Drosophila Research Articles

Point Mutations in the M Domain of PCID2 Impair Its Function in mRNA Export in Drosophila melanogaster.

The PCID2 protein is a component of the eukaryotic TREX-2 complex, which is responsible for mRNA export from the nucleus into the cytoplasm. We have previously shown that Drosophila melanogaster PCID2 is involved in specific mRNA recognition and identified the key amino acids responsible for its interaction with the ras2 RNA. In this work, point mutations of the amino acids were shown to disrupt the PCID2 interaction with cell RNAs and to distort the export of polyA-containing mRNAs from the nucleus into the cytoplasm in Drosophila cells.

High-affinity biomolecular interactions are modulated by low-affinity binders

The strength of molecular interactions is characterized by their dissociation constants (KD). Only high-affinity interactions (KD ≤ 10−8 M) are extensively investigated and support binary on/off switches. However, such analyses have discounted the presence of low-affinity binders (KD > 10−5 M) in the cellular environment. We assess the potential influence of low-affinity binders on high-affinity interactions. By employing Gillespie stochastic simulations and continuous methods, we demonstrate that the presence of low-affinity binders can alter the kinetics and the steady state of high-affinity interactions. We refer to this effect as ‘herd regulation’ and have evaluated its possible impact in two different contexts including sex determination in Drosophila melanogaster and in signalling systems that employ molecular thresholds. We have also suggested experiments to validate herd regulation in vitro. We speculate that low-affinity binders are prevalent in biological contexts where the outcomes depend on molecular thresholds impacting homoeostatic regulation.

Drosophila melanogaster pigmentation demonstrates adaptive phenotypic parallelism but genomic unpredictability over multiple timescales.

Shifts in global climate conditions have heightened our need to understand the dynamics and pace of adaptation in natural populations. In order to anticipate the population-level response to rapidly changing environmental conditions, we need to understand whether trait evolution is predictable over short timescales, and whether the genetic basis of adaptation is shared or distinct across multiple timescales. Here, we explored parallelism in the adaptive response of a complex phenotype, D. melanogaster pigmentation, to shared conditions that varied over multiple spatiotemporal scales. Our results demonstrate that while phenotypic adaptation proceeds as a predictable response to environmental gradients, even over short timescales, the genetic basis of the adaptive response is variable and nuanced across spatial and temporal contexts.

A large-scale in vivo screen to investigate the roles of human genes in Drosophila melanogaster.

Understanding the signaling pathways in which genes participate is essential for discovering the etiology of diseases in humans. The model organism, Drosophila melanogaster, has been crucial in understanding the signaling pathways in humans, given the evolutionary conservation of a significant number of genes between the two species. Genetic screens using Drosophila are a useful way of testing large number of genes to study their function and roles within signaling pathways. We conducted a large-scale genetic screen to identify which human genes cause an alteration in the morphology of the Drosophila eye. The GMR-Gal4 was employed to activate a single UAS-human gene in the eye tissue. In total, we screened 802 UAS-human gene stocks, corresponding to 787 human protein-coding genes, for the ability to influence eye development. We found that overexpression of 64 human genes were capable of disrupting eye development, as determined by phenotypic changes in eye texture, size, shape, bristle morphology, and ommatidia organization. Subsequent analysis revealed that the fly genome encodes proteins that are homologous to a majority of the 64 human genes, raising the possibility that overexpression of these transgenes altered eye development by altering the activity of evolutionarily conserved developmental signaling pathways. Consistent with this hypothesis, a secondary screen demonstrated that overexpression of fly homologs produced phenotypes that mimicked those produced by overexpression of the human gene. Our screening has identified 64 human genes capable of inducing phenotypes in the fly, offering a foundation for ongoing research aimed at understanding functionally conserved pathways across species.

A newly evolved gene is essential for efficient sperm entry into eggs in Drosophila melanogaster.

New genes arise through a variety of evolutionary processes and provide raw material for adaptation in the face of both natural and sexual selection. De novo evolved genes emerge from previously non-protein-coding DNA sequences, and many such genes are expressed in male reproductive structures. In Drosophila melanogaster, several putative de novo genes have evolved essential roles in spermatogenesis, but whether such genes can also impact sperm function beyond the male has not been investigated. We identified a putative de novo gene, katherine johnson (kj), that is required for high levels of male fertility. Males that do not express kj produce and transfer sperm that are stored normally in females, but sperm from these males enter eggs with severely reduced efficiency. Using a tagged transgenic rescue construct, we observed that KJ protein localizes to the nuclear periphery in various stages of spermatogenesis, but is not detectable in mature sperm. These data suggest that kj exerts an effect on sperm development, the loss of which results in reduced fertilization ability. While previous bioinformatic analyses suggested the kj gene was restricted to the melanogaster group of Drosophila, we identified putative orthologs with conserved synteny, male-biased expression, and predicted protein features across the genus, as well as instances of gene loss in some lineages. Thus, kj potentially arose in the Drosophila common ancestor and subsequently evolved an essential role in D. melanogaster. Our results demonstrate a new aspect of male reproduction that has been shaped by new gene evolution and provide a molecular foothold for further investigating the mechanism of sperm entry into eggs in Drosophila.

Drosophila ring chromosomes interact with sisters and homologs to produce anaphase bridges in mitosis.

Ring chromosomes are known in many eukaryotic organisms, including humans. They are typically associated with a variety of maladies, including abnormal development and lethality. Underlying these phenotypes are anaphase chromatin bridges that can lead to chromosome loss, nondisjunction and breakage. By cytological examination of ring chromosomes in Drosophila melanogaster we identified five causes for anaphase bridges produced by ring chromosomes. Catenation of sister chromatids is the most common cause and these bridges frequently resolve during anaphase, presumably by the action of topoisomerase II. Sister chromatid exchange and chromosome breakage followed by sister chromatid union also produce anaphase bridges. Mitotic recombination with the homolog was rare, but was another route to generation of anaphase bridges. Most surprising, was the discovery of homolog capture, where the ring chromosome was connected to its linear homolog in anaphase. We hypothesize that this is a remnant of mitotic pairing and that the linear chromosome is connected to the ring by multiple wraps produced through the action of topoisomerase II during establishment of homolog pairing. In support, we showed that in a ring/ring homozygote the two rings are frequently catenated in mitotic metaphase, a configuration that requires breaking and rejoining of at least one chromosome.

Exploring the Mutated Kinases for Chemoenzymatic Synthesis of N4-Modified Cytidine Monophosphates.

Nucleosides, nucleotides, and their analogues are an important class of molecules that are used as substrates in research of enzymes and nucleic acid, or as antiviral and antineoplastic agents. Nucleoside phosphorylation is usually achieved with chemical methods; however, enzymatic phosphorylation is a viable alternative. Here, we present a chemoenzymatic synthesis of modified cytidine monophosphates, where a chemical synthesis of novel N4-modified cytidines is followed by an enzymatic phosphorylation of the nucleosides by nucleoside kinases. To enlarge the substrate scope, multiple mutant variants of Drosophila melanogaster deoxynucleoside kinase (DmdNK) (EC:2.7.1.145) and Bacillus subtilis deoxycytidine kinase (BsdCK) (EC:2.7.1.74) have been created and tested. It has been determined that certain point mutations in the active sites of the kinases alter their substrate specificities noticeably and allow phosphorylation of compounds that had been otherwise not phosphorylated by the wild-type DmdNK or BsdCK.

Assessing the role of mitonuclear interactions on mitochondrial function and organismal fitness in natural Drosophila populations

Abstract Mitochondrial function depends on the effective interactions between proteins and RNA encoded by the mitochondrial and nuclear genomes. Evidence suggests that both genomes respond to thermal selection and promote adaptation. However, the contribution of their epistatic interactions to life history phenotypes in the wild remains elusive. We investigated the evolutionary implications of mitonuclear interactions in a real-world scenario that sees populations adapted to different environments, altering their geographical distribution while experiencing flow and admixture. We created a Drosophila melanogaster panel with replicate native populations from the ends of the Australian east-coast cline, into which we substituted the mtDNA haplotypes that were either predominant or rare at each cline-end, thus creating putatively mitonuclear matched and mismatched populations. Our results suggest that mismatching may impact phenotype, with populations harboring the rarer mtDNA haplotype suffering a trade-off between aerobic capacity and key fitness aspects such as reproduction, growth, and survival. We discuss the significance of mitonuclear interactions as modulators of life history phenotypes in the context of future adaptation and population persistence.

Neural Control of Naturalistic Behavior Choices.

In the natural world, animals make decisions on an ongoing basis, continuously selecting which action to undertake next. In the lab, however, the neural bases of decision processes have mostly been studied using artificial trial structures. New experimental tools based on the genetic toolkit of model organisms now make it experimentally feasible to monitor and manipulate neural activity in small subsets of neurons during naturalistic behaviors. We thus propose a new approach to investigating decision processes, termed reverse neuroethology. In this approach, experimenters select animal models based on experimental accessibility and then utilize cutting-edge tools such as connectomes and genetically encoded reagents to analyze the flow of information through an animal's nervous system during naturalistic choice behaviors. We describe how the reverse neuroethology strategy has been applied to understand the neural underpinnings of innate, rapid decision making, with a focus on defensive behavioral choices in the vinegar fly Drosophila melanogaster.

Engineered fungus containing a caterpillar gene kills insects rapidly by disrupting their ecto- and endo-microbiomes

Similar to the physiological importance of gut microbiomes, recent works have shown that insect ectomicrobiotas can mediate defensive colonization resistance against fungal parasites that infect via cuticle penetration. Here we show that engineering the entomopathogenic fungus Metarhizium robertsii with a potent antibacterial moricin gene from silkworms substantially enhances the ability of the fungus to kill mosquitos, locusts, and two Drosophila species. Further use of Drosophila melanogaster as an infection model, quantitative microbiome analysis reveals that engineered strains designed to suppress insect cuticular bacteria additionally disrupt gut microbiomes. An overgrowth of harmful bacteria such as the opportunistic pathogens of Providencia species is detected that can accelerate insect death. In support, quantitative analysis of antimicrobial genes in fly fat bodies and guts indicates that topical fungal infections result in the compromise of intestinal immune responses. In addition to providing an innovative strategy for improving the potency of mycoinsecticides, our data solidify the importance of both the ecto- and endo-microbiomes in maintaining insect wellbeing.

Selection for greater dispersal in early life increases rate of age-dependent decline in locomotor activity and shortens lifespan.

Locomotor activity is one of the major traits that is affected by age. Greater locomotor activity is also known to evolve in the course of dispersal evolution. However, the impact of dispersal evolution on the functional senescence of locomotor activity is largely unknown. We addressed this knowledge gap using large outbred populations of Drosophila melanogaster selected for increased dispersal. We tracked locomotor activity of these flies at regular intervals until a late age. The longevity of these flies was also recorded. We found that locomotor activity declines with age in general. However interestingly, the activity level of dispersal-selected populations never drops below the ancestry-matched controls, despite the rate of age-dependent decline in activity of the dispersal-selected populations being greater than their respective controls. The dispersal-selected population was also found to have a shorter lifespan as compared to its control, a potential cost of elevated level of activity throughout their life. These results are crucial in the context of invasion biology as contemporary climate change, habitat degradation, and destruction provide congenial conditions for dispersal evolution. Such controlled and tractable studies investigating the ageing pattern of important functional traits are important in the field of biogerontology as well.

The role of uncertainty and negative feedback loops in the evolution of induced immune defenses.

Organisms use constitutive or induced defenses against pathogens and other external threats. Constitutive defenses are constantly on, whereas induced defenses are activated when needed. Each of these strategies has costs and benefits, which can affect the type of defense that evolves in response to pathogens. In addition, induced defenses are usually regulated by multiple negative feedback mechanisms that prevent overactivation of the immune response. However, it is unclear how negative feedback affects the costs, benefits, and evolution of induced responses. To address this gap, we developed a mechanistic model of the well-characterized Drosophila melanogaster immune signaling network that includes 3 separate mechanisms of negative feedback as a representative of the widespread phenomenon of multilevel regulation of induced responses. We show that, under stochastic fly-bacteria encounters, an induced defense is favored when bacterial encounters are rare or uncertain, but in ways that depend on the bacterial proliferation rate. Our model also predicts that the specific negative regulators that optimize the induced response depend on the bacterial proliferation rate, linking negative feedback mechanisms to the factors that favor induction.

Mechanisms for controlling Dorsal nuclear levels.

Formation of the Dorsal nuclear-cytoplasmic gradient is important for the proper establishment of gene expression patterns along the dorsal-ventral (DV) axis during embryogenesis in Drosophila melanogaster. Correct patterning of the DV axis leads to formation of the presumptive mesoderm, neurogenic ectoderm, dorsal ectoderm, and amnioserosa, which are tissues necessary for embryo viability. While Toll signaling is necessary for Dorsal gradient formation, a gradient still forms in the absence of Toll, suggesting there are additional mechanisms required to achieve correct nuclear Dorsal levels. Potential mechanisms include post-translational modification, shuttling, and nuclear spacing. Post-translational modification could affect import and export rates either directly through modification of a nuclear localization sequence or nuclear export sequence, or indirectly by affecting interactions with binding partners that alter import and export rates. Shuttling, which refers to the facilitated diffusion of Dorsal through its interaction with its cytoplasmic inhibitor Cactus, could regulate nuclear levels by delivering more Dorsal ventrally. Finally, nuclear spacing could result in higher nuclear levels by leaving fewer nuclei in the ventral domain to uptake Dorsal. This review details how each of these mechanisms may help establish Dorsal nuclear levels in the early fly embryo, which serves as a paradigm for understanding how the dynamics of graded inputs can influence patterning and target gene expression. Furthermore, careful analysis of nuclear Dorsal levels is likely to provide general insights as recent studies have suggested that the regulation of nuclear import affects the timing of gene expression at the maternal-to-zygotic transition.

Digest: Evaluating sexual conflict in the genome.

Audet et al. (2024) investigate the genomic basis of sexual conflict in response to sexually discordant size selection in Drosophila melanogaster. They report interesting morphological changes in sexual dimorphism and multivariate allometry. Although they do not find any genetic variants that individually show a strong effect on these traits, as expected for a polygenic trait such as body size, they do find a region on chromosome 3L showing signs of sexually discordant selection (i.e., conflict between males and females). This study highlights potential genomic regions involved in sexual conflict, offering insights into sex-specific adaptations.

The impact of altered dietary adenine concentrations on the gut microbiota in Drosophila.

The gut microbiota influences host metabolism and health, impacting diseases. Research into how diet affects gut microbiome dynamics in model organisms is crucial but underexplored. Herein, we examined how dietary adenine affects uric acid levels and the gut microbiota over five generations of Drosophila melanogaster. Wild-type W1118 flies consumed diets with various adenine concentrations (GC: 0%, GL: 0.05%, and GH: 0.10%), and their gut microbiota were assessed via Illumina MiSeq sequencing. Adenine intake significantly increased uric acid levels in the GH group > the GC group. Despite no significant differences in the alpha diversity indices, there were significant disparities in the gut microbiota health index (GMHI) and dysbiosis index (MDI) among the groups. Adenine concentrations significantly altered the diversity and composition of the gut microbiota. High adenine intake correlated with increased uric acid levels and microbial population shifts, notably affecting the abundances of Proteobacteria and Firmicutes. The gut microbiota phenotypes included mobile elements, gram-positive bacteria, biofilm-forming bacteria, and gram-negative bacteria. The significantly enriched KEGG pathways included ageing, carbohydrate metabolism, and the immune system. In conclusion, adenine intake increases uric acid levels, alters gut microbiota, and affects KEGG pathways in Drosophila across generations. This study highlights the impact of dietary adenine on uric acid levels and the gut microbiota, providing insights into intergenerational nutritional effects.

Ancestral lineages of dietary exposure to an endocrine disrupting chemical drive distinct forms of transgenerational subfertility in an insect model

Across the globe, many species of insects are facing population decline. This is largely driven by anthropogenic changes to the environment, including the widespread exposure of invertebrates to endocrine disrupting chemicals (EDCs), which impair fertility. To test whether generations of Drosophila melanogaster born from parents exposed to a common dietary EDC, equol, could recover reproductive function, we quantified the reproductive capacity of the two subsequent generations. Using a novel suite of flow cytometry assays to assess sperm functionality in real time, we find that sperm function is compromised across three generations, even after non-exposed in individuals contribute to the breeding population. Though the sex ratio alters in response to EDC exposure, favouring the survival of female offspring, most lineages with ancestral EDC exposure exhibit persistent subfertility in both the male and female. Male offspring with ancestral EDC exposure present with reduced fertility and dysfunctional spermatozoa, whereby spermatozoa are metabolically stressed, lack DNA integrity and present with permanent epigenetic alterations. Across generations, male and female offspring demonstrate distinct patterns of reproductive characteristics, depending upon the specific lineage of EDC exposure. Our results illustrate how dietary EDCs present in agricultural plants could promote transgenerational subfertility and contribute to declining insect populations.

Subsets of Nanometer Vesicles in the Fly Release Differential Fractions of Vesicular Serotonin Content during Exocytosis.

Serotonin, a monoamine neurotransmitter, is important in both the central nervous system (CNS) and the peripheral nervous system. Malfunction of serotonin signaling leads to various disorders. We studied serotonin signaling from serotonergic neurons inside the ventral nerve cord of Drosophila melanogaster. Serotonergic neurons and stimulated release were visualized and achieved with mCherry and channelrhodopsin-2 (an optogenetically transfected ion channel), respectively, and two electrochemical techniques quantified serotonin release and vesicular content. Mean vesicular serotonin content released during exocytosis from these neurons was 84 %, considerably higher than reported in previous studies regarding octopamine (4.5 %) and glutamate release (31 %). Serotonin content within all vesicles is uniformly changed when serotonin concentration is inhibited or enhanced. However, serotonin release exhibits two Gaussian distributions: higher frequency of small release events, and similar or slightly higher frequency of large events, resulting in differential release fractions ranging from partial (13-18 %) to full (100 %) release after treatment with agents to either enhance or diminish release. This is the first example of consistent full exocytotic release events we have observed in any system. We suggest one pool of vesicles can release significantly diverse fractions of transmitter load during exocytosis, a potentially novel pathway to regulate exocytosis and neuronal signaling.

Enhanced expression of the myogenic factor Myocyte enhancer factor-2 in imaginal disc myoblasts activates a partial, but incomplete, muscle development program

The Myocyte enhancer factor-2 (MEF2) transcription factor plays a vital role in orchestrating muscle differentiation. While MEF2 cannot effectively induce myogenesis in naïve cells, it can potently accelerate myogenesis in mesodermal cells. This includes in Drosophila melanogaster imaginal disc myoblasts, where triggering premature muscle gene expression in these adult muscle progenitors has become a paradigm for understanding the regulation of the myogenic program. Here, we investigated the global consequences of MEF2 overexpression in the imaginal wing disc myoblasts, by combining RNA-sequencing with RT-qPCR and immunofluorescence. We observed the formation of sarcomere-like structures that contained both muscle and cytoplasmic myosin, and significant upregulation of muscle gene expression, especially genes essential for myofibril formation and function. These transcripts were functional since numerous myofibrillar proteins were detected in discs using immunofluorescence. Interestingly, muscle genes whose expression is restricted to the adult stages were not activated in these adult myoblasts. These studies confirm a broad activation of the myogenic program in response to MEF2 expression and suggest that additional regulatory factors are required for promoting the adult muscle-specific program. Our findings contribute to understanding the regulatory mechanisms governing muscle development and highlight the multifaceted role of MEF2 in orchestrating this intricate process.

Neurodevelopmental changes in Drosophila melanogaster are restored by treatment with lutein-loaded nanoparticles: Positive modulation of neurochemical and behavioral parameters

Neurodevelopmental disorders, such as autism spectrum disorder (ASD) and attention deficit hyperactivity disorder (ADHD), are characterized by persistent changes in communication and social interaction, as well as restricted and stereotyped patterns of behavior. The complex etiology of these disorders possibly combines the effects of multiple genes and environmental factors. Hence, exposure to insecticides such as imidacloprid (IMI) has been used to replicate the changes observed in these disorders. Lutein is known for its anti-inflammatory and antioxidant properties and is associated with neuroprotective effects. Therefore, the aim of this study was to evaluate the protective effect of lutein-loaded nanoparticles, along with their mechanisms of action, on Drosophila melanogaster offspring exposed to IMI-induced damage. To simulate the neurodevelopmental disorder model, flies were exposed to a diet containing IMI for 7 days. Posteriorly, their offspring were exposed to a diet containing lutein-loaded nanoparticles for a period of 24 h, and male and female flies were subjected to behavioral and biochemical evaluations. Treatment with lutein-loaded nanoparticles reversed the parameters of hyperactivity, aggressiveness, social interaction, repetitive movements, and anxiety in the offspring of flies exposed to IMI. It also protected markers of oxidative stress and cell viability, in addition to preventing the reduction of Nrf2 and Shank3 immunoreactivity. These results demonstrate that the damage induced by exposure to IMI was restored through treatment with lutein-loaded nanoparticles, elucidating lutein's mechanisms of action as a therapeutic agent, which, after further studies, can become a co-adjuvant in the treatment of neurodevelopmental disorders, such as ASD and ADHD.

Mitochondrial dysfunction and NDUFS3: Insights from a PINK1B9 Drosophila model in Parkinson’s disease pathogenesis

PTEN-induced kinase1 (PINK1) mutation is the main cause of autosomal recessive inheritance and early-onset Parkinson’s disease. Mitochondrial respiratory chain complex I (CI) functional impairment has been considered to be an important factor in the pathogenesis of PD in recent years. In addition, NDUFS3 (nicotinamide adenine dinucleotide deoxylase iron-thionein 3) is one of the core subunits of mitochondrial CI. Therefore, this study explored the role of NDUFS3 gene in PINK1B9 transgenic Drosophila and its possible related mechanisms. In this study, the PD transgenic Drosophila model of MHC-Gal4/UAS system was selected to specifically activate the expression of PINK1B9 gene in the chest muscle tissue of Drosophila melanogaster. NDUFS3 RNAi interference was used to interfere with PINK1B9 transgenic Drosophila melanogaster and its effect on PD transgenic flies was studied. The results suggest that down-regulation of NDUFS3 gene expression may have a protective effect on PINK1B9 transgenic Drosophila melanogaster, and we speculate that down-regulation of NDUFS3 gene expression to reduce oxidative stress and restore mitochondrial function may be related to mitochondrial stress response.