The effect of larval nutrition on female fertility in honey bees is a focus for both scientific studies and for practical applications in beekeeping. In general, morphological traits are standards for classifying queens and workers and for evaluating their quality. In recent years, in vitro rearing techniques have been improved and used in many studies; they can produce queen-like and worker-like bees. Here, we questioned whether queens and workers reared in vitro are the same as queens and workers reared in a natural hive environment. We reared workers and queens both in vitro and naturally in beehives to test how these different environments affect metabolic physiology and candidate genes in newly emerged queens and workers. We found that sugar (glucose and trehalose) levels differed between queens and workers in both in vitro and in-hive-reared bees. The in vitro-reared bees had significantly higher levels of lipids in the abdomen. Moreover, hive reared queens had almost 20 times higher levels of vitellogenin than in vitro-reared queens, despite similar morphologies. In addition, hive-reared bees had significantly higher levels of expression of mrjp1 In conclusion, in vitro rearing produces queens and workers that differ from those reared in the hive environment at physiological and gene expression levels.This article has an associated First Person interview with the first author of the paper.

Highly social insects are characterized by caste dimorphism, with distinct size differences of reproductive organs between fertile queens and the more or less sterile workers. An abundance of nutrition or instruction via diet-specific compounds has been proposed as explanations for the nutrition-driven queen and worker polyphenism. Here, we further explored these models in the honeybee (Apis mellifera) using worker nutrition rearing and a novel mutational screening approach using the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) method. The worker nutrition-driven size reduction of reproductive organs was restricted to the female sex, suggesting input from the sex determination pathway. Genetic screens on the sex determination genes in genetic females for size polyphenism revealed that doublesex (dsx) mutants display size-reduced reproductive organs irrespective of the sexual morphology of the organ tissue. In contrast, feminizer (fem) mutants lost the response to worker nutrition-driven size control. The first morphological worker mutants in honeybees demonstrate that the response to nutrition relies on a genetic program that is switched "ON" by the fem gene. Thus, the genetic instruction provided by the fem gene provides an entry point to genetically dissect the underlying processes that implement the size polyphenism.