lab, Laura Jones. We described the process of pesticide leaching into the soil by using cotton and tinted water that went down a PVC plant. There was a native bee nest on the area that represented the soil to represent a possible scenario in the wild or agricultural lands. We also talked about measures that farmers and the general public can take in order to have a lesser impact on native bee populations.
A translation of the extension publication “Integrated Crop Pollination for Squashes, Pumpkins, and Gourds” was created and published in November. The fact sheet was updated to Penn State Extension under the Spanish title “Polinizacion integrada de cultivos de calabaza”.
I am preparing to present a poster at the 2020 Mid-Atlantic Fruit and Vegetable Convention to describe the summer work and a second poster about neonicotinoid impacts in bee health. No preliminary results can be presented for the greenhouse experiments because those have been delayed.
The project has not been completed, there has been no impact yet. However, we expect that the results of the project, once shared with farmers, will serve as a guideline regarding the use of rented or bought bumble bee colonies.
Although the project has not been completed, I know that I want to continue a career doing research that allows to be in contact with farmers, the general public or both.