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.

CRISPR/Cas9, a predominant gene-editing tool, has been utilised to dissect the gene function in Apis mellifera. However, only the genomic region containing NGG PAM could be recognised and edited in A. mellifera, seriously hampering the application of CRISPR technology in honeybees. In this study, we carried out the bioinformatics analysis for genome-wide targeting sites of NGG, TTN, and NNGRRT to determine the potential expansion of the SpCas9, SaCas9, Cpf1, and it was found that the targetable spectrum of the CRISPR editing system could be markedly extended via the integrated gene manipulation system. Meanwhile, the single guide RNA (sgRNA)/crRNA of different novel gene editing systems and the corresponding CRISPR proteins were co-injected into honeybee embryos, and their feasibility was tested in A. mellifera. The sequencing data revealed that both SaCas9 and Cpf1 are capable of mediating mutation in A. mellifera, albeit with relatively lower mutagenesis rates for Cpf1 and unstable editing for SaCas9. To our knowledge, our results provide the first demonstration that SaCas9 and Cpf1 can function to induce genome sequence alternation, which extended the editing scope to the targets with TTN and NNGRRT and enabled CRISPR-based genome research in a broader range in A. mellifera.

In insects, tyrosine hydroxylase (TH) plays crucial roles in a wide variety of biological processes. However, its function in Apis mellifera remains unknown. Here, AmTH (A. mellifera TH) was cloned and characterized. Expression profiles at different stages showed that AmTH was highly expressed in 2-day-old eggs and during the pupal stage. Tissue expression profiles of newly emerged workers revealed that AmTH was highest expressed in the wings, followed by the legs and antennae. To explore the function of AmTH, gene-edited worker larvae were generated using CRISPR/Cas9 technology, and the edited individuals died at the larval and prepupal stages. In addition, a TH inhibitor (3-IT) was administered via larval feeding and the survival rates were significantly lower in groups fed with 800 μM, 3200 μM, and 12,800 μM of 3-IT in a dose-dependent manner. To investigate the role of AmTH in honeybee immunity, 8-day-old worker bees were fed with 3-IT and subsequently injected with lipopolysaccharide. The results showed that the mRNA expression of Relish, antimicrobial peptide-related genes, TH and DDC were significantly downregulated, whereas laccase 2 and yellow-y were significantly upregulated. These findings indicate that AmTH is indispensable for larval development and adult immunity in honeybees.