Progress 01/01/17 to 12/31/19
Outputs
Target Audience:Target Audience The primary target audience for this project were honeybee researchers and beekeepers. Many of the questions and experiments executed were established to test fundamental research hypotheses concerning the long-term effects of early developmental stress (high heat shock and oxidative stress) on honeybee workers and thus there is potentially limited applied uses. The exemption however being in Varroa treatment via heat. The basis of this technique is that Varroa destructor, a parasitic mite, has a lower critical temperature than bees. Heating hives at 40°C for an extended period of time has been shown to kill varroa. Varroa is usually present in developmental stages, particularly pupation. Thus, my research elucidates the potential side-effects of heat-treating varroa. Given this perspective, there might be some applied uses for beekeepers from my research in form of advice on the long-term effect of heat treatments. Effort I dedicated 100% effort to the scientific research conducted during my scholarship. Additionally, as part of my school's and department's goals as a public institution, there has been a great push to expose science to the general public. I have participated in several university sponsored programs that sought to educate the general public in science. Additionally, I participated in a pre-college orientation where I discussed the nature of our lab to incoming high school students. During these education and general public events, my role was to establish, monitor and execute a diverse set of interactive activities. There are still ongoing efforts to reach the scientific and honeybee keeper communities. Mainly though the preparation and execution of both written manuscripts in peer-review journals and oral presentations in conferences. Changes/Problems:Our original intention was to set up a Radio Frequency Identification (RFID) system in order to determine flight behavior between individuals that had been stressed during early development. The system was set up properly in our bee, stations and we were able to set up the instruments. However, there were unforseen complications. Primarily, it was that the chips were not properly secured to the bees. We made a small pilot where we tested different adhesives and tried to determine which was more effective. We determined that superglued chips were not removed even when subjected to violent shaking. However, when we attempted to introduce the marked bee they were instantly rejected from the colony. At this point it was to late in the season to continue experiments. Fortunately, however, we managed to get data regarding the long-term survivability after acute heat shock exposure for the 8 developmental stages. Additionally, our results supported the potential hormetic effect, beneficial effects of being exposed to low-dosages of stress, and as a result we did an additional set of experiment to investigate this phenomenom. What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?Results have been disseminated via international and local conferences. Such as the Entomological Society of America, The South Appalachian Honeybee Keeper Association. Additionally, results are currently being compiled and synthesized for the Journal of Experimental Biology as a publication. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Impact My project was able to evaluate the long-term effects of two stressors: the pesticide paraquat that induces oxidative stress and a high, but short heat exposure. The results of all experiments were radically different reinforcing the notion that each long-term effect might be stressor specific. My investigations would suggest that worker brood exposed to an acute, one-time dose of the herbicide paraquat were no different in behavioral and life-history patterns than non-exposed hive-mates. Despite the lack of phenotypic effects, gene expression analyses revealed that many genes associated with oxidative-stress response and general cellular maintenance were upregulated by stress. The results varied across different worker life history stages, which would suggest each worker caste exhibits unique compensatory mechanism to these early life stressors. Data collected from the second set of experiments, where heat shock was used showed both differences in observed behavior as well as in the molecular signatures. Individuals exposed to a 1-hour heat shock regimen showed increased thermal-resistance when compared to their untreated siblings. These particular results could suggest potential long-term benefits to early life exposure at least within the early part of adult worker life. Overall goals and accomplishments Objective 1 a) Major activities completed / experiments conducted: Behavioral recordings For the main study cohorts in the observation hives, behavioral observations were conducted daily from 8:00 - 17:00, following a previously established scan-sampling protocol. All observers were trained in advance to classify worker behavior according to 31 unique behavioral definitions, which were collapsed to 14 behavioral categories based on biological function in subsequent analyses Other hives in the UNCG bee yard were checked periodically for marked bees that had drifted into those colonies. ?c) Summary statistics and discussion of results Behavior and life history effects During the observation period of 19 days (cohort 1) and 31 days (cohort 2), no significant effect of treatment on the mortality of experimental workers was found (Figure 1A; Cohort 1: Χ2 = 1.6, df=1, p=0.2; Cohort 2: Χ2= 1.1, df =1, p= 0.3). Likewise, no statistically significant difference between experimental groups was found in the Age of First Foraging in either cohort (Figure 1B; Cohort 1: Χ2 = 1.3, df =1, p = 0.3; Cohort 2: Χ2 = 3.1, df=1, p=0.06). Overall, 52,445 behavioral observations were performed in the first replicate and 50,134 in the second. No significant differences were observed between treatments in the overall behavioral profiles in both replicates (Replicate 1, MANOVA: F28,413 = 1.5, p = 0.07 Replicate 2, MANOVA: F28,240 = 1.04, p = 0.4). Collapsed behavior analysis yielded similar results (Replicate 1, MANOVA: F14,427 = 1.18, p = 0.3; Replicate 2, MANOVA: F13,255 = 1.38, p = 0.2). Corresponding to the similarity in overall behavioral profiles between stressed and control groups, differences in single behavioral categories were not significant in either replicate (Table 2). No significant difference in the probability of reversing from foraging to nursing activities was found between stressed and control bees when measured in the first replicate (X2 = 0.157, df=1, p=0.69). d) Key outcomes or other accomplishments realized. Developmental stress affects later life in a number of organisms and this topic is also of practical relevance in the context of honeybee health. Our initial predictions that stress negatively affects adult behavior and life history were not confirmed. Specifically, this might be due to us not studying very old individuals which may be sensitive to early life stress. Social buffering of early life stress effects in social species has been documented and might explain why no phenotypic effect was visible but our paraquat exposure did not affect survival in isolation either. Thus, this study has uncovered stage-specific molecular compensatory mechanisms occurring weeks after stress exposure, that deserve further investigation. Objective 2 a) Major activities completed / experiments conducted: Sublethal heat exposure To determine a sublethal 1hr heat exposure temperature, frames of 5th instar larvae were placed at one of the following temperatures: 40°C, 45°C, 50°C and 55°C. After 1hr, frames were returned to their hives and each frame was checked a day before emergence, on day 20 after the egg was laid, according to the previously marked transparency sheet. A capped cell at this time point was interpreted as successful survival of the stress, while an empty cell was taken as indication for removal of the individual due to its death.? Resistance to increasing temperatures All emerging workers were subjected to increasing (0.2°C per minute) ambient temperatures, starting at 35°C until the last bee died. A hybridization oven (Model# 7930-00110 from the BellCo Glass Inc.) with manual temperature control was used. The machine was operated with a custom rotary speed, about 7 full rotations per minute. Standard 50 ml centrifuge tubes were modified to fit into the holders of the machine: The tubes were divided into four, 2 cm long sections to house 4 separate workers simultaneously. Observations were made after each minute to evaluate effects of the gradual heating. The temperature and time were recorded when each bee was knocked-out (KoT), defined as laying on her side unable to locomote or walk but with antennae or legs still visibly moving. In addition, the death temperature (DT), defined as complete cessation of movement, and the difference between knock-out-to-death time (terminal infirmity period, TIP) were determined. All three variables were compared among the four experimental groups.? c) Summary statistics and discussion of results Determining a Sublethal heat exposure at 5th instar and its effect on developmental rate Multiple temperatures were tested to determine the highest survivable 1hr heat exposure on 5th larval instars of three unrelated colonies. For all three colonies, survival of the heat stress varied among temperatures (Colony 1: 40°C:481/489, 45°C:408/508 , 50°C:102/409 , 55°C:0/333; Colony 2: 40°C:360/390, 45°C:402/442 , 50°C:18/318 , 55°C:0/350; Colony 3: 40°C:300/310, 45°C:347/431 , 50°C: 0/336, 55°C:0/408). Logistical regression showed significant effect of Temperature, but not Colony. Additionally, no interaction effects were found between Temperature and Colony. A one hour's exposure of 45°C was our selected stress treatment for subsequent experiments. Determining most and least heat susceptible developmental stages 100% removal was observed in all stages younger than 2nd instar (Figure 7). 2nd larval instars showed some survival but it was statistically lower than any other larval and pupal stages. No difference in removal was seen on developmental stages stressed after the 3rd instar. A one hour's exposure of 45°C was our selected stress treatment for subsequent experiments. Resistance to increasing temperatures Nested ANOVA results showed similar patterns, and confirmed treatment effects on KOT (Nested ANOVA: F3,59=4.1, p-value=0.0073), DT (Nested ANOVA: F3,59=2.9, p-value=0.033), DT (Nested ANOVA: F3,59=2.9, p-value=0.033), but not in TIP (Nested ANOVA: F3,59=0.17, p-value=0.92). Post hoc results showed similar results in all variables, with previously stressed workers, regardless of developmental stages, showing higher KT and DT than unstressed workers. (Figure 10A and 10B).
Publications
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Progress 01/01/17 to 12/31/17
Outputs
Target Audience:My project is primarily focused on answering fundamental questions and thus has limited applied uses. The exemption however is in a varroa treatment via heat. The basis of this technique is that Varroa has a lower survival temperature than brood. Heating brood at 40 degree for an extended period of time has been shown to kill off varroa. Varroa is usually present in early stages particularly pupations during ages 9-20. Thus, my research could give rise to the potential side-effects of heat-treating varroa. Target audiences:The project did not have any particular audience inmind. However, I desire to educate the public about science as well as collaborate with my scientific peers.I have disciminated my finding to the scientific community. This has been done by attending multiple conference such as the Entomological Society of America, which appealed to the general entomologist community. Efforts:As part of my schools and department'sgoals as a public institution, there has been a great push to expose science to the general public. I have participated in several university sponsored programs that sought to educate the general public in science. Additionally, participated in a pre-college orientation where I discussed the nature of our lab to incoming high-school students. Changes/Problems:Our original intention was to set up an Radio Frequency Identification (RFID) system in order to determine flight behavior between individuals that had been stressed during early development. The system was set up properly in our bee, stations and we were able to set up the instruments. However, there were unforseen complications. Primarily, it was that the chips were not properly secured to the bees. We made a small pilot where we tested different adhesives and tried to determine which was more effective. We determined that superglued chips were not removed even when subjected to violent shaking. However, when we attempted to introduce the marked bee they were instantly rejected from the colony. At this point it was to late in the season to continue experiments. Fortunately, however, we managed to get data regarding the long-term survivability after acute heat shock exposure for the 8 developmental stages. Additionally, our results supported the potential hormetic effect, beneficial effects of being exposed to low-dosages of stress, and as a result we did an additional set of experiment to investigate this phenomenom. What opportunities for training and professional development has the project provided?Due to being in a high Undergraduate Institution, the project has been used as a venuew to train undergraduates not only in basic bee keeping but also in proper scientific practices. I trained a Bio499 (Reseach credit) student in beekeeping techniques as well as data collection for my particular project. How have the results been disseminated to communities of interest?Results have been disseminated via international and local conferences. Such as the Entomological Society of America, The South Apalachian Honeybee Keeper Association. What do you plan to do during the next reporting period to accomplish the goals?We are currently determening whether a second field season will be needed. I am planning on graduating on December 2018. So most of my time will be placed to writing both for publications regarding both aims and to finish my thesis.
Impacts
What was accomplished under these goals?
1) We were able to finalize all the data analisys of this particular objective. Our findings did not find any particular differences between behavior repetoir between the different treatment groups, at least not severely different (Anova, p= 0.045). There was no differencealso in the age of foraging initiation between the different treatment groups as well. Our Rna seq experiment however did find some molecular differences. Mostly the findings show, that there is no consistent change between all the developmental stages. We did not find any genes that were consistently differentially expressed in stressed individuals, and thus no long-term adaptive molecular fixation. The results suggest that there might be long-term compensatory mechanism, that are unique to each caste. 2)My initial prediction involved that early developmental stress would have had a negative effect. However, we decided to expand and check for potential homertic effects. Hormesis occurs when exposed to low dosage of stress to actually achieve a hightened or beneficial effect. We ended up modifying a small incubation chamber and then collected individuals that had been stressed at three developmental stages (3rd, 5th, 14days) and tested whether there was any later life hormetic effect in the form of heat resistance. Our preliminary studies showed that individuals that were heat stressed at 13 days of age died in hotter temperatures.
Publications
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