Progress 03/01/23 to 02/28/25
Outputs
Target Audience:We continuously collaborated with the five beekeeping associations that are present in Southern California, which are the (Long Beach Beekeepers (LBB), the Beekeeping Association of Southern California (BASC), the Orange County Beekeeper Association (OCBA), the San Diego Beekeeping Society (SDBS) and the Los Angeles County Beekeepers Association (LACBA). They represent over 1400 local beekeepers and benefited from the research outlined in our proposal and were highly interested and supportive of the project. We attended more than 50 beekeeper meetings since the start of the project to provide research talks and/or updates about activities and projects that we run at the Center for Integrative Bee Research (CIBER). We also organized yearly bee health conference at the University of California Riverside in September 2022, 2023 and 2024, which was attended by more than 120 delegates, mostly beekeepers. We used these conferences to update beekeepers about our research activities, asked for feedback, and discussed future projects and research plans. As a result of our close links to the beekeeping community, our MS student Emilia Burnham who was working on this project was nominated as the 2024 Californian honey queen and was consequently selected as the National honey bee princess for 2025. She currently promotes bees and bee research throughout the United States. Changes/Problems:Over the past 5 months, the asctivities conducted at the Center for Integrative Bee Research (CIBER) weretargeted multiple times. They ranged from the theft of beekeeping equipment, attempted theft, the distribution of unfounded acquisitions of individuals to be engaged in scientific misconduct or the involvement into criminal activities, multiple cases of identity theft as well as phone-based harassments. A fire destroyed significant amounts of additional beekeeping equipment, the cause of the fire could so far not be determined. Such continuous attacks are not acceptable, and some are of criminal nature. They have impacted the work conducted at CIBER including activities that have been supported through this NIFA grant. It certainly delayed the completion of this project as well as the publication of manuscripts. What opportunities for training and professional development has the project provided?This project has provided substantial training and professional development opportunities for both undergraduate and graduate students. Several students were directly involved and supported through this work, receiving hands-on training in experimental design, data collection, chemical analysis, and interpretation. One-on-one mentorship was provided by both PIs, ensuring individualized guidance in key scientific and technical skills. Three undergraduate students, Nicole Salguero, Imelda Larreta, and Sam Shamie have been actively engaged in a subproject examining Varroa destructor reproductive success in larvae from survivor vs managed colonies. To address this, these students used gelatine capsules to isolate infested brood and quantified mite reproduction under controlled conditions. This work not only contributed to our broader research goal of the project, but also provided students with direct experience in hypothesis driven experimentation and a exciting opportunity to study host-parasite interactions. In addition, the graduate students participating in the project gained experience in mentoring, as they supervised and supported the training of undergraduate researchers. Though these activities, students developed critical skills in research coordination, scientific communication, and collaborative problem solving, preparing them for future roles in academic or applied research settings. How have the results been disseminated to communities of interest?PI Baer attended 28 beekeeper meetings over the past year and provided regular research talks and/or updates about activities and projects that we run as part of this project as well as other activities at the Center for Integrative Bee Research (CIBER). These efforts were highly appreciated by beekeepers and three associations offered him and other team members life long honorary memberships. We also organized our yearly bee health conference at the University of California Riverside in September 2024, which was attended again by more than 125 delegates, mostly beekeepers from the 5 participating beekeeper associations. We used the conference to update beekeepers about our various research activities, asked for feedback and discussed future projects and research plans. Based on feedback from beekeepers at our 2023 conference, we also offered workshops to discuss the idea to form a Southern Californian Honey Bee Collective, that would unite all Southern Californian Beekeepers to better support local beekeepers and address common concerns. Based on feedback provided during the conference, we also set up a CIBER advisory board where representatives from the beekeeping community meet with CIBER researchers on a regular basis for feedback, planning and the discussion of upcoming issues. Over the last 6 month we have written up monthly summaries of research, extension and teaching activities conducted at CIBER that has been published in separate articles in the journal BeeCraft, which is the largest beekeeping journal in the UK. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
During the second and final year of the project, we obtained several complete datasets that are currently written for publication or have already been submitted. We completed all experimental work where we inoculated commercial and local Southern California honeybees with heat-killed (dead) and live Nosema ceranae spores to trigger natural infections. The main aim of this work, conducted by MS student Emilia Burnham was to test whether survivor and managed honey bees respond differently to infections. As part of this work, we also dissected these bees to collect hemolymph to quantify the enzymatic activities of juvenile hormone, vitellogenin, chitinase, and phenol oxidase, which are all linked to immune responses in honey bees. This allowed us to test for differences in immune responses between honey bee genotypes and the effects of live versus dead spore exposures. We found that survivor managed honey bee genotypes have different immune system responses to pathogens such as Nosema ceranae heat-killed spores. Interestingly, managed honey bees showed increased tolerance for Nosema ceranae, measured by higher infection intensities and mortality compared to survivor bees. Juvenile hormone expression did not differ between genotype and treatments but was somewhat elevated 6 and 12 days after the treatments. Chitinase activity differed between managed and survivor bees 6 days after the treatments were applied and was higher in bees that received heat killed dead spores compared to bees we inoculated with living Nosema spores. In summary, we found the surprising result that managed honey bee genotypes seem better able to cope with N. ceranae infections compared to survivor bee genotypes. This is an important finding, because it implies that tolerance traits found in survivor bees against Nosema became inherited through commercial bee stock rather than though their African ancestry. We also conducted additional analyses to study the chemical communication among honey bee larvae in response to Varroa destructor infestation. To investigate whether the presence of mites triggers a chemical signal in larvae, we conducted a lab-based experiment using larvae from eight colonies (four survivor and four managed). Larvae were placed in sealed chambers either alone or in the presence of mites, and volatile compounds were collected across three sequential time points (2h, 3h, and 13h) over an 18h period. Using GC-MS, we detected over 9,000 volatile features across the entire dataset. Survivor colonies emitted the highest number of compounds in the first 2h after the start of the experiment, with a sharp decline in later samples, suggesting a rapid and transient response. In contrast, larvae of the managed genotype emitted few to no volatiles during the first 2 hours after exposure to mites, but they started to release an increasing number and amount of volatiles 3 hours after the start of the exposure experiment, consistent with a delayed response to mite infestations. Overall, we detected 12 unique compounds in survivor colonies, and 11 in managed colonies. After filtering for consistency across replicates, three compounds were retained as candidate biomarkers: 1,3-Diacetylbenzene, β-Curcumene, and 1-Heptatriacotanol. These findings suggest that survivor larvae mount a faster, possibly tolerance-based chemical response to Varroa infestations, which may contribute to their resilience. These results are under further investigation and manuscript preparation is underway. These results are currently written up for publication and will be part of a thesis chapter of PhD student Genesis Chong. In addition, we conducted a proteomic study to investigate the early immune response of honey bee larvae to Varroa destructor exposure. Using a well-controlled experimental design, larvae from both survivor and manages genotypes were exposed to mites in petri dishes for 2 hours. Hemolymph was then extracted for LC-MS/MS proteomic profiling. A total of 705 protein groups and 3,963 peptides were identified across all samples with 1% FDR threshold. Comparisons between treatment and control groups revealed clear proteomic shifts following mite exposure. Among the analyzed group, 41 proteins were differentially expressed and 14 downregulated in mite-exposed individuals. In the survivor genotype, and additionally 25 proteins were differentially expressed under the same treatment, indicating genotype-specific response. Principal component and PLS-DA analyses showed distinct clustering by treatment condition, confirming consistent proteomic shifts. Go enrichments of differential proteins highlighted functions related to stress response, immune system processes, and metabolic reprogramming, while KEGG pathway analysis pointed to alterations in lysosome, oxidative phosphorylation, and endocytosis pathways, all of which have known roles in innate immunity and cellular stress adaptation. Taken together, these findings suggest that Varroa exposure elicits a rapid immune-related proteomic response in honey bee larvae, with genotype-dependent differences in both magnitude and pathway specificity. Survivor colonies may exhibit more coordinated or robust molecular response to infestation, which aligns with our previous findings on volatile emission and behavioral tolerance. These proteomic results complement our ongoing work on chemical communication and social immunity and will be integrated into a comprehensive manuscript in preparation for peer-review. We also conducted several behavioral experiments to study how healthy worker bees interact with infected nest mates, which was part of the doctoral thesis of Sakshi Watts. We set up arenas using 15-cm Petri dishes to better understand how the disease spreads within a colony among colony members and whether this differs between the two genotypes. To do this, we conducted behavioral assays to record and analyze all contact-based interactions between sick and healthy bees under red light in lab conditions. Pilot data we initially obtained indicated that managed bees increase both friendly (antennation, trophallaxis) and aggressive (biting, stinging) behaviors towards sick bees. In contrast, survivor bees engage in more friendly interactions among healthy individuals but exhibit higher aggression toward sick bees. To confirm these differences statistically we currently increase the samples sizes of these behavioral assays, especially to test additional colonies for each genotype. As part of this work, Sakshi Watts has also investigated the differences in the volatile chemical profiles of healthy and Nosema ceranae-infected adult honey bees during two critical phases of infection: the early infection stage around day 7, and the peak infection phase on day 12. We were interested to identify potential chemical markers that are associated with infection status and whether they differ between managed and survivor bee genotypes. All experiments have been conducted, and a first statistical analysis revealed the presence of several volatile compounds that are uniquely present in infected bees. One of them was detected exclusively in infected survivor bees, suggesting genotype-specific responses to infection. We have started to write these results up as a manuscript that will be submitted to a peer reviewed journal before the end of the year.
Publications
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Progress 03/01/23 to 02/29/24
Outputs
Target Audience:We continued to collaborateclosely with thefive beekeeping groups that are present in Southern California (Long Beach Beekeepers (LBB), Beekeeping Association of Southern California (BASC), Orange County Beekeeper Association (OCBA), the San Diego Beekeeping Society (SDBS) and the Los Angeles County Beekeepers Association (LACBA) that benefit from the research outlined in our proposal and are highly interestedand supportive of the project.We attended more than 25 beekeepermeetings since the start of the project to provide researchtalksand/or updates about activities and projects that we run at the Center for Integrative Bee Research (CIBER).We also organized our yearlybee health conference at the University of California Riverside in September 2023, whichwas attended by more than 120 delegates, mostly beekeepers. We used the conference to update beekeepers about our research activities, asked for feedback, and discussed future projects and research plans. PI Baer was invited to the yearly conferenceof the California State Beekeeper Association where he provided a plenary talk about the research conducted at CIBER(including activities funded through this grant) and was a panel member discussing bee health-related issues. The MS student (Emilia Burnham) working on thisproject was nominated by a beekeeper as the 2025 honey queen and was consequently selected as the California representative. She promotes bees and bee research regularly (seehttps://insideucr.ucr.edu/awards/2024/07/15/ca-honey-queen-ucr-entomology-graduate-student?fbclid=IwY2xjawEfPFxleHRuA2FlbQIxMQABHXOm66SvlA2Zz4AAM17FGBAKBl0RNoLp4uUujAFWnEM46sLyPxpEhJUs3g_aem_qVozuAE2w-AakkierQY3Dg) and intends to become the national honey queen in 2025. Changes/Problems:Due to admistrative reasons,the project outlined in in this grant applicationstarted with a delay of several months. No other issues occured since and the project is on track. What opportunities for training and professional development has the project provided?Several undergradaute and graduate students became invoilved in the project and were supportted through this project. They were trained on a one to one basis by teh two PIs in the planning of experiments, teh methodologies used and teh analyses of data. Postgraduate students gained experience in the supervision of undergraduate students. How have the results been disseminated to communities of interest?We attended more than 25 beekeepermeetings since the start of the project to provided researchtalksand/or updates about activities and projects that we run at the Center for Integrative Bee Research (CIBER).We also organized our yearlybee health conference at the University of California Riverside in September 2023 whichwas attended by more then 120 delegates, mostly beekeepers. We used the conference to update beekeepers about our research activities, asked for feedback and discussed future projects and research plans. PI Baer was invited to the yearly conferenceof the California State Beekeeper Association where he provided a plenary talk about the research conducted at CIBER(including activities funded through this grant) and was a panel member discussing bee health related issues. What do you plan to do during the next reporting period to accomplish the goals?We will continue to run experiments as outlined in the proposal, finalise analyses of data colelcted so far, especially the statitical analyses of the data we obtained from our proteomic and metabolomic anlayses and write findings up for publication in peer revoewed journals.
Impacts
What was accomplished under these goals?
We conducted a series of experiments to quantify responses of survivor and managed honeybees to parasite infections. This work was done by 2 PhD students (Sakshi Watts and Genesis Chong) and MS student (Emilia Burnham) with the help of several undergraduate students. Experiment 1:We identified key volatile compounds emitted by survivor and managed honeybee larvae when exposed to mite parasites. To do this, we collected 7-day-old larvae from four managed and four survivor colonies, grouped them into control and treatment groups, and used 200 bee larvae per colony. We conducted a total of 8 rounds of volatile collections with a push-pull air system method. We collected samples at three time points (minutes 1-60, minutes 61-180, and minutes 181-780 minutes). We collected a total of 288 samples, and eluted compounds for metabolite identification. We used GC-MS and identified a total of 9087 compounds over three different sampling times and 8 experiments. We found significant effects of volatile profiles between treatments (i.e. between larvae exposed to the mites versus non-exposed controls) and identified 23 compounds that are responsive to mite infestation of larvae. Survivor larvae appear to have a distinct emission patterns for some of these compounds, particularly when exposed to mites, and managed larvae show a delayed response. Key compounds identified in the survivor larval response include beta-Curcumene, 1,3-Diacetylbenzene, and 1-Heptatriacotanol. Experiment 2:We quantified protein expression in survivor and managed honeybee larvae after exposure to Varroa mites. To do this we collected 120 7-day-old larvae per colony from 4 managed and 4 survivor colonies and randomly allocated them to 2 treatment groups (mite exposed versus control). We conducted a total of 6 rounds of experiments using petri dishes as arenas to conduct the exposure experiments. To do this we used groups of 20 larvae as control and exposed 20 additional larvae to 10 mites as treatment. We conducted three technical replicates (petri dishes) per assay and sampled bees 2, 5, and 10 hours after mite exposure. To quantify proteomic responses, we collected the hemolymph and extracted proteins using standard methods, followed by MS-MS and protein identification. We found a total of 443 proteins and are currently conducting statistical analyses to compare protein expression levels in the 2 genotypes. Preliminary analyses revealed the presence of 16 immune-related proteins that were differentially expressed among treatments. They included three proteins with known antimicrobial activity (abaecin precursor, apidaecin precursor, defensin precursor, and one protein part of the innate immune system (venom protein). Interestingly we also founddifferentially expressed heat shock proteins (Hsp70, Hsp60, Hsp90, Hsp83, and Hsp cognate 3 precursor). Experiment 3:We compared volatile profiles in response to fungal infections in managed and survivor bee genotypes. To do this, we provided 2 colonies with capped brood frames for bees to emerge in an incubator. We inoculated bees withNosema ceranaespores or sugar water controls and conducted volatile collections after 7 and 12 days, representing early and maximal infection stages. Volatile collections were conducted in a darkroom with a flow rate of 1 l/min of clean air, initially passed through a charcoal filter, and pulled through the cups at 0.5 l/min after passing through the filters. Collections ran for 6 hours, after which collected volatiles were eluted from filters and analyzed using GC-MS. We found that infected bees emitted several unique volatiles relative to uninoculated bees, and survivor bees emit unique compounds relative to managed bees. We are currently confirming identifications of approximately 20 compounds of interest. Experiment 4: We quantified Immune response differences between managed and survivor honeybees following Nosema infections.We triggered an immune response in the two bee genotypes using heat-killed spores compared to infections with live spores or inoculation containing no spores as a control. To do this we fed newly hatched individual bees with a 1:1 sucrose and water solution that contained live, dead, or no spores. We sampled bees at 6, 12, and 15 days after inoculation and extracted their midguts to quantify Nosema infections. We found higher spore counts in managed compared to survivor honeybees. However, whereas managed honeybees clear infections after 15 days, survivor bees remained infected. Ongoing work is now using these bee samples to quantify the dynamics of hormones and proteins involved in immune responses, including juvenile hormone, vitellogenin protein, and chitinase enzyme.
Publications
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