Genomics analysis of Drosophila sechellia response to Morinda citrifolia fruit diet

Abstract

Drosophila sechellia is an island endemic host specialist that has evolved to consume the toxic fruit of Morinda citrifolia, also known as noni fruit. Recent studies by our group and others have examined genome-wide gene expression responses of fruit flies to individual highly abundant compounds found in noni responsible for the fruit's unique chemistry and toxicity. In order to relate these reductionist experiments to the gene expression responses to feeding on noni fruit itself, we fed rotten noni fruit to adult female D. sechellia and performed RNA-sequencing. Combining the reductionist and more wholistic approaches, we have identified candidate genes that may contribute to each individual compound and those that play a more general role in response to the fruit as a whole. Using the compound specific and general responses, we used transcription factor prediction analyses to identify the regulatory networks and specific regulators involved in the responses to each compound and the fruit itself. The identified genes and regulators represent the possible genetic mechanisms and biochemical pathways that contribute to toxin resistance and noni specialization in D. sechellia.

Keywords: Morinda citrifolia; RNA-seq; host specialization; noni; toxin resistance.

Fig. 1.

Experimental design and bioinformatics pipeline. Female D. sechellia were exposed to either control food or food supplemented with rotten noni fruit. RNA was extracted, underwent polyA selection, library preparation, and sequencing. Raw-sequencing reads were checked for quality using FastQC, and then aligned to the D. sechellia reference genome-with Bowtie2. Differential expression testing was performed using Cuffdiff, and expression data were analyzed using R, Gene ontology, and icis-Target.

Fig. 2.

Noni treatment alters genome-wide gene expression in adult D. sechellia. a) After RNA-sequencing of D. sechellia females fed noni fruit, expression of genes (FPKM) in control are shown on the X-axis, with expression of each gene on the Y-axis. Genes that are significantly differentially expressed in noni treatment are shown in red. b) Volcano plot of DEGs are shown, with Log₂(control/noni) on the X-axis, and -Log₁₀(q-value) on the Y-axis. Significantly DEGs are shown in red.

Fig. 3.

GO analysis of DEGs in response to noni fruit. See Supplementary Tables 7 and 8 for complete lists. a and b) Upregulated DEGs analyzed for enriched GO processes. a) Upregulated DEGs are enriched for several cellular components, including nonmembrane-bound organelle, external encapsulating structure, egg chorion, and chromosome. b) Upregulated DEGs are enriched for several biological processes, including sexual reproductive processes, eggshell formation, and cell cycle processes. c and d) Downregulated DEGs analyzed for enriched GO processes. c) Downregulated DEGs and enriched for several cellular components, including smooth septate junction, plasma membrane, nucleus, extracellular region, and cell surface. d) Downregulated DEGs are enriched for several molecular functions, including hydrolase activity and alkaline phosphatase activity.

Fig. 4.

Overlap of significantly DEGs in response to components of noni fruit. DEGs changing expression in response to OA (white), HA (gray), noni (blue), and l-DOPA (red). Overlaps of shared DEGs are shown, as are DEGs specific to each treatment.

Fig. 5.

Several TFs are predicted to be involved in the response to multiple components of noni fruit. TFs predicted by i-cisTarget analysis to regulate DEG expression in noni, l-DOPA, HA, and OA treatments in D. sechellia are shown. The GATA family of TFs (pnr, grn, GATAe, GATAd, and srp) is predicted to regulate DEG expression in both noni and l-DOPA treatment, with srp also being predicted in HA treatment. Zelda is predicted to regulate expression of DEGs in all 4 treatments. Single-minded is predicted to regulate DEG expression in noni, l-DOPA, and OA treatment. Rel, Hsf, and Blimp-1 are predicted to regulate DEG expression in both OA and HA treatments. Predicted regulatory networks for each treatment are found in Supplementary Fig. 3.