David M. Rand

Evolutionary divergence in gene regulation is a major source of phenotypic novelty, yet understanding how deeply conserved, pleiotropic signaling pathways evolve under selective constraint while maintaining essential cellular functions remains an important challenge. Here, we investigate the evolutionary divergence in transcriptional regulation mediated by the mechanistic target of rapamycin (mTOR) pathway in two related species, Drosophila melanogaster and Drosophila simulans . mTOR is a highly conserved central regulator of cellular growth and metabolism, making it well-suited for the study of regulatory evolution under functional constraint. Using a fully factorial RNA-seq design, we quantified transcriptional responses to mTOR inhibition by rapamycin across three tissues (head, thorax, and abdomen) and both sexes. Despite highly conserved tissue-specific expression – reflecting the close phylogenetic relationship between species – mTOR-mediated transcriptional responses showed clear evidence of evolutionary divergence. Divergence varied across tissues and sexes: heads of both sexes and female thoraces and abdomens showed the most rapid gene-level divergence, suggesting stronger directional selection, whereas male thorax and abdomen exhibited comparatively conserved responses, consistent with stabilizing selection. Gene-level divergence patterns were consistent across multiple metrics and generally mirrored pathway-level divergence, except in the female abdomen, which showed relative pathway-level conservation despite extensive gene-level divergence. Species-by-treatment interaction analyses further revealed divergence in core mTOR-regulated biological processes. Together, our results suggest that regulatory modularity may allow even highly conserved signaling pathways to evolve under context-specific selective pressures while maintaining critical functionality. Significance statement: The evolution of gene regulation is increasingly recognized as a major contributor to biological diversification. However, how highly pleiotropic and deeply conserved pathways evolve new regulatory effects without disrupting essential cellular functions remains an open question in evolutionary biology. Using the nutrient-sensing mTOR pathway as a model, we show that gene regulation mediated by this core, selectively-constrained signaling network has diverged between two closely related fruit fly species in a tissue- and sex-specific manner. Our findings suggest that even pleiotropic pathways like mTOR can diverge over short evolutionary timescales, and that regulatory modularity may facilitate the evolution of novel transcriptional effects in specific contexts while preserving essential functions.

Maternal inheritance allows selection to act on mtDNA-encoded effects in females but prevents direct selection on mtDNA in males. Mutations that are deleterious in males but neutral or beneficial in females can persist in populations. This predicts that mtDNA-based phenotypic variation should be more common among males than among females, a pattern referred to as Mothers Curse (MC). Most studies of MC place alternative mtDNAs on common homozygous nuclear chromosomal backgrounds, a condition not common in nature. Moreover, it is not known whether MC effects accumulate as mtDNAs acquire nucleotide substitutions between populations or species. We tested the MC hypothesis using mtDNAs from Drosophila melanogaster (OreR, Zimbabwe or w1118), D. simulans (siI and siII) and D. yakuba each placed on several D. melanogaster nuclear backgrounds heterozygous for different chromosomal deficiencies paired with a common w1118 chromosome set. Females and males were tested for starvation resistance, climbing speed, and flight performance. In the majority of chromosomal backgrounds the variance among mtDNA genotypes was greater in females than in males, opposite from the central prediction of Mothers Curse. This suggests that additive and dominance variation across the nuclear genome may provide nuclear blessings that can counter the curse of maternally inherited mtDNA.

The interdependent relationship between mitochondrial and nuclear genomes is a powerful model for understanding how epistasis shapes the architecture and evolution of complex traits. Once considered a neutral marker, mitochondrial DNA variation is now recognized as critical to phenotypic evolution because of its epistatic interactions and history of coevolution with the nuclear genome. A central challenge in evolutionary genetics is to quantify the relative importance of stabilizing and directional selection shaping complex trait distributions within and among species. Both can act on interacting and/or co-evolving genes contributing to quantitative traits, but resolving their relative roles is complicated by the complex architecture of most traits. Here, we use a panel of 90 Drosophila mitonuclear genotypes to quantify the relative contributions of mitochondrial, nuclear, and environmental variation and their interactions to four metabolically demanding complex traits. We sample both within-species and between-species mitochondrial variation and observe stronger interaction effects attributable to within-species variation, consistent with stabilizing selection maintaining mitonuclear function. Additionally, culturing the flies on a mitochondrial Complex I inhibitor, rotenone, reveals significant genotype x environment (G×E and G×G×E) interaction effects, providing insight into how genetic variation can be maintained across changing environments. Our results have broader implications in medicine, where mitochondrial DNA donors with longer purifying selection histories may be safer for mitochondrial replacement therapies.

Background: Many common diseases exhibit uncontrolled mTOR signaling, prompting considerable interest in the therapeutic potential of mTOR inhibitors, such as rapamycin, to treat a range of conditions, including cancer, aging-related pathologies, and neurological disorders. Despite encouraging preclinical results, the success of mTOR interventions in the clinic has been limited by off-target side effects and dose-limiting toxicities. Improving clinical efficacy and mitigating side effects require a better understanding of the influence of key clinical factors, such as sex, tissue, and genomic background, on the outcomes of mTOR-targeting therapies. Results: We assayed gene expression with and without rapamycin exposure across three distinct body parts (head, thorax, abdomen) of D. melanogaster flies, bearing either their native melanogaster mitochondrial genome or the mitochondrial genome from a related species, D. simulans. The fully factorial RNA-seq study design revealed a large number of genes that responded to the rapamycin treatment in a sex-dependent and tissue-dependent manner, and relatively few genes with the transcriptional response to rapamycin affected by the mitochondrial background. Reanalysis of an earlier study confirmed that mitochondria can have a temporal influence on rapamycin response. Conclusions: We found significant and wide-ranging effects of sex and body part, alongside a subtle, potentially time-dependent, influence of mitochondria on the transcriptional response to rapamycin. Our findings suggest a number of pathways that could be crucial for predicting potential side effects of mTOR inhibition in a particular sex or tissue. Further studies of the temporal response to rapamycin are necessary to elucidate the effects of the mitochondrial background on mTOR and its inhibition.

Resistance to starvation is a classic complex trait where genetic and environmental variables can greatly modify an animal’s ability to survive without nutrients. Genetic analyses in Drosophila have shown that starvation resistance is highly polygenic with different genetic architectures in different mapping populations. In this study we sought to dissect the genetic basis of starvation resistance among a set of mitonuclear genotypes carrying different mtDNAs paired with specific nuclear genomes from the Drosophila Genetic Reference Panel (DGRP). We focused on differences between one of the most sensitive strains (DGRP-765) and a strain with more moderate resistance (DGRP-315) whose starvation phenotypes appeared to be modified by alternative mtDNAs. Using complementary pooled-sequencing and forward genetic mapping approaches, we identified regions of chromosomes 2L, 3L and 3R contributing to starvation sensitivity and localize a major effect locus modifying starvation resistance to the coding region of phospholipase iPLA2-VIA. These analyses further confirm that the alternative mtDNAs had little influence on variation in starvation resistance between the genotypes studied. The sensitive line shows a starvation-dependent depletion of glucose and glycogen that is modified by hemi- and heterozygosity in the iPLA2-VIA region. These findings contribute to our understanding of the complex genetic relationship between resistance to starvation stress and nutrient metabolism.

Manipulation of host phenotypes by parasites is hypothesized to be an adaptive strategy enhancing parasite transmission across hosts and generations. Characterizing the molecular mechanisms of manipulation is important to advance our understanding of host–parasite coevolution. The trematode (Levinseniella byrdi) is known to alter the colour and behaviour of its amphipod host (Orchestia grillus) presumably increasing predation of amphipods which enhances trematode transmission through its life cycle. We sampled 24 infected and 24 uninfected amphipods from a salt marsh in Massachusetts to perform differential gene expression analysis. In addition, we constructed novel genomic tools forO. grillusincluding a de novo genome and transcriptome. We discovered that trematode infection results in upregulation of amphipod transcripts associated with pigmentation and detection of external stimuli, and downregulation of multiple amphipod transcripts implicated in invertebrate immune responses, such as vacuolar ATPase genes. We hypothesize that suppression of immune genes and the altered expression of genes associated with coloration and behaviour may allow the trematode to persist in the amphipod and engage in further biochemical manipulation that promotes transmission. The genomic tools and transcriptomic analyses reported provide new opportunities to discover how parasites alter diverse pathways underlying host phenotypic changes in natural populations.

We demonstrate that transcriptomes expressed in lung homogenates also differ between the mouse strains and after acute (3 wk) exposure of animals to cigarette smoke (CS) and/or to lipopolysaccharide. Mouse strains also differed in physiologic, pathologic, and transcriptomic, responses to more prolonged (6 wk) exposure to CS. These data support a genetic basis for enhanced susceptibility to acute and chronic lung injury among humans who smoke cigarettes.

MtDNA sequences can be incorporated into the nuclear genome and produce nuclear mitochondrial fragments (NUMTs), which resemble mtDNA in their sequence but are transmitted biparentally, like the nuclear genome. NUMTs can be mistaken as real mtDNA and may lead to the erroneous impression that mtDNA is biparentally transmitted. Here, we report a case of mtDNA heteroplasmy in a Drosophila melanogaster DGRP line, in which the one haplotype was biparentally transmitted in an autosomal manner. Given the sequence identity of this haplotype with the mtDNA, the crossing experiments led to uncertainty about whether heteroplasmy was real or an artifact due to a NUMT. More specific experiments revealed that there is a large NUMT insertion in the X chromosome of a specific DGRP line, imitating biparental inheritance of mtDNA. Our result suggests that studies on mtDNA heteroplasmy and on mtDNA inheritance should first exclude the possibility of NUMT interference in their data.

Mitochondria evolved from a union of microbial cells belonging to distinct lineages that were likely anaerobic. The evolution of eukaryotes required a massive reorganization of the 2 genomes and eventual adaptation to aerobic environments. The nutrients and oxygen that sustain eukaryotic metabolism today are processed in mitochondria through coordinated expression of 37 mitochondrial genes and over 1000 nuclear genes. This puts mitochondria at the nexus of gene-by-gene (G×G) and gene-by-environment (G×E) interactions that sustain life. Here we use a Drosophila model of mitonuclear genetic interactions to explore the notion that mitochondria are environments for the nuclear genome, and vice versa. We construct factorial combinations of mtDNA and nuclear chromosomes to test for epistatic interactions (G×G), and expose these mitonuclear genotypes to altered dietary environments to examine G×E interactions. We use development time and genome-wide RNAseq analyses to assess the relative contributions of mtDNA, nuclear chromosomes, and environmental effects on these traits (mitonuclear G×G×E). We show that the nuclear transcriptional response to alternative mitochondrial “environments” (G×G) has significant overlap with the transcriptional response of mitonuclear genotypes to altered dietary environments. These analyses point to specific transcription factors (e.g., giant) that mediated these interactions, and identified coexpressed modules of genes that may account for the overlap in differentially expressed genes. Roughly 20% of the transcriptome includes G×G genes that are concordant with G×E genes, suggesting that mitonuclear interactions are part of an organism’s environment.

A central challenge of quantitative genetics is partitioning phenotypic variation into genetic and non-genetic components. These non-genetic components are usually interpreted as environmental effects; however, variation between genetically identical individuals in a common environment can still exhibit phenotypic variation. A trait’s resistance to variation is called robustness, though the genetics underlying it are poorly understood. Accordingly, we performed an association study on a previously studied, whole organism trait: robustness for flight performance. Using 197 of the Drosophila Genetic Reference Panel (DGRP) lines, we surveyed variation across single nucleotide polymorphisms, whole genes, and epistatic interactions to find genetic modifiers robustness for flight performance. There was an abundance of genes involved in the development of sensory organs and processing of external stimuli, supporting previous work that processing proprioceptive cues is important for affecting variation in flight performance. Additionally, we tested insertional mutants for their effect on robustness using candidate genes found to modify flight performance. These results suggest several genes involved in modulating a trait mean are also important for affecting trait variance, or robustness, as well. Article Summary We sought to understand the genetic architecture of robustness (variation in a trait caused by non-genetic factors) for flight performance. We used 197 Drosophila Genetic Reference Panel (DGRP) lines to find significant individual variants and pairs of epistatic interactions, many of which were involved in proprioception. Additionally, we validated significant genes identified from a prior study for the mean of flight performance, showing genes affecting trait means may also affect trait robustness.