Identification and function analysis of two new gustatory receptors related to silkworm monophagy.

Silkworm is a typical monophagous insect that can only feed on fresh mulberry leaves. The mechanism for this monophagous nature is not fully understood. One bitter gustatory receptor (GR) GR66 located on the maxilla of the mouthpart has been reported to be an important factor influencing the feeding preference of silkworm. However, the preference of GR66 mutants for a nonhost plant was very low, suggesting that other factors related to silkworm monophagy need to be further explored. In this study, 10 bitter GRs were screened out based on their specific high expression in the maxilla of silkworm, and the 4 most expressed GRs (GR15, GR43, GR69, and GR66) were knocked out by clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated nuclease 9. Feeding experiments showed that except for GR69-/-, the host range of GR43-/-, GR15-/-, and GR66-/- expanded remarkably, and both GR43-/- and GR66-/- revealed the highest preference for the nonhost plants. Moreover, the strict preference for mulberry leaves was almost eliminated in the double mutant silkworms of GR43-/- and GR66-/-. These results imply that it is likely that a variety of different bitter GRs are involved in determining the monophagy of silkworm larvae. In addition, GR15 mutation significantly inhibited ovarian development, resulting in a significant decrease in oviposition. These findings enhance our understanding of the monophagous nature of silkworms and provide a possibility for the molecular breeding of polyphagous silkworms in the future.