Bee declines pose a serious risk to human food supply, crop production, wild plant diversity, and the commercial bee industry. Pathogens, including RNA viruses and microsporidian fungi, have been primarily documented in honey bees and to some extent bumble bees and solitary bees, with data suggesting these pathogens are shared to some degree in the bee community (Singh et al. 2010; Fürst et al. 2014; McMahon et al. 2015; Ravoet et al. 2014; Dolezal et al. 2016). However, our understanding of pathogen epidemiology and transmission within the bee community is limited (Graystock et al. 2015).
The purpose of this research is to better understand the epidemiology and transmission of pathogens within the bee community by collecting empirical data to develop a predictive model of pathogen prevalence. Under this mission, we more specifically address (1) the seasonal prevalence of pathogens in bee communities, (2) how overwintering strategies impact pathogen prevalence in honey bees and bumble bees, (3) how the influence of pathogens differs between honey bees, bumble bees, and solitary bees, (4) the relationship between foraging experience and pathogen prevalence, and (5) the spatial properties of pathogen prevalence in bee communities. Through integrating information on spatial properties of pathogen communities, likelihood of horizontal transmission, and the seasonal prevalence of pathogens across different types of bees (honey bees, bumble bees, and solitary bees) we can better understand the patterns of pathogen transmission and the threats that these pathogens may pose to pollinator communities. With this improved understanding, we can define applicable control strategies for bee pathogens to ensure sustained and high productivity of crops and reduce the price of pollination services.
Honey bees and bumble bees (Bombus impatiens) were collected across two overwintering periods (fall 2015 to spring 2016 and fall 2016 and spring 2017) and one full active season (early spring to fall of 2016) at six sites within Centre County, Pennsylvania. Samples are being screened for the following pathogens: Deformed Wing Virus (DWV), Black Queen Cell Virus (BQCV), Crithidia, Nosema bombi, Nosema ceranae, and Ascosphaera. Preliminary data suggests that there are seasonal trends for DWV and BQCV and that these trends can be spatially heterogeneous. DWV peaks in the fall in both honey bees and bumble bees, however, throughout the season there are time points where the seasonal trend deviates between honey bees and bumble bees. BQCV peaks in early summer in both honey bees and bumble bees and the trends between lineages is consistent throughout the season. Overall, for both viruses honey bees consistently had a higher prevalence than bumble bees. Examining the trends through the overwintering period between fall and spring, the prevalence of DWV and BQCV significantly decreases for bumble bees, but not honey bees. This suggests that bumble bee populations can purge pathogens through overwintering and honey bees may be retaining the pathogens in the pollinator community.
Agricultural sustainability hinges on pollination services. Worldwide, 75% of the crops that humans consume require animal pollination (Klein et al. 2007). These pollinators are also essential for wild plant pollination; 80% of wild plant species rely on pollinators for successful reproduction (Potts et al. 2010). The majority of pollination services are provided by bees (Free et al. 1993). Pollination services in agriculture have relied heavily on commercial honey bees, nonetheless native bees, including bumble bees and the thousands of species of solitary bees, perform vital pollination services. Native bees can be more efficient pollinators than honey bees, enhance crop production alongside honey bees, and can provide sufficient pollination in lieu of honey bees for many of our agricultural crops (Garibaldi et al. 2013; Winfree et al. 2008). The pollination services of native bees alone is valued at $ 3.07 billion in the United States (Losey and Vaughan 2006).
In the last decade, there have been precipitous losses of honey bee colonies (VanEngelsdorp and Meixner 2010). This has raised concerns about how the demand for pollination services will be met and also has had economic repercussions, as the cost of renting honey bees has increased substantially (Calderone 2012). Adding to the concern, native bee communities have witnessed range reductions and declines (Koh et al. 2016). For both honey bees and native bees, pathogens are considered a leading factor driving these declines (Cameron et al. 2011).
However, our understanding of pathogen epidemiology and transmission among pollinators is limited (Graystock et al. 2015). Many factors can affect the dynamics of pathogen prevalence and spread within the bee community. For example, seasonality, host life history, spatial properties, and transmission ability and rates.
Seasonality is the cyclic trend in pathogen prevalence in response to seasonal changes such as temperature or precipitation (Altizer et al. 2006). Improving our understanding of seasonality of pathogens affecting bees will provide us with a better idea of host-pathogen interactions, how best to implement control strategies, and the effect climate change may have on pathogen dynamics.
Life cycle differences may be important for how pathogens persist in bee populations and for how they spread through a community. The life cycles of honey bees (Apis mellifera), bumble bees (Bombus spp.), and solitary bees differ. Honey bee colonies are perennial, meaning they are active throughout the entire year, although populations are reduced during the winter. Bumble bees have an annual cycle which means that every winter the whole colony except for the next year’s queens dies. This overwintering period is a stressful time for bumble bees, whereby bumble bees with poor nutrition and pathogens may not survive. Solitary bees have an annual cycle similar to bumble bees, but they have a shorter active period and do not live in social colonies (Linsley 1958). Given these dynamics we might expect honey bees to harbor pathogens year round, for bumble bees to purge pathogens yearly through mortality of overwintering queens, and solitary bees to be less likely to harbor pathogens overall.
Furthermore, transmission rates can differ between species based on their connectivity to the pathogen host species within the pollinator network, evolutionary relatedness, or spatial properties of the landscape (Fenton and Pedersen 2005). Bumble bees and honey bees are more closely related to each other than to the other solitary bees (Peters et al. 2017). Therefore, it may be easier for pathogens to spillover between honey bees and bumble bees. However, given that these pathogens have been found in solitary bees means that these pathogens likely have wide host ranges. The type of landscape can impact pathogen transmission. A lower quality landscape may present poor nutrition and thus amplify pathogen loads through bee stress. A high-quality landscape may promote better bee health but also may promote higher abundance and thus increased potential for transmissions. Moreover, a fragmented landscape may have only a few areas with good resources that colonies can reasonably reach creating a hot spot for transmission. Therefore, understanding the variation in pathogen prevalence among bee lineages, the spatial properties of the landscape as well as the relationship between foraging experience and pathogen prevalence will improve our understanding of spatial properties and transmission ability and rates.
Given the significant risk of pathogens to pollinators, it is important that we fully understand the epidemiology and transmission of pathogens within the whole bee community. It is vital that we understand the players and mechanisms behind the bee pathogen network. This includes the spatial properties of the landscape, seasonal patterns of pathogen prevalence, the impact of this pathogens on different bee taxa, and the impact of horizontal transmission outside of the colony.
The overall goal of this project is to develop a predictive model of pathogen prevalence within bee communities. Utilizing this information, we will be able to create more informed management strategies and decisions. To achieve this overall goal, we need to understand when pathogens occur and how pathogens are being transmitted within the pollinator community. Therefore, we will address the following questions:
- Is there a seasonal pattern to pathogen prevalence in bees and does this differ between bee taxa (honey bees, bumble bees, solitary bees)?
- Does overwintering purge pathogens in bumble bees but not honey bees?
- Does overall pathogen prevalence differ between honey bees, bumble bees, and solitary bees suggesting that life history and limited transmissibility between taxa may impact pathogen spread?
- What are the spatial properties of bee pathogen prevalence?
- Is there a relationship between foraging experience and pathogen prevalence?
To accomplish these goals, the project is separated into the following tasks:
- Collect bumble bees and honey bees for two overwintering periods (fall 2015 to spring 2016 and fall 2016 and spring 2017) and a full active season (early spring to fall 2016). Samples will be collected at 6 sites each year (2 agricultural, 2 urban, and 2 natural).
- Assess the pathogens present in the bee types and communities. The pathogens examined include DWV, BQCV, Crithidia, N. bombi, N. ceranae, and Ascosphaera.
- Utilize the data collected to parameterize a predictive model of disease patterns in bee communities.
Sample collection: Bumble bees, honey bees, and solitary bees were net collected while nest searching or foraging during the active season across three years (2015, 2016, and 2017). In 2015, bumble bees and honey bees were collected at 5 sites (Bellefonte, Colyer Lake Area, Penn State Campus, Russell E Larson Agricultural Research Farm, and Tussey Mountain) at three time points (spring (late April), summer (July), and fall (late September). In 2016, bumble bees, honey bees were collected at six sites (Airport Area, Bellefonte, Colyer Lake Area, Penn State Campus, Russell E Larson Agricultural Research Farm, and Tussey Mountain) at four time points (spring (late March/early April), summer 1 (early June), summer 2 (mid-late July), and fall (mid-September/late October)). Furthermore, at three sites (Airport Area, Penn State, and Russell E Larson Agricultural Research Farm) approximately 15 Xylocopa virginica were collected during the summer 1 collection period. In 2017, bumble bees and honey bees were collected at four sites (Colyer Lake Area, Penn State Campus, Russell E Larson Agricultural Research Farm, and Tussey) at one time period (spring (April)). All sites were 2 km2 and were located in Centre County (Figure 12). All samples were stored at -80oC for later analyses. Our spring bumble bees were newly emerged queens whereas those collected in other seasons were workers, although a few fall queens were collected.
Assessing the prevalence and seasonal incidence of pathogens: RNA (Qiazol) and DNA extractions (EZNA Tissue DNA Kit) are being performed on 15 bees per time period per site. The RNA extraction is performed on the abdominal cavity with the gut removed and the DNA extraction is performed on the gut. To assess for RNA viruses, RT-PCR is performed on converted bee cDNA using established primers for Deformed Wing Virus (DWV), Black Queen Cell Virus (BQCV), and Chronic Bee Paralysis Virus (CBPV) (Singh et al. 2010; Muli et al. 2014). To detect the presence of other pathogens, such as microsporidian and fungi, PCR using known primers for Nosema ceranae, N. bombi, Crithidia bombi, Ascosphaera apis is conducted (Chen et al. 2008; Klee et al. 2006; Meeus et al. 2011; James and Skinner 2005). Following RT-PCR and PCR, gel electrophoresis is utilized to detect presence and absence of each pathogen.
Assessment of foraging experience and body size: Using wing wear, we will estimate bumble bee’s foraging experience/age. During the dissection of the bumble bees, the right wing was removed by cutting at the base of the wing to avoid causing any damage to the outer edge. Using Photoshop, we measured the total area of the wing remaining, the marginal cell length, and estimated the area of the wing missing. The marginal cell is a good estimate of body size (Medler 1962; Plowright and Jay 1968) and, thus, can be used as a measurement of foraging range because larger bees can typically forage farther distances (Greenleaf et al. 2007). To account for differences in wing size, we divided the area of the wing missing by the sum of the total area of the wing remaining and the area of the wing missing.
While the results of this project are not complete, the preliminary results shed some light on the implications of pathogen spillover between honey bees and bumble bees as well as provides important information regarding the impact of horizontal transmission in pathogen spread and the best times of the year for management strategies to decrease the negative impacts of pathogens on bee communities.
Seasonal Variation of DWV and BQCV in bumble bees and honey bees: Preliminary results suggest that the seasonal peak for DWV is in the fall. Furthermore, while the seasonal trends look similar between honey bees and bumble bees, the patterns are not significantly correlated (Linear Regression: R2=0.309, p=0.25) (Figure 1). Furthermore, the results for BQCV differed from those of DWV. Preliminary results suggest that the seasonal peak for BQCV is in early summer. In addition, the seasonal trend of BQCV is correlated between lineages (Linear Regression: R2=0.758, p=0.024) (Figure 2). These results suggest that seasonality does affect pathogen prevalence for both pathogens. When the pathogen prevalence across time is assessed at the level of the site, the same trend is not observed at every site suggesting there is spatial heterogeneity. Overall, for both viruses honey bees consistently had a higher prevalence than bumble bees (Figures 1 and 2).
Figure 1: Seasonal prevalence of DWV for honey bees (solid line) and bumble bees (dashed line). Fall was September/October. Spring was March or April. Summer 1 was early June. Summer 2 was mid-July.
Figure 2: Seasonal prevalence of BQCV for honey bees (solid line) and bumble bees (dashed line). Fall was September/October. Spring was March or April. Summer 1 was early June. Summer 2 was mid-July.
Effect of overwintering strategies in bumble bees and honey bees on pathogen prevalence: Using a maximum a posteriori estimation of pathogen prevalence comparing all time points and lineages, our preliminary results suggest that pathogen prevalence in honey bees does not significantly change through the overwintering period (from fall to spring) for both pathogens, whereas it does change significantly for bumble bees (Figure 3). Therefore, it seems that bumble bee populations purge pathogens through overwintering and honey bee populations actually retain pathogens in the community. Therefore, the data suggest the bumble bee community has seasonal pathogen reduction and that the retention of these pathogens in honey bees may negatively impact levels in native bee communities thereafter.
Figure 3: Viral prevalence of DWV and BQCV in honey bees (blue) and bumble bees (pink) over two overwintering periods (Fall 2015 to Spring 2016 and Fall 2016 to Spring 2017).
Effect of functional traits (foraging experience and body size) on pathogen prevalence: Our preliminary data suggests that foraging experience does not have a significant impact on pathogen prevalence in bumble bees (DWV: T-test p=0.253, BQCV: T-test p=0.347). However, while the result is not significant, the average wing wear is higher for individuals with pathogens (Figure 4). Furthermore, body size does not significantly influence pathogen prevalence, however, there is a slight trend towards larger bees having more pathogens (Figure 5).
Figure 4: Effect of foraging experience (wing wear) in B. impatiens on pathogen absence or presence. There was no significant difference in wing wear between disease absent or present for either pathogen (BQCV: p=0.347; DWV: p=0.253).
Figure 5: Effect of body size of B. impatiens on pathogen absence or presence.
Education & Outreach Activities and Participation Summary
Preliminary findings from this research have been presented at two international conferences, a national meeting, and a local meeting by the project coordinator:
- Ezray B, Hines H. (2017) Pathogen dynamics in bee communities. Annual Meeting of the Entomological Society of America Student Oral Ten Minute Paper Competition. Denver, CO.
- Ezray B, Hines H. (2016) Seasonality of pathogens in bumble bees and honey bees. The Pennsylvania State University Center for Pollinator Research Spring 2016 Symposium. State College, PA.
- Ezray B, Hines H. (2016) Understanding pathogen dynamics in bee communities.2016 XXV International Congress of Entomology Student Oral Ten Minute Paper Competition. Orlando, FL.
- Ezray B, Hines H. (2016) Understanding pathogen dynamics in bee communitiesPoster Presented at: 3rd International Conference on Pollinator Biology, Health and Policy.State College, PA.
Presenting the preliminary results of this research at the above meetings has sparked interest and conversation about the implications of honey bees in spreading pathogens to native bees as well as about the best potential management strategies.
An outreach activity to teach children about the many factors that are causing bee declines was created and utilized at the Pennsylvania State University 2017 Great Insect Fair (Figure 1). The outreach activity used a piece of yellow tissue paper with a bee drawn on it to represent a bee colony and a variety of knick knacks to represent different stressors. For example, diseases were signified by bouncy balls, varroa mites were symbolized as basketball shaped erasers, pesticides were represented by a water spray bottle, and poor nutrition was exemplified by removing a pillar of fake flowers from underneath the bee colony. Visitors could place the different stressors on the tissue paper until it eventually ripped. Then by talking through what the visitors had just done, we were able to explain both verbally and visually how it is the interaction of stressors and not one stressor alone that is likely causing bee declines. While this concept can be difficult to grasp, especially for children, this outreach activity successfully portrayed this point and it engaged children of all ages. Furthermore, we created and distributed the following handout which provides ways to help support our native pollinators (Figure 6).
Figure 6:Handout discussing how to support Pennsylvania bumble bees.