OBP56h modulates social interactions in Drosophila

Social interactions in insects are strongly driven by conspecific chemical signals that are detected via chemoreceptors of olfactory and gustatory neurons. Odorant binding proteins (Obps) transport volatile odorants to chemosensory receptors, but their pleiotropic effects on behaviors and other traits are not well characterized. Here, we knocked down expression of 14 Drosophila melanogaster Obp genes using RNAi and found that seven had reduced male aggressive behavior, particularly Obp56h. RNAi knockdown of Obp56h gene expression also reduced courtship latency; significantly impacted cuticular hydrocarbon production, with a major reduction in 5-Tricosene (5-T); and affected expression of many other genes, including Or19b, Gr97a and several genes associated with lipase activity. We propose that Obp56h plays a dual role in pheromone production and perception and that 5-T, an inhibitory sex pheromone produced by males that increases latency for early stages of courtship, is implicated as a possible ligand for Obp56h. Overall design: We used RNAseq to quantify differences in gene expression in heads and bodies of males and females of Dll-GAL4 x UAS-Obp56h and Dll-GAL4 x control F1 individuals. F1 individuals with CyO or TM3 balancer genotypes were discarded. Flies were aged for 5-6 days in a mixed sex environment at a density of approximately 20 in a vial. Flies were flash frozen over dry ice between 8:00 a.m. and 11:00 a.m. and 30 heads and bodies per sex and genotype were manually dissected and collected over three days in a randomized design, with four biological replicates per sex, genotype and tissue. We extracted total RNA with Trizol with the RNAeasy Mini Kit (Qiagen Inc.). rRNA was depleted using the Ribo-Zero™ Gold Kit (Epicentre Inc.) with 5ug total RNA input. Depleted mRNA was fragmented and converted to first strand cDNA. During the synthesis of second strand cDNA, dUTP instead of dTTP was incorporated to label the second strand cDNA. cDNA from each RNA sample was used to produce barcoded cDNA libraries using NEXTflex™ DNA Barcodes (Bioo Scientific Inc.) with an Illumina TrueSeq compatible protocol. Library size was selected using Agencourt Ampure XP Beads (Beckman Coulter Inc.) and centered on 250bp with average insert size around 130bp. Second strand DNA was digested with Uracil-DNA glycosylase before amplification to produce directional cDNA libraries. Libraries were quantified using Qubit dsDNA HS Kits (Life Technologies Inc.) and Bioanalyzer (Agilent Technologies Inc.) to calculate molarity. They were then diluted to equal molarity and re-quantified, and 32 libraries were pooled. Pooled library samples were quantified to calculate final molarity and finally denatured and diluted to 14pM. Pooled library samples were clustered on an Illumina cBot and sequenced on an Illumina Hiseq2500 using 125bp single-read v4 chemistry on each of two lanes. The quality of the RNA-seq data was assessed using FASTQC (Andrews, 2010). Following quality assessment, adapter sequences were trimmed using Cutadapt (Martin, 2011). Ribosomal reads were filtered against a database of the most common ribosomal sequences using fast BWA alignment BWA-0.7.10 (Li & Durbin, 2009). The remaining reads were aligned to the Dmel_r5.57_FB2014_03 genome and transcriptome using STAR_2.4.1d (Dobin et al., 2013). Read count was performed using HTSeq-count HTSeq-0.6.1p1 (Anders et al., 2015). R software was used for further quality assessment and statistical analysis (R-Core-Team, 2012). The EDASeq package was used to plot principal components, and one replicate sample (HRNAiF1) was identified as a technical outlier, removed, and the remaining 31 samples were used for analysis. The edgeR package was used to calculate differential expression analysis for pairwise comparisons between the control and RNAi sample for sex and tissue as well as the interaction between genotype and tissue for each sex (Robinson et al., 2010). Biological pathway and gene ontology enrichment analyses were performed using DAVID (Huang et al., 2009).