high-throughput-method-for-measuring-alcohol-sedation-time-individual

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

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.

Instinctive behaviors are genetically programmed behaviors that occur independent of experience. How genetic programs that give rise to the manifestation of such behaviors evolve remains an unresolved question. I propose that evolution of species-specific innate behaviors is accomplished through progressive modifications of pre-existing genetic networks composed of allelic variants. I hypothesize that changes in frequencies of one or more constituent allelic variants within the network leads to changes in gene network connectivity and the emergence of a reorganized network that can support the emergence of a novel behavioral phenotype and becomes stabilized when key allelic variants are driven to fixation.

Evolution of Epistatic Networks and the Genetic Basis of Innate Behaviors.

Behaviors associated with reproduction are major contributors to the evolutionary success of organisms and are subject to many evolutionary forces, including natural and sexual selection, and sexual conflict. Successful reproduction involves a range of behaviors, from finding an appropriate mate, courting, and copulation, to the successful production and (in oviparous animals) deposition of eggs following mating. As a consequence, behaviors and genes associated with reproduction are often under strong selection and evolve rapidly. Courtship rituals in flies follow a multimodal pattern, mediated through visual, chemical, tactile, and auditory signals. Premating behaviors allow males and females to assess the species identity, reproductive state, and condition of their partners. Conflicts between the "interests" of individual males, and/or between the reproductive strategies of males and females, often drive the evolution of reproductive behaviors. For example, seminal proteins transmitted by males often show evidence of rapid evolution, mediated by positive selection. Postmating behaviors, including the selection of oviposition sites, are highly variable and  species span the spectrum from generalists to obligate specialists. Chemical recognition features prominently in adaptation to host plants for feeding and oviposition. Selection acting on variation in pre-, peri-, and postmating behaviors can lead to reproductive isolation and incipient speciation. Response to selection at the genetic level can include the expansion of gene families, such as those for detecting pheromonal cues for mating, or changes in the expression of genes leading to visual cues such as wing spots that are assessed during mating. Here, we consider the evolution of reproductive behavior in  at two distinct, yet complementary, scales. Some studies take a microevolutionary approach, identifying genes and networks involved in reproduction, and then dissecting the genetics underlying complex behaviors in  Other studies take a macroevolutionary approach, comparing reproductive behaviors across the genus  and how these might correlate with environmental cues. A full synthesis of this field will require unification across these levels.

Evolution of Reproductive Behavior.

The ability to respond to chemosensory cues is critical for survival of most organisms. Among insects, Drosophila melanogaster has the best characterized olfactory system, and the availability of genome sequences of 30 Drosophila species provides an ideal scenario for studies on evolution of chemosensation. Gene duplications of chemoreceptor genes allow for functional diversification of the rapidly evolving chemoreceptor repertoire. Although some species of the genus Drosophila are generalists for host plant selection, rapid evolution of olfactory receptors, gustatory receptors, odorant-binding proteins, and cytochrome P450s has enabled diverse host specializations of different members of the genus. Here, I review diversification of the chemoreceptor repertoire among members of the genus Drosophila along with co-evolution of detoxification mechanisms that may have enabled occupation of diverse host plant ecological niches.

Chemosensation and Evolution of Drosophila Host Plant Selection.

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.

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.

Article Total : 2	Year
Publication title	2020
Publication title	2021

Article	Year
A Centered Genetic Network Contributes to Alcohol-Induced Variation in Drosophila Development. G3 (Bethesda, Md.)\| PubMed ID: 29871898	2018
Evolution of Epistatic Networks and the Genetic Basis of Innate Behaviors. Trends in genetics : TIG\| PubMed ID: 31706688	2020
Evolution of Reproductive Behavior. Genetics\| PubMed ID: 31907301	2020
Chemosensation and Evolution of Drosophila Host Plant Selection. iScience\| PubMed ID: 31923648	2020
Genotype by environment interaction for gene expression in Drosophila melanogaster. Nature communications\| PubMed ID: 33116142	2020
Functional Diversification, Redundancy, and Epistasis among Paralogs of the Drosophila melanogaster Obp50a-d Gene Cluster. Molecular biology and evolution\| PubMed ID: 33560417	2021
The brain on cocaine at single-cell resolution. Genome research\| PubMed ID: 34035044	2021
Genetic basis of variation in cocaine and methamphetamine consumption in outbred populations of . Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 34074789	2021

Robert R. H. Anholt

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