Odorant and gustatory receptors synergistically modulate feeding behaviors that underlie food preference and consumption in the silkworm

Feeding behavior determines insect survival and reproductive success. As insects process multimodal olfactory and gustatory cues to guide their feeding decisions, appetitive odors not only attract insects to food sources but also directly increase food ingestion. However, the mechanisms that coordinate chemosensory integration to regulate these behaviors remain poorly characterized. The silkworm mainly feeds on mulberry leaves. We previously identified GS01 as a natural mutant that consumes an expanded range of non-mulberry diets and exhibits compromised olfactory specificity for mulberry leaves. Thus, GS01 is an excellent model for studying the genetic basis of interactions between olfaction and gustation. Combining the results of molecular mapping, transcriptome sequencing, and DNA sequencing, we determined that the gustatory receptor Gr66, and odorant receptors Or39 and OrJ are involved in the euryphagous phenotype of GS01. Next, we generated a series of mutants by single or combined gene knockout using CRISPR/Cas9 and studied the interactions between the affected genes in regulating feeding behavior. We showed that a single knockout of Gr66 increases intake of an artificial diet containing no mulberry leaves, while individual or simultaneous knockouts of genes Or39 and OrJ do not increase consumption of this diet. However, the combined knockout of genes Gr66, Or39, and OrJ significantly enhances uptake of the artificial diet beyond levels exhibited by the Gr66 knockout alone. This study demonstrates the synergy between gustatory and odorant receptors in regulating silkworm feeding preference and food uptake, and sheds light on understanding the complexity of regulation of feeding behavior in insects.