Identification of yellow gene family in Agrotis ipsilon and functional analysis of Aiyellow-y by CRISPR/Cas9

Abstract

The yellow gene family has been identified in several model insects, but yellow genes were poorly identified in non-model insects and the functions of yellow genes are largely unknown. In this study, we identified seven yellow genes in an important agricultural pest Agrotis ipsilon. Each gene encodes a protein containing a major royal jelly domain. Phylogenetic analysis defined these genes as yellow-y, -b, -b2, -c, -d, -e, and –h, respectively. The A. ipsilon yellow genes yellow-b, -b2, and –c were stably expressed in all developmental stages and tissues analyzed, whereas the other four yellow genes had unique expression patterns, suggesting distinct physiological roles of each gene. Using the CRISPR/Cas9 system, we successfully disrupted yellow-y in A. ipsilon and obtained G0 insects with somatic mutations. Unlike the black of wild-type newly hatched larvae and of adults, the mutants were yellow, although in the pupal stage mutant coloration did not differ from wild-type coloration. This phenotype was inherited by G1 offspring. The G0 mutants did not show any growth deficiency compared with control insects; however, a dehydration-like phenotype was observed in newly hatched G1 larvae from sibling crossed mutants. Our results indicate that A. ipsilon yellow-y gene plays a role in body pigmentation and also might function in waterproofing.