Progress 01/01/21 to 12/04/24

Outputs
Target Audience:The target audiences that were the focus of the effort for the duration of the project include the honey bee research community, stakeholders (beekeepers andcrop growers), and the general public.This project incorporates fundamental and applied research that aimed to provide the framework necessary to develop novel products capable of being used by the beekeeping industry to increase sustainability and profitability. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Martin Ewert, A. Honey Bee Colony Health 101. Capital Area Beekeepers Association monthly meeting. Baton Rouge, LA. Outreach presentation delivered to over 25 local beekeepers. January 2024. Martin Ewert A, McMenamin A, Rainey V, Adjaye D, Ricigliano V. Engineered microalgae feed to bolster honey bee disease resistance. American Bee Research Conference (ABRC). New Orleans, LA. 10-minute oral presentation.January 2024. Martin Ewert A,McMenamin A, Simone-Finstrom M, Ricigliano V. Microalgal feed additives for improved honey bee health. "More than the sum of their parts: Sociobiology and health of eusocial insects" symposium. Entomological Society of America (ESA) Southeastern Branch (SEB) Meeting. Augusta, GA. Invited 12-minute oral presentation.March 2024. Martin Ewert A, McMenamin A, Adjaye D, Rainey V, Husseneder C, Ricigliano V. Enhancing honey bee immunity with microalgal feed additives. LSU Entomology Graduate Student Symposium. Baton Rouge, LA. Poster. October 2024. Martin Ewert, A. Improving pollen subs with microalgae. LBA and USDA-ARS Joint Fall Field Day. Baton Rouge, LA. Outreach presentation delivered to nearly 200 Louisiana beekeepers. October 2024. Martin Ewert, A. Evidence-based beekeeping. Capital Area Beekeepers Association monthly meeting. Baton Rouge, LA. Outreach presentation delivered to over 30 local beekeepers. November 2024. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? We made significant progress during the final reporting period of this project. Project objectives were met and have peer reviewed publications and/or a patent associated with each objective. The 2024 reporting period focused on characterizing the immunomodulatory effects of microalgae in honey bees.Two honey bee genetic stocks (Russian and Pol-line) were fed diets consisting of sucrose-only, pollen,Chlorellaor spirulina. Nutrigenetic variation was assessed by body weight measurements followed by transcriptome sequencing. Standard differential gene expression analysis and supervised machine learning algorithms were used to measure genotype-specific transcriptional responses to these diets.This approach identified well-known (i.e.,vitellogeninandJhe) and novel (i.e.,drop dead,takeoutandGlob1) biomarkers of nutritional health that showed a significant genetics-by-diet interaction. Furthermore, it revealed that Russian bees showed a signature of tighter transcriptional control and delayed behavioral maturation as compared to Pol-line bees, suggesting alternative nutrient assimilation strategies between the two stocks. Lastly, we observed higher expression of well-described humoral and cellular immune genes in microalgae-fed bees, though spirulina was significantly more immunogenic thanChlorella.Sustainable and secure food production hinges on effective management of livestock pollinators. This study shows that there are unique benefits of two nutritious microalgae species for honey bees, but that genetics are an important consideration for optimal diet formulation. Another projectinvestigated the impacts of microalgal feed additives on honey bee immunity and longevity. Caged bees were fed a control artificial diet, or the same diet supplemented with pollen, spirulina, or chlorella for one week, after which gene expression and longevity were measured. Both spirulina- and chlorella-fed bees lived longer than controls. Spirulina-fed bees exhibited significantly higher expression of several antimicrobial peptide-encoding genes and had superior bacterial clearing ability when injected with liveE. colicells. This observed increased immunocompetence likely resulted from elevated AMP levels. Overall, our findings indicate that spirulina can support greater honey bee health and immune function with no apparent health costs when used as a component of supplemental feed. Additional work is needed to further understand these effects in a more field-realistic setting, but we propose that spirulina-containing feeds have potential to better support honey bee resilience in the context of modern beekeeping.

Publications

• Type: Peer Reviewed Journal Articles Status: Published Year Published: 2021 Citation: Ricigliano, V.A., Ihle, K.E. & Williams, S.T. Nutrigenetic comparison of two Varroa-resistant honey bee stocks fed pollen and spirulina microalgae. Apidologie 52, 873886 (2021). https://doi.org/10.1007/s13592-021-00877-3
• Type: Peer Reviewed Journal Articles Status: Published Year Published: 2021 Citation: Ricigliano, V. A., Cank, K. B., Todd, D. A., Knowles, S. L., & Oberlies, N. H. (2022). Metabolomics-guided comparison of pollen and microalgae-based artificial diets in honey bees. Journal of agricultural and food chemistry, 70(31), 9790-9801.
• Type: Peer Reviewed Journal Articles Status: Published Year Published: 2022 Citation: Nichols, B. J., & Ricigliano, V. A. (2022). Uses and benefits of algae as a nutritional supplement for honey bees. Frontiers in Sustainable Food Systems, 6, 1005058.
• Type: Peer Reviewed Journal Articles Status: Published Year Published: 2023 Citation: McMenamin, A., Weiss, M., Meikle, W., & Ricigliano, V. (2023). Efficacy of a microalgal feed additive in commercial honey bee colonies used for crop pollination. ACS Agricultural Science & Technology, 3(9), 748-759.
• Type: Peer Reviewed Journal Articles Status: Published Year Published: 2024 Citation: Ricigliano, V.A., McMenamin, A., Martin Ewert, A. et al. Green biomanufacturing of edible antiviral therapeutics for managed pollinators. npj Sustain. Agric. 2, 4 (2024). https://doi.org/10.1038/s44264-024-00011-7

Progress 01/01/23 to 12/31/23

Outputs
Target Audience:Target audiences reached by our efforts this reporting periodinclude beekeeper stakeholders, farmers, pollinator researchers, and the broader scientific community. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Grad student AllysonMartin Ewert received the following awards based on her participation in this project: The John and Grace Roussel Graduate Fellowship Award in Entomology (PhD).Presented by the Louisiana State University Department of Entomology, Oct. 2023. 1stplace winner of the 10-minute oralpresentationstudent competition (PhD). LSU Department of Entomology Graduate Symposium, Oct. 2023. 2ndplace winner of the 10-minute oralpresentationstudent competition (PBT: Apiculture).Entomological Society of America Annual Meeting, Nov. 2023. Foundation for the Preservation of Honey Bees (FPHB) Scholarship.Presented by FPHB. Nov. 2023. How have the results been disseminated to communities of interest?Project-relevant presentations delivered by lab members: ?Martin Ewert, A.,McMenamin, A., Rainey, V., Adjaye, D., Ricigliano, V. Engineered microalgae feed to bolster honey bee disease resistance. 10-minute oralpresentation. Entomological Society of America (ESA) Annual Meeting.Nov. 2023. National Harbor, MD.2ndPlace(Grad Competition PBT: Apiculture, 10-minute oralpresentation). Pathogenic diseases threaten the global beekeeping industry by weakening honey bee (Apis mellifera) health and productivity. To combat record colony losses, beekeepers urgently need access to effective, practical, and sustainable treatments against pathogens. Addressing this problem, we have genetically engineered microalgae to express dsRNA specific to honey bee pathogens. When consumed, these dsRNA-producing engineered strains provide dietary amino acids that support honey bee nutrition and also induce an RNAi immune response against target pathogens. In previous work, we determined that adult bees fed engineered microalgae that produces DWV-targeting dsRNA show reduced viral load after injection with DWV compared to controls. Here, we test whether this microalgae-induced disease resistance can also be conferred to younger life stages. In this study, honey bee larvae were rearedin vitroon royal jelly diets containing either non-engineered microalgae (Vector), non-specific dsRNA-producing engineered microalgae (YFP), or DWV-targeting engineered microalgae (DWV1 and DWV2). The resulting pupae who were reared on DWV-targeting microalgae diets had lower DWV load 48 and 72 hours post injection compared to controls. Our results show that engineered microalgae feed additives have potential to benefit whole colonies by dually supporting proper nutrition and improving disease resistance across all life stages of bees. Engineered microalgae feed additives represent a highly scalable, sustainable, and effective opportunity to bolster honey bee health. Martin Ewert, A.,McMenamin, A., Rainey, V., Adjaye, D., Ricigliano, V. Engineered microalgae feed to bolster honey bee disease resistance. 10-minute oralpresentation. LSU Entomology Graduate Student Symposium.Oct. 2023. Baton Rouge, LA.1stPlace(PhD 10-minute oralpresentation). Pathogenic diseases threaten the global beekeeping industry by weakening honey bee (Apis mellifera) health and productivity. To combat record colony losses, beekeepers urgently need access to effective, practical, and sustainable treatments against pathogens. Addressing this problem, we have genetically engineered microalgae to express dsRNA specific to honey bee pathogens. When consumed, these dsRNA-producing engineered strains provide dietary amino acids that support honey bee nutrition and also induce an RNAi immune response against target pathogens. In previous work, we determined that adult bees fed engineered microalgae that produces DWV-targeting dsRNA show reduced viral load after injection with DWV compared to controls. Here, we test whether this microalgae-induced disease resistance can also be conferred to younger life stages. In this study, honey bee larvae were rearedin vitroon royal jelly diets containing either non-engineered microalgae (Vector), non-specific dsRNA-producing engineered microalgae (YFP), or DWV-targeting engineered microalgae (DWV1 and DWV2). The resulting pupae who were reared on DWV-targeting microalgae diets had lower DWV load 48 and 72 hours post injection compared to controls. Our results show that engineered microalgae feed additives have potential to benefit whole colonies by dually supporting proper nutrition and improving disease resistance across all life stages of bees. Engineered microalgae feed additives represent a highly scalable, sustainable, and effective opportunity to bolster honey bee health. Martin Ewert, A.Natural products for honey bee health. 10-minute oralpresentation.Louisiana Beekeepers Association and USDA-ARS Honey Bee Breeding, Genetics, and Physiology Laboratory Annual Fall Field Day. Oct. 2023. Baton Rouge, LA. Martin Ewert, A.,McMenamin, A., Ricigliano, V. Engineered microalgae feed to bolster honey bee disease resistance. 10-minute paperpresentation.Entomological Society of America (ESA) Southeastern Branch Meeting. Mar. 2023. Little Rock, AR, USA. Pathogenic diseases threaten the global beekeeping industry by weakening honey bee health and productivity. To combat record colony losses, beekeepers urgently need access to effective, practical, and sustainable treatments against pathogens. Addressing this problem, we have genetically engineered microalgae to express dsRNA specific to honey bee pathogens. When consumed, these engineered strains provide dietary amino acids and induce an RNAi immune response against target pathogens. Here, we tested the efficacy of diets augmented with engineered microalgae to reduce pathogen loads and increase survival of pathogen-challenged bees. When challenged with deformed wing virus (DWV), bees fed microalgae targeting DWV had greater survival and significantly reduced viral titers relative to controls. Additionally, larvae rearedin vitroon an artificial diet containing the same microalgae strains and injected with DWV as pupae had fewer disease deformities and greater survival relative to those reared on the control diet. Our ongoing experiments will test whether engineered microalgae targeting the microsporidian gut parasiteNosema ceranaecan confer disease resistance to bees. Our current results show that engineered microalgae are promising feed additives to improve honey bee disease resistance. With our ongoingNosemastudies, we will better understand whether algae feed-based therapies can be applied to other types of honey bee pathogens. What do you plan to do during the next reporting period to accomplish the goals?We will continue developing improved strains of antiviral microalgae and scaling up growth in our 100 L photobioreactor for larger experimental designs involving virus-infected bees.

Impacts
What was accomplished under these goals? The primary objective of this project is to systematically characterize the nutritional value and functional properties ofmicroalgae in honey bees while developing engineered strains to improve targeted aspects of bee nutrition and immunity. Significant progress was made towards accomplishing project goals this reporting period. We developed multiple strains of enginered microalgae (cyanobacteria / blue-green algae) that express double stranded RNAs (dsRNAs) against honey bee viruses. Here, we developed a novel antiviral platform for bees using the edible photosynthetic cyanobacterium Synechococcus elongatus UTEX 2973. Cyanobacterial strains were engineered to induce RNA interference (RNAi) immune responses when fed to bees. Treatments targeting deformed wing virus - a notorious pathogen - suppressed viral infection and improved survival in honey bees. This design presents a versatile and sustainable therapeutic that can be directly incorporated into supplemental feeds for managed pollinators to mitigate viruses and support global food security. Cyanobacteria are promising cellular factories for carbon-negative biomanufacturing due to their photosynthetic growth and easy scalability. Cyanobacterial biomass has historically been part of human diets and is nutritious to livestock animals, including honey bees. We engineered S2973 to express recombinant dsRNAs that require no purification prior to delivery, are stable when mixed into feed, and protected during gastric transport. Next, we developed antiviral strains and tested their efficacy in virus-challenged honey bees. Cyanobacteria have been used for the manufacture and oral delivery of protein therapeutics but have not yet been developed for RNAi applications. We designed a set of modular genetic parts to engineer dsRNA production in S2973. Parts consisted of two synthetic inverted promoters lacking ribosome binding sites that flank the target sequence for dsRNA production. A dsRNA expression cassette containing the yellow fluorescent protein (YFP) coding sequence was introduced into S2973 by homologous recombination with an integrative vector to generate strain S2973-YFP. Once genomic integration was confirmed using primers flanking the integration site , the strain was maintained up to 19 months without selection and RNA expression was periodically checked by RT-qPCR. dsRNA accumulation was stabilized by CRISPR-Cas9-mediated knockout of RNase III , a non-essential dsRNA-specific endonuclease.Cyanobacteria have been used for the manufacture and oral delivery of protein therapeutics but have not yet been developed for RNAi applications. We designed a set of modular genetic parts to engineer dsRNA production in S2973. Parts consisted of two synthetic inverted promoters lacking ribosome binding sites that flank the target sequence for dsRNA production.A dsRNA expression cassette containing the yellow fluorescent protein (YFP) coding sequence was introduced into S2973 by homologous recombination with an integrative vector to generate strain S2973-YFP. Once genomic integration was confirmed using primers flanking the integration site , the strain was maintained up to 19 months without selection and RNA expression was periodically checked by RT-qPCR. dsRNA accumulation was stabilized by CRISPR-Cas9-mediated knockout of RNase III , a non-essential dsRNA-specific endonuclease. We engineered S2973 to induce RNAi against DWV, a notorious pathogen linked to the deaths of millions of honey bee colonies worldwide. Two dsRNA-expressing strains (S2973-DWV1 and S2973-DWV2) targeted different regions of the DWV genome. Adult bees were fed the formulated strains ad libitum for 4 days and then injected with DWV. This assay mimics the natural route of DWV transmission via parasitic mites feeding on bees. Preliminary trials indicated that the S2973-DWV2 strain can limit virus replication and increase bee survival . In a larger experiment, dsRNA strains significantly reduced DWV levels and upregulated gene expression of dicer, the initiating step of the RNAi pathway. These results indicate immune modulation by dsRNA strains, but not by S2973-NR . Honey bees have a non-specific dsRNA-mediated antiviral response, which may explain dicer upregulation and DWV reduction in bees fed S2973-YFP. Strain S2973-DWV2 had the strongest antiviral effects and kept bees alive the longest. After DWV injection, median survival was 19 days for bees fed S2973-DWV2 and 11 days for bees fed S2973-NR (no dsRNA control). Therefore, engineered S2973 treatments can induce sequence-specific antiviral RNAi against DWV.

Publications

• Type: Journal Articles Status: Awaiting Publication Year Published: 2024 Citation: Ricigliano, V. A. et al. Green biomanufacturing of edible antiviral therapeutics for managed pollinators. npj Sustainable Agriculture. In Press (2024).

Progress 01/01/22 to 12/31/22

Outputs
Target Audience:The target audience for this project are researchers focused on applied beekeeping, large-scalecommercial beekeepers, hobby beekeepers, and, more broadly, agriculturalresearchers.During this reporting period, we published one scientific article related to the project in ACS Agricultural and Food Chemistry, which is an agricultural and chemistry journal. We also published a review article in Frontiers in Sustainable Food Systems, which is aligned with the UN Sustainable Development Goals, whichexplores the intersection of food systems, science and practice of sustainability. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Through the ORISE DOE postdoc position listed in the budget, we have hired postdoctoral researcher AlexanderMcmennamin who is currently being trained in recombinant DNA technologies, honey bee nutrition, andalgae biotechnologyby PI VincentRicigliano. How have the results been disseminated to communities of interest?Results from this project in this reporting period were published in the journal ACS Agricultural and Food Chemistry, which is read by applied beekeeping researchs and, more broadly, agricultural researchers.Ricigliano, V. A., Cank, K. B., Todd, D. A., Knowles, S. L., & Oberlies, N. H. (2022). Metabolomics-Guided Comparison of Pollen and Microalgae-Based Artificial Diets in Honey Bees.Journal of Agricultural and Food Chemistry,70(31), 9790-9801. We published a review article highlighting the uses and benefits of algae as a nutritional supplement for bees. This work was published in Frontiers in Sustainable Food Systems, which is aligned with the UN Sustainable Development Goals, andexplores the intersection of food systems, science and sustainability. Nichols BJ, Ricigliano VA. Uses and benefits of algae as a nutritional supplement for honey bees. Frontiers in Sustainable Food Systems 2022; 6 This year, this work was presented to beekeeper stake holders at the following meetings: Ricigliano V. Uses and benefits of algae as a nutritional supplement for honey bees. Louisiana State Beekeepers Association Annual Meeting 2022 Ricigliano V. (keynote speaker). Engineered microalgae as a novel platform to improve honey bee pathogen resistance. California StateBeekeepers Association Annual Meeting 2022 McMenamin A. Engineered microalgae as a novel pollen substitute and therapeutic delivery system. American Beekeeping Research Conference 2022 What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? The primary objective of this project is to systematically characterize the nutritional value and functional properties ofmicroalgae in honey bees while developing engineered strains to improve targeted aspects of bee nutrition and immunity.This year, we madeconsiderable progress towards these objectives. We first tested and compared multiple differentspecies and strains of microalgae for their nutritional value and immune-modulatingeffects. We formulated novel microalgae diets usingChlorella vulgarisandArthrospira platensis(spirulina) biomass and fed them to young adult honey bee workers. Diet-induced changes in bee metabolite profiles were studied relative to a natural pollen diet using liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS) metabolomics. Untargeted analyses of pollen- and microalgae-fed bees revealed significant overlap, with 248 shared features determined by LC-MS and 87 shared features determined by GC-MS. Further metabolomic commonalities were evident upon subtraction of unique diet features. Twenty-five identified metabolites were influenced by diet, which included complex lipids, essential fatty acids, vitamins, and phytochemicals. The metabolomics results were useful to understand mechanisms underlying favorable growth performance as well as increased antioxidant and heat shock protein gene expression in bees fed the microalgae diets. We concluded that the tested microalgae have potential as sustainable feed additives and as a source of bee health-modulating natural products. Further, metabolomics-guided diet development could eventually help tailor feed interventions to achieve precision nutrition in honey bees and other livestock animals. This work was published under the following citation:Ricigliano, V. A., Cank, K. B., Todd, D. A., Knowles, S. L., & Oberlies, N. H. (2022). Metabolomics-Guided Comparison of Pollen and Microalgae-Based Artificial Diets in Honey Bees.Journal of Agricultural and Food Chemistry,70(31), 9790-9801. We developed multiple strains of enginered microalgae that express double stranded RNAs (dsRNAs) against honey beepathogens. In order to do this we developed modular genetic parts for the expression of targetdsRNA.Weformulateddiets that contain dsRNA-expressing algae strains and fed them topathogen challenged honeybees. Bees fed engineered diets targeting the genome of Deformed Wing Virus had improved resistance and survival outcomes. We are currently testing these diets against additonal viruses and pathogens. We aim to submit a peer-reviewed manuscript before the end of 2023.

Publications

• Type: Journal Articles Status: Published Year Published: 2022 Citation: Ricigliano, V. A., Cank, K. B., Todd, D. A., Knowles, S. L., & Oberlies, N. H. (2022). Metabolomics-Guided Comparison of Pollen and Microalgae-Based Artificial Diets in Honey Bees. Journal of Agricultural and Food Chemistry, 70(31), 9790-9801.
• Type: Other Status: Published Year Published: 2022 Citation: Nichols BJ, Ricigliano VA. Uses and benefits of algae as a nutritional supplement for honey bees. Frontiers in Sustainable Food Systems 2022; 6.
• Type: Conference Papers and Presentations Status: Under Review Year Published: 2023 Citation: McMenamin A, Weiss M, Meikle W, Ricigliano V. Microalgae-augmented feed improves performance of commercially managed honey bee colonies used for crop pollination. ACS Agricultural Science and Technology
• Type: Conference Papers and Presentations Status: Awaiting Publication Year Published: 2023 Citation: McMenamin A, Weiss M, Meikle W, Ricigliano V., Engineered microalgae as a novel pollen substitute and therapeutic delivery system. American Bee Research Conference

Progress 01/01/21 to 12/31/21

Outputs
Target Audience:The target audience for this project are researchers focsed on applied beekeeping as well as commercial beekeepers. During this reporting period, we have published one scientific article related to the project in the journal Apidologie, which is bee-centric journal that is read by researchers as well as beekeepers. Changes/Problems:In the process of generating recombinant microlagae strains to express dsRNAs against bee pathogens, we encountered problems related to genetic transformation of target strains. Specifically, we have been attempting to integrate DNA constructs into neutral sites of the host algae genome, which have been found to not interfere with growth performance. However, we have been recovering largely single recombinantion events, instead of the preferred double recombination events in which the target genome region is completely replaced by the introduced gene construct. We are in the process of troubleshooting this by attempting different methods of DNA introduction into the host algae genomes including linearization followed by electroporation as well as triparental mating conjugation with donor E.coli strains. What opportunities for training and professional development has the project provided?Through the ORISEDOE postdoc position listed in the budget, we have hired postdoctoral researcher Alexander Mcmennamin who is currently being trained in recombinant DNA technologies and algae maintenence by PI Vincent Ricigliano. How have the results been disseminated to communities of interest?Results from this project in this reporting period were published in the journal Apidologie, which is read by applied beekeeping researchers and beekeepers alike. Ricigliano, V. A., Ihle, K. E., & Williams, S. T. (2021). Nutrigenetic comparison of two Varroa-resistant honey bee stocks fed pollen and spirulina microalgae. Apidologie, 52(4), 873-886. Chicago Further, findings from the published study were highlighted in a USDA-ARS press release: https://www.ars.usda.gov/news-events/news/research-news/2021/breeding-honey-bees-for-adaptation-to-regionalized-plants-and-artificial-diets/ What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? The primary objective of this project is to systematically characterize the nutritional value and functional properties of microalgae in honey bees while developing augmented strains to improve targeted aspects of bee nutrition and immunity. Despite the hurdles of the COVID-19 pandemic, we were able to make considerable progress towards these objectives. First, we characterized the nutritional value of Arthrospira (spirulina) microalgae biomass in two different honey bee stocks using cage bioassays. We found that spirulina diets led to improved physiological markers including increased body weight, vitellogenin and abdominal lipid stores. Spirulina-fed bees were not significantly different from pollen-fed bees based on these metrics. Further, this study revealed evidence for honey bee genotype-dependent responses to both spirulina and pollen diets, suggesting thatgenotype-dependent nutritional responses are present in honey bees, with promising implications for breeding efforts and tailored approaches to diet and health. This work was published in the journal Apidologie (Ricigliano, V. A., Ihle, K. E., & Williams, S. T. (2021). Nutrigenetic comparison of two Varroa-resistant honey bee stocks fed pollen and spirulina microalgae. Apidologie, 52(4), 873-886.) Second, we have begun to test additional species and strains of microalgae for their nutritional value and immune-modulating effects. In particular, we have begun testing the green alga Chlorella vulgaris, which appears to be of higher nutritional value than spirulina, likely due to its high levels of complex lipids andessential fatty acids. Chlorella-fed bees consistently had higher levels of the nutritional storage protein Vitellogenin relative to spirulina-fed bees. We have also begun to characterize and compare the microbiome and metabolomes of Chlorella- and spirulina-fed bees alongside a natural pollen diet to understand the differences and similarities among the different diet types. We are completing a manuscript that reports physiological and metabolomic comparison of Chlorella, spirulina and pollen diets in young adult honey bees. This manuscript will be submitted to the Journal of Agricultural and Food Chemistry by mid-April 2022. Third, we have been working to develop microalgae strains that express double stranded RNAs (dsRNAs) against honey bee pathogens, with the ultimate goal of formulating diets that contain dsRNA algae strains to feed to pathogen challenged honey bees. We have built gene constructs for introduction into algae, which rely on type-II restriction enzyme assemblies and allow for complex part assemblies. The gene constructs have been confirmed by PCR, sequencing, and restriction digest and we will soon introduce them into wild type algae via bacterial conjugation and homologous recombination.

Publications

• Type: Journal Articles Status: Accepted Year Published: 2021 Citation: Ricigliano, V. A., Ihle, K. E., & Williams, S. T. (2021). Nutrigenetic comparison of two Varroa-resistant honey bee stocks fed pollen and spirulina microalgae. Apidologie, 52(4), 873-886.