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Tetracycline (Tet) and derivative chemicals (e.g., doxycycline or Dox) have gained widespread recognition for their antibiotic properties since their introduction in the late 1970s, but recent work with these chemicals in the lab has shifted to include multiple techniques in all genetic model systems for the precise control of gene expression. The most widely used Tet‐modulated methodology is the Tet‐On/Tet‐Off gene expression system. Tet is generally considered to have effects specific to bacteria; therefore, it should have few off‐target effects when used in eukaryotic systems, and a previous study in the yeast Saccharomyces cerevisiae found that Dox had no effect on genome‐wide gene expression as measured by microarray. In contrast, another study found that the use of Dox in common cell lines and several model organisms led to mitonuclear protein imbalance, suggesting an inhibitory role of Dox in eukaryotic mitochondria. Recently, a new Dox derivative, 4‐epidoxycycline (4‐ED) was developed that was shown to have less off‐target consequences on mitochondrial health. To determine the best tetracycline family chemical to use for gene expression control in S. cerevisiae, we performed RNA sequencing (RNA‐seq) on yeast grown on standard medium compared with growth on media supplemented with Tet, Dox or 4‐ED. We found each caused dozens of genes to change expression, with Dox eliciting the greatest expression responses, suggesting that the specific tetracycline used in experiments should be tailored to the specific gene(s) of interest when using the Tet‐On/Tet‐Off system to reduce the consequences of confounding off‐target responses.

Drosophila sechellia is a dietary specialist fruit fly that evolved from a generalist ancestor to specialize on the toxic fruit of Morinda citrifolia. This species pair has been the subject of numerous studies where the goal has largely been to determine the genetic basis of adaptations associated with host specialization. Because one of the most striking features of M. citrifolia fruit is the production of toxic volatile compounds that kill insects, most genomic studies in D. sechellia to date have focused on gene expression responses to the toxic compounds in its food. In this study, we aim to identify new genes important for host specialization by profiling gene expression response to 3,4-dihydroxyphenylalanine (L-DOPA). Recent work found it to be highly abundant in M. citrifolia, critical for reproductive success of D. sechellia, and supplementation of diet with the downstream pathway product dopamine can influence toxin resistance phenotypes in related species. Here we used a combination of functional genetics and genomics techniques to identify new genes that are important for D. sechellia ecological adaptation to this new niche. We show that L-DOPA exposure can affect toxin resistance phenotypes, identify genes with plastic responses to L-DOPA exposure, and functionally test an identified candidate gene. We found that knock-down of Esterase 6 (Est6) in a heterologous species alters toxin resistance suggesting Est6 may play an important role in D. sechellia host specialization.

The underlying genetic basis of adaptive phenotypic changes is generally poorly understood, yet a growing number of case studies are beginning to shed light on important questions about the molecular nature and pleiotropy of such changes. We use Drosophila sechellia, a dietary specialist fruit fly that evolved to specialize on a single toxic host plant, Morinda citrifolia, as a model for adaptive phenotypic change and seek to determine the genetic basis of traits associated with host specialization in this species. The fruit of M. citrifolia is toxic to other drosophilids, primarily due to high levels of the defense chemical octanoic acid (OA), yet D. sechellia has evolved resistance to OA. Our prior work identified three Osiris family genes that reside in a fine‐mapped QTL for OA resistance: Osiris 6 (Osi6), Osi7, and Osi8, which can alter OA resistance in adult D. melanogaster when knocked down with RNA interference suggesting they may contribute to OA resistance in D. sechellia. Genetic mapping identified overlapping genomic regions involved in larval and adult OA resistance in D. sechellia, yet it remains unknown whether Osiris genes contribute to resistance in both life stages. Furthermore, because multiple genomic regions contribute to OA resistance, we aim to identify other gene(s) involved in this adaptation. Here, we identify candidate larval OA resistance genes using RNA sequencing to measure genome‐wide differential gene expression in D. sechellia larvae after exposure to OA and functionally test identified genes for a role in OA resistance. We then test the Osiris genes previously shown to alter adult OA resistance for effects on OA resistance in larvae. We found that Osi8 knockdown decreased OA resistance in D. melanogaster larvae. These data suggest that evolved changes in Osi8 could impact OA resistance in multiple life stages while Osi6 and Osi7 may only impact adult resistance to OA.

The dietary specialist fruit fly Drosophila sechellia has evolved to specialize on the toxic fruit of its host plant Morinda citrifolia. Toxicity of Morinda fruit is primarily due to high levels of octanoic acid (OA). Using RNA interference (RNAi), prior work found that knockdown of Osiris family genes Osiris 6 (Osi6), Osi7, and Osi8 led to increased susceptibility to OA in adult D. melanogaster flies, likely representing genes underlying a Quantitative Trait Locus (QTL) for OA resistance in D. sechellia. While genes in this major effect locus are beginning to be revealed, prior work has shown at least five regions of the genome contribute to OA resistance. Here, we identify new candidate OA resistance genes by performing differential gene expression analysis using RNA-sequencing (RNA-seq) on control and OA-exposed D. sechellia flies. We found 104 significantly differentially expressed genes with annotated orthologs in D. melanogaster, including six Osiris gene family members, consistent with previous functional studies and gene expression analyses. Gene ontology (GO) term enrichment showed significant enrichment for cuticle development in upregulated genes and significant enrichment of immune and defense responses in downregulated genes, suggesting important aspects of the physiology of D. sechellia that may play a role in OA resistance. In addition, we identified five candidate OA resistance genes that potentially underlie QTL peaks outside of the major effect region, representing promising new candidate genes for future functional studies.