Loss of the Dβ1 nicotinic acetylcholine receptor subunit disrupts bursicon-driven wing expansion and diminishes adult viability in Drosophila melanogaster

Abstract

Cholinergic signaling dominates the insect central nervous system, contributing to numerous fundamental pathways and behavioral circuits. However, we are only just beginning to uncover the diverse roles different cholinergic receptors may play. Historically, insect nicotinic acetylcholine receptors have received attention due to several subunits being key insecticide targets. More recently, there has been a focus on teasing apart the roles of these receptors, and their constituent subunits, in native signaling pathways. In this study, we use CRISPR-Cas9 genome editing to generate germline and somatic deletions of the Dβ1 nicotinic acetylcholine receptor subunit and investigate the consequences of loss of function in Drosophila melanogaster. Severe impacts on movement, male courtship, longevity, and wing expansion were found. Loss of Dβ1 was also associated with a reduction in transcript levels for the wing expansion hormone bursicon. Neuron-specific somatic deletion of Dβ1 in bursicon-producing neurons (CCAP-GAL4) was sufficient to disrupt wing expansion. Furthermore, CCAP-GAL4-specific expression of Dβ1 in a germline deletion background was sufficient to rescue the wing phenotype, pinpointing CCAP neurons as the neuronal subset requiring Dβ1 for the wing expansion pathway. Dβ1 is a known target of multiple commercially important insecticides, and the fitness costs exposed here explain why field-isolated target-site resistance has only been reported for amino acid replacements and not loss of function. This work reveals the importance of Dβ1-containing nicotinic acetylcholine receptors in CCAP neurons for robust bursicon-driven wing expansion.

Drosophila nicotinic acetylcholine receptor subunits and their native interactions with insecticidal peptide toxins

“…The locomotor defects we observed are in agreement with previously reported neuronal phenotypes with nAChR subunit genes, including sleep disruption, defective jump response, memory impairment or locomotor defects ( Fayyazuddin et al, 2006 ; Rohde et al, 2016 ; Somers et al, 2017 ; Tackenberg et al, 2020 ). Locomotor defects in climbing assays were previously shown in nAChRβ1 mutant strains ( Homem et al, 2020 ; Christesen et al, 2021 ).…”

“…In both studies, null mutations in the nAChRβ1 gene were inviable as stocks. However, Christesen and colleagues report lethality primarily at the pre-pupal/pupal stage with a low percentage of individuals surviving to adulthood ( Christesen et al, 2021 ). In contrast, we never recovered homozygous adults or pupae and when outcrossing to a 3rd chromosome balancer with a dominant larval marker did not find homozygous nAChRβ1 3rd instar larvae.…”