Protein craving links larval signals to food provisioning in honey bees
Zhenfang Li https://orcid.org/0009-0002-5766-8936, Yashuai Wu https://orcid.org/0009-0008-3936-289X, Jiaming Liu https://orcid.org/0000-0001-7571-8760, Chengfeng Yang https://orcid.org/0009-0009-1460-3236, Shuai Wang, Min Huang, Shiqi Luo https://orcid.org/0000-0002-0506-2230, and Xin Zhou https://orcid.org/0000-0002-1407-7952Authors Info & Affiliations
Abstract
The provisioning of royal jelly for developing larvae by nurse bees is fundamental to social interaction in honey bee colonies. While royal jelly production is regulated by collective larval demand, it remains unclear how colony-level needs are translated into individual worker behavior. Here, we show that the larval pheromone E-β-ocimene (EBO), a volatile compound also used by pollinators as a floral food cue, elicits an intrinsic craving for protein in nurse bees that drives increased pollen consumption. Through in vitro and in vivo experiments, we demonstrate that this response is mediated by the leucokinin (Lk) and leucokinin receptor (Lkr) system, acting through the PKA-CREB-IRS signaling pathway to modulate the expression of the insulin receptor substrate gene (Irs). Elevated pollen intake then promotes the enlargement of the hypopharyngeal glands and enhanced production of major royal jelly proteins. Our findings uncover a molecular mechanism linking larval signaling to worker nutrition, highlighting how social bonds between honey bee larvae and nurses are rooted in ancestral pathways of protein hunger that predate eusociality.
INTRODUCTION
The division of labor and altruistic brood care form the foundation of honey bee sociality. Worker bees contribute to colony success through tasks such as nursing and foraging, which support brood development. Of the two principal food sources, pollen is the main source of protein required for larval growth (1, 2). Because direct pollen consumption by larvae is negligible, larvae rely on nurse bees to ingest and metabolize pollen into protein-rich brood food (3–6). Thus, nurse bees serve as the colony’s primary consumers of pollen (4, 7–10). Pollen-derived nutrients are converted into royal jelly components, which are synthesized and secreted by the hypopharyngeal glands (HPGs) (11, 12). In this way, HPGs provide the proteins necessary for larval growth and colony development (3, 5, 12). The stimulatory effect of pollen on HPG development is conserved across social bees, including Apis mellifera (12, 13), Apis cerana (14), and the stingless bee Tetragonula pagdeni (15). Moreover, the size of HPG development in nurse bees correlates strongly with larval survival and developmental capacity (16), making it a key indicator of larval food quality (17).
Larvae and nurses interact closely to balance brood nutritional needs with worker behavior. Colony-level protein demands are met through behavioral adjustments of individual workers (18, 19), mediated by nurse inspections of brood cells (20) and brood pheromones (19, 21, 22). Among these mechanisms, pheromones provide particularly effective signals of collective larval nutritional demand. Honey bee brood produce two major pheromones, E-β-ocimene (EBO) and brood ester pheromone (23, 24), with EBO being the most abundant (25). EBO is synthesized primarily by second- and third-instar larvae (24) and is released at elevated levels when larvae are hungry (19). Because EBO accumulation scales with larval numbers, its concentration modulates worker behaviors, particularly pollen foraging and brood care (1, 19, 26).
However, the mechanistic link between elevated larval EBO and increased food provisioning by nurses is still poorly understood. In particular, it remains unclear how larval signals are translated into molecular and behavioral responses in workers. Given that EBO is also naturally released by flowers to attract pollinators (27), we hypothesize that intensified larval EBO increases protein cravings in nurse bees. This response would increase pollen consumption and promote HPGs to produce royal jelly. Previous studies suggest roles for neuromodulators and insulin signaling in the regulation of feeding behavior (28), but their integration with larval signaling remains unresolved. In our recent work on A. cerana, we identified the leucokinin receptor (Lkr) as a key regulator of worker feeding motivation (29). We therefore propose that larval EBO acts through the leucokinin and leucokinin receptor (Lk/Lkr) system and its downstream signaling components to couple brood nutritional demand with nurse provisioning behavior.
Here, we demonstrate that honey bee larvae exploit ancestral neuropeptide and nutrient-sensing mechanisms to convey amino acid demand to worker bees. This signal stimulates intrinsic protein hunger in nurses, thereby driving pollen consumption and facilitating HPG enlargement, which enhances the synthesis and secretion of royal jelly. At the molecular level, we identify the Lk/Lkr system as the central hub that transduces larval EBO into worker responses by activating the insulin receptor substrate gene (Irs) pathway via a signaling cascade involving protein kinase A (PKA) and cAMP-response element binding protein (CREB). Our results reveal that collective larval demand directs individual worker behavior, offering insights into the molecular basis of honey bee sociality.
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