Neurodevelopmental and transcriptomic effects of CRISPR/Cas9-induced somatic orco mutation in honey bees

Abstract

In insects, odorant receptors facilitate olfactory communication and require the functionality of the highly conserved co-receptor gene orco. Genome editing studies in a few species of ants and moths have revealed that orco can also have a neurodevelopmental function, in addition to its canonical role in adult olfaction, discovered first in Drosophila melanogaster. To extend this analysis, we determined whether orco mutations also affect the development of the adult brain of the honey bee Apis mellifera, an important model system for social behavior and chemical communication. We used CRISPR/Cas9 to knock out orco and examined anatomical and molecular consequences. To increase efficiency, we coupled embryo microinjection with a laboratory egg collection and in vitro rearing system. This new workflow advances genomic engineering technologies in honey bees by overcoming restrictions associated with field studies. We used Sanger sequencing to quickly select individuals with complete orco knockout for neuroanatomical analyses and later validated and described the mutations with amplicon sequencing. Mutant bees had significantly fewer glomeruli, smaller total volume of all the glomeruli, and higher mean individual glomerulus volume in the antennal lobe compared to wild-type controls. RNA-Sequencing revealed that orco knockout also caused differential expression of hundreds of genes in the antenna, including genes related to neural development and genes encoding odorant receptors. The expression of other types of chemoreceptor genes was generally unaffected, reflecting specificity of CRISPR activity in this study. These results suggest that neurodevelopmental effects of orco are related to specific insect life histories.

Keywords:

• antennal lobe
• development
• genome editing
• Orco
• OR
• olfaction
• RNA-seq
• social insects

Acknowledgements

We thank P. Hanke for assistance with Cas9 protein preparation, L. Licitis and M. Seyller for assistance with the bees, A. Hamilton, K. Torres, N. Beach and J. Fine for support and suggestions on in vitro rearing, and members of the Robinson lab for helpful discussion.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

The work was funded by an NSF NeuroNex grant NSF-DBI 1707221 (PI: Y. Ben-Shahar) and a grant from the Defense Advanced Research Projects Agency #HR0011-16–2-0019 (to GER and H. Zhao).