Pleiotropic Effects of Loss of the Dα1 Subunit in Drosophila melanogaster: Implications for Insecticide Resistance

Abstract

Nicotinic acetylcholine receptors (nAChRs) are a highly conserved gene family that form pentameric receptors involved in fast excitatory synaptic neurotransmission. The specific roles individual nAChR subunits perform in Drosophila melanogaster and other insects are relatively uncharacterized. Of the 10 D. melanogaster nAChR subunits, only three have described roles in behavioral pathways; Dα3 and Dα4 in sleep, and Dα7 in the escape response. Other subunits have been associated with resistance to several classes of insecticides. In particular, our previous work has demonstrated that an allele of the Dα1 subunit is associated with resistance to neonicotinoid insecticides. We used ends-out gene targeting to create a knockout of the Dα1 gene to facilitate phenotypic analysis in a controlled genetic background. To our knowledge, this is the first report of a native function for any nAChR subunits known to be targeted by insecticides. Loss of Dα1 function was associated with changes in courtship, sleep, longevity, and insecticide resistance. While acetylcholine signaling had previously been linked with mating behavior and reproduction in D. melanogaster, no specific nAChR subunit had been directly implicated. The role of Dα1 in a number of behavioral phenotypes highlights the importance of understanding the biological roles of nAChRs and points to the fitness cost that may be associated with neonicotinoid resistance.

Keywords: behavior; neonicotinoids; nicotinic acetylcholine receptors; resistance; sleep.

Figure 1

Courtship initiation and copulation initiation of wild-type and Dα1^KO mutant males measured when paired with either wild-type or mutant females. Mutant males consistently took longer to initiate both courtship and copulation compared to wild-type males, regardless of the genotype of the female partner. Wild-type males initiated courtship with both wild-type and mutant females with equal vigor; however, copulation initiation took significantly longer with mutant females compared to wild-type females. Error bars display 95% confidence intervals (** P < 0.01, Student’s t-test).

Figure 2

Rescue of courtship and copulation initiation by Dα1 expression using the elav::GAL4 driver. Left panels are pairs consisting of wild-type male flies crossed to control or rescue female flies, right panels are pairs consisting of wild-type female flies crossed to control or rescue male flies. No significant differences in courtship initiation of wild-type males were observed regardless of the female’s genotype. Wild-type females were courted significantly faster by rescue male flies compared to control male. Copulation initiation was significantly faster for both rescue crosses compared to the appropriate controls. Error bars display 95% confidence intervals (** P < 0.01, Student’s t-test).

Figure 3

Analysis of Dα1^KO sleeping behavior. (A) No significant difference in total mean sleep is observed between wild-type and mutant flies; however, mutant flies sleep significantly less at night and significantly more during the day. (B and C) Mutant flies experience significantly less and shorter sleep episodes during the night and significantly more episodes during the day. Error bars display 95% confidence intervals (* P < 0.05, ** P < 0.01, Student’s t-test).

Figure 4

Modulation of Dα1^KO sleeping behavior by Dα1 expression using the elav::GAL4 driver. (A) Total mean sleep is significantly increased, as is mean sleep during the day and night, for rescue flies compared to both control genotypes. (B) Sleep episode lengths are significantly increased in rescue flies compared to control genotypes, particularly at night. (C) No significant difference is observed in the number of day sleep episodes rescue flies experience and a reduced number of night episodes was observed. Error bars display 95% confidence intervals (* P < 0.05, ** P < 0.01, Student’s t-test).

Figure 5

Kaplan–Meier survival plot of Dα1^KO. The Dα1^KO mutant had significantly shorter median survivorship of 34 days compared to that of the control with a median survivorship of 52 days. (P < 0.01, Mantel–Cox test).