1/ A technical semi-field methodology to measure the effect of nutrition on honeybee brood rearing

ABSTRACT A honeybee colony’s well-being is its ability to nurture larvae into healthy adults. Understanding how nutrition supports brood rearing is crucial for developing diets that could aid against environmental threats. Nutritional research on whole colony brood development has been historically challenging due to difficulties documenting the diet’s impact on brood production over time. We describe a novel semi-field method to study the influence of nutrition on brood rearing using standardised small colonies formed de novo (ca. 1500 nurse-age bees and a queen) housed in adapted mating-nucs, placed inside an enclosure and limited to feeding on chemically defined diets. Complete assessments were conducted every fifteen days, assisted by a bespoke device to photograph every frame to measure cell contents. A novel metric describes the number of bees generated per gram of diet consumed, measuring the impact of nutrition on brood rearing and overall colony size. Competing Interest Statement The authors, Rui FS Goncalves, Raquel T. de Sousa, Daniel Stabler, Geraldine A. Wright, and Sharoni Shafir, are shareholders in Apix Biosciences, a bee-nutrition university spin-off registered in Belgium. The authors declare that they have no other competing interests. David MS Pinto has no competing interests to declare. All authors have read and approved the manuscript for submission. Footnotes DS - d.stabler{at}soton.ac.uk, DMSP - david.pinto{at}eng.ox.ac.uk, GAW - geraldine.wright{at}biology.ox.ac.uk, SS - sharoni.shafir{at}mail.huji.ac.il We addressed the concerns raised by the reviewers by clarifying some methodological details, such as disease management procedures and the limitations of our semi-field system. We added explanations of how Varroa load was visually assessed and kept at zero through routine monitoring and treatments, and clarified that nurse bees were sourced only from healthy, virus-free colonies. We specified that all queens were sister queens, described in detail how they were introduced and released, and clarified that all colonies began the experiment with a fully built comb. To standardise colony size, we detailed our volumetric method of measuring nurse bees (700 mL, approx. 1,580 workers per mini-nuc). We expanded the Discussion to address limitations associated with small colony size, behavioural flexibility, variation in nurse nutritional stores, mortality in enclosures, and restricted foraging. Finally, due to figure-number limits, step-by-step visual guidance on establishing mini-nucs has been moved to the supplementary materials. These revisions improve clarity, reproducibility, and transparency of the method.