Hemolymph transfusions transfer heritable learned novel odor preferences to naive larvae of Bicyclus anynana butterflies

The mechanisms whereby environmental experiences of parents are transmitted to their offspring to impact their behavior and fitness are poorly understood. Previously, we showed that naive Bicyclus anynana butterfly larvae, whose parents fed on a normal plant feed but coated with a novel odor, inherited a learned preference towards that odor, which had initially elicited avoidance in the naive parents. Here, we performed simple hemolymph transfusions from odor-fed and control-fed larvae to naive larval recipients. We found that larvae injected with hemolymph from odor-fed donors stopped avoiding the novel odor, and their naive offspring preferred the odor more, compared to the offspring of larvae injected with control hemolymph. These results indicate that factors in the hemolymph, potentially the odor molecule itself, play an important role in odor learning and preference transmission across generations. Furthermore, this mechanism of odor preference inheritance, mediated by the hemolymph, bypasses the peripheral odor-sensing mechanisms taking place in the antennae, mouthparts, or legs, and may mediate host plant switching and diversification in the Lepidoptera or more broadly across insects.