Transcriptional Profiles of Diploid Mutant Apis mellifera Embryos after Knockout of csd by CRISPR/Cas9

SIMPLE SUMMARY: In honey bees, males are haploid while females are diploid, leading to a fundamental difference in genetic materials between the sexes. In order to better control the comparison of gene expression between males and females, diploid mutant males were generated by knocking out the sex-determining gene, complementary sex determiner (csd), in fertilized embryos. The diploid mutant drones had male external morphological features, as well as male gonads. RNA sequencing was performed on the diploid mutant embryos and one-day-old larvae. The transcriptome analysis showed that several female-biased genes, such as worker-enriched antennal (Wat), vitellogenin (Vg), and some venom-related genes, were down-regulated in the diploid mutant males. In contrast, some male-biased genes, like takeout and apolipophorin-III-like protein (A4), were up-regulated. Moreover, the co-expression gene networks suggested that csd might interact very closely with fruitless (fru), feminizer (fem) might have connections with hexamerin 70c (hex70c), and transformer-2 (tra2) might play roles with troponin T (TpnT). Foundational information about the differences in the gene expression caused by sex differentiation was provided in this study. It is believed that this study will pave the ground for further research on the different mechanisms between males and females in honey bees. ABSTRACT: In honey bees, complementary sex determiner (csd) is the primary signal of sex determination. Its allelic composition is heterozygous in females, and hemizygous or homozygous in males. To explore the transcriptome differences after sex differentiation between males and females, with genetic differences excluded, csd in fertilized embryos was knocked out by CRISPR/Cas9. The diploid mutant males at 24 h, 48 h, 72 h, and 96 h after egg laying (AEL) and the mock-treated females derived from the same fertilized queen were investigated through RNA-seq. Mutations were detected in the target sequence in diploid mutants. The diploid mutant drones had typical male morphological characteristics and gonads. Transcriptome analysis showed that several female-biased genes, such as worker-enriched antennal (Wat), vitellogenin (Vg), and some venom-related genes, were down-regulated in the diploid mutant males. In contrast, some male-biased genes, such as takeout and apolipophorin-III-like protein (A4), had higher expressions in the diploid mutant males. Weighted gene co-expression network analysis (WGCNA) indicated that there might be interactions between csd and fruitless (fru), feminizer (fem) and hexamerin 70c (hex70c), transformer-2 (tra2) and troponin T (TpnT). The information provided by this study will benefit further research on the sex dimorphism and development of honey bees and other insects in Hymenoptera.