Abstract
The European honeybee (Apis mellifera L.) exhibits various social behaviors. The molecular and neural mechanisms underlying these behaviors have long been explored, but causal relations between genes or neurons and behaviors remain to be elucidated because effective gene manipulation methods in the honeybee have not been available until recently. We recently established a basic technology to produce mutant honeybee drones using CRISPR/Cas9. Here we produced mutant drones using CRISPR/Cas9 targeting mKast, which is preferentially expressed in a certain subtype of class I Kenyon cells that comprise the mushroom bodies in the honeybee brain. By immunoblot analysis, we showed that mKast protein expression was completely lost in the mutant drone heads. In addition, during the production process of homozygous mutant workers, we demonstrated that heterozygous mutant workers could be produced by artificial insemination of wild-type queens with the sperm of mutant drones, indicating that mKast mutant drones were sexually mature. These results demonstrate that mKast is dispensable for normal development and sexual maturation in drone honeybees, and allow us to proceed with the production of homozygous mutant workers for the analysis of a particular gene by gene knockout in the future.
Highlights
The European honeybee (Apis mellifera L.) exhibits various social behaviors, and has long been used as an experimental animal for the study of social behaviors[1,2,3]
We recently found that the number of class I Kenyon cells (KCs) subtypes increases from one to two and from two to three with the behavioral evolution from solitary phytophagous lifestyle to solitary parasitism, and to nidification (Aculeate Hymenoptera, including the honeybee), suggesting that the subdivision of KC subtypes is related to behavioral evolution in hymenopteran insects[23]
A specific single-guide RNA (sgRNA) target site was designed in the first open-reading frame (ORF) of middle-type Kenyon cell-preferential arrestin-related protein (mKast) as far upstream as possible to induce the complete loss of mKast protein function by frameshift mutation (Supplementary Fig. S1)
Summary
The European honeybee (Apis mellifera L.) exhibits various social behaviors, and has long been used as an experimental animal for the study of social behaviors[1,2,3]. The molecular and neural mechanisms underlying honeybee social behaviors, especially the functions of genes preferentially expressed in each KC subtype, have remained mostly unknown because effective gene manipulation methods for the honeybee were not available until recently. We conducted gene knockout using CRISPR/Cas[9] methods targeting middle-type Kenyon cell-preferential arrestin-related protein (mKast), which is preferentially expressed in the mKCs in honeybee MBs22. Quantitative reverse transcription- polymerase chain reaction (PCR) analysis revealed its adult brain-specific expression[26]. These mKast expression patterns imply that mKast is related to higher-order function of the adult brain by regulating the processing of various types of sensory information. To analyze the function of mKast in the regulation of honeybee social behaviors, it is necessary to produce mKast knockout workers through production of the mKast knockout drones (see Fig. 1A). mKast is useful as a target gene because mKast knockout may not cause embryonic lethality, considering its adult brain-specific expression
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