Final report for GNE19-216
Project Information
Bee decline has been linked to stressors like pesticide exposure and pathogen infections. This is problematic because many crops depend on pollinators to produce fruit. While bees need to visit flowers to collect pollen and nectar for their developing brood, flowers are also a hub for pathogen transmission between and within bees as they often defecate on them after collecting floral rewards. These pathogens can be lethal for bees or make their foraging and reproduction less effective. In some cases, gut parasites of bees make them defecate more frequently potentially increasing the risk of pathogen transmission via flowers. Thus, bee defecation on flowers can impact the health of the pollinator community in the farm by possibly providing a diverse array of pathogens that the bees can become infected with.
Another important stressor of bees is exposure to pesticides via flowers. Among the most widely used insecticides are neonicotinoids (a class of insecticides that includes thiamethoxam, imidacloprid, clothianidin, etc.), which also negatively impact bee foraging, fitness, and lifespan. The combination of these two stressors has synergistic effects on bees, which can be exacerbated if bees increase consumption of floral resources when exposed to neonicotinoids on flowers. The goal of this study was to determine if the exposure to a neonicotinoid increases nectar consumption in bees that could lead to an increase in the presence of pathogens on the flowers. In addition, we investigated if farm management practices influenced pathogen deposition on flowers in field conditions.
We approached our questions with field and laboratory assays. First, we set up an experiment to determine if nectar consumption of a pesticide was different between bees infected with a pathogen or with the concentration of the pesticide in the food. We used the same experiment to determine if bees infected with a pathogen defecated more when consuming different pesticide diets and determined their survival under the different conditions in the experiment. The experimental design included feeding microcolonies (groups of five bees from one source colony) different doses of a pesticide (0, 2.4, 10 ppb). Half of these microcolonies were infected with the gut parasite Crithidia bombi. We found that infection had no statistically significant effect in consumption, but bees that fed on low pesticide dose consumed more sugar. Survival in one of the rounds of the experiment was significantly lower in the infected bees. These results suggest that the combination of pathogens and pesticide have an overall negative impact on pollinators. Despite the fact that farmers do not have direct control over the amount of pathogens that pollinators share while using crop flowers, developing recommendations that farmers can use to reduce bees' exposure to pathogens would be valuable. The interaction between pathogens and pesticides requires more studies before specific recommendations to farmers can be made.
In an attempt to develop farm management recommendations that can reduce pollinator pathogen pressure, we studied the abundance of two bee pathogens on the surface of pumpkin flowers in 14 fields with different farm management practices. We collected male pumpkin and squash flowers from the edge and the interior of the fields. Those flowers were washed and analyzed for two pathogens (Nosema and Crithidia). We also developed a farmer survey to collect detailed information about their farm management practices. We found a higher number of pathogens on flowers that were collected in farms with little to no cover crops. These results suggest that the presence of cover crops like thistle and clover can dilute the presence of pathogens in crop flowers and that way reduce pathogen pressure on crop pollinators.
These studies clearly show that bee pathogens and pesticide exposure are common stressors for bees foraging on pumpkin fields and that the use of certain cover flowers can reduce the presence of pathogens on the crops. This could impact the way in which farmers decide which cover crops, if any, they incorporate into their cropping systems. Our results also impact our understanding of how bees are exposed to pathogens in agricultural areas and ways in which we could alter bee-pathogen interactions. In addition, we provide a basic understanding of the interaction between pesticides and bee infection that can serve as a base for future studies and recommendations for farm management practices that help maintain pollinators in the fields for longer.
We aimed 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 hypothesized that bumble bees would forage longer on neonicotinoid treated plants and would have a shorter lifespan than bumble bees that foraged 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 hypothesized that pathogen load would increase as bees spend more time foraging on the flowers.
3. Are pathogen loads on pumpkin flowers variable between organic and conventional pumpkin farms? We hypothesized that the pathogen loads would be higher in farms that used neonicotinoids for pest management than farms that used organic approaches.
These questions were addressed through greenhouse, field, and laboratory experiments.
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 was 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 were 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 aimed to determine the effects of bumble bees’ preferential behavior towards neonicotinoids and how that preference impacts pathogen transmission. I hypothesized 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 were used to suggest more sustainable management practices in pumpkin fields (and other crops that use neonicotinoids for pest control) and fill a void in the literature by defining the relationship between insecticide use and pathogen transmission.
Research
Proposed methodology (please see Updated methodology below):
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 flowers?
I completed a fully-crossed greenhouse experiment using neonicotinoid treated plants, untreated plants, and bumble bees infected and uninfected with the gut parasite Crithidia bombi. This experiment was attempted in February, 2020. After three failed trials of the methodology related to objectives one and two, we decided to change our approach (as discussed in updated methodology below).
Experiment: I created enclosures to hold Bombus impatiens (bumble bee) microcolonies (see next section) and two pumpkin plants. There were eight enclosures in total, four with neonicotinoid treated plants and four with untreated plants. Bumble bee microcolonies were infected with C. bombi and four were left uninfected as controls. Of the four enclosures with neonicotinoid treated plants, two were given infected microcolonies and two
had C. bombi free colonies. The four enclosures with untreated plants were treated with bumble bee microcolonies the same way treated plants did (two had infected microcolonies and two had C. bombi free microcolonies). The bees were allowed to forage from 8:00 to 10:00 am for ten days. Behavioral data was 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 was recorded each day. The bee’s feces on the surface of the flowers were collected using an ethanol wash and a pipette at the end of the foraging assay each day. C. bombi amounts, when present, were quantified by doing Microscopy. After each experiment, bumble bees had died and we were not able to maintain them in the lab for the intended 10 more days to estimate survival curves for the colonies that were foraging on
plants from the 2 different treatments. This experiment was replicated and attempted three times.
Bumble bee microcolonies and pathogens: I purchased three Bombus impatiens (bumble bees) colonies from BioBest Sustainable Crop Management and marked the queen upon arrival. I tested a sub-sample of the bees per colony for presence of C. bombi in the guts. To do so, I randomly selected ten bees from each colony and kept them in separate falcon tubes in the fridge. I then proceeded to dissect the bees’ guts and macerated them in 1000ul of
distilled water (five bees per 1000ul). I left the solution at room temperature for three to four hours. Then, I used a hemocytometer to look for C. bombi in 10ul of solution using a compound microscope. Once I confirmed that the bees were not infected with C. bombi, I proceeded with the creation of microcolonies for the experiments.
To create microcolonies, I marked newly emerged bees with water-based markers every day on the dorsal side of the thorax and placed them back in the colony. The day after marking the bees, I removed them from the main colony and added them to a microcolony container and proceeded to mark the newly emerged bees that I saw that day. The bee marking and microcolony creation continued until enough were created for the experiments (a total of 24 for all the experiments, eight per experimental run).
After I created eight microcolonies, I infected the bees in half of the colonies with C. bombi. I collected five bees from an infected colony kept in the lab as a source of C. bombi, dissected the guts, and macerated them in distilled water. The solution was left at room temperature for three to four hours. I used a hemocytometer to count C. bombi cells and estimated the amounts of cells present in 1000ul of solution. I calculated the amount of
sucrose solution and C. bombi suspension needed to have 6000 C. bombi cells per 10ul of inoculum [14]. While the C. bombi suspension rested at room temperature, I removed the sugar water source from the microcolonies that were infected. After four hours of starvation, I separated the bees into individual falcon tubes with holes in the lids. Each bumble bee was fed 10ul of inoculum. To ensure consumption; I let a drop of inoculum in the tip of the pipette touch the antennae of the bee, then released the inoculum slowly near the bumble bees’ head once I saw proboscis extension and observed until the inoculum had been consumed.
To quantify the thiamethoxam in the nectar of insecticide treated plants, I collected nectar samples from two male flowers of each plant in the experiment. The nectar was collected by inserting microcapillary tubes in the flowers’ nectaries. Nectar from all plants in a treatment were pooled to obtain the 500ul needed for the chemical analysis of the nectar. A control sample from four untreated plants were collected and analyzed as a control.
The samples were 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 visited 14 farms with different management practices and quantified the Crithidia and Nosema in a subset of flowers to know how much of it exists in fields.
Interviews to farmers: I electronically and physically distributed a survey to 14 pumpkin farmers to collect detailed information about the management practices they use in their pumpkin fields. This information was correlated with the field collected data using the statistical analyses outlined below.
Field Experiments: I visited 14 cucurbit fields with another graduate student. Bee visitation data was collected during standardized 10 minute sampling periods. Data on bee visitation, visit type, flower sex, and species visiting the flowers was collected. Observations were made from 6:30 am to 9:00am by standing in front of five flowers in the edge or interior sections on the field. Then, flowers of eight plants plants per field were inspected for feces and collected. These samples were analyzed using a compound Microscope available to use in the lab.
Statistical Analyses for all objectives: I used 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 used a Cox proportional-hazards model to analyze the survival data of objective 1. All the data was analyzed in the R environment. A generalized linear model using flower location on the field and management information (flowers on the edge, size of the field, and bee counts) as fixed effects was done for objective two.
Was not done:
For objective 3; 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).
Updated methodology:
For objective one: Do bumble bees forage longer in flowers that have been treated with neonicotinoids? If so, do longer visits impact their survival?
This experiment was done twice with the same number of experimental units. We created 18 microcolonies (containers with five bees each, explained in the proposed methodology) from two source colonies for round one and three source colonies for round two. These bees were purchased with relocated funds from the molecular part of the budget. Instead of placing the bees in flight arenas with neonicotinoid treated flowers, we offered the bees sucrose with a dose of thiamethoxam in it. Thiamethoxam is one of the neonicotinoids that can be the active ingredient in neonicotinoid pesticides, others are clothianidin, imidacloprid, etc. The pesticide we intended to use for these objectives had thiamethoxam as active ingredient, hence, we decided to use a thiamethoxam solution to create the sucrose treatments for the bees. There were three doses 0ppm, 2.4ppm, and 10ppm. Half of the microcolonies were infected with Crithidia, giving the following experimental units:
Infected bees X 0ppm (Control): 3
Infected bees X 2.4ppm (low dose): 3
Infected bees X 10ppm (high dose): 3
Same units for uninfected bees (infection controls).
This modification allowed us to asses consumption of the insecticides by Crithidia infected bees. Survival was assessed by counting living bees each day of the experiment. Although we could not determine foraging times, we were able to determine the impact continuous exposure to thiamethoxam has on sucrose consumption. Consumption was determined by weighing the sucrose container (treatment) before placing it in the microcolony container and after 24 hours for ten days.
The bees that survived round two of the experiment were collected and qPCR analysis was done to confirm infection with Crithidia by the end of the experiment.
For objective two: 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?
Due to the lack of flowers in the new experimental design, we place a 3.5cm platform around the sucrose container. This piece of filter paper was taped to the container below the feeding orifice. We intended to collect any defecation the bees might deposit on a surface while they feed. To observe the feces we used a bee-safe fluorescent dye that was added to the treatment mixtures and later observed under black light. The platforms were changed every 24 hours -at the same time the sucrose treatments were exchanged. The platforms were washed with ethanol and Crithidia presence and prevalence was assessed using a compound microscope.
The experiment for objective one and two was replicated twice. The first replicate happened in July and the second in October. Due to COVID 19 related delays the end time of our grant got extended to February 2021.
For objective three: Are pathogen loads on pumpkin flowers variable between organic and
conventional pumpkin farms?
Molecular (qPCR) was not 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 $1035 from Materials and Supplies Budget to obtain Colonies needed for the Greenhouse assessments. These colonies were needed because of the death of the colonies we had previously.
In each field, bee visitation was assessed via direct observation on flowers. Bee visitation per site was estimated from three ten-minute intervals at the edge and interior of each field, with 30 min wait time between counts each day. A total of four to five flowers were observed per interval, when individual visitors were counted and cataloged.
The visitors were identified as bumble bees, honey bees, male squash bees, and female squash bees. An average of the number of visits per ten-minute interval was calculated for each type of visitor from edge and interior counts.
I collected six to eight male flowers from a determine transect on the edge of the field and the interior (12m from the edge of field). The flowers were then transported to the lab on ice and washed with 100% ethanol. The wash was then observed with a compound microscope to determine presence (yes or no for pathogens) and abundance (number of pathogens found per sample).
For objective one: Do bumble bees forage longer in flowers that have been treated with neonicotinoids? If so, do longer visits impact their survival?
Insecticide dose had a significant effect on sucrose consumption (F-value = 5.63, p-value = 0.008). Infection status did not have a significant effect on sucrose consumption (F-value = 0.55, p-value = 0.46). The interaction of infection status and insecticide dose did not have a significant effect on sucrose consumption (F-value = 1.26, p-value = 0.29). We found a significant reduction of sucrose consumption for the low and high pesticide dose [2.4ppm (t-value = -2.44, p-value = 0.02) 10.0ppm (t-value = -2.00, p-value = 0.05)]. Post hoc analyses showed marginally significant difference in sucrose consumption between the 0ppm dose and the 10ppm dose in both infected (p-value = 0.05) and uninfected (p-value = 0.07) microcolonies. In infected microcolonies, consumption of the 2.4ppm dose was marginally different from the 10.0ppm dose (p-value = 0.07). Although we saw a trend to increased consumption of the low dose by infected bees, it is not significantly different from the controls.
All treatment groups showed significant differences in survival (Health: 𝑋2 =32.98, p<0.01; Treatment: 𝑋 2= 10.37, p<0.01). Crithidia infected bees experienced significantly lower survival (p<0.01), with an increased hazard factor of 4.36 (336% more likely to die during the experiment). Neonicotinoid treatments significantly increased bee survival in the high thiamethoxam concentrations (by a factor of 0.38 = 62%) compared to the control treatment. The low neonicotinoid treatment did not show a reduction in survival compared to the bees in the control treatment (p>0.05). The bees on the second round of experiments survived, hence survival analysis was not possible.
For objective two: 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?
The number of Crithidia cells found on the platform wash was not different by treatments. Likewise, the number of frass droplets on the platforms was not significantly different by treatment, health, or their interaction.
For objective three: Are pathogen loads on pumpkin flowers variable between organic and conventional pumpkin farms?
Due to only having two organic farms available for our study, we created a more complex set of farm characteristics based on the survey farmers answered for us about management.
From these, roughly 74% of the flowers showed the presence of Crithidia and 83% showed Nosema. The prevalence of Nosema spores was not correlated with average bee abundance or location on the field. Variation in pathogen abundance was correlated with the presence of edge crops, field size, and female squash bee average abundance. Crithidia abundance was marginally reduced with the presence of thistle and clover at the edge of the field during flowering times (p-value=0.03, p-value <0.01 respectively). Size of the farm in acres was negatively correlated with Crithidia abundance on flowers at marginally significant level (t-value = -1.85, p-value = 0.06). Female squash bee average abundance showed marginal reduction on Crithidia abundance (t-value=-1.80, p-value=0.07). Crithidia abundance was variable between sites. Abundance of Nosema spores on samples was negatively correlated with the presence of clover at the edge of the field (t-value=-2.68, p-value <0.01). Size of the field was negatively correlated with abundance of Nosema spores (t-value=-2.53, p-value = 0.01). Nosema abundance was negatively correlated with average abundance of female squash bees (t-value=-2.61, p-value< 0.01). Abundance of Nosema was variable between sites.
For objective one: Do bumble bees forage longer in flowers that have been treated with neonicotinoids? If so, do longer visits impact their survival?
It appears that bumble bees consumed more sucrose when treated with 2.4ppm of thiamethoxam than the control and the 10ppm doses. Infection was not statistically significant for consumption, however, a trend was observed. These two stressors did not have a combined effect on consumption, contrary to our expectations. It is important to note that bees in this study were continually exposed to thiamethoxam, which might not be the case in the field. More research regarding the interaction between stressors could inform the impact these might have on the bees during continuous exposure. Since survival was low on replicate one and high on replicate two, it is not possible to determine the impact these stressors had for the totality of the bees in the experiments. However, in round one, infection seemed to cause the highest mortality between all treatments. The bees showed a slightly higher survival in the pesticide treatments. We would recommend a study on the effects of thiamethoxam in Crithidia infections is done before drawing conclusions of its effects in bee survival.
For objective two: 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?
Defecation of Crithidia by bumble bees did not seem to be impacted by thiamethoxam exposure. Exposure to this pesticide in our experimental set up did not have an effect on its deposition on platforms. It is likely that defection, like survival, is more impacted by infection than it is by food (thiamethoxam) consumed. However, these are not flowers and more studies should be conducted to determine how broad the defecation effects (or lack there of) of thiamethoxam on bee are for other crops.
For objective three: Are pathogen loads on pumpkin flowers variable between organic and
conventional pumpkin farms?
This was the first study to quantify pathogen abundance on flowers in agricultural areas and to investigate how farm management practices may impact the abundance of these pathogens in the field. Our results corroborate recent studies suggesting that a diverse array of floral resources dilutes the prevalence and abundance of bee pathogens on flowers and pollinators (Figueroa et al. 2020). We found that the presence of edge crops decreases pathogen abundance on flowers. This suggest that an increase of available floral resources decreases the probability of pathogen transmission via flowers. Specifically, we found that the presence of thistle and clover reduced the abundance of Crithidia and Nosema on Cucurbita (pumpkin and squash) flowers. These results suggest that having a variety of floral resources near crops can dilute the presence of parasites on individual flowers and potentially their spread across pollinator communities. However, these results also indicate that not all edge flowers are equally effective on diluting the abundance of Crithidia and Nosema. Hence, if landscape modifications are used to facilitate pathogen deposition dilution in agro-ecosystems, the plant species selected for edge flower plant should be carefully chosen as an appeasing option to the bees present in the field.
Education & Outreach Activities and Participation Summary
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 a range of information about bumble bee and neonicotinoid impacts on pollinator behavior. At the Pollinator In-Service Meeting I interacted with Master Gardeners, approximately 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 show 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 presented a poster at the 2020 Mid-Atlantic Fruit and Vegetable Convention to describe the experimental design to examine bee behavior when exposed to neonicotinoids in pumpkin plants. The poster was presented to about 30 participants of the pollinator identification workshop Penn State professors offered in the conference. No preliminary results were presented for the greenhouse experiments because those were delayed and later cancelled.
Project Outcomes
I think this project will contribute to sustainability by having laid the ground work for future research on the impact of a particular pesticide on Crithidia infections. Also, by having found that certain edge crops can be beneficial to reduce the number of pathogens on flowers in pumpkin and squash fields. That information can be used to determine which edge crops farmers use to keep their bees healthy and coming back to their fields. It can also protect the investment some farmers make on honey bee hives or bumble bee quads by helping the farmers provide an environment that provides the bees with food sources that might not harbor as much pathogens as others.
During the course of this project I became more aware of the versatility of sustainable agriculture and was able to discuss its benefits with gardeners, farmers, beekeepers, and the general public during outreach events. I also learned about how diverse agricultural lands and management can be, as well as the conscious effort by farmers to maintain their land and crops healthy and profitable. This project helped me narrow my interest into a career that helps support sustainable agriculture. I am searching for opportunities to use the skills I learned to either to hands-on work with farmers focused on research and improvements, with people focused on outreach and accessibility of educational materials, or a combination of both.
Our methodology suffered big changes for objectives one and two: 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 flowers?
Although the proposed methodology for this was achieved in three occasions, we had survival issues of the bees in the pesticide and infected treatments. In the first attempt, we were able to complete the ten days of experiment, but the bees that visited the flowers were in pesticide free (control) cages. In the second and third round of the experiments we suffered survival issues for infected and uninfected bees as well as the pesticide treatment cages. This made data collection not possible and, after having two microcolonies die within three days of starting the third attempt of the experiment, we decided to do a laboratory experiment. We understand that the bees not flying inside the cages might be related to the light or temperature inside of the greenhouse. However, we do not have data to point to a specific reason.
We also decided to not do PCR on the samples we obtained for objective three of this grant. When following the protocol to do DNA extractions (step before PCR) we were unable to obtain clean and good quantities of DNA. We realized this after I did a trial run with the first flower samples from the farm visits following the microscopy analysis done before saving the rest of the sample for PCR. Due to time constrains, we decided that microscopy was enough to determine the presence and abundance of the target pathogens because of their relatively easy visual identification. Perhaps pooling the samples would have helped with the DNA extractions and PCR analyses. However, our experimental design did not permit us to do so.