Knockout of the EgriBLOS2 gene results in the transparent integuments of Ectropis grisescens larvae

Abstract

• CRISPR/Cas9-mediated EgriBLOS2 knockout resulted in larval transparent integuments of Ectropis grisescens . • As many as 70% of the transparent larvae were obtained in G0 generation. • EgriBLOS2 was involved in the formation of larval interment colors. The body colors of insects have evolved specialized roles in body protection, signaling and physiology. In some case, the larvae can camouflage their body colors to adapt the habitants and protect themselves. However, the genetic and molecular basis on larval body colors of the tea geometrid, Ectropis grisescens , remains poorly known. Here, we reported an effect of the lysosome-related organelles complex-1, subunit 2 ( EgriBLOS2 ) gene knockout on larval integuments of E. grisescens , by using a clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system. Combining transcriptomic data and PCR approaches, we first identified the EgriBLOS2 gene from E. grisescens , which encoded 137 amino acids and comprised 3 introns. In the phylogenetic analysis, EgriBLOS2 clustered in the Lepidoptera clade with high conservation to members in other lepidopteran species. Developmental expression profiles revealed that EgriBLOS2 was constantly transcribed at each stage, in which its expression was significantly lower in 2nd instar larvae than that of other instars. After injecting EgriBLOS2 -specific guide RNA and Cas9 messenger RNA into eggs, 70% of larvae showed the translucent integuments in G0 generation, with an emphasis on black splayed patterns in the 2nd and 8th segments of abdomens. However, some typical characteristics of larvae were not obviously changed in mutant instars, such as ocelli, mouthparts and other appendants. This study has unraveled the roles of EgriBLOS2 in the formation of larval integument colors, and provides an alternative strategy for pest management based on the colors in this species .