Single-Cell Hi-C Analysis Workflow with Pairtools.

Single-cell Hi-C (scHi-C) is a collection of protocols for studying genomic interactions within individual cells. Although data analysis for scHi-C resembles data analysis for bulk Hi-C, the unique challenges of scHi-C, such as high noise and protocol-specific biases, require specialized data processing strategies. In this tutorial chapter, we focus on using pairtools, a suite of tools optimized for scHi-C data, demonstrating its application on a Drosophila snHi-C dataset. While centered on pairtools for snHi-C data, the principles outlined are applicable across scHi-C variants with minor adjustments. This educational chapter aims to guide researchers in using open-source tools for scHi-C analysis, emphasizing critical steps of contact pair extraction, detection of ligation junctions, filtration, and deduplication.

Animal flight: Fly gyros get a new spin.

A new study exploits genetic approaches available in Drosophila to record the neural activity within the specialized mechanosensory fields of halteres, the unique equilibrium organs of flies. The results challenge the traditional explanation for how these rapidly oscillating structures encode angular velocity during flight.

Advancing rational pesticide development against Drosophila suzukii: bioinformatics tools and applications-a systematic review.

CONTEXT: Drosophila suzukii (Matsumura, 1931) is a widespread agricultural pest responsible for significant damage to various soft-skinned fruit hosts. The revolutionary potential of bioinformatics in agriculture emerges from its ability to provide extensive information on pests, fungi, chemical resistance, implications of non-target species, and other critical aspects. This wealth of information allows researchers to engage in projects and applied research in diverse agricultural domains that face these challenges. In this context, bioinformatics tools play a fundamental role. The negative impact of pests on crops, resulting in substantial economic losses, has highlighted the importance of in silico methods. METHODS: To achieve this, we conducted a systematic search in scientific databases using as keywords "Drosophila suzukii," "biopesticides," "simulations computational," and "in-silico." After applying the filters of relevance and publication date, we organized the articles and prioritized those that directly addressed that matched the keywords and the use of bioinformatics tools. Additionally, we included studies focusing on in silico assays of biopesticides, such as molecular docking. Our review aimed to present a collection of recent literature on biopesticides against Drosophila suzukii, emphasizing bioinformatics methods. Through this work, we strive to contribute to the literature of new perspectives on the development and efficiency of biopesticides, along with to advance research that may improve pest control strategies. RESULTS: In the results of the systematic review, we found 2734 articles related to the selected keywords. Six of these articles directly address Drosophila suzukii and the use of bioinformatics tools in the search for alternatives in pest control. In the selected studies, we observed that two articles tend to focus on phylogenetic approaches, searching for gene sequences, amino acids, and constructing phylogenetic trees. The other three articles used molecular modeling and docking of receptors such as GABA and TRP with plant-derived and synthetic compounds to study intermolecular interactions. However, we identified gaps in these studies that could lead to further research in the biorational development of biopesticides using bioinformatics tools.

Regulation of proteostasis by sleep through autophagy in Drosophila models of Alzheimer's disease.

Sleep and circadian rhythm dysfunctions are common clinical features of Alzheimer's disease (AD). Increasing evidence suggests that in addition to being a symptom, sleep disturbances can also drive the progression of neurodegeneration. Protein aggregation is a pathological hallmark of AD; however, the molecular pathways behind how sleep affects protein homeostasis remain elusive. Here we demonstrate that sleep modulation influences proteostasis and the progression of neurodegeneration in Drosophila models of tauopathy. We show that sleep deprivation enhanced Tau aggregational toxicity resulting in exacerbated synaptic degeneration. In contrast, sleep induction using gaboxadol led to reduced toxic Tau accumulation in neurons as a result of modulated autophagic flux and enhanced clearance of ubiquitinated Tau, suggesting altered protein processing and clearance that resulted in improved synaptic integrity and function. These findings highlight the complex relationship between sleep and regulation of protein homeostasis and the neuroprotective potential of sleep-enhancing therapeutics to slow the progression or delay the onset of neurodegeneration.

Glial ferritin maintains neural stem cells via transporting iron required for self-renewal in Drosophila.

Stem cell niche is critical for regulating the behavior of stem cells. Drosophila neural stem cells (Neuroblasts, NBs) are encased by glial niche cells closely, but it still remains unclear whether glial niche cells can regulate the self-renewal and differentiation of NBs. Here, we show that ferritin produced by glia, cooperates with Zip13 to transport iron into NBs for the energy production, which is essential to the self-renewal and proliferation of NBs. The knockdown of glial ferritin encoding genes causes energy shortage in NBs via downregulating aconitase activity and NAD+ level, which leads to the low proliferation and premature differentiation of NBs mediated by Prospero entering nuclei. More importantly, ferritin is a potential target for tumor suppression. In addition, the level of glial ferritin production is affected by the status of NBs, establishing a bicellular iron homeostasis. In this study, we demonstrate that glial cells are indispensable to maintain the self-renewal of NBs, unveiling a novel role of the NB glial niche during brain development.

Iron is an essential nutrient for almost all living organisms. For example, iron contributes to the replication of DNA, the generation of energy inside cells, and the transport of oxygen around the body. Iron deficiency is the most common of all nutrient deficiencies, affecting over 40% of children worldwide. This can lead to anemia and also impair how the brain and nervous system develop, potentially resulting in long-lasting cognitive damage, even after the deficiency has been treated. It is poorly understood how iron contributes to the development of the brain and nervous system. In particular, whether and how it supports nerve stem cells (or NSCs for short) which give rise to the various neural types in the mature brain. To investigate, Ma et al. experimentally reduced the levels of ferritin (a protein which stores iron) in the developing brains of fruit fly larvae. This reduction in ferritin led to lower numbers of NSCs and a smaller brain. Unexpectedly, this effect was largest when ferritin levels were reduced in glial cells which support and send signals to NSCs, rather than in the stem cells themselves. Ma et al. then used fluorescence microscopy to confirm that glial cells make and contain a lot of ferritin which can be transported to NSCs. Adding iron supplements to the diet of flies lacking ferritin did not lead to normal numbers of stem cells in the brains of the developing fruit flies, whereas adding compounds that reduce the amount of iron led to lower numbers of stem cells. Together, this suggests that ferritin transports iron from glial cells to the NSCs. Without ferritin and iron, the NSCs could not produce enough energy to divide and make new stem cells. This caused the NSCs to lose the characteristics of stem cells and prematurely turn into other types of neurons or glial cells. Together, these findings show that when iron cannot move from glial cells to NSCs this leads to defects in brain development. Future experiments will have to test whether a similar transport of iron from supporting cells to NSCs also occurs in the developing brains of mammals, and whether this mechanism applies to stem cells in other parts of the body.

DBT is a metabolic switch for maintenance of proteostasis under proteasomal impairment.

Proteotoxic stress impairs cellular homeostasis and underlies the pathogenesis of many neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). The proteasomal and autophagic degradation of proteins are two major pathways for protein quality control in the cell. Here, we report a genome-wide CRISPR screen uncovering a major regulator of cytotoxicity resulting from the inhibition of the proteasome. Dihydrolipoamide branched chain transacylase E2 (DBT) was found to be a robust suppressor, the loss of which protects against proteasome inhibition-associated cell death through promoting clearance of ubiquitinated proteins. Loss of DBT altered the metabolic and energetic status of the cell and resulted in activation of autophagy in an AMP-activated protein kinase (AMPK)-dependent mechanism in the presence of proteasomal inhibition. Loss of DBT protected against proteotoxicity induced by ALS-linked mutant TDP-43 in Drosophila and mammalian neurons. DBT is upregulated in the tissues of ALS patients. These results demonstrate that DBT is a master switch in the metabolic control of protein quality control with implications in neurodegenerative diseases.

Spatial organization of translation and translational repression in two phases of germ granules.

Most RNA-protein condensates are composed of heterogeneous immiscible phases. However, how this multiphase organization contributes to their biological functions remains largely unexplored. Drosophila germ granules, a class of RNA-protein condensates, are the site of mRNA storage and translational activation. Here, using super-resolution microscopy and single-molecule imaging approaches, we show that germ granules have a biphasic organization and that translation occurs in the outer phase and at the surface of the granules. The localization, directionality, and compaction of mRNAs within the granule depend on their translation status, translated mRNAs being enriched in the outer phase with their 5'end oriented towards the surface. Translation is strongly reduced when germ granule biphasic organization is lost. These findings reveal the intimate links between the architecture of RNA-protein condensates and the organization of their different functions, highlighting the functional compartmentalization of these condensates.

Two novel DnaJ chaperone proteins CG5001 and P58IPK regulate the pathogenicity of Huntington's disease related aggregates.

Huntington's disease (HD) is a rare neurodegenerative disease caused due to aggregation of Huntingtin (HTT) protein. This study involves the cloning of 40 DnaJ chaperones from Drosophila, and overexpressing them in yeasts and fly models of HD. Accordingly, DnaJ chaperones were catalogued as enhancers or suppressors based on their growth phenotypes and aggregation properties. 2 of the chaperones that came up as targets were CG5001 and P58IPK. Protein aggregation and slow growth phenotype was rescued in yeasts, S2 cells, and Drosophila transgenic lines of HTT103Q with these overexpressed chaperones. Since DnaJ chaperones have protein sequence similarity across species, they can be used as possible tools to combat the effects of neurodegenerative diseases.

A dystroglycan-laminin-integrin axis coordinates cell shape remodeling in the developing Drosophila retina.

Cell shape remodeling is a principal driver of epithelial tissue morphogenesis. While progress continues to be made in our understanding of the pathways that control the apical (top) geometry of epithelial cells, we know comparatively little about those that control cell basal (bottom) geometry. To examine this, we used the Drosophila ommatidium, which is the basic visual unit of the compound eye. The ommatidium is shaped as a hexagonal prism, and generating this 3D structure requires ommatidial cells to adopt specific apical and basal polygonal geometries. Using this model system, we find that generating cell type-specific basal geometries starts with patterning of the basal extracellular matrix, whereby Laminin accumulates at discrete locations across the basal surface of the retina. We find the Dystroglycan receptor complex (DGC) is required for this patterning by promoting localized Laminin accumulation at the basal surface of cells. Moreover, our results reveal that localized accumulation of Laminin and the DGC are required for directing Integrin adhesion. This induces cell basal geometry remodeling by anchoring the basal surface of cells to the extracellular matrix at specific, Laminin-rich locations. We propose that patterning of a basal extracellular matrix by generating discrete Laminin domains can direct Integrin adhesion to induce cell shape remodeling in epithelial morphogenesis.

Fundamental Patterns of Structural Evolution Revealed by Chromosome-Length Genomes of Cactophilic Drosophila.

A thorough understanding of adaptation and speciation requires model organisms with both a history of ecological and phenotypic study as well as a complete set of genomic resources. In particular, high-quality genome assemblies of ecological model organisms are needed to assess the evolution of genome structure and its role in adaptation and speciation. Here, we generate new genomes of cactophilic Drosophila, a crucial model clade for understanding speciation and ecological adaptation in xeric environments. We generated chromosome-level genome assemblies and complete annotations for seven populations across Drosophila mojavensis, Drosophila arizonae, and Drosophila navojoa. We use these data first to establish the most robust phylogeny for this clade to date, and to assess patterns of molecular evolution across the phylogeny, showing concordance with a priori hypotheses regarding adaptive genes in this system. We then show that structural evolution occurs at constant rate across the phylogeny, varies by chromosome, and is correlated with molecular evolution. These results advance the understanding of the D. mojavensis clade by demonstrating core evolutionary genetic patterns and integrating those patterns to generate new gene-level hypotheses regarding adaptation. Our data are presented in a new public database (cactusflybase.arizona.edu), providing one of the most in-depth resources for the analysis of inter- and intraspecific evolutionary genomic data. Furthermore, we anticipate that the patterns of structural evolution identified here will serve as a baseline for future comparative studies to identify the factors that influence the evolution of genome structure across taxa.

bHLH family proteins control the timing and completion of transition from neuroepithelial cells into neural stem cells.

The number of neural stem cells reflects the total number of neurons in the mature brain. As neural stem cells arise from neuroepithelial cells, the neuroepithelial cell population must be expanded to secure a sufficient number of neural stem cells. However, molecular mechanisms that regulate timely differentiation from neuroepithelial to neural stem cells are largely unclear. Here, we show that TCF4/Daughterless is a key factor that determines the timing of the differentiation in Drosophila. The neuroepithelial cells initiated but never completed the differentiation in the absence of TCF4/Daughterless. We also found that TCF4/Daughterless binds to the Notch locus, suggesting that Notch is one of its downstream candidate genes. Consistently, Notch expression was ectopically induced in the absence of TCF4/Daughterless. Furthermore, ectopic activation of Notch signaling phenocopied loss of TCF4/Daughterless. Our findings demonstrate that TCF4/Daughterless directly inactivates Notch signaling pathway, resulting in completion of the differentiation from neuroepithelial cells into neural stem cells with optimal timing. Thus, the present results suggest that TCF4/Daughterless is essential for determining whether to move to the next state or stay in the current state in differentiating neuroepithelial cells.

Anti-aging and immunomodulatory role of caffeine in Drosophila larvae.

Drug repurposing is a promising approach to identify new pharmacological indications for drugs that have already been established. However, there is still a limitation in the availability of a high-throughput in vivo preclinical system that is suitable for screening and investigating new pharmacological indications. The aim of this study was to introduce the application of Drosophila larvae as an in vivo platform to screen drug candidates with anti-aging and immunomodulatory activities. To determine whether Drosophila larvae can be utilized for assessing anti-aging and immunomodulatory activities, phenotypical and molecular assays were conducted using wildtype and mutant lines of Drosophila. The utilization of mutant lines (PGRP-LBΔ and Psh[1];;ModSP[KO]) mimics the autoinflammatory and immunodeficient conditions in humans, thereby enabling a thorough investigation of the effects of various compounds. The phenotypical assay was carried out using survival and locomotor observation in Drosophila larvae and adult flies. Meanwhile, the molecular assay was conducted using the RT-qPCR method. In vivo survival analysis revealed that caffeine was relatively safe for Drosophila larvae and exhibited the ability to extend Drosophila lifespan compared to the untreated controls, suggesting its anti-aging properties. Further analysis using the RT-qPCR method demonstrated that caffeine treatment induced transcriptional changes in the Drosophila larvae, particularly in the downstream of NF-κB and JAK-STAT pathways, two distinct immune-related pathways homologue to humans. In addition, caffeine enhanced the survival of Drosophila autoinflammatory model, further implying its immunosuppressive activity. Nevertheless, this compound had minimal to no effect on the survival of Staphylococcus aureus-infected wildtype and immunodeficient Drosophila, refuting its antibacterial and immunostimulant activities. Overall, our results suggest that the anti-aging and immunosuppressive activities of caffeine observed in Drosophila larvae align with those reported in mammalian model systems, emphasizing the suitability of Drosophila larvae as a model organism in drug repurposing endeavors, particularly for the screening of newly discovered chemical entities to assess their immunomodulatory activities before proceedings to investigations in mammalian animal models.

High sugar diet promotes tumor progression paradoxically through aberrant upregulation of pepck1.

High dietary sugar (HDS), a contemporary dietary concern due to excessive intake of added sugars and carbohydrates, escalates the risk of metabolic disorders and concomitant cancers. However, the molecular mechanisms underlying HDS-induced cancer progression are not completely understood. We found that phosphoenolpyruvate carboxykinase 1 (PEPCK1), a pivotal enzyme in gluconeogenesis, is paradoxically upregulated in tumors by HDS, but not by normal dietary sugar (NDS), during tumor progression. Targeted knockdown of pepck1, but not pepck2, specifically in tumor tissue in Drosophila in vivo, not only attenuates HDS-induced tumor growth but also significantly improves the survival of Ras/Src tumor-bearing animals fed HDS. Interestingly, HP1a-mediated heterochromatin interacts directly with the pepck1 gene and downregulates pepck1 gene expression in wild-type Drosophila. Mechanistically, we demonstrated that, under HDS conditions, pepck1 knockdown reduces both wingless and TOR signaling, decreases evasion of apoptosis, reduces genome instability, and suppresses glucose uptake and trehalose levels in tumor cells in vivo. Moreover, rational pharmacological inhibition of PEPCK1, using hydrazinium sulfate, greatly improves the survival of tumor-bearing animals with pepck1 knockdown under HDS. This study is the first to show that elevated levels of dietary sugar induce aberrant upregulation of PEPCK1, which promotes tumor progression through altered cell signaling, evasion of apoptosis, genome instability, and reprogramming of carbohydrate metabolism. These findings contribute to our understanding of the complex relationship between diet and cancer at the molecular, cellular, and organismal levels and reveal PEPCK1 as a potential target for the prevention and treatment of cancers associated with metabolic disorders.

Vitamin B12 ameliorates gut epithelial injury via modulating the HIF-1 pathway and gut microbiota.

Inflammatory bowel diseases (IBDs) are immune chronic diseases characterized by recurrent episodes, resulting in continuous intestinal barrier damage and intestinal microbiota dysbiosis. Safe strategies aimed at stabilizing and reducing IBDs recurrence have been vigorously pursued. Here, we constructed a recurrent intestinal injury Drosophila model and found that vitamin B12 (VB12), an essential co-factor for organism physiological functions, could effectively protect the intestine and reduce dextran sulfate sodium-induced intestinal barrier disruption. VB12 also alleviated microbial dysbiosis in the Drosophila model and inhibited the growth of gram-negative bacteria. We demonstrated that VB12 could mitigate intestinal damage by activating the hypoxia-inducible factor-1 signaling pathway in injured conditions, which was achieved by regulating the intestinal oxidation. In addition, we also validated the protective effect of VB12 in a murine acute colitis model. In summary, we offer new insights and implications for the potential supportive role of VB12 in the management of recurrent IBDs flare-ups.

Dysregulated FOXO1 activity drives skeletal muscle intrinsic dysfunction in amyotrophic lateral sclerosis.

Amyotrophic Lateral Sclerosis (ALS) is a multisystemic neurodegenerative disorder, with accumulating evidence indicating metabolic disruptions in the skeletal muscle preceding disease symptoms, rather than them manifesting as a secondary consequence of motor neuron (MN) degeneration. Hence, energy homeostasis is deeply implicated in the complex physiopathology of ALS and skeletal muscle has emerged as a key therapeutic target. Here, we describe intrinsic abnormalities in ALS skeletal muscle, both in patient-derived muscle cells and in muscle cell lines with genetic knockdown of genes related to familial ALS, such as TARDBP (TDP-43) and FUS. We found a functional impairment of myogenesis that parallels defects of glucose oxidation in ALS muscle cells. We identified FOXO1 transcription factor as a key mediator of these metabolic and functional features in ALS muscle, via gene expression profiling and biochemical surveys in TDP-43 and FUS-silenced muscle progenitors. Strikingly, inhibition of FOXO1 mitigated the impaired myogenesis in both the genetically modified and the primary ALS myoblasts. In addition, specific in vivo conditional knockdown of TDP-43 or FUS orthologs (TBPH or caz) in Drosophila muscle precursor cells resulted in decreased innervation and profound dysfunction of motor nerve terminals and neuromuscular synapses, accompanied by motor abnormalities and reduced lifespan. Remarkably, these phenotypes were partially corrected by foxo inhibition, bolstering the potential pharmacological management of muscle intrinsic abnormalities associated with ALS. The findings demonstrate an intrinsic muscle dysfunction in ALS, which can be modulated by targeting FOXO factors, paving the way for novel therapeutic approaches that focus on the skeletal muscle as complementary target tissue.

Molecular regulation of axon termination in mechanosensory neurons.

Spatially and temporally accurate termination of axon outgrowth, a process called axon termination, is required for efficient, precise nervous system construction and wiring. The mechanosensory neurons that sense low-threshold mechanical stimulation or gentle touch have proven exceptionally valuable for studying axon termination over the past 40 years. In this Review, we discuss progress made in deciphering the molecular and genetic mechanisms that govern axon termination in touch receptor neurons. Findings across model organisms, including Caenorhabditis elegans, Drosophila, zebrafish and mice, have revealed that complex signaling is required for termination with conserved principles and players beginning to surface. A key emerging theme is that axon termination is mediated by complex signaling networks that include ubiquitin ligase signaling hubs, kinase cascades, transcription factors, guidance/adhesion receptors and growth factors. Here, we begin a discussion about how these signaling networks could represent termination codes that trigger cessation of axon outgrowth in different species and types of mechanosensory neurons.

Ca2+ channel and active zone protein abundance intersects with input-specific synapse organization to shape functional synaptic diversity.

Synaptic heterogeneity is a hallmark of nervous systems that enables complex and adaptable communication in neural circuits. To understand circuit function, it is thus critical to determine the factors that contribute to the functional diversity of synapses. We investigated the contributions of voltage-gated calcium channel (VGCC) abundance, spatial organization, and subunit composition to synapse diversity among and between synapses formed by two closely related Drosophila glutamatergic motor neurons with distinct neurotransmitter release probabilities (Pr). Surprisingly, VGCC levels are highly predictive of heterogeneous Pr among individual synapses of either low- or high-Pr inputs, but not between inputs. We find that the same number of VGCCs are more densely organized at high-Pr synapses, consistent with tighter VGCC-synaptic vesicle coupling. We generated endogenously tagged lines to investigate VGCC subunits in vivo and found that the α2δ-3 subunit Straightjacket along with the CAST/ELKS active zone (AZ) protein Bruchpilot, both key regulators of VGCCs, are less abundant at high-Pr inputs, yet positively correlate with Pr among synapses formed by either input. Consistently, both Straightjacket and Bruchpilot levels are dynamically increased across AZs of both inputs when neurotransmitter release is potentiated to maintain stable communication following glutamate receptor inhibition. Together, these findings suggest a model in which VGCC and AZ protein abundance intersects with input-specific spatial and molecular organization to shape the functional diversity of synapses.

Actomyosin contraction in the follicular epithelium provides the major mechanical force for follicle rupture during Drosophila ovulation.

Ovulation is critical for sexual reproduction and consists of the process of liberating fertilizable oocytes from their somatic follicle capsules, also known as follicle rupture. The mechanical force for oocyte expulsion is largely unknown in many species. Our previous work demonstrated that Drosophila ovulation, as in mammals, requires the proteolytic degradation of the posterior follicle wall and follicle rupture to release the mature oocyte from a layer of somatic follicle cells. Here, we identified actomyosin contraction in somatic follicle cells as the major mechanical force for follicle rupture. Filamentous actin (F-actin) and nonmuscle myosin II (NMII) are highly enriched in the cortex of follicle cells upon stimulation with octopamine (OA), a monoamine critical for Drosophila ovulation. Pharmacological disruption of F-actin polymerization prevented follicle rupture without interfering with the follicle wall breakdown. In addition, we demonstrated that OA induces Rho1 guanosine triphosphate (GTP)ase activation in the follicle cell cortex, which activates Ras homolog (Rho) kinase to promote actomyosin contraction and follicle rupture. All these results led us to conclude that OA signaling induces actomyosin cortex enrichment and contractility, which generates the mechanical force for follicle rupture during Drosophila ovulation. Due to the conserved nature of actomyosin contraction, this work could shed light on the mechanical force required for follicle rupture in other species including humans.

A highly conserved A-to-I RNA editing event within the glutamate-gated chloride channel GluClα is necessary for olfactory-based behaviors in Drosophila.

A-to-I RNA editing is a cellular mechanism that generates transcriptomic and proteomic diversity, which is essential for neuronal and immune functions. It involves the conversion of specific adenosines in RNA molecules to inosines, which are recognized as guanosines by cellular machinery. Despite the vast number of editing sites observed across the animal kingdom, pinpointing critical sites and understanding their in vivo functions remains challenging. Here, we study the function of an evolutionary conserved editing site in Drosophila, located in glutamate-gated chloride channel (GluClα). Our findings reveal that flies lacking editing at this site exhibit reduced olfactory responses to odors and impaired pheromone-dependent social interactions. Moreover, we demonstrate that editing of this site is crucial for the proper processing of olfactory information in projection neurons. Our results highlight the value of using evolutionary conservation as a criterion for identifying editing events with potential functional significance and paves the way for elucidating the intricate link between RNA modification, neuronal physiology, and behavior.

Host and venom evolution in parasitoid wasps: does independently adapting to the same host shape the evolution of the venom gland transcriptome?

BACKGROUND: Venoms have repeatedly evolved over 100 occasions throughout the animal tree of life, making them excellent systems for exploring convergent evolutionary novelty. Growing evidence supports that venom evolution is predominantly driven by prey or host-related selection pressures, and the expression patterns of venom glands reflect adaptive evolution. However, it remains elusive whether the evolution of expression patterns in venom glands is likewise a convergent evolution driven by their prey/host species. RESULTS: We utilized parasitoid wasps that had independently adapted to Drosophila hosts as models to investigate the convergent evolution of venom gland transcriptomes in 19 hymenopteran species spanning ~ 200 million years of evolution. Comparative transcriptome analysis reveals that the global expression patterns among the venom glands of Drosophila parasitoid wasps do not achieve higher similarity compared to non-Drosophila parasitoid wasps. Further evolutionary analyses of expression patterns at the single gene, orthogroup, and Gene Ontology (GO) term levels indicate that some orthogroups/GO terms show correlation with the Drosophila parasitoid wasps. However, these groups rarely include genes highly expressed in venom glands or putative venom genes in the Drosophila parasitoid wasps. CONCLUSIONS: Our study suggests that convergent evolution may not play a predominant force shaping gene expression levels in the venom gland of the Drosophila parasitoid wasps, offering novel insights into the co-evolution between venom and prey/host.