In species that care for their young, provisioning has profound effects on offspring fitness. Provisioning is important in honeybees because nutritional cues determine whether a female becomes a reproductive queen or sterile worker. A qualitative difference between the larval diets of queens and workers is thought to drive this divergence; however, no single compound seems to be responsible. Diet quantity may have a role during honeybee caste determination yet has never been formally studied. Our goal was to determine the relative contributions of diet quantity and quality to queen development. Larvae were reared in vitro on nine diets varying in the amount of royal jelly and sugars, which were fed to larvae in eight different quantities. For the middle diet, an ad libitum quantity treatment was included. Once adults eclosed, the queenliness was determined using principal component analysis on seven morphological measurements. We found that larvae fed an ad libitum quantity of diet were indistinguishable from commercially reared queens, and that queenliness was independent of the proportion of protein and carbohydrate in the diet. Neither protein nor carbohydrate content had a significant influence on the first principle component 1 (PC1), which explained 64.4% of the difference between queens and workers. Instead, the total quantity of diet explained a significant amount of the variation in PC1. Large amounts of diet in the final instar were capable of inducing queen traits, contrary to the received wisdom that queen determination can only occur in the third instar. These results indicate that total diet quantity fed to larvae may regulate the difference between queen and worker castes in honeybees.
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