High-Throughput Method for Measuring Alcohol Sedation Time of Individual <em>Drosophila melanogaster</em>

Drosophila melanogaster provides an excellent model to study the genetic underpinnings of alcohol sensitivity. In contrast to studies in human ...

A High Throughput Microplate Feeder Assay for Quantification of Consumption in <em>Drosophila</em>

Quantifying food intake in Drosophila is used to study the genetic and physiological underpinnings of consumption-associated traits, their environmental ...

Clemson University

Prenatal exposure to ethanol causes a wide range of adverse physiological, behavioral and cognitive consequences. However, identifying allelic variants and genetic networks associated with variation in susceptibility to prenatal alcohol exposure is challenging in human populations, since time and frequency of exposure and effective dose cannot be determined quantitatively and phenotypic manifestations are diverse. Here, we harnessed the power of natural variation in the  Genetic Reference Panel (DGRP) to identify genes and genetic networks associated with variation in sensitivity to developmental alcohol exposure. We measured development time from egg to adult and viability of 201 DGRP lines reared on regular or ethanol- supplemented medium and identified polymorphisms associated with variation in susceptibility to developmental ethanol exposure. We also documented genotype-dependent variation in sensorimotor behavior after developmental exposure to ethanol using the startle response assay in a subset of 39 DGRP lines. Genes associated with development, including development of the nervous system, featured prominently among genes that harbored variants associated with differential sensitivity to developmental ethanol exposure. Many of them have human orthologs and mutational analyses and RNAi targeting functionally validated a high percentage of candidate genes. Analysis of genetic interaction networks identified C (C) as a central, highly interconnected hub gene.  encodes a protein kinase associated with cell cycle regulation and is prominently expressed in ovaries. Thus, exposure to ethanol during development of  might serve as a genetic model for translational studies on fetal alcohol spectrum disorder.

A  Centered Genetic Network Contributes to Alcohol-Induced Variation in Drosophila Development.

Circadian rhythms influence physiological processes from sleep-wake cycles to body temperature and are controlled by highly conserved cycling molecules. Although the mechanistic basis of the circadian clock has been known for decades, the extent to which circadian rhythms vary in nature and the underlying genetic basis for that variation is not well understood. We measured circadian period (Ʈ) and rhythmicity index in the Drosophila Genetic Reference Panel (DGRP) and observed extensive genetic variation in both. Seven DGRP lines had sexually dimorphic arrhythmicity and one line had an exceptionally long Ʈ. Genome-wide analyses identified 584 polymorphisms in 268 genes. We observed differences among transcripts for nine genes predicted to interact among themselves and canonical clock genes in the long period line and a control. Mutations/RNAi knockdown targeting these genes also affected circadian behavior. Our observations reveal that complex genetic interactions influence high levels of variation in circadian phenotypes.

Genome-Wide Association Study of Circadian Behavior in Drosophila melanogaster.

Testing the assumptions of parametric linear models: the need for biological data mining in disciplines such as human genetics.

Limited lifespan and senescence are near-universal phenomena. These quantitative traits exhibit variation in natural populations due to the segregation of many interacting loci and from environmental effects. Due to the complexity of the genetic control of lifespan and senescence, our understanding of the genetic basis of variation in these traits is incomplete. Here, we analyzed the pattern of genetic divergence between long-lived (O)  lines selected for postponed reproductive senescence and unselected control (B) lines. We quantified the productivity of the O and B lines and found that reproductive senescence is maternally controlled. We therefore chose 57 candidate genes that are expressed in ovaries, 49 of which have human orthologs, and assessed the effects of RNA interference in ovaries and accessary glands on lifespan and reproduction. All but one candidate gene affected at least one life history trait in one sex or productivity week. In addition, 23 genes had antagonistic pleiotropic effects on lifespan and productivity. Identifying evolutionarily conserved genes affecting increased lifespan and delayed reproductive senescence is the first step toward understanding the evolutionary forces that maintain segregating variation at these loci in nature and may provide potential targets for therapeutic intervention to delay senescence while increasing lifespan.

Genetic Basis of Increased Lifespan and Postponed Senescence in .

Spontaneous grooming behavior is a component of insect fitness. We quantified spontaneous grooming behavior in 201 sequenced lines of the  Genetic Reference Panel and observed significant genetic variation in spontaneous grooming, with broad-sense heritabilities of 0.25 and 0.24 in females and males, respectively. Although grooming behavior is highly correlated between males and females, we observed significant sex by genotype interactions, indicating that the genetic basis of spontaneous grooming is partially distinct in the two sexes. We performed genome-wide association analyses of grooming behavior, and mapped 107 molecular polymorphisms associated with spontaneous grooming behavior, of which 73 were in or near 70 genes and 34 were over 1 kilobase from the nearest gene. The candidate genes were associated with a wide variety of gene ontology terms, and several of the candidate genes were significantly enriched in a genetic interaction network. We performed functional assessments of 29 candidate genes using RNA interference, and found that 11 affected spontaneous grooming behavior. The genes associated with natural variation in Drosophila grooming are involved with glutamate metabolism () and transport (); interact genetically with () or are in the same gene family as () genes previously implicated in grooming behavior; are involved in the development of the nervous system and other tissues; or regulate the Notch and Epidermal growth factor receptor signaling pathways. Several DGRP lines exhibited extreme grooming behavior. Excessive grooming behavior can serve as a model for repetitive behaviors diagnostic of several human neuropsychiatric diseases.

Genetic Basis of Natural Variation in Spontaneous Grooming in .

The genetics of phenotypic responses to changing environments remains elusive. Using whole-genome quantitative gene expression as a model, here we study how the genetic architecture of regulatory variation in gene expression changed in a population of fully sequenced inbred Drosophila melanogaster strains when flies developed in different environments (25 °C and 18 °C). We find a substantial fraction of the transcriptome exhibited genotype by environment interaction, implicating environmentally plastic genetic architecture of gene expression. Genetic variance in expression increases at 18 °C relative to 25 °C for most genes that have a change in genetic variance. Although the majority of expression quantitative trait loci (eQTLs) for the gene expression traits in the two environments are shared and have similar effects, analysis of the environment-specific eQTLs reveals enrichment of binding sites for two transcription factors. Finally, although genotype by environment interaction in gene expression could potentially disrupt genetic networks, the co-expression networks are highly conserved across environments. Genes with higher network connectivity are under stronger stabilizing selection, suggesting that stabilizing selection on expression plays an important role in promoting network robustness.

Genotype by environment interaction for gene expression in Drosophila melanogaster.

Large multigene families, such as the insect odorant-binding proteins (OBPs), are thought to arise through functional diversification after repeated gene duplications. Whereas many OBPs function in chemoreception, members of this family are also expressed in tissues outside chemosensory organs. Paralogs of the Obp50 gene cluster are expressed in metabolic and male reproductive tissues, but their functions and interrelationships remain unknown. Here, we report the genetic dissection of four members of the Obp50 cluster, which are in close physical proximity without intervening genes. We used CRISPR technology to excise the entire cluster while introducing a PhiC31 reintegration site to reinsert constructs in which different combinations of the constituent Obp genes were either intact or rendered inactive. We performed whole transcriptome sequencing and assessed sexually dimorphic changes in transcript abundances (transcriptional niches) associated with each gene-edited genotype. Using this approach, we were able to estimate redundancy, additivity, diversification, and epistasis among Obp50 paralogs. We analyzed the effects of gene editing of this cluster on organismal phenotypes and found a significant skewing of sex ratios attributable to Obp50a, and sex-specific effects on starvation stress resistance attributable to Obp50d. Thus, there is functional diversification within the Obp50 cluster with Obp50a contributing to development and Obp50d to stress resistance. The deletion-reinsertion approach we applied to the Obp50 cluster provides a general paradigm for the genetic dissection of paralogs of multigene families.

Functional Diversification, Redundancy, and Epistasis among Paralogs of the Drosophila melanogaster Obp50a-d Gene Cluster.

In cats, mutations in myosin binding protein C (encoded by the MYBPC3 gene) have been associated with hypertrophic cardiomyopathy (HCM). However, the molecular mechanisms linking these mutations to HCM remain unknown. Here, we establish Drosophila melanogaster as a model to understand this connection by generating flies harboring MYBPC3 missense mutations (A31P and R820W) associated with feline HCM. The A31P and R820W flies displayed cardiovascular defects in their heart rates and exercise endurance. We used RNA-seq to determine which processes are misregulated in the presence of mutant MYBPC3 alleles. Transcriptome analysis revealed significant downregulation of genes encoding small nucleolar RNA (snoRNAs) in exercised female flies harboring the mutant alleles compared to flies that harbor the wild-type allele. Other processes that were affected included the unfolded protein response and immune/defense responses. These data show that mutant MYBPC3 proteins have widespread effects on the transcriptome of co-regulated genes. Transcriptionally differentially expressed genes are also candidate genes for future evaluation as genetic modifiers of HCM as well as candidate genes for genotype by exercise environment interaction effects on the manifestation of HCM; in cats as well as humans.

Heat shock proteins and small nucleolar RNAs are dysregulated in a Drosophila model for feline hypertrophic cardiomyopathy.

Whereas the neurological effects of cocaine have been well documented, effects of acute cocaine consumption on genome-wide gene expression across the brain remain largely unexplored. This question cannot be readily addressed in humans but can be approached using the  model, where gene expression in the entire brain can be surveyed at once. Flies exposed to cocaine show impaired locomotor activity, including climbing behavior and startle response (a measure of sensorimotor integration), and increased incidence of seizures and compulsive grooming. To identify specific cell populations that respond to acute cocaine exposure, we analyzed single-cell transcriptional responses in duplicate samples of flies that consumed fixed amounts of sucrose or sucrose supplemented with cocaine, in both sexes. Unsupervised clustering of the transcriptional profiles of a total of 86,224 cells yielded 36 distinct clusters. Annotation of clusters based on gene markers revealed that all major cell types (neuronal and glial) as well as neurotransmitter types from most brain regions were represented. The brain transcriptional responses to cocaine showed profound sexual dimorphism and were considerably more pronounced in males than females. Differential expression analysis within individual clusters indicated cluster-specific responses to cocaine. Clusters corresponding to Kenyon cells of the mushroom bodies and glia showed especially large transcriptional responses following cocaine exposure. Cluster specific coexpression networks and global interaction networks revealed a diverse array of cellular processes affected by acute cocaine exposure. These results provide an atlas of sexually dimorphic cocaine-modulated gene expression in a model brain.

The  brain on cocaine at single-cell resolution.

We used  to map the genetic basis of naturally occurring variation in voluntary consumption of cocaine and methamphetamine. We derived an outbred advanced intercross population (AIP) from 37 sequenced inbred wild-derived lines of the  Genetic Reference Panel (DGRP), which are maximally genetically divergent, have minimal residual heterozygosity, are not segregating for common inversions, and are not infected with  We assessed consumption of sucrose, methamphetamine-supplemented sucrose, and cocaine-supplemented sucrose and found considerable phenotypic variation for consumption of both drugs, in both sexes. We performed whole-genome sequencing and extreme quantitative trait locus (QTL) mapping on the top 10% of consumers for each replicate, sex, and condition and an equal number of randomly selected flies. We evaluated changes in allele frequencies among high consumers and control flies and identified 3,033 variants significantly ( < 1.9 × 10) associated with increased consumption, located in or near 1,962 genes. Many of these genes are associated with nervous system development and function, and 77 belong to a known gene-gene interaction subnetwork. We assessed the effects of RNA interference (RNAi) on drug consumption for 22 candidate genes; 17 had a significant effect in at least one sex. We constructed allele-specific AIPs that were homozygous for alternative candidate alleles for 10 single-nucleotide polymorphisms (SNPs) and measured average consumption for each population; 9 SNPs had significant effects in at least one sex. The genetic basis of voluntary drug consumption in  is polygenic and implicates genes with human orthologs and associated variants with sex- and drug-specific effects.

Trudy F. C. Mackay

Department of Genetics and Biochemistry and Center for Human Genetics

Clemson University

High-Throughput Method for Measuring Alcohol Sedation Time of Individual Drosophila melanogaster

A High Throughput Microplate Feeder Assay for Quantification of Consumption in Drosophila

Trudy F. C. Mackay

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Genetics and Biochemistry and Center for Human Genetics

Clemson University

High-Throughput Method for Measuring Alcohol Sedation Time of Individual Drosophila melanogaster

A High Throughput Microplate Feeder Assay for Quantification of Consumption in Drosophila

Department of Genetics and Biochemistry and Center for Human Genetics