Boosting the bee: Leveraging the structural capabilities of the lipoprotein Vitellogenin to support honey bee (Apis mellifera) health.

Vitellogenin (Vg) is an important protein: it is as old as multicellular animal life on Earth, about 700 million years old. Today, it is present in almost all animals that lay eggs. Vg is essential for eggs to be viable. But what is more: Our previous work revealed that it also can be a major player in the immune system, nutritional health, and behavior of adult animals. Our research has focused on honey bees, which are important as producers of honey and as pollinators of crops like berries, fruits and vegetables. Our most recent work led to the discovery of at least 121 Vg variants (unique protein shapes) in honey bees. With this project, we study specific variants of Vg that are predicted to vary in their effect on the immunity, nutritional health and behavior of honey bees. We have successfully overcome the first hurdle in testing the predictions by establishing a honey bee colony that carries the standard variant of honey bee Vg. The standard Vg will be used as a reference to quantify the effects caused by amino acid-altering protein variants. The work resulted in an improved sequencing protocol that leverages AI-driven protein structure prediction algorithms and barcoded samples to identify short and unique Vg gene signatures, saving time and costs. The work was in collaboration with the USDA (Ihle). We are preparing a manuscript presenting our successful state-of-the-art breeding concept. In collaboration with UCL (Fraternali and Orengo), we developed an advanced bioinformatic pipeline to navigate the selection of Vg variants. Two datasets highlight functional Vg regions that play important roles in behavioral and nutritional health. We have selected a Vg target site predicted to affect the lipid binding potential, and breeding of homozygotic honey bee queens with this variant is underway at the USDA. We have collected hemolymph (insect blood) from 450 honey bees at NMBU to map out the diversity in the lipid-binding potential for honey bee Vg. Purification approaches and precipitation assays for the Vg-lipid complexes are now being developed at the UiB (Halskau), in preparation for lipidomic analysis. To understand a Vg functional region’s dynamics, which regulates honey bees' behavioral health, we simulated its movements in solution. This novel study is the first to identify the dynamic behavior of a Vg domain across different taxa. A manuscript is being prepared to show how the Vg region is affected by a protein variant. The simulation data will also be used to investigate how the region interacts with DNA. In addition to our computational studies, we have made experimental progress. In collaboration with the UiB, we have generated a traceable Vg construct of the functional region responsible for the translocation of Vg into honey bee tissues, cell nuclei, and recognition of DNA. The preliminary test on stability and the in vitro functionality of the construct and its fluorescence marker are promising. The introduction of the construct into honey bee tissues is ongoing at ASU. Amdam, Halskau, and Leipart have submitted a Disclosure of Invention as a first step toward patent application for the delivery system. The postdoc secured funding for a 12-month overseas research grant from RCN (350231) at the UCL. The stay allows her to improve their computational skills and learn state-of-the-art bioinformatic tools, reflected in the project's progress. In addition to project-specific tasks, the stay at UCL has allowed the postdoc to participate in two structural bioinformatic and biomolecular simulation courses, present her work to the institute and several symposiums (for which one was granted a 2nd place poster prize), given the role as chair for a session during an institute retreat, co-wrote a symposium report and assisted in an interview process. Vg and the allelic dataset have been used as teaching material for advanced master's and bachelor's courses at the UiB and Wageningen University. Leipart was an invited lecturer to the MOL310 course at the UiB to teach the state-of-the-art methodology AlphaFold using honey bee Vg as an example. Halskau has presented a master’s student project on the von Willebrand factor of Vg. Overall, our results have gained traction in the scientific field for stakeholders and the general audience. We were awarded the “Top Downloaded Article during the first 12 months of publication” and shortlisted for the FEBS Open Bio Article prize for our FEBS Open Bio article. The results have caused Honey Bee Vg to be highlighted as a poster child of the structural biology revolution from DeepMind and in Nature. Leipart’s PhD thesis was awarded the Alf Bjørseth Inspirational Award 2023 (News articles from NMBU and UCL). Ihle and Amdam have presented our work for several Beekeeping Associations, and Amdam is featured in Aftenposten A-magasinet.

Vitellogenin (Vg) is an important yolk protein in most egg-laying animals?from sponges to chickens. We have studied Vg for 20 years, and during this time, the functional characterization of the protein expanded: From simple "yolk precursor", to a multifunctional protein important for health and behavior. Our previous work was a key to this paradigm shift. We now propose to use our unique knowledge base to leverage a new finding: 121 Vg variants in honey bees (Apis mellifera). By leveraging these protein variants, we will deliver results at the forefront of molecular biology while addressing a challenge to agriculture and society: the need to alleviate stresses on bees from diseases and poor nutrition. To achieve our objectives, we launch a multidisciplinary initiative that involves 6 academic partners as well as stakeholder groups. Our project is structured around two task areas: cutting-edge basic research (WP1-3) and cutting-edge applied research (WP4-5). Under WP1-3 we utilize specific Vg variants or Vg-inspired synthetic constructs to resolve questions about Vg multifunctionality; in pathogen-, lipid- and DNA-binding. Under WP4 we test how specific Vg variants perform in bee colonies in the USA, in collaboration and synergy with the USDA's Baton Rouge lab. This lab is a world-authority on bee health and genetic stock development. Our applied research is taken to a next level in WP5, where stakeholders form interdisciplinary teams to develop simple "at-home" quantification tools for Vg. Our project outputs are positioned to boost the molecular understanding of Vg (with implications for the many taxa that rely on this protein), and to modernize the use of Vg in veterinary diagnostics in the service of bees and beekeepers.