Reporters are widely used in Drosophila genetics to visualize gene expression and cell lineages. However, uncharacterized limitations in specific reporter lines can lead to data misinterpretation. Here, we identify a consistent, driver-independent tdTomato signal in the adult proventriculus from the widely used lexAop-tdTomato.nls reporter line. This signal was observed across multiple lexA driver combinations and was directly detectable in lexAop-tdTomato.nls responder-alone adult proventriculi lacking any lexA driver and without antibody staining. In contrast, no comparable native red fluorescence was detected in larval proventriculi under the same no-antibody imaging condition. Mouse and rabbit anti-RFP immunostaining further supported the presence of proventriculus-associated tdTomato/RFP antigen in adult responder-alone animals. In larval responder-alone proventriculi, antibody-amplified staining was antibody-source-dependent: a detectable signal was observed only with rabbit anti-RFP, whereas mouse and rat anti-RFP produced no reliable detectable signal under the same staining condition. A driver-matched comparison using lexAop-RFP.nls did not reproduce the proventricular signal, arguing against detectable ectopic activity of the tested lexA driver in this tissue. However, because lexAop-tdTomato.nls and lexAop-RFP.nls differ in reporter/transgene architecture and possibly genomic insertion context, the underlying cause cannot be assigned specifically to the lexAop sequence. Our findings highlight the necessity of including driver-negative and no-antibody controls when using this reporter line in adult Drosophila proventriculus and gut studies.
Pharmaceutical waste has been recognized as an emerging contaminant that is increasingly detected in the environment and may pose ecotoxicological risks. This review aims to describe the global development of research on pharmaceutical waste within the field of ecotoxicology and to examine the role of insects in trophic food-chain exposure pathways. A bibliometric analysis was conducted using the Scopus database, comprising 1,051 articles published between 2010 and 2025. The results show a consistent upward trend in publications, with pharmaceutical ecotoxicology research predominantly focused on aquatic environments, environmental risk assessment, contaminant monitoring, and toxicological evaluation. Keyword analysis further indicates that terrestrial exposure pathways remain comparatively less emphasized within the current bibliometric landscape. The narrative review indicates that pharmaceutical waste can accumulate in organisms and be transferred through the food chain, leading to various physiological and systemic toxic effects, even at low environmental concentrations. In this context, insects play a strategic role as mediators of trophic exposure. Hermetia illucens is relevant in waste management and biotransformation processes, while Drosophila melanogaster is a well-established model organism for investigating physiological and molecular responses to pharmaceutical compound exposure. Thus, this review discusses the potential of an insect-based ecotoxicology framework in which H. illucens may serve as a bioconversion model for waste processing and D. melanogaster as a mechanistic model for toxicological studies. We also highlight the need for a more integrative ecotoxicological approach to pharmaceutical waste that explicitly considers insect involvement and food chain - based exposure pathways.
Organophosphates (OPs) cause acute cholinergic toxicity, oxidative stress, DNA damage, and delayed axonopathy through neuropathy target esterase (NTE) inhibition. Efficient in vivo models capturing this toxicity spectrum are essential for risk assessment and therapeutic discovery. Here, we review Drosophila melanogaster as a versatile platform for OP toxicology across: (1) wild-type assays using dietary and vapour exposures with behavioural endpoints (survival, locomotion, negative geotaxis) and biochemical markers (acetylcholinesterase activity, oxidative stress indices); (2) genotoxicity assessment via the Somatic Mutation and Recombination Test (SMART) and comet assay; and (3) the swiss cheese (sws) mutant model - the Drosophila ortholog of human PNPLA6/NTE - enabling investigation of organophosphate-induced delayed neuropathy (OPIDN) independent of cholinergic effects. Together, these approaches provide hazard characterization and support discovery of protective strategies targeting both cholinergic and non-cholinergic pathways.
Although several hundred continuous cell lines have been generated from Drosophila spp. fruit flies over the past half-century, nearly all are derived from a single species, Drosophila melanogaster, and none are derived from neotropical flies. To address this deficit, a simplified protocol was used to generate three primary cell cultures from larvae of Drosophila willistoni originating from Costa Rica. All three primary cultures developed into continuous cell lines, and all three cell lines, designated DWL/LULS68, DWL/LULS70 and DWL/LULS72, were found to be persistently infected with the bacterial endosymbiont Wolbachia. Sublines free of Wolbachia were generated from all three cell lines by prolonged tetracycline treatment. Molecular analysis, karyotyping and fluorescence in situ hybridization confirmed species origin of the cells as D. willistoni and identified the Wolbachia as the strain wWil. Wolbachia wWil was successfully transferred from D. willistoni cells to heterologous cell lines derived from the sand fly Lutzomyia longipalpis, the biting midge Culicoides sonorensis and the tsetse fly Glossina morsitans, but not to cell lines derived from ticks or triatomine bugs. The new D. willistoni cell lines are expected to facilitate many aspects of research into this species and its bacterial symbionts.
Chemical insecticides have long been used to control agricultural pests, but their widespread application has driven resistance and caused significant ecological and health impacts. CRISPR/Cas9-based genetic biocontrol technologies, including the precision-guided sterile insect technique (pgSIT) and homing gene drives (HGDs), offer targeted alternatives for suppressing pest populations with reduced environmental cost. pgSIT produces sterile males without radiation and achieves high mating competitiveness without multigenerational persistence. In contrast, HGDs bias inheritance to enable sustained population suppression through disruption of essential fertility or viability genes, albeit with greater ecological and regulatory considerations. Experimental applications in multiple agricultural pest species demonstrate robust suppression efficacy. Emerging innovations, including temperature-inducible pgSIT systems, may further streamline mass-rearing and deployment. Together, these approaches have the potential to reduce crop losses and reliance on chemical insecticides while lowering long-term management costs. Their successful integration into agricultural systems will depend on rigorous risk assessment, regulatory oversight, and stakeholder engagement.
This review examines the efficacy and toxicity of the major insecticide groups in current use, with particular reference to neonicotinoids, which are one of the most widely used pesticides in the world. Mammalian toxicity and hazards posed during insecticide application will be discussed, along with their ecotoxicology, effects on non-target organisms and on the environment. However, toxicity must be balanced by usefulness in crop protection, first in terms of integrated pest management (IPM) and then by detailing national registration processes according to international normative frameworks to achieve the appropriate balance between efficacy and toxicity. Critical in ensuring that this balance is met are controls on pesticides in use, enforcement of pesticide regulations, and food safety measures related insecticide residues and maximum residue limit (MRL) setting. International trade conventions and trade issues are of direct relevance to import and export not only of agricultural products protected by the use of pesticides, but also of the pesticides themselves along with the waste products from their manufacture or use.
Sulphur-containing amino acids (SAAs), including methionine and cysteine, play crucial roles in antioxidant defence, anti-ageing, cytoprotection, and anti-inflammatory responses. Previous studies have shown that SAAs promote peroxisome elevation and fat loss by inducing the expression of the peroxisome-related gene CG33474 in the Drosophila fat body. However, the underlying regulatory mechanism remains unclear. In this study, we demonstrated that the transsulphuration pathway contributes to CG33474 induction, as supplementation with specific downstream metabolites of this pathway recapitulates this effect. Moreover, we found that SAAs upregulate not only CG33474 but also several neighbouring genes - including CG11825, Prx2540-1, Prx2540-2, and CG12896 - suggesting coordinated regulation within this genomic locus. Through fluorescence reporter assays, we discovered that a ~1 kb genomic region upstream of CG33474 harbours the cis-regulatory element mediating SAA responsiveness and that this responsiveness is fat body-specific. Finally, our data suggest that induction of CG33474 may play a role in resistance to different stresses and in regulating ageing as fat body-specific overexpression of CG33474 significantly extends lifespan in Drosophila. Together, our findings reveal that SAAs modulate the expression of CG33474 and its adjacent genes through the transsulphuration pathway, providing an additional mechanistic basis for the antioxidant effects of SAAs.
A conserved cohort of signalling pathways orchestrate development and adult homoeostasis. Deregulation of these pathways underlies many diseases. A key set of signals is the family of Wnt ligands. Members of this family are conserved, but a clear understanding of the unique and redundant roles is lacking. Previous efforts to study Wnt ligand function in Drosophila have been hampered by the difficulty of generating , functional transgenes. To address this, we have created a complete set of synthesized constructs in an insulated expression system, integrated into the same genomic location, enabling reliable gain-of-function analyses across multiple tissues. Distinct phenotypic outcomes were observed, reflecting both shared and unique features of individual ligands. To define the canonicity of Wnt signalling, we monitored canonical targets such as Dfz3, notum, and the ‘naked cuticle’ phenotype in developing tissues and the adult gut. Our findings revealed strong evidence of canonical responses from not only wg, but also DWnt6 and DWnt10 in the embryo, wing disc, larval gut, and adult gut. In addition, DWnt2, DWnt4, and WntD produced phenotypes distinct from the control with DWnt2 and DWnt4, showing context-dependent evidence of some canonical activity. While previous studies have suggested regulatory features between wg and DWnt6, our work provides functional evidence that Wg, DWnt6, and DWnt10 each induce expression of canonical signalling reporters in vivo. These findings refine our understanding of redundancy and specificity within the Drosophila Wnt family and demonstrate that multiple Wnt ligands can act similarly within the canonical pathway depending on tissue context.
Drosophila melanogaster Nora virus (DmNV), a positive-sense single stranded RNA virus related to picornaviruses. Given its genetic and structural similarity to neurotropic picornaviruses, such as poliovirus, we sought to determine whether DmNV could be found within the head and brain of D. melanogaster. RNA was extracted from heads of chronically DmNV-infected stocks, as well as from uninfected controls, and assayed using reverse transcription-polymerase chain reaction (RT-PCR) for DmNV open reading frame 1 (ORF1). The results showed that DmNV genomic material can be isolated from the heads of DmNV-infected D. melanogaster, which suggests that the virus reaches the head during the course of infection. To determine whether DmNV infects the brain tissue itself, small-molecule RNA fluorescence in situ hybridization (smRNA FISH) experiments on whole brains dissected from DmNV-infected and uninfected D. melanogaster were done. The smRNA FISH detection method was validated by identifying DmNV RNA in gut tissue, but there was no evidence of DmNV localization in any brain specimens examined. These findings suggest an alternative explanation for why DmNV may be present in dissected head specimens. Additionally, we highlight the effectiveness of smRNA FISH as a highly specific and accessible method for detecting RNA viruses in Drosophila, offering an alternative to antibody-based or transgenic fluorescence approaches. Together, our results refine the understanding of DmNV tissue tropism and provide methodological insights for future studies using insect RNA viruses.
Angelman syndrome (AS) is a rare neurogenetic disorder characterized by developmental delay, speech impairment, ataxia, epilepsy, and in some cases hyperphagic feeding behavior. AS is caused by loss of function mutations, loss of expression, or maternal allele deletion of the E3 ubiquitin ligase UBE3A. Recent work has identified a connection between UBE3A and the mechanosensitive ion channel PIEZO2, raising the possibility that UBE3A may regulate PIEZO-dependent satiety signaling. In this study, we investigated the role of the Drosophila UBE3A ortholog, Dube3a, in Piezo-associated feeding behaviors. Single-cell RNA-sequencing data revealed overlapping expression of Dube3a and Piezo within crop and enterocyte populations of the gut, identifying a relevant cellular context for this pathway to occur. We developed a novel feeding assay using GFP-expressing yeast to quantify food intake and gut distention in vivo. Dube3a loss-of-function (Dube3a15b) flies exhibited hyperphagia and gut distention nearly identical to Piezo knockout flies. Analysis of chromosomal deficiency lines spanning the Dube3a locus further supported a requirement for Dube3a in normal satiety signaling. Finally, biochemical analyses demonstrated that Dube3a knockdown results in decreased Piezo protein levels, consistent with an indirect regulatory relationship. Together, these findings identify Dube3a as a critical regulator of Piezo-dependent satiety pathways and suggest that dysregulation of mechanosensory signaling may contribute to hyperphagia observed in AS. Further work is needed to define the intermediate factors linking UBE3A activity to Piezo stability and function.
Ribosomal protein (RP) gene haploinsufficiency is a conserved form of ribosome dysfunction across species and underlies a class of disorders known as ribosomopathies. In Drosophila, RP gene haploinsufficiency manifests as the Minute phenotype, characterized by thinner and shorter mechanosensory bristles. The development of both bristles and proprioceptive campaniform sensilla (CS) is initiated by the bHLH proneural proteins Achaete (Ac) and Scute (Sc). By analysing genetic interactions between ac sc mutants and Minute mutants of varying severity, we identified a novel bristle-promoting effect that occurs only in the strongly affected Minutes in which the average bristle length is shorter than a threshold. This threshold-dependent effect also promotes ectopic CS formation in the strong Minutes. Transcriptomic analyses comparing the sensory organ - promoting and non-promoting Minutes revealed significant differences in stress-response pathways, including differentially elevated expression of the Xrp1-Irbp18 transcriptional dimer. Notably, mutation of Xrp1 suppresses the ectopic CS phenotype, indicating a positive regulatory role. These findings reveal a previously unrecognized threshold effect in RP gene haploinsufficiency, in which excessive Xrp1 activity promotes supernumerary sensory organ formation, suggesting a compensatory mechanism that modulates neurogenesis under severe ribosomal stress.
Drosophila Futsch is a key microtubule-associated protein (fly homolog of MAP1B) that regulates microtubule organization, synaptic terminal growth, and neuronal development. Functional analysis of futsch has long been limited by the inability to clone and express a full-length futsch transgene, owing to its exceptional size (~16.5 kb) and extensive repetitive sequences. Here, I present an efficient and reproducible method for cloning both wild-type and mutated full-length Drosophila futsch cDNA (16,488 bp) using Gibson Assembly. These resulting cDNAs were used to generate UAS-futsch transgenes. When expressed in neurons, the wild‑type transgenic Futsch associated with microtubule and rescued the synaptic morphological defects observed in futschK68 mutants. This approach substantially reduces the time and complexity compared with traditional cloning techniques. Furthermore, I highlight common pitfalls encountered during the cloning process and provide practical solutions to enhance cloning efficiency. This protocol offers a broadly applicable and cost-effective framework for cloning otherwise intractable large cDNAs from Drosophila and other organisms.
Drosophila melanogaster is an incredible model system, providing tools and technologies that allow careful, effective, and reproducible research. This experimental approach, and the genetic tools and techniques available in Drosophila are desperately needed for the study of other insects, a hugely diverse group of huge importance to natural and productive ecosystems. For those of you with the skills and ‘Drosophila mindset’, studying other insects may help us understand diversity, improve the security of food production, and help avoid the current, worrying, insect apocalypse.
UCH-L1 (Ubiquitin Carboxyl-terminal Hydrolase - L1) is a protein that plays a critical role in the ubiquitin-proteasome system. Previous studies have demonstrated a link between UCH-L1 and various diseases, including neurodegenerative disorders, diabetes, and cancer. However, the role of UCH-L1 in development remains unclear. To investigate the functions of UCH-L1 in a living organism, taking advantage of the Drosophila model, and to explore the correlation between Drosophila UCH (dUCH) and human UCH-L1, we established a GAL4/UAS-targeted expression system to examine the effect of dUCH on Drosophila eye development. We found that knockdown of dUCH resulted in a rough eye phenotype associated with the MAPK pathway. In this study, for the first time, we revealed that loss of dUCH function leads to a reduction in EGFR protein levels. Additionally, dUCH knockdown downregulated Spitz (spi), a ligand of EGFR, as well as Draf, a key component of the MAPK pathway. Furthermore, under dUCH knockdown conditions, several genes known to play critical roles in eye cell differentiation were affected, including the downregulation of sens, salm, lz, barth1/2, and salm, which are essential for the differentiation of R2/5, R3/4, and R1/6 photoreceptor cells. Interestingly, dUCH was found to be involved not only in the MAPK pathway but also in the regulation of pros, lz, barth1/2, and sev gene expression, suggesting its role in R7 photoreceptor differentiation. Taken together, these findings highlight the important role of dUCH in regulating genes associated with eye cell differentiation and its involvement in EGFR signalling in Drosophila melanogaster.
Three decades of research aimed at understanding the basis for autosomal recessive primary microcephaly (MCPH), a human clinical disorder defined by a significant reduction in head and brain size, has uncovered a suite of ~30 genes that participate in this process. Work in both vertebrate and invertebrate model systems have been instrumental in attempting to link MCPH gene function to the brain growth phenotype. However, we still lack definitive evidence as to what these functions are for many of these genes. In this review, we summarize recent work in Drosophila aimed at overcoming these limitations in our knowledge of MCPH gene function that may be applicable to humans. We discuss the clinical features of MCPH, parallels between human and Drosophila neurogenesis modes with a particular focus on the fly optic lobe, and highlight four of the most well-studied Drosophila MCPH orthologs: abnormal spindle (asp)/MCPH5, Microcephalin/MCPH1, WD Repeat-Containing Protein 62 (Wdr62)/MCPH2, and Ankryin Repeat-and LEM Domain- Containing Protein 2 (ANKLE2)/MCPH16. We focus on the multifunctional roles for these proteins that may underlie the microcephaly phenotype and advocate for the use of flies as a relevant model for human MCPH.
One hundred years ago, two reports appeared of tetraploid D. melanogaster females - curiosities that had never been seen before. The authors, Calvin Bridges and Lilian Morgan, were among the famed founders of fly genetics in T.H. Morgan's lab at Columbia University. Sadly, their findings have faded into the fog of ancient fly lore. This review exhumes those relics in order to offer modern fly-pushers some possible avenues for polyploid research. That subfield is undergoing a revival that may interest them.
Neurodegenerative diseases are devastating conditions characterized by progressive cognitive decline with few available treatments. Neurodegeneration can be quantified in vertebrate and invertebrate models of disease by analysis of vacuolation - the formation of empty spaces within brain tissue. Previous approaches for quantifying this phenotype have required time-consuming methods such as manual counting and measuring of vacuole dimensions, which can be subjective. Here we describe VacQuant, a novel application that can be paired with existing machine learning software to automatically measure the area of vacuolation in brain tissue. Using Drosophila brain sections from tauopathy model flies, a well-described model of dementia-related neurodegeneration, we quantified a significant increase in brain vacuolation at several timepoints in adult flies with the aid of VacQuant. When compared with quantification by five blinded volunteers, the machine learning method positively correlated with their group average, confirming its accuracy and functionality. This automated method developed with VacQuant removes human bias and measurement variation, providing a consistent threshold for all brain sections and experiments. This automated pipeline will be particularly useful for high-throughput screening for genetic modifiers or therapeutic compounds in animal models of neurodegeneration.
This study investigates the effects of polyglutamine (polyQ) expansions on the locomotion of Drosophila larvae, focusing on the role of class IV dendritic arborization (da) neurons. PolyQ expansions are associated with neurodegenerative diseases like Huntington’s disease, and Drosophila is a valuable model organism for studying these diseases due to its genetic tractability and short generation time. We found that expressing a polyQ protein in class IV da neurons caused significant locomotion deficits. Specifically, larvae with polyQ expression exhibited slower crawling speed and increased turn frequency, indicating impaired movement. The most intriguing finding of our study was that electrically silencing class IV da neurons completely rescued the locomotion deficits caused by polyQ expression. By expressing a potassium channel that makes the neurons less active, we effectively reversed the locomotion defects. This suggests that modulating the activity of these neurons could be a promising therapeutic approach for treating polyQ diseases. Our findings have significant implications for understanding polyQ diseases and developing new therapeutic approaches. By electrically silencing these neurons, we may be preventing the harmful effects of polyQ-induced cation channels, which are thought to disrupt cellular function. This opens up exciting possibilities for exploring electrical silencing as a potential treatment for polyQ diseases, offering hope for future therapies that target the underlying mechanisms of these devastating conditions.
