Pleiotropic fitness effects of a Drosophila odorant-binding protein

Abstract

Insect odorant-binding proteins (OBPs) are members of a rapidly evolving multigene family traditionally thought to facilitate chemosensation. However, studies on Drosophila have shown that members of this family have evolved functions beyond chemosensation, as evident from their expression in reproductive tissues and the brain. Previous studies implicated diverse functions of Obp56h, a member of the largest gene cluster of the D. melanogaster Obp repertoire. Here, we examined the effect of CRISPR/Cas9-mediated deletion of Obp56h on 2 fitness phenotypes, on resistance to starvation stress and heat stress, and on locomotion and sleep phenotypes. Obp56h-/- mutants show a strong sexually dimorphic effect on starvation stress survival, with females being more resistant to starvation stress than the control. In contrast, Obp56h-/- females, but not males, are highly sensitive to heat stress. Both sexes show changes in locomotion and sleep patterns. Transcriptional profiling of RNA from heads of Obp56h-/- flies and the wildtype control reveals differentially expressed genes, including gene products associated with antimicrobial immune responses and members of the Turandot family of stress-induced secreted peptides. In addition, differentially expressed genes of unknown function were identified in both sexes. Genes encoding components of the mitochondrial electron transport chain, cuticular proteins, gene products associated with regulation of feeding behavior (Lst and CCHa2), ribosomal proteins, lncRNAs, snoRNAs, tRNAs, and snRNAs show changes in transcript abundances in Obp56h-/- females. These differentially expressed genes are likely to contribute to Obp56h-mediated effects on the diverse phenotypes that arise upon deletion of this OBP.

Keywords: Obp56h; CRISPR-Cas9; RNA sequencing; chemosensation; fitness; heat stress; pleiotropy; sexual dimorphism; sleep; starvation resistance.

Fig. 1.

CRISPR/Cas9-mediated deletion of Obp56h. a) Construction of the Obp56h null allele. Dark boxes represent exons of the Obp56h gene, and light boxes indicate the intron and 5′ and 3′ untranslated sequences. We designed guide RNAs flanking the Obp56h gene for CRISPR/Cas9-mediated deletion at the cut sites, indicated by the scissor symbols, in the CSB genetic background (Supplementary Table 1). We replaced the gene with a cassette that contains a DsRed fluorescent marker under the control of an eyespecific 3XP3 promoter and with SV40 polyadenylation sequences, loxP sites for Cre-mediated removal of the insert, and an attP site. b) Average normalized Obp56h cpm expression counts from whole genome RNA sequencing for males and females for Obp56h deletion flies and the CSB control (see also Supplementary Tables 3 and 4).

Fig. 2.

Pleiotropic effects of Obp56h alleles on fitness-related quantitative traits. a) Survival curves under starvation stress. The black survival curve represents the CSB control, and the red survival curve represents the Obp56h^−/− flies. b) Heat shock survival. Males are indicated in blue and females in pink. ***P < 0.0001 (Supplementary Table 2).

Fig. 3.

Effects of Obp56h alleles on activity and sleep phenotypes. Box plots showing total activity (a), sleep proportion during the day (b), and sleep proportion during the night (c) for sexes separately. *P < 0.05; **P < 0.001; ***P < 0.0001 (Supplementary Table 2).

Fig. 4.

Venn diagram of differentially expressed genes across both sexes in Obp56h^−/− flies compared to the control.