Multi-omics Analysis Uncovers Lifespan Effects of Polyethylene and Polystyrene Microplastics Coexposure in Drosophila melanogaster

Multi-omics Analysis Uncovers Lifespan Effects of Polyethylene and Polystyrene Microplastics Coexposure in Drosophila melanogaster | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Multi-omics Analysis Uncovers Lifespan Effects of Polyethylene and Polystyrene Microplastics Coexposure in Drosophila melanogaster Yingyu Liu, Cheng Wang, Caixia Wang, Longhuan Fu, Yunbo Zhang, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7459263/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Microplastics (MPs) are ubiquitous global contaminants, posing a long-term exposure risk to both the entire ecosystem and human health. Although increasing researches have indicated that individual MPs generally exhibit biotoxicity, the combined effects of multiple MPs exposure on biological lifespan and the mechanisms involved remain largely unrevealed. Here we employed Drosophila melanogaster , subsequently referred to Drosophila , as a biological model to investigate the impact of polyethylene (PE, irregular shape, 14.55 ± 5.98 µm) and polystyrene (PS, sphere, 1.86 ± 0.89 µm) microplastics co-exposure on lifespan at both low concentrations (10 and 100 mg/L) and high concentrations (10, 20 and 50 g/L). Furthermore, we delved into the underlying mechanism through metabolomics and transcriptomics analysis. Our results demonstrated PE and PS MPs co-exposure with greatly high concentrations significantly reduced the lifespan of Drosophila and influenced age-related phenotypes such as climbing ability, intestinal barrier and hunger resistance. We found that differential metabolites were engaged in various metabolic pathways, including ABC transporters, alanine, aspartate and glutamate metabolism. Differentially expressed genes (DEGs) were closely related to Toll and Imd signaling pathway and Longevity regulating pathway. A combined metabolomics and transcriptomics analysis revealed that PE and PS MPs co-exposure induced alterations in gene expression and metabolites related to the immune system and energy metabolism, thereby affecting Drosophila lifespan. The findings provided a mechanistic understanding for the effects of PE and PS MPs co-exposure on Drosophila’s lifespan. microplastics co-exposure lifespan age-related phenotypes Drosophila Multi-omics Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Synopsis Co-exposure to PE and PS MPs with high concentrations induces changes in gene expression and metabolites associated with immune system and energy metabolism in Drosophila , thereby affecting their lifespan. Introduction Plastic has become an integral part of industrial manufacture and daily lives.(Thompson et al., 2024 ) Due to its widespread use and resistance to degradation, plastic accumulates rapidly within ecosystems. The interplay of physical, chemical, and biological processes lead to the disintegration of larger plastic items into microplastics (MPs) and nanoplastics (NPs), defined as particles with at least one dimension less than 5 millimeters. Nowadays, MPs have been detected in various environmental compartments, including soil, atmosphere, water bodies, and even in drinking water and food.(Vethaak and Legler, 2021 ; Yang et al., 2024 ) A mere one square centimeter of plastic container surface can release MPs in staggering quantities, ranging from millions to billions, following microwave heating or extended storage periods in fridge.(Hussain et al., 2023 ) The pervasive presence of MPs pollution highlights the long-term exposure risks faced by both the entire ecosystem and human beings, as evidenced by the detection of MPs in human whole blood, placenta and fluids.(Chen and Lin, 2024 ; Leslie et al., 2022 ; Ragusa et al., 2021 ; Zuri et al., 2023 ) Nonetheless, our understanding of the biological hazards associated with MPs exposure remains incomplete.(Ramsperger et al., 2020 ) The biotoxicity of MPs is well-documented in aquatic organism species,(Purayil et al., 2024 ; Yakubu et al., 2024 ; Yong et al., 2020 ) with effects including reduced survival, lowered body mass, weakened immunity, diminished reproductive capacity, intestinal dysfunction, behavioral changes metabolic disruption, and heightened oxidative stress.(Bhat et al., 2024 ; Hirt and Body-Malapel, 2020 ; Kumar et al., 2024 ) Compared to aquatic systems, the ecological impacts of MPs on terrestrial ecosystems have been largely overlooked even though MPs contamination on land could be 4 to 23-fold higher than those in the oceans.(Horton et al., 2017 ; Wang et al., 2021 ) Evidence suggests that MPs interact with a range of terrestrial organisms, including invertebrates,(Jemec Kokalj et al., 2022 ) fungi,(de Souza Machado et al., 2018 ) and pollinating animals. Ingestion or adherence to MPs by these organisms may facilitate their transfer through food chains, potentially affecting ecosystem services and functions.(Liang et al., 2024 ; Liu et al., 2022 ) Drosophila , a well-established terrestrial model organism, is widely used in toxicity studies due to its short lifespan and manageable care. Meanwhile, approximately 75% of human disease-related genes possess functional homologs in Drosophila , substantiating the organism's suitability and dependability for modeling human diseases.(Brandt and Vilcinskas, 2013 ) To investigate the biotoxicity of MPs on Drosophila , various types of MPs have been introduced into the food culture medium, including polystyrene (PS), polyethylene (PE), polyethylene terephthalate (PET), polyamide (PA), and polylactic acid (PLA). The concentrations used ranged from 0.005 mg/L to 25 g/L, and the particle sizes varied from MPs (≥ 1 µm) to NPs (< 1 µm). PET-MPs and PA-MPs with the concentrations of 10 and 20 g/L could reduce triglyceride, protein and glucose levels in male Drosophila , suggesting an effect on energy metabolism.(Shen et al., 2021 ; Zhong et al., 2022 ) PS was also verified to affect survival rates and alter the ultrastructure of the midgut, ovaries and testes in Drosophila .(Urbisz et al., 2024 ) As a sustainable alternative to traditional plastics, PLA-NPs nonetheless induce structural and molecular disruptions in Drosophila larvae.(Alaraby et al., 2024 ) Additionally, Drosophila serves as a promising model for the rapid assessment of MPs mediated toxicity such as cadmium.(Zhang et al., 2020b ) Diverse factors influence the biotoxicity of MPs, including type, particle size, shape, surface charge, exposure concentration and duration, and developmental stage and gender of exposed organisms.(Liang et al., 2022 ; Liu et al., 2022 ) However, the toxicological mechanisms associated with these factors remains limited. To clarify the reproductive toxicity mechanism of PS-NPs, transcriptomic analysis was conducted on the ovaries of Drosophila following multi-generational exposure. This analysis identified 102 and 208 DEGs in the 1 mg/L and 100 mg/L PS-NPs treatment groups, respectively, compared to the control group.(Tu et al., 2023 ) Similar findings have been observed in the expression of the immune function genes in mussels and oysters.(Détrée and Gallardo-Escárate, 2018 ; Gardon et al., 2020 ) Currently, researches predominantly examine the effects of short-term exposure to a single type of MPs on the physiology and reproduction of Drosophila , and the underlying mechanism is still insufficient. This study pioneers the investigation of long-term and mixed MPs exposure effects on Drosophila’s lifespan and the related mechanisms, integrating transcriptome and metabolome analyses. We have selected PE and PS MPs for this investigation based on the ubiquitous use of PS and PE, their frequent detection in environment and human body, and their prevalence in studies reflecting a distribution of PS > PE > PET ≌ PVC. (Kögel et al., 2020 ) We hypothesized that irregularly sized and shaped PE and PS MPs can impair the intestinal barrier, and long-term exposure to high concentration PE and PS MPs mixture may affect gene expression and metabolism activity, consequently impacting the lifespan and related phenotypes of Drosophila . The outcomes are expected to bolster our capacity to evaluate the risks posed by MPs. 2. Materials and methods 2.1 Obtention and characterization of PE and PS MPs Solid powder of PE and PS with a mesh size of 2000 were purchased from China Petroleum and Chemical Corporation Limited. The morphology of PE and PS MPs were characterized by Scanning Electron Microscope (SEM, Tescan MAGNA, The Czech Republic) and the SEM images were analyzed with ImageJ software to determine the average size of MPs. A Raman spectrometer (Renishaw, UK) was used to identify the types of MPs. 2.2 Drosophila strains and PE and PS exposure In this study, wild type w 1118 Drosophila , obtained from Fungene Biotechnology Co., LTD, were cultured on standard corn medium in an incubator with a relative humidity of 60%, a temperature of 25 ± 1°C and a light cycle of 12:12 hours.(Tu et al., 2023 ) This medium contained corn flour, sucrose, brown sugar, agar and yeast. The study established co-exposure for PE and PS MPs at both low concentrations (10 and 100 mg/L) and high concentrations (10, 20 and 50 g/L) to reflect the multi-pathway, continuous, and accumulative effect of MPs on organisms. To achieve this, a specific, equal mass ratio of mixed PS and PE MPs were added to a solution consisting of 10 ml of pure water and 10 ml of anhydrous ethanol. The mixed MPs solution was sonicated for 30 minutes to ensure uniform distribution. After cooling the medium to 60°C, the MPs solution was thoroughly mixed with 100 mL of standard corn medium, continuously stirred to integrate. A control group was prepared with standard corn medium containing 10 ml water and 10 ml ethanol. The entire experimental procedure followed the methods of a previous study.(Zhong et al., 2022 ) 2.3 PE and PS uptake A TGA/DSC thermogravimetric analyzer (STA449F5, Netzsch, Germany) couple with a mass spectrometer (QMS 403 Quadro, Netzsch, Germany) were used to analyse PE and PS in the whole body of Drosophila . The body tissue of Drosophila in control group, 10 mg/L and 10g/L PE and PS treatment group were dried at 60 d°C and ground into a powder. The dried powder samples of 10mg were thermally decomposed in a nitrogen atmosphere (50mL/min). The temperature was increased from 30 to 800°C at 10°C/min. All the TGA analyses were blank curve corrected. The mass spectrometer detector, operated in Single Ion Monitoring Mode (SIM), measured the ions m/z 56 for PE and m/z 104 for PS. To assess the ingestion and distribution of PE and PS MPs in Drosophila , we conducted Raman spectroscopic analysis on sliced tissue. The specimens were fixed in 4% paraformaldehyde for over 24 hours, dehydrated through a graded alcohol series, infiltrated with wax, embedded in paraffin blocks, and sectioned to 4 µm thickness. The sections were smoothed in 40°C warm water, then mounted on slides, dehydrated at 60°C to remove moisture and wax, and stored at room temperature for analysis.(Urbisz et al., 2024 ) Using a Renishaw in Via Micro-Raman spectrometer (λ = 532 nm), we analyzed the particles' distribution in a 120 µm × 120 µm area of the Drosophila abdomen via Raman mapping, collecting spectral data from 1600 points at 3 µm intervals. Characteristic peaks at 1296 cm⁻¹ for PE and 1002 cm⁻¹ for PS identified their distribution. ImageJ software determined the average fluorescence intensity in the scanned area. 2.4 Measurement of lifespan-related indicators 2.4.1 Assessment of lifespan For the longevity test, every freshly hatched Drosophila was carefully collected and categorized by gender. Each experimental group contained 100 Drosophila , with three replicates for reliability. The flies were transferred to fresh culture medium every three days to maintain optimal conditions. The dead Drosophila number was recorded everyday.(Urbisz et al., 2024 ) The median life span was determined by calculating the median survival duration across all flies. The survival rates were analyzed using the log-rank test. 2.4.2 Climbing ability test To evaluate the climbing ability of Drosophila , 30 individuals were carefully selected from each group after 20 days of cultivation. They were gently tapped and vibrated to encourage descent to the test tube's base. We recorded the number of flies that successfully climbed beyond 13 cm mark within 7 second.(Qiu et al., 2020 ) This test was repeated three times per group, with a 30-minute rest between trials to prevent fatigue from affecting performance. 2.4.3 Measurement of adult body weight and length Adult Drosophila , 20-day post-emergence, were carefully collected and placed onto filter paper for the measurement of their weight and length.(Krittika and Yadav, 2022 ) The experiments were conducted in triplicate to ensure accuracy, with the average values being reported for each parameter. 2.4.4 Evaluation of anti-starvation ability For the anti-starvation ability test, 20-day-old Drosophila of both genders were introduced into a 0.9% agarose medium, which provided hydration without nutritional content.(Zhang et al., 2020a ) The daily mortality rate was meticulously recorded throughout the experiment. Each replicate involved 100 flies, and the experiment was repeated three times to ensure reliable results. 2.4.5 Assessment of intestinal barrier function Following a 20-day exposure, both male and female Drosophila were transferred to a medium containing 2.5% brilliant blue (Erioglaucine disodium salt) to evaluate intestinal barriers integrity. They were divided equally among 10 tubes per group, each containing 10 flies. After 5 days, the percentage of flies exhibiting a blue coloration for each group, referred to as "Smurfs," was determined as an intestinal permeability indicator.(Rera et al., 2011 ) 2.5 Metabolomics Analysis For the metabolomics study, whole-body Drosophila tissue in control and PE + PS group after 20 days feeding were analyzed with triplicates for each group. Samples were extracted with 50% methanol, and analyzed via UPLC-MS on a Vanquish Flex UPLC system (Thermo Fisher Scientific, Bremen, Germany) utilizing an ACQUITY UPLC T3 column (100 mm × 2.1 mm, 1.8 µm, Waters, Milford, USA) for reversed-phase separations at a controlled oven temperature of 35°C. The mobile phase consisted of solvent A (water, 0.1% formic acid) and solvent B (Acetonitrile, 0.1% formic acid). The flow rate was 0.4 ml/min. Metabolites were identified using a Q-Exactive mass spectrometer (Thermo Scientific) and annotated against Kyoto Encyclopedia of Genes and Genomes (KEGG) and Human Metabolome Database (HMDB) databases with a 10 ppm mass tolerance. Data was preprocessed with XCMS software and analyzed by PCA, t-tests, and PLS-DA, identifying differential metabolites based on VIP, P value, and fold change. We followed established protocols from LC-Bio Technology CO., Ltd (Hangzhou, China) for metabolomic analyses. More details are fully described in the supplementary material. 2.6 Transcriptomic analysis Total RNA was extracted from the whole-body tissue of Drosophila in control and PE + PS group after 20 days feeding, using the trizol reagent (Thermo Fisher, 15596018). The quality and quantity of the extracted RNA were further validated using a Bioanalyzer 2100 and RNA 6000 Nano LabChip Kit (Agilent, CA, USA, 5067 − 1511). High-quality RNA, with RNA Integrity Number value exceeding 7.0, was selected for sequencing library construction. mRNA was purified using Dynabeads Oligo (Thermo Fisher, CA, USA) and sequenced on an Illumina NovaseqTM 6000 platform. Transcript expression levels were quantified using StringTie and ballgown package, with DEGs identified through DESeq2 and edgeR software, classified by false discovery rate (FDR) < 0.05 and fold change ≥ 2. These DEGs were analyzed for Gene Ontology (GO) and KEGG pathways enrichment. Transcriptomic analyses also followed the established protocols from LC-Bio Technology CO., Ltd (Hangzhou, China), as shown in the Supporting Material. 2.7 Correlation analysis of transcriptomic and metabolomic We conducted a correlation analysis utilizing data from both transcriptomic and metabolomic profiles to investigate the relationship between DEGs and identified metabolites. A signed clustering correlation heat map was generated using the OmicStudio platform. 2.8 Statistical analyses Statistical analyses were performed using GraphPad Prism software, version 7. The lifespan and starvation survivorship data were analyzed using log-rank assays. Differences among groups were assessed employing one-way ANOVA, followed by Turkeys' post hoc test. A P < 0.05 was considered significant for all statistical analyses. 3. Results and Discussion 3.1 Characterization and internalization of PE and PS MPs SEM images demonstrated a uniform dispersion and irregular morphology of PE MPs (Fig. 1 A), with an average particle size of 14.55 ± 5.97 µm (Fig. 1 C). While PS MPs displayed a more regular shape (Fig. 1 B) and a notably smaller average size of 1.86 ± 0.89 µm (Fig. 1 D). Raman spectroscopy confirmed their distinct chemical identities by analyzing the unique spectral peaks for PE and PS standard substance (Fig. 1 E). The TGA-MS analysis (Fig. S1 ) demonstrated distinct peak intensities at m/z 56 for PE and at m/z 104 for PS in their respective standard substances. Notably, the signal intensity for both PE and PS in flies cultured in a 10 g/L mixture of PE and PS for 50 days showed a modest increase, while the flies cultured in a 10 mg/L PE and PS exhibited no significant difference from the control group. To confirm the internalization of MPS, Raman spectroscopy was used for high concentration group. In the randomly selected scanning regions (Fig. 2 A to 2 C), the size and brightness of Raman signal spot increased with exposure concentration at the characteristic peaks of 1296 cm − 1 for PE and 1002 cm − 1 for PS (Fig. 2 D to 2 I). ImageJ analysis revealed that the average fluorescence intensity for PE across the control, 10 g/L and 50 g/L group were 101.12, 159.45 and 163.23, respectively. For PS, the intensities were 88.39, 156.78 and 171.61, respectively. These results showed that both PE and PS MPs were ingested by Drosophila , with ingestion levels rising in tandem with exposure concentration. 3.2 Effects of PE and PS MPs on lifespan of Drosophila Long-term exposure diet enriched with high levels of PE and PS MPs substantially reduced the life span of Drosophila . Notably, when subjected to the MPs mixture at concentrations of 10, 20 and 50 g/L, the median lifespan of female flies decreased to 32.67 ± 2.31, 27.33 ± 2.08, and 19.33 ± 3.88 days, respectively, compared with the 51.32 ± 1.00 days in control group. In contrast, exposure to low concentrations of PE and PS MPs at 10 and 100 mg/L did not significantly alter the lifespan of Drosophila with median lifespans of 46.94 ± 2.06 and 48.23 ± 1.28 days, respectively. A comparable pattern was noted in male flies, with median lifespans ranging from 46.53 ± 0.73 to 23.67 ± 2.31 days as the concentration of MPs increased (Fig. 3 A to 3 D). Sex-specific response to MPs was apparent at lower concentrations with females showing a higher survival rate compared to males at equivalent lifespan stages. This result is consistent with the research from Urbisz et al.(Urbisz et al., 2024 ) It is recognized that Drosophila , along with many other insects, exhibit sex-specific responses to a variety of physiological, environmental and ecological stressors.(Parkash and Ranga, 2013 ; Shen et al., 2023 ) However, at higher concentrations of 20 and 50 g/L, these sex differences were unconspicuous. Liang et al. investigated effects of long-term exposure to PET MPs (2 µm) on Drosophila’s lifespan at the concentration of 1, 10 and 20 g/L, results presented that 10 g/L PET-MPs caused 20.42% decrease in female mean lifespan, and 20 g/L PET MPs caused 16.01% decrease in male mean lifespan. Co-exposure to PE and PS has a more pronounced impact on lifespan than exposure to PET alone, which may be related to the type of MPs, because PET is more bioinert as it is often used in food packaging.(Lee et al., 2022 ) Considering the minimal impact of low-concentration (10 and 100 mg/L) PE and PS MPs co-exposure on the lifespan of Drosophila , further investigation was conducted to explore the effects of mixed PE and PS MPs exposure on lifespan-related phenotypes within the high concentration group (10, 20 and 50 g/L). 3.3 Effects of PE and PS MPs on age-related phenotypes of Drosophila Continuous exposure to MPs profoundly influenced age-related phenotype characteristics of Drosophila . Male flies subjected to PS and PE MPs showed a notable reduction in body weight and length, contrasting with the control group, while females displayed an increase (Fig. 4 A, B). The climbing assay, a standard measure of Drosophila 's spontaneous activity, revealed a dose-dependent reduction in motor function upon exposure to PS and PE MPs (Fig. 4 C). The study results reported by Zhang et al. also showed that exposure to PS alone could reduce the climbing ability of Drosophila , and this effect was more serious after combined exposure to Cd.(Zhang et al., 2020b ) Similarly, the survival curve under starvation conditions indicated a diminished capacity for hunger resistance with increasing MPs concentrations (Fig. 4 E, F), and the anti-starvation ability of females was stronger than that of males. Exposure to PET and PA MPs also had similar results, accompanying with a reduced triglyceride, protein and glucose content in males, indicating that energy metabolism of male Drosophila was affected by MPs ingestion.(Shen et al., 2021 ; Zhong et al., 2022 ) In Drosophila , lifespan reduction is often associated with intestinal epithelial barrier dysfunction.(Guo et al., 2014 ) The Smurf assay corroborated this, demonstrating a decline in gut barrier integrity post-exposure to PS and PE MPs (Fig. 4 D), which was consistent with the previous study.(Zhang et al., 2020b ) Based on our findings, escalating exposure to PS and PE MPs induced a series of dose-dependent reduction in Drosophila lifespan and manifestation of age-related phenotypes. To maximize the identification of DEGs and metabolites between the treatment and control, as well as between genders, this study concentrated on analyzing the effects of the highest PE and PS concentration on Drosophila 's transcriptome and metabolome profile. 3.4 Effects of PE and PS MPs on global metabolomics profiles in the Drosophila The PCA scores plot, with QC samples tightly clustered as purple dots, demonstrated a high stability and reliability of our metabolomics platform (Supporting Material Fig. S2). PLS-DA analysis distinctly segregated the two groups of samples (Fig. S3 A and C). R2Y and Q2 values exceeding the threshold of 0.5 indicated the model's goodness of fit and predictive ability, with specific values of 0.998 and 0.733 for females, and 0.999 and 0.853 for males, respectively. The comparison of R2Y and Q2 values in Fig. S3 B and D, illustrating lower values on the left side versus higher on the right, confirmed the model's comprehensive explanatory and robust predictive capabilities for the observed sample variations. Metabonomic analysis revealed significant metabolic disparities between the control and PE + PS groups, identifying 24 distinct metabolites in males and 7 in females, as depicted in the clustering heat maps Fig. 5 and Supporting Material Table S1 . In males, exposure to PE and PS MPs led to significant changes in organic acids and derivatives, organic heterocyclic compounds, lipid molecules, phenylpropanoids and polyketides, organic oxygen compounds, nucleosides, nucleotides and analogues. Among these, Lysophosphatidylcholine (Lyso PC 18:0, LPC), a glycerophospholipid and lipid-like molecule, is recognized for its anti-inflammatory properties.(Hung et al., 2011 ) LPC containing docosahexaenoic acid at the sn-1 position and the 18:0 LPC can inhibit inflammatory mediator release, such as TNF-α, IL-6, IL-α, IL-1β, and IL-10.(Huang et al., 2010 ; Yan et al., 2004 ) Furthermore, the balance between pro-inflammatory cytokines and anti-inflammatory cytokines is crucial for delaying aging. In females, exposure to PE and PS MPs induced significant alterations in organic heterocyclic compounds, lipid molecules, organic acids and derivatives, as well as nucleosides, nucleotides and analogues. Notably, glutamine, categorized under organic acids and derivative, is intricately linked to aging process due to its vital role for cell proliferation and survival.(Meynial-Denis, 2016 ; Salabei et al., 2015 ) Glutamine serves as a precursor for essential molecules including amino acids and nucleotide. It is instrumental in the synthesis of nicotinamide adenine dinucleotide phosphate (NADPH), glutathione (GSH) and adenosine triphosphate (ATP), which are crucial for redox homeostasis and energy supply.(Xiao et al., 2016 ) Meanwhile, aging is commonly characterized by energy generation reduction(Giezenaar et al., 2016 ) and redox homeostasis dysfunction.(Meng et al., 2017 ) Therefore, changes of LPE, LPC, L-Glutathione and glutamine maybe one reason of aging through affecting the energy metabolism. Enrichment analysis visualized via bubble diagram revealed distinct KEGG pathways of metabolites enrichments in male and female flies. In males, the most significantly enriched pathways included ABC transporters, alanine, aspartate and glutamate metabolism, and purine metabolism (Fig. 6A). Females showed predominant enrichment in ABC transporters, choline metabolism in cancer, and metabolism of alanine, aspartate, glutamate, cysteine and methionine (Fig. 6B). Notably, ABC transporters were the most enriched pathway in both genders, highlighting their crucial role in insect physiology.(Huang et al., 2014 ) This pathway involves a family of integral membrane proteins essential for the transmembrane transport of various molecules.(Campos et al., 2013 ) Fig. 6 Metabolic pathways analysis of PS + PE exposure on Drosophila for the male flies (A) and female flies (B). The top 10 pathways of significant of the up-regulated and down-regulated DEMs on KEGG. The X-axis was the rich factor, the Y-axis represents the name of the pathway. The bubbles size represents the number of differential expression metabolites involved. The bubbles color indicates the enrichment degree of pathway. 3.5 Effects of PE and PS MPs on global transcriptomics profiles in the Drosophila In our study, we detected significant changes in gene expression profiles, with a total of 286 DEGs in males and 205 DEGs in females, when comparing the PE + PS exposure group to the control group. Notably, the majority of these DEGs were implicated in the regulation of energy metabolism (Fig. 7 ). Phosphoenopyruvate carboxykinase (PEPCK), a pivotal regulatory enzyme in gluconeogenesis, glyceroneogenesis, serine synthesis, and amino acid metabolism, is stringently regulated at the transcriptional level.(Xiong et al., 2011 ) UDP-glycosyltransferases (UGTs), a widespread superfamily of enzymes, mediate biotransformation and play crucial roles in detoxifying harmful exogenous chemicals and modulating signaling molecules.(Ramírez et al., 2015 ) The immune response to MPs may exacerbate energy constraints, diverting energy from growth and reproduction to maintenance, which includes energetically costly cellular processes.(Olsen et al., 2015 ) These metabolic and immune disruptions caused by MPs could increase the susceptibility of organism to further stress from pathogens, other pollutants, and environmental factors, potentially affecting lifespan. Heat shock proteins (Hsps) are crucial in regulating protein structure and folding.(Kim et al., 2007 ) They essential for Drosophila 's stress resistance, including thermal and environmental stressors.(Feder and Krebs, 1997 ) Hsp70, a predominant Hsp family member, serves as a sensitive bioindicator for environmental monitoring due to its role in cellular defense mechanisms under pollutant stress.(Liu et al., 2017 ) In this study, a significant upregulation of the Hsp70 gene in female flies was observed after exposure to PS and PE MPs, indicating that such exposure triggers a stress response. This upregulation is likely a compensatory mechanism to mitigate the stress induced by PE and PS MPs. This is consistent with observations that female Drosophila exhibit a greater climbing ability, a enhanced hunger resistance, and a higher survival rate at 20 days compared to male flies. Our findings align with those of Brandts et al., who noted an increase in Hsp70 gene expression in mussels' digestive glands exposed to PS nanoplastic.(Brandts et al., 2018 ) Given proteotoxicity's role in aging, Hsps may enhance Drosophila 's stress resistance by mediating protein refolding or degradation.(Hagymasi et al., 2022 ) These DEGs were categorized across 201 biological processes, 70 cellular components, and 138 molecular functions (Fig. 8 ). In male flies, KEGG enrichment analysis revealed interference in the “Toll and Imd signaling pathways” after PE + PS co-exposure, along with perturbations in Lysosome, Neuroactive ligand-receptor interaction, and Drug metabolism - cytochrome P450 pathways. Female flies displayed a distinct enrichment in pathways such as Insect hormone biosynthesis, Longevity regulating pathways - multiple species, Spliceosome, and Autophagy-animal. Network analysis of these pathways indicated that some genes participate in multiple signaling pathways simultaneously (Fig. 8 ). Notably, in Drosophila , humoral immunity is primarily mediated by the “Toll and Imd signaling pathway”.(Yu et al., 2022 ) The Toll pathway, activated in response to systemic infections, plays a key role in adipose tissue growth, metabolism, and hemocyte activation.(Myllymäki et al., 2014 ) The Imd pathway is essential for bacterial defense,(Mussabekova et al., 2017 ) controlling the expression of most antimicrobial peptides, highlighting its role in immune homeostasis.(Lemaitre et al., 1995 ) The intestinal epithelium is a recognized immunoactive tissue, playing a key role in longevity regulation.(Franceschi et al., 2018 ) Microplastics can induce an immune response through mechanical damage to the intestinal epithelium, while excessive immune activation can hasten degeneration, cause inflammation, and reduce lifespan.(Guo et al., 2014 ) The Smurf assay results provided strong confirmation of this association. 3.6 Combined analysis of transcriptomics and metabolomics Combined analysis has revealed the involvement of multiple DEGs and metabolites in energy metabolism pathways (Fig. 9 ). A pronounced interconnection between immune and metabolic processes is particularly evident in the gut, where intestinal cells collaborate to prevent microbial invasion.(Miguel-Aliaga et al., 2018 ) Studies on model organisms have shown that diet significantly influences aging, with nutritional status being closely associated with the incidence of age-related diseases. Cellular senescence represents both a cause and a consequence of metabolic dysregulation, and disruptions in macronutrient metabolism can influence aging and longevity by impacting the onset and characteristics of the senescent state.(Nehme et al., 2023 ) Our study detected a dose-dependent decline in gut barrier function in Drosophila after exposure to PE and PS MPs. Transcriptomics analysis also revealed a significant disruption in Drosophila’s immune defense function after mixed microplastics exposure. Therefore, we propose that PE and PS co-exposure impairs Drosophila’s gut barrier function, triggering immune stress, disrupting energy metabolism, and ultimately leading to a shortened lifespan in Drosophila . 4. Conclusions This study presents the first comprehensive examination of the molecular-level toxicity resulting from co-exposure to PE and PS MPs in terrestrial invertebrates. Our findings show that chronic exposure to PE and PS MPs with very high concentration profoundly affects the lifespan and several phenotypic biomarkers of Drosophila , encompassing climbing ability, stress resistance to hunger, and intestinal barrier function. Following co-exposure to PS and PE MPs, we observed DEGs and altered metabolites in Drosophila , predominantly associated with immune function and energy metabolism. These insights significantly enhance our comprehension of the molecular mechanisms that underlie the physiological and behavioral impairments observed in Drosophila when subjected to MPs exposure. Declarations Competing Interests The authors have no relevant financial or non-financial interests to disclose. Author contributions Yingyu Liu: Data curation, Investigation. Cheng Wang: Formal analysis. Caixia Wang: Writing- Original draft. Longhuan Fu: Investigation. Yunbo Zhang: Writing- Reviewing and Editing. Zhuo Gao: Visualization. Zhugen Yang: Writing- Reviewing and Editing. Fanyu Meng: Conceptualization, Methodology, Supervision. Acknowledgments The authors gratefully acknowledge funding from Project LH2021E097 supported by the Natural Science Foundation of Heilongjiang Province. ZY thanks UKRI NERC Fellowship (NE/R013349/2) and The Leverhulme Trust Research Leaderships Awards (RL-2022-041). Data Availability Data will be made available on request. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7459263","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":507586478,"identity":"d126df01-2d0f-4a1d-826a-21d6c3f6e969","order_by":0,"name":"Yingyu Liu","email":"","orcid":"","institution":"Harbin Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yingyu","middleName":"","lastName":"Liu","suffix":""},{"id":507586479,"identity":"2a8f2b91-dc9d-4a22-b8a8-c68e7005bafe","order_by":1,"name":"Cheng Wang","email":"","orcid":"","institution":"Harbin Medical University","correspondingAuthor":false,"prefix":"","firstName":"Cheng","middleName":"","lastName":"Wang","suffix":""},{"id":507586480,"identity":"97cbbb62-7205-482e-904f-e03dbb551821","order_by":2,"name":"Caixia Wang","email":"","orcid":"","institution":"Harbin Medical University","correspondingAuthor":false,"prefix":"","firstName":"Caixia","middleName":"","lastName":"Wang","suffix":""},{"id":507586481,"identity":"a1609092-d76f-43a3-a1a4-0402edd49555","order_by":3,"name":"Longhuan Fu","email":"","orcid":"","institution":"Harbin Medical University","correspondingAuthor":false,"prefix":"","firstName":"Longhuan","middleName":"","lastName":"Fu","suffix":""},{"id":507586482,"identity":"4aac897f-27fe-4509-87fe-3fbb22d402f8","order_by":4,"name":"Yunbo Zhang","email":"","orcid":"","institution":"Harbin Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yunbo","middleName":"","lastName":"Zhang","suffix":""},{"id":507586483,"identity":"de22677a-028e-422c-b82b-168093fc67fb","order_by":5,"name":"Zhuo Gao","email":"","orcid":"","institution":"Harbin Medical University","correspondingAuthor":false,"prefix":"","firstName":"Zhuo","middleName":"","lastName":"Gao","suffix":""},{"id":507586484,"identity":"644a0daa-592e-4de6-850c-99d54d74224d","order_by":6,"name":"Zhugen Yang","email":"","orcid":"","institution":"Cranfield University","correspondingAuthor":false,"prefix":"","firstName":"Zhugen","middleName":"","lastName":"Yang","suffix":""},{"id":507586485,"identity":"c7f0235a-93f4-48a7-9d20-52c33f6a254a","order_by":7,"name":"FanYu Meng","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABG0lEQVRIiWNgGAWjYLCCChDBzHzwgUTFAQYDsBAbAS1nQAQ7W7KBxRmStPDzmElUthGhRbe99/CLAzV3Ejcc5jE2uDnvTr652BkDhg9lhxn4Zzdg1WJ25lyaxYFjz4Ba2Aofztz2zHLn7BwDxhnnDjNI3DmAXcuNHDPjD2yHgVqYNxtLbjtsYHA7x4CZt+0wg4FEAnYt99+YGRz4B9LCYCb9dw5Uy198Wm7wGD842AbSwmImIdkA1cKIT8uZHDOGg32HjWceBgayxLHDBpaz0woO9pxL55G4gUPL8TPGHw58Oyzbd/4wMCprDhuYSydvfPCjzFqOfwZ2LUDAJgEkHBuQhQ4AMQ8u9UDA/AFI2ONRMApGwSgYBSMdAAB7Lm6IYKqIbAAAAABJRU5ErkJggg==","orcid":"","institution":"Harbin Medical University","correspondingAuthor":true,"prefix":"","firstName":"FanYu","middleName":"","lastName":"Meng","suffix":""}],"badges":[],"createdAt":"2025-08-26 06:12:41","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7459263/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7459263/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":91001257,"identity":"76dac3ae-af8c-4317-8787-57f3ec2a830c","added_by":"auto","created_at":"2025-09-10 13:46:19","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":342223,"visible":true,"origin":"","legend":"\u003cp\u003eSEM images of PE-MPs (A) and PS-MPs (B); Histogram of size frequencies of PE-MPs (C) and PS-MPs (D); Raman spectra of PE and PS (E).\u003c/p\u003e","description":"","filename":"Figure1.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7459263/v1/c54b69a22cf68c06cf1e9caa.jpg"},{"id":91001256,"identity":"00e3c31f-5062-407f-b57b-d2917580420b","added_by":"auto","created_at":"2025-09-10 13:46:19","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":334582,"visible":true,"origin":"","legend":"\u003cp\u003eRaman analysis of ingested PE and PS. Raman mapping test area of different exposure concentrations of 50 g/L (A), 10 g/L (B), and control (C). Raman signal strength at peak 1296\u003csup\u003e-1\u003c/sup\u003e for PE of 50 g/L (D), 10 g/L (E), and control (F). Raman signal strength at peak 1002\u003csup\u003e-1\u003c/sup\u003e for PS of different exposure concentrations of 50 g/L (G), 10 g/L (H), and control (I).\u003c/p\u003e","description":"","filename":"Figure2.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7459263/v1/365215eb16877f0c957172be.jpg"},{"id":91002386,"identity":"c5ca6c5e-dab9-45e2-b622-a6d622ab636f","added_by":"auto","created_at":"2025-09-10 13:54:19","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":6160942,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of PS+PE on the lifespan of \u003cem\u003eDrosophila\u003c/em\u003e. Statistical analysis for survival curves of male flies (A) and female flies (C) was performed by log-rank test (\u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01, \u003csup\u003e***\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001). (B, D) Summary of median survival for independent lifespan experiments (n=3) of male flies (B) and female flies (D). Data are presented as box-and-whisker plots (min-max error bars), analyzed by one-way ANOVA with Tukey' post hoc test (n/s, \u003cem\u003eP\u003c/em\u003e\u0026gt; 0.05, \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01, \u003csup\u003e***\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"Figure3.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7459263/v1/6a95693f59aeecb1710c378c.jpg"},{"id":91001258,"identity":"2d0d888a-eff8-4ea3-acb8-ae499702d101","added_by":"auto","created_at":"2025-09-10 13:46:19","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":970959,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of different concentrations of PS+PE on \u003cem\u003eDrosophila\u003c/em\u003e body weight, body length, climbing ability, intestinal barrier, starvation resistance. (A) Effect of PS+PE on the body weight. (B) Effect of PS+PE on the body length. (C) Effect of PS+PE on the climbing ability. (D) Effect of PS+PE on the intestinal barrier. (E, F) Effect of PS+PE on the starvation resistance of flies. (E) Male flies. (F) Female flies. Data are presented as box-and-whisker plots (min-max error bars), analyzed by one-way ANOVA with Tukey' post hoc test. Statistical analysis for survival curves (E, F) was performed by log-rank test (n/s, \u003cem\u003eP \u003c/em\u003e\u0026gt; 0.05, \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01, \u003csup\u003e***\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"Figure4.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7459263/v1/426fbd63595c8d4055d19701.jpg"},{"id":91001259,"identity":"5edb3342-a2b7-4de9-bd22-05d0f90d24df","added_by":"auto","created_at":"2025-09-10 13:46:19","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":455282,"visible":true,"origin":"","legend":"\u003cp\u003eHeatmaps of differential metabolites compared between control and PS+PE exposure groups in male flies (A) and female flies (B).\u003c/p\u003e","description":"","filename":"Figure5.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7459263/v1/b9cbdbb5b53985f7af58c030.jpg"},{"id":91001262,"identity":"a703d966-1f4a-489c-aaf7-0a2d1894d62c","added_by":"auto","created_at":"2025-09-10 13:46:19","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":484383,"visible":true,"origin":"","legend":"\u003cp\u003eMetabolic pathways analysis of PS+PE exposure on \u003cem\u003eDrosophila\u003c/em\u003e for the male flies (A) and female flies (B). The top 10 pathways of significant of the up-regulated and down-regulated DEMs on KEGG. The X-axis was the rich factor, the Y-axis represents the name of the pathway. The bubbles size represents the number of differential expression metabolites involved. The bubbles color indicates the enrichment degree of pathway.\u003c/p\u003e","description":"","filename":"Figure6.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7459263/v1/db35ddd65130681669db32cc.jpg"},{"id":91001261,"identity":"50d68035-446c-4b84-ab2e-d313c4360756","added_by":"auto","created_at":"2025-09-10 13:46:19","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":538373,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of PS+PE exposure on global transcriptomics profiles. Volcano plot of distribution trends for differentially expressed genes between control and PS+PE exposure groups. The negative log10-transformed P-values of the t-test were plotted against the log2-transformed fold change in the control-treatment experiment. Red circle and blue circle, up- and down-regulation of DEGs, respectively. Grey dots indicate that the genes were not differentially expressed. (A) Male flies. (B) Female flies.\u003c/p\u003e","description":"","filename":"Figure7.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7459263/v1/a964dbacf45fd514c8214d84.jpg"},{"id":91001267,"identity":"4fd55ca7-ea76-4b39-9b7f-c4bbfdd52ca0","added_by":"auto","created_at":"2025-09-10 13:46:19","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1166770,"visible":true,"origin":"","legend":"\u003cp\u003eGO enrichment of significant DEGs and the top ten significantly enriched KEGG pathways in transcriptomic analysis. The vertical coordinate is the negative log10 P-value of the GO enrichment analysis. The horizontal coordinate is the Z-Score, Z-Score = (up-down)/(S\u003csup\u003e0.5\u003c/sup\u003e), which represents the degree of difference in the number of up-and down-regulated genes. Bubble size represents the number of genes assigned to the specific process, and bubble color indicates the classification of GO term. The colors of the blue represent different pathways, and the colors of the pink represent different genes in the Network analysis. The larger the pathway node is, the greater the number of enriched genes in the pathway. (A) Male flies. (B) Female flies.\u003c/p\u003e","description":"","filename":"Figure8.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7459263/v1/7e49af77f043228792789898.jpg"},{"id":91001268,"identity":"dafd9b91-a554-45e7-aab6-238e6721d2fd","added_by":"auto","created_at":"2025-09-10 13:46:19","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":853672,"visible":true,"origin":"","legend":"\u003cp\u003eDifferential genes and metabolites related to energy metabolism. (A) Male flies. (B) Female flies.\u003c/p\u003e","description":"","filename":"Figure9.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7459263/v1/a74c9cf94b8bb51d3920b97f.jpg"},{"id":91149333,"identity":"5c20e51c-7a62-444d-9668-b208ee8f9887","added_by":"auto","created_at":"2025-09-12 06:48:54","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":12271697,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7459263/v1/efd59aee-7770-486f-be57-00eedfa0d1e2.pdf"},{"id":91002389,"identity":"871a6bf0-a321-478b-9c41-117057153e99","added_by":"auto","created_at":"2025-09-10 13:54:19","extension":"docx","order_by":13,"title":"","display":"","copyAsset":false,"role":"supplement","size":1962024,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-7459263/v1/b71ce92b07f2cfb0076468aa.docx"}],"financialInterests":"","formattedTitle":"Multi-omics Analysis Uncovers Lifespan Effects of Polyethylene and Polystyrene Microplastics Coexposure in Drosophila melanogaster","fulltext":[{"header":"Synopsis","content":"\u003cp\u003eCo-exposure to PE and PS MPs with high concentrations induces changes in gene expression and metabolites associated with immune system and energy metabolism in \u003cem\u003eDrosophila\u003c/em\u003e, thereby affecting their lifespan.\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003ePlastic has become an integral part of industrial manufacture and daily lives.(Thompson et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) Due to its widespread use and resistance to degradation, plastic accumulates rapidly within ecosystems. The interplay of physical, chemical, and biological processes lead to the disintegration of larger plastic items into microplastics (MPs) and nanoplastics (NPs), defined as particles with at least one dimension less than 5 millimeters. Nowadays, MPs have been detected in various environmental compartments, including soil, atmosphere, water bodies, and even in drinking water and food.(Vethaak and Legler, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Yang et al., \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) A mere one square centimeter of plastic container surface can release MPs in staggering quantities, ranging from millions to billions, following microwave heating or extended storage periods in fridge.(Hussain et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) The pervasive presence of MPs pollution highlights the long-term exposure risks faced by both the entire ecosystem and human beings, as evidenced by the detection of MPs in human whole blood, placenta and fluids.(Chen and Lin, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Leslie et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Ragusa et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Zuri et al., \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) Nonetheless, our understanding of the biological hazards associated with MPs exposure remains incomplete.(Ramsperger et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2020\u003c/span\u003e)\u003c/p\u003e\u003cp\u003eThe biotoxicity of MPs is well-documented in aquatic organism species,(Purayil et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Yakubu et al., \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Yong et al., \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) with effects including reduced survival, lowered body mass, weakened immunity, diminished reproductive capacity, intestinal dysfunction, behavioral changes metabolic disruption, and heightened oxidative stress.(Bhat et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Hirt and Body-Malapel, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Kumar et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) Compared to aquatic systems, the ecological impacts of MPs on terrestrial ecosystems have been largely overlooked even though MPs contamination on land could be 4 to 23-fold higher than those in the oceans.(Horton et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) Evidence suggests that MPs interact with a range of terrestrial organisms, including invertebrates,(Jemec Kokalj et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) fungi,(de Souza Machado et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) and pollinating animals. Ingestion or adherence to MPs by these organisms may facilitate their transfer through food chains, potentially affecting ecosystem services and functions.(Liang et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Liu et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2022\u003c/span\u003e)\u003c/p\u003e\u003cp\u003e\u003cem\u003eDrosophila\u003c/em\u003e, a well-established terrestrial model organism, is widely used in toxicity studies due to its short lifespan and manageable care. Meanwhile, approximately 75% of human disease-related genes possess functional homologs in \u003cem\u003eDrosophila\u003c/em\u003e, substantiating the organism's suitability and dependability for modeling human diseases.(Brandt and Vilcinskas, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) To investigate the biotoxicity of MPs on \u003cem\u003eDrosophila\u003c/em\u003e, various types of MPs have been introduced into the food culture medium, including polystyrene (PS), polyethylene (PE), polyethylene terephthalate (PET), polyamide (PA), and polylactic acid (PLA). The concentrations used ranged from 0.005 mg/L to 25 g/L, and the particle sizes varied from MPs (\u0026ge;\u0026thinsp;1 \u0026micro;m) to NPs (\u0026lt;\u0026thinsp;1 \u0026micro;m). PET-MPs and PA-MPs with the concentrations of 10 and 20 g/L could reduce triglyceride, protein and glucose levels in male \u003cem\u003eDrosophila\u003c/em\u003e, suggesting an effect on energy metabolism.(Shen et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Zhong et al., \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) PS was also verified to affect survival rates and alter the ultrastructure of the midgut, ovaries and testes in \u003cem\u003eDrosophila\u003c/em\u003e.(Urbisz et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) As a sustainable alternative to traditional plastics, PLA-NPs nonetheless induce structural and molecular disruptions in \u003cem\u003eDrosophila\u003c/em\u003e larvae.(Alaraby et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) Additionally, \u003cem\u003eDrosophila\u003c/em\u003e serves as a promising model for the rapid assessment of MPs mediated toxicity such as cadmium.(Zhang et al., \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2020b\u003c/span\u003e)\u003c/p\u003e\u003cp\u003eDiverse factors influence the biotoxicity of MPs, including type, particle size, shape, surface charge, exposure concentration and duration, and developmental stage and gender of exposed organisms.(Liang et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Liu et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) However, the toxicological mechanisms associated with these factors remains limited. To clarify the reproductive toxicity mechanism of PS-NPs, transcriptomic analysis was conducted on the ovaries of \u003cem\u003eDrosophila\u003c/em\u003e following multi-generational exposure. This analysis identified 102 and 208 DEGs in the 1 mg/L and 100 mg/L PS-NPs treatment groups, respectively, compared to the control group.(Tu et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) Similar findings have been observed in the expression of the immune function genes in mussels and oysters.(D\u0026eacute;tr\u0026eacute;e and Gallardo-Esc\u0026aacute;rate, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Gardon et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2020\u003c/span\u003e)\u003c/p\u003e\u003cp\u003eCurrently, researches predominantly examine the effects of short-term exposure to a single type of MPs on the physiology and reproduction of \u003cem\u003eDrosophila\u003c/em\u003e, and the underlying mechanism is still insufficient. This study pioneers the investigation of long-term and mixed MPs exposure effects on \u003cem\u003eDrosophila\u0026rsquo;s\u003c/em\u003e lifespan and the related mechanisms, integrating transcriptome and metabolome analyses. We have selected PE and PS MPs for this investigation based on the ubiquitous use of PS and PE, their frequent detection in environment and human body, and their prevalence in studies reflecting a distribution of PS\u0026thinsp;\u0026gt;\u0026thinsp;PE\u0026thinsp;\u0026gt;\u0026thinsp;PET ≌ PVC. (K\u0026ouml;gel et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) We hypothesized that irregularly sized and shaped PE and PS MPs can impair the intestinal barrier, and long-term exposure to high concentration PE and PS MPs mixture may affect gene expression and metabolism activity, consequently impacting the lifespan and related phenotypes of \u003cem\u003eDrosophila\u003c/em\u003e. The outcomes are expected to bolster our capacity to evaluate the risks posed by MPs.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Obtention and characterization of PE and PS MPs\u003c/h2\u003e\u003cp\u003eSolid powder of PE and PS with a mesh size of 2000 were purchased from China Petroleum and Chemical Corporation Limited. The morphology of PE and PS MPs were characterized by Scanning Electron Microscope (SEM, Tescan MAGNA, The Czech Republic) and the SEM images were analyzed with ImageJ software to determine the average size of MPs. A Raman spectrometer (Renishaw, UK) was used to identify the types of MPs.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 \u003cem\u003eDrosophila\u003c/em\u003e strains and PE and PS exposure\u003c/h2\u003e\u003cp\u003eIn this study, wild type w\u003csup\u003e\u003cem\u003e1118\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eDrosophila\u003c/em\u003e, obtained from Fungene Biotechnology Co., LTD, were cultured on standard corn medium in an incubator with a relative humidity of 60%, a temperature of 25\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C and a light cycle of 12:12 hours.(Tu et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) This medium contained corn flour, sucrose, brown sugar, agar and yeast.\u003c/p\u003e\u003cp\u003eThe study established co-exposure for PE and PS MPs at both low concentrations (10 and 100 mg/L) and high concentrations (10, 20 and 50 g/L) to reflect the multi-pathway, continuous, and accumulative effect of MPs on organisms. To achieve this, a specific, equal mass ratio of mixed PS and PE MPs were added to a solution consisting of 10 ml of pure water and 10 ml of anhydrous ethanol. The mixed MPs solution was sonicated for 30 minutes to ensure uniform distribution. After cooling the medium to 60\u0026deg;C, the MPs solution was thoroughly mixed with 100 mL of standard corn medium, continuously stirred to integrate. A control group was prepared with standard corn medium containing 10 ml water and 10 ml ethanol. The entire experimental procedure followed the methods of a previous study.(Zhong et al., \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2022\u003c/span\u003e)\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 PE and PS uptake\u003c/h2\u003e\u003cp\u003eA TGA/DSC thermogravimetric analyzer (STA449F5, Netzsch, Germany) couple with a mass spectrometer (QMS 403 Quadro, Netzsch, Germany) were used to analyse PE and PS in the whole body of \u003cem\u003eDrosophila\u003c/em\u003e. The body tissue of \u003cem\u003eDrosophila\u003c/em\u003e in control group, 10 mg/L and 10g/L PE and PS treatment group were dried at 60 d\u0026deg;C and ground into a powder. The dried powder samples of 10mg were thermally decomposed in a nitrogen atmosphere (50mL/min). The temperature was increased from 30 to 800\u0026deg;C at 10\u0026deg;C/min. All the TGA analyses were blank curve corrected. The mass spectrometer detector, operated in Single Ion Monitoring Mode (SIM), measured the ions m/z 56 for PE and m/z 104 for PS.\u003c/p\u003e\u003cp\u003eTo assess the ingestion and distribution of PE and PS MPs in \u003cem\u003eDrosophila\u003c/em\u003e, we conducted Raman spectroscopic analysis on sliced tissue. The specimens were fixed in 4% paraformaldehyde for over 24 hours, dehydrated through a graded alcohol series, infiltrated with wax, embedded in paraffin blocks, and sectioned to 4 \u0026micro;m thickness. The sections were smoothed in 40\u0026deg;C warm water, then mounted on slides, dehydrated at 60\u0026deg;C to remove moisture and wax, and stored at room temperature for analysis.(Urbisz et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) Using a Renishaw in Via Micro-Raman spectrometer (λ\u0026thinsp;=\u0026thinsp;532 nm), we analyzed the particles' distribution in a 120 \u0026micro;m \u0026times; 120 \u0026micro;m area of the \u003cem\u003eDrosophila\u003c/em\u003e abdomen via Raman mapping, collecting spectral data from 1600 points at 3 \u0026micro;m intervals. Characteristic peaks at 1296 cm⁻\u0026sup1; for PE and 1002 cm⁻\u0026sup1; for PS identified their distribution. ImageJ software determined the average fluorescence intensity in the scanned area.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Measurement of lifespan-related indicators\u003c/h2\u003e\u003cdiv id=\"Sec7\" class=\"Section3\"\u003e\u003ch2\u003e2.4.1 Assessment of lifespan\u003c/h2\u003e\u003cp\u003eFor the longevity test, every freshly hatched \u003cem\u003eDrosophila\u003c/em\u003e was carefully collected and categorized by gender. Each experimental group contained 100 \u003cem\u003eDrosophila\u003c/em\u003e, with three replicates for reliability. The flies were transferred to fresh culture medium every three days to maintain optimal conditions. The dead \u003cem\u003eDrosophila\u003c/em\u003e number was recorded everyday.(Urbisz et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) The median life span was determined by calculating the median survival duration across all flies. The survival rates were analyzed using the log-rank test.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section3\"\u003e\u003ch2\u003e2.4.2 Climbing ability test\u003c/h2\u003e\u003cp\u003eTo evaluate the climbing ability of \u003cem\u003eDrosophila\u003c/em\u003e, 30 individuals were carefully selected from each group after 20 days of cultivation. They were gently tapped and vibrated to encourage descent to the test tube's base. We recorded the number of flies that successfully climbed beyond 13 cm mark within 7 second.(Qiu et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) This test was repeated three times per group, with a 30-minute rest between trials to prevent fatigue from affecting performance.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section3\"\u003e\u003ch2\u003e2.4.3 Measurement of adult body weight and length\u003c/h2\u003e\u003cp\u003eAdult \u003cem\u003eDrosophila\u003c/em\u003e, 20-day post-emergence, were carefully collected and placed onto filter paper for the measurement of their weight and length.(Krittika and Yadav, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) The experiments were conducted in triplicate to ensure accuracy, with the average values being reported for each parameter.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section3\"\u003e\u003ch2\u003e2.4.4 Evaluation of anti-starvation ability\u003c/h2\u003e\u003cp\u003eFor the anti-starvation ability test, 20-day-old \u003cem\u003eDrosophila\u003c/em\u003e of both genders were introduced into a 0.9% agarose medium, which provided hydration without nutritional content.(Zhang et al., \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2020a\u003c/span\u003e) The daily mortality rate was meticulously recorded throughout the experiment. Each replicate involved 100 flies, and the experiment was repeated three times to ensure reliable results.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section3\"\u003e\u003ch2\u003e2.4.5 Assessment of intestinal barrier function\u003c/h2\u003e\u003cp\u003eFollowing a 20-day exposure, both male and female \u003cem\u003eDrosophila\u003c/em\u003e were transferred to a medium containing 2.5% brilliant blue (Erioglaucine disodium salt) to evaluate intestinal barriers integrity. They were divided equally among 10 tubes per group, each containing 10 flies. After 5 days, the percentage of flies exhibiting a blue coloration for each group, referred to as \"Smurfs,\" was determined as an intestinal permeability indicator.(Rera et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2011\u003c/span\u003e)\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Metabolomics Analysis\u003c/h2\u003e\u003cp\u003eFor the metabolomics study, whole-body \u003cem\u003eDrosophila\u003c/em\u003e tissue in control and PE\u0026thinsp;+\u0026thinsp;PS group after 20 days feeding were analyzed with triplicates for each group. Samples were extracted with 50% methanol, and analyzed via UPLC-MS on a Vanquish Flex UPLC system (Thermo Fisher Scientific, Bremen, Germany) utilizing an ACQUITY UPLC T3 column (100 mm \u0026times; 2.1 mm, 1.8 \u0026micro;m, Waters, Milford, USA) for reversed-phase separations at a controlled oven temperature of 35\u0026deg;C. The mobile phase consisted of solvent A (water, 0.1% formic acid) and solvent B (Acetonitrile, 0.1% formic acid). The flow rate was 0.4 ml/min. Metabolites were identified using a Q-Exactive mass spectrometer (Thermo Scientific) and annotated against Kyoto Encyclopedia of Genes and Genomes (KEGG) and Human Metabolome Database (HMDB) databases with a 10 ppm mass tolerance. Data was preprocessed with XCMS software and analyzed by PCA, t-tests, and PLS-DA, identifying differential metabolites based on VIP, P value, and fold change. We followed established protocols from LC-Bio Technology CO., Ltd (Hangzhou, China) for metabolomic analyses. More details are fully described in the supplementary material.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e2.6 Transcriptomic analysis\u003c/h2\u003e\u003cp\u003eTotal RNA was extracted from the whole-body tissue of \u003cem\u003eDrosophila\u003c/em\u003e in control and PE\u0026thinsp;+\u0026thinsp;PS group after 20 days feeding, using the trizol reagent (Thermo Fisher, 15596018). The quality and quantity of the extracted RNA were further validated using a Bioanalyzer 2100 and RNA 6000 Nano LabChip Kit (Agilent, CA, USA, 5067\u0026thinsp;\u0026minus;\u0026thinsp;1511). High-quality RNA, with RNA Integrity Number value exceeding 7.0, was selected for sequencing library construction. mRNA was purified using Dynabeads Oligo (Thermo Fisher, CA, USA) and sequenced on an Illumina NovaseqTM 6000 platform. Transcript expression levels were quantified using StringTie and ballgown package, with DEGs identified through DESeq2 and edgeR software, classified by false discovery rate (FDR)\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and fold change\u0026thinsp;\u0026ge;\u0026thinsp;2. These DEGs were analyzed for Gene Ontology (GO) and KEGG pathways enrichment. Transcriptomic analyses also followed the established protocols from LC-Bio Technology CO., Ltd (Hangzhou, China), as shown in the Supporting Material.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e2.7 Correlation analysis of transcriptomic and metabolomic\u003c/h2\u003e\u003cp\u003eWe conducted a correlation analysis utilizing data from both transcriptomic and metabolomic profiles to investigate the relationship between DEGs and identified metabolites. A signed clustering correlation heat map was generated using the OmicStudio platform.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e2.8 Statistical analyses\u003c/h2\u003e\u003cp\u003eStatistical analyses were performed using GraphPad Prism software, version 7. The lifespan and starvation survivorship data were analyzed using log-rank assays. Differences among groups were assessed employing one-way ANOVA, followed by Turkeys' post hoc test. A \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered significant for all statistical analyses.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results and Discussion","content":"\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e3.1 Characterization and internalization of PE and PS MPs\u003c/h2\u003e\u003cp\u003eSEM images demonstrated a uniform dispersion and irregular morphology of PE MPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA), with an average particle size of 14.55\u0026thinsp;\u0026plusmn;\u0026thinsp;5.97 \u0026micro;m (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). While PS MPs displayed a more regular shape (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB) and a notably smaller average size of 1.86\u0026thinsp;\u0026plusmn;\u0026thinsp;0.89 \u0026micro;m (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Raman spectroscopy confirmed their distinct chemical identities by analyzing the unique spectral peaks for PE and PS standard substance (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe TGA-MS analysis (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) demonstrated distinct peak intensities at m/z 56 for PE and at m/z 104 for PS in their respective standard substances. Notably, the signal intensity for both PE and PS in flies cultured in a 10 g/L mixture of PE and PS for 50 days showed a modest increase, while the flies cultured in a 10 mg/L PE and PS exhibited no significant difference from the control group.\u003c/p\u003e\u003cp\u003eTo confirm the internalization of MPS, Raman spectroscopy was used for high concentration group. In the randomly selected scanning regions (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA to \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC), the size and brightness of Raman signal spot increased with exposure concentration at the characteristic peaks of 1296 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for PE and 1002 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for PS (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD to \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI). ImageJ analysis revealed that the average fluorescence intensity for PE across the control, 10 g/L and 50 g/L group were 101.12, 159.45 and 163.23, respectively. For PS, the intensities were 88.39, 156.78 and 171.61, respectively. These results showed that both PE and PS MPs were ingested by \u003cem\u003eDrosophila\u003c/em\u003e, with ingestion levels rising in tandem with exposure concentration.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e3.2 Effects of PE and PS MPs on lifespan of \u003cem\u003eDrosophila\u003c/em\u003e\u003c/h2\u003e\u003cp\u003eLong-term exposure diet enriched with high levels of PE and PS MPs substantially reduced the life span of \u003cem\u003eDrosophila\u003c/em\u003e. Notably, when subjected to the MPs mixture at concentrations of 10, 20 and 50 g/L, the median lifespan of female flies decreased to 32.67\u0026thinsp;\u0026plusmn;\u0026thinsp;2.31, 27.33\u0026thinsp;\u0026plusmn;\u0026thinsp;2.08, and 19.33\u0026thinsp;\u0026plusmn;\u0026thinsp;3.88 days, respectively, compared with the 51.32\u0026thinsp;\u0026plusmn;\u0026thinsp;1.00 days in control group. In contrast, exposure to low concentrations of PE and PS MPs at 10 and 100 mg/L did not significantly alter the lifespan of \u003cem\u003eDrosophila\u003c/em\u003e with median lifespans of 46.94\u0026thinsp;\u0026plusmn;\u0026thinsp;2.06 and 48.23\u0026thinsp;\u0026plusmn;\u0026thinsp;1.28 days, respectively. A comparable pattern was noted in male flies, with median lifespans ranging from 46.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.73 to 23.67\u0026thinsp;\u0026plusmn;\u0026thinsp;2.31 days as the concentration of MPs increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA to \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Sex-specific response to MPs was apparent at lower concentrations with females showing a higher survival rate compared to males at equivalent lifespan stages. This result is consistent with the research from Urbisz et al.(Urbisz et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) It is recognized that \u003cem\u003eDrosophila\u003c/em\u003e, along with many other insects, exhibit sex-specific responses to a variety of physiological, environmental and ecological stressors.(Parkash and Ranga, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Shen et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) However, at higher concentrations of 20 and 50 g/L, these sex differences were unconspicuous. Liang et al. investigated effects of long-term exposure to PET MPs (2 \u0026micro;m) on \u003cem\u003eDrosophila\u0026rsquo;s\u003c/em\u003e lifespan at the concentration of 1, 10 and 20 g/L, results presented that 10 g/L PET-MPs caused 20.42% decrease in female mean lifespan, and 20 g/L PET MPs caused 16.01% decrease in male mean lifespan. Co-exposure to PE and PS has a more pronounced impact on lifespan than exposure to PET alone, which may be related to the type of MPs, because PET is more bioinert as it is often used in food packaging.(Lee et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2022\u003c/span\u003e)\u003c/p\u003e\u003cp\u003eConsidering the minimal impact of low-concentration (10 and 100 mg/L) PE and PS MPs co-exposure on the lifespan of \u003cem\u003eDrosophila\u003c/em\u003e, further investigation was conducted to explore the effects of mixed PE and PS MPs exposure on lifespan-related phenotypes within the high concentration group (10, 20 and 50 g/L).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003e3.3 Effects of PE and PS MPs on age-related phenotypes of \u003cem\u003eDrosophila\u003c/em\u003e\u003c/h2\u003e\u003cp\u003eContinuous exposure to MPs profoundly influenced age-related phenotype characteristics of \u003cem\u003eDrosophila\u003c/em\u003e. Male flies subjected to PS and PE MPs showed a notable reduction in body weight and length, contrasting with the control group, while females displayed an increase (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, B). The climbing assay, a standard measure of \u003cem\u003eDrosophila\u003c/em\u003e's spontaneous activity, revealed a dose-dependent reduction in motor function upon exposure to PS and PE MPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). The study results reported by Zhang et al. also showed that exposure to PS alone could reduce the climbing ability of \u003cem\u003eDrosophila\u003c/em\u003e, and this effect was more serious after combined exposure to Cd.(Zhang et al., \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2020b\u003c/span\u003e) Similarly, the survival curve under starvation conditions indicated a diminished capacity for hunger resistance with increasing MPs concentrations (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE, F), and the anti-starvation ability of females was stronger than that of males. Exposure to PET and PA MPs also had similar results, accompanying with a reduced triglyceride, protein and glucose content in males, indicating that energy metabolism of male \u003cem\u003eDrosophila\u003c/em\u003e was affected by MPs ingestion.(Shen et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Zhong et al., \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) In \u003cem\u003eDrosophila\u003c/em\u003e, lifespan reduction is often associated with intestinal epithelial barrier dysfunction.(Guo et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) The Smurf assay corroborated this, demonstrating a decline in gut barrier integrity post-exposure to PS and PE MPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD), which was consistent with the previous study.(Zhang et al., \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2020b\u003c/span\u003e)\u003c/p\u003e\u003cp\u003eBased on our findings, escalating exposure to PS and PE MPs induced a series of dose-dependent reduction in \u003cem\u003eDrosophila\u003c/em\u003e lifespan and manifestation of age-related phenotypes. To maximize the identification of DEGs and metabolites between the treatment and control, as well as between genders, this study concentrated on analyzing the effects of the highest PE and PS concentration on \u003cem\u003eDrosophila\u003c/em\u003e's transcriptome and metabolome profile.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003e3.4 Effects of PE and PS MPs on global metabolomics profiles in the \u003cem\u003eDrosophila\u003c/em\u003e\u003c/h2\u003e\u003cp\u003eThe PCA scores plot, with QC samples tightly clustered as purple dots, demonstrated a high stability and reliability of our metabolomics platform (Supporting Material Fig. S2). PLS-DA analysis distinctly segregated the two groups of samples (Fig. S3 A and C). R2Y and Q2 values exceeding the threshold of 0.5 indicated the model's goodness of fit and predictive ability, with specific values of 0.998 and 0.733 for females, and 0.999 and 0.853 for males, respectively. The comparison of R2Y and Q2 values in Fig. S3 B and D, illustrating lower values on the left side versus higher on the right, confirmed the model's comprehensive explanatory and robust predictive capabilities for the observed sample variations.\u003c/p\u003e\u003cp\u003eMetabonomic analysis revealed significant metabolic disparities between the control and PE\u0026thinsp;+\u0026thinsp;PS groups, identifying 24 distinct metabolites in males and 7 in females, as depicted in the clustering heat maps Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e and Supporting Material Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. In males, exposure to PE and PS MPs led to significant changes in organic acids and derivatives, organic heterocyclic compounds, lipid molecules, phenylpropanoids and polyketides, organic oxygen compounds, nucleosides, nucleotides and analogues. Among these, Lysophosphatidylcholine (Lyso PC 18:0, LPC), a glycerophospholipid and lipid-like molecule, is recognized for its anti-inflammatory properties.(Hung et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2011\u003c/span\u003e) LPC containing docosahexaenoic acid at the sn-1 position and the 18:0 LPC can inhibit inflammatory mediator release, such as TNF-α, IL-6, IL-α, IL-1β, and IL-10.(Huang et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Yan et al., \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2004\u003c/span\u003e) Furthermore, the balance between pro-inflammatory cytokines and anti-inflammatory cytokines is crucial for delaying aging. In females, exposure to PE and PS MPs induced significant alterations in organic heterocyclic compounds, lipid molecules, organic acids and derivatives, as well as nucleosides, nucleotides and analogues. Notably, glutamine, categorized under organic acids and derivative, is intricately linked to aging process due to its vital role for cell proliferation and survival.(Meynial-Denis, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Salabei et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) Glutamine serves as a precursor for essential molecules including amino acids and nucleotide. It is instrumental in the synthesis of nicotinamide adenine dinucleotide phosphate (NADPH), glutathione (GSH) and adenosine triphosphate (ATP), which are crucial for redox homeostasis and energy supply.(Xiao et al., \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) Meanwhile, aging is commonly characterized by energy generation reduction(Giezenaar et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) and redox homeostasis dysfunction.(Meng et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) Therefore, changes of LPE, LPC, L-Glutathione and glutamine maybe one reason of aging through affecting the energy metabolism.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eEnrichment analysis visualized via bubble diagram revealed distinct KEGG pathways of metabolites enrichments in male and female flies. In males, the most significantly enriched pathways included ABC transporters, alanine, aspartate and glutamate metabolism, and purine metabolism (Fig.\u0026nbsp;6A). Females showed predominant enrichment in ABC transporters, choline metabolism in cancer, and metabolism of alanine, aspartate, glutamate, cysteine and methionine (Fig.\u0026nbsp;6B). Notably, ABC transporters were the most enriched pathway in both genders, highlighting their crucial role in insect physiology.(Huang et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) This pathway involves a family of integral membrane proteins essential for the transmembrane transport of various molecules.(Campos et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) \u003cb\u003eFig.\u0026nbsp;6\u003c/b\u003e Metabolic pathways analysis of PS\u0026thinsp;+\u0026thinsp;PE exposure on \u003cem\u003eDrosophila\u003c/em\u003e for the male flies (A) and female flies (B). The top 10 pathways of significant of the up-regulated and down-regulated DEMs on KEGG. The X-axis was the rich factor, the Y-axis represents the name of the pathway. The bubbles size represents the number of differential expression metabolites involved. The bubbles color indicates the enrichment degree of pathway.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003e3.5 Effects of PE and PS MPs on global transcriptomics profiles in the \u003cem\u003eDrosophila\u003c/em\u003e\u003c/h2\u003e\u003cp\u003eIn our study, we detected significant changes in gene expression profiles, with a total of 286 DEGs in males and 205 DEGs in females, when comparing the PE\u0026thinsp;+\u0026thinsp;PS exposure group to the control group. Notably, the majority of these DEGs were implicated in the regulation of energy metabolism (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Phosphoenopyruvate carboxykinase (PEPCK), a pivotal regulatory enzyme in gluconeogenesis, glyceroneogenesis, serine synthesis, and amino acid metabolism, is stringently regulated at the transcriptional level.(Xiong et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2011\u003c/span\u003e) UDP-glycosyltransferases (UGTs), a widespread superfamily of enzymes, mediate biotransformation and play crucial roles in detoxifying harmful exogenous chemicals and modulating signaling molecules.(Ram\u0026iacute;rez et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) The immune response to MPs may exacerbate energy constraints, diverting energy from growth and reproduction to maintenance, which includes energetically costly cellular processes.(Olsen et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) These metabolic and immune disruptions caused by MPs could increase the susceptibility of organism to further stress from pathogens, other pollutants, and environmental factors, potentially affecting lifespan. Heat shock proteins (Hsps) are crucial in regulating protein structure and folding.(Kim et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2007\u003c/span\u003e) They essential for \u003cem\u003eDrosophila\u003c/em\u003e's stress resistance, including thermal and environmental stressors.(Feder and Krebs, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e1997\u003c/span\u003e) Hsp70, a predominant Hsp family member, serves as a sensitive bioindicator for environmental monitoring due to its role in cellular defense mechanisms under pollutant stress.(Liu et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) In this study, a significant upregulation of the Hsp70 gene in female flies was observed after exposure to PS and PE MPs, indicating that such exposure triggers a stress response. This upregulation is likely a compensatory mechanism to mitigate the stress induced by PE and PS MPs. This is consistent with observations that female \u003cem\u003eDrosophila\u003c/em\u003e exhibit a greater climbing ability, a enhanced hunger resistance, and a higher survival rate at 20 days compared to male flies. Our findings align with those of Brandts et al., who noted an increase in Hsp70 gene expression in mussels' digestive glands exposed to PS nanoplastic.(Brandts et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) Given proteotoxicity's role in aging, Hsps may enhance \u003cem\u003eDrosophila\u003c/em\u003e's stress resistance by mediating protein refolding or degradation.(Hagymasi et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2022\u003c/span\u003e)\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThese DEGs were categorized across 201 biological processes, 70 cellular components, and 138 molecular functions (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e8\u003c/span\u003e). In male flies, KEGG enrichment analysis revealed interference in the \u0026ldquo;Toll and Imd signaling pathways\u0026rdquo; after PE\u0026thinsp;+\u0026thinsp;PS co-exposure, along with perturbations in Lysosome, Neuroactive ligand-receptor interaction, and Drug metabolism - cytochrome P450 pathways. Female flies displayed a distinct enrichment in pathways such as Insect hormone biosynthesis, Longevity regulating pathways - multiple species, Spliceosome, and Autophagy-animal. Network analysis of these pathways indicated that some genes participate in multiple signaling pathways simultaneously (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e8\u003c/span\u003e). Notably, in \u003cem\u003eDrosophila\u003c/em\u003e, humoral immunity is primarily mediated by the \u0026ldquo;Toll and Imd signaling pathway\u0026rdquo;.(Yu et al., \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) The Toll pathway, activated in response to systemic infections, plays a key role in adipose tissue growth, metabolism, and hemocyte activation.(Myllym\u0026auml;ki et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) The Imd pathway is essential for bacterial defense,(Mussabekova et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) controlling the expression of most antimicrobial peptides, highlighting its role in immune homeostasis.(Lemaitre et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e1995\u003c/span\u003e) The intestinal epithelium is a recognized immunoactive tissue, playing a key role in longevity regulation.(Franceschi et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) Microplastics can induce an immune response through mechanical damage to the intestinal epithelium, while excessive immune activation can hasten degeneration, cause inflammation, and reduce lifespan.(Guo et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) The Smurf assay results provided strong confirmation of this association.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003e3.6 Combined analysis of transcriptomics and metabolomics\u003c/h2\u003e\u003cp\u003eCombined analysis has revealed the involvement of multiple DEGs and metabolites in energy metabolism pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e9\u003c/span\u003e). A pronounced interconnection between immune and metabolic processes is particularly evident in the gut, where intestinal cells collaborate to prevent microbial invasion.(Miguel-Aliaga et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) Studies on model organisms have shown that diet significantly influences aging, with nutritional status being closely associated with the incidence of age-related diseases. Cellular senescence represents both a cause and a consequence of metabolic dysregulation, and disruptions in macronutrient metabolism can influence aging and longevity by impacting the onset and characteristics of the senescent state.(Nehme et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) Our study detected a dose-dependent decline in gut barrier function in \u003cem\u003eDrosophila\u003c/em\u003e after exposure to PE and PS MPs. Transcriptomics analysis also revealed a significant disruption in \u003cem\u003eDrosophila\u0026rsquo;s\u003c/em\u003e immune defense function after mixed microplastics exposure. Therefore, we propose that PE and PS co-exposure impairs \u003cem\u003eDrosophila\u0026rsquo;s\u003c/em\u003e gut barrier function, triggering immune stress, disrupting energy metabolism, and ultimately leading to a shortened lifespan in \u003cem\u003eDrosophila\u003c/em\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Conclusions","content":"\u003cp\u003eThis study presents the first comprehensive examination of the molecular-level toxicity resulting from co-exposure to PE and PS MPs in terrestrial invertebrates. Our findings show that chronic exposure to PE and PS MPs with very high concentration profoundly affects the lifespan and several phenotypic biomarkers of \u003cem\u003eDrosophila\u003c/em\u003e, encompassing climbing ability, stress resistance to hunger, and intestinal barrier function. Following co-exposure to PS and PE MPs, we observed DEGs and altered metabolites in \u003cem\u003eDrosophila\u003c/em\u003e, predominantly associated with immune function and energy metabolism. These insights significantly enhance our comprehension of the molecular mechanisms that underlie the physiological and behavioral impairments observed in \u003cem\u003eDrosophila\u003c/em\u003e when subjected to MPs exposure.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eCompeting Interests\u003c/h2\u003e\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eAuthor contributions\u003c/h2\u003e\u003cp\u003eYingyu Liu: Data curation, Investigation. Cheng Wang: Formal analysis. Caixia Wang: Writing- Original draft. Longhuan Fu: Investigation. Yunbo Zhang: Writing- Reviewing and Editing. Zhuo Gao: Visualization. Zhugen Yang: Writing- Reviewing and Editing. Fanyu Meng: Conceptualization, Methodology, Supervision.\u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h2\u003e\u003cp\u003eThe authors gratefully acknowledge funding from Project LH2021E097 supported by the Natural Science Foundation of Heilongjiang Province. ZY thanks UKRI NERC Fellowship (NE/R013349/2) and The Leverhulme Trust Research Leaderships Awards (RL-2022-041).\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eData will be made available on request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAlaraby, M., Abass, D., Farre, M., Hern\u0026aacute;ndez, A. and Marcos, R. 2024. Are bioplastics safe? Hazardous effects of polylactic acid (PLA) nanoplastics in Drosophila. Sci Total Environ 919, 170592.\u003c/li\u003e\n\u003cli\u003eBhat, R.A.H., Sidiq, M.J. and Altinok, I. 2024. Impact of microplastics and nanoplastics on fish health and reproduction. 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Environ Res 237(Pt 1), 116966.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"microplastics co-exposure, lifespan, age-related phenotypes, Drosophila, Multi-omics","lastPublishedDoi":"10.21203/rs.3.rs-7459263/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7459263/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMicroplastics (MPs) are ubiquitous global contaminants, posing a long-term exposure risk to both the entire ecosystem and human health. Although increasing researches have indicated that individual MPs generally exhibit biotoxicity, the combined effects of multiple MPs exposure on biological lifespan and the mechanisms involved remain largely unrevealed. Here we employed \u003cem\u003eDrosophila melanogaster\u003c/em\u003e, subsequently referred to \u003cem\u003eDrosophila\u003c/em\u003e, as a biological model to investigate the impact of polyethylene (PE, irregular shape, 14.55\u0026thinsp;\u0026plusmn;\u0026thinsp;5.98 \u0026micro;m) and polystyrene (PS, sphere, 1.86\u0026thinsp;\u0026plusmn;\u0026thinsp;0.89 \u0026micro;m) microplastics co-exposure on lifespan at both low concentrations (10 and 100 mg/L) and high concentrations (10, 20 and 50 g/L). Furthermore, we delved into the underlying mechanism through metabolomics and transcriptomics analysis. Our results demonstrated PE and PS MPs co-exposure with greatly high concentrations significantly reduced the lifespan of \u003cem\u003eDrosophila\u003c/em\u003e and influenced age-related phenotypes such as climbing ability, intestinal barrier and hunger resistance. We found that differential metabolites were engaged in various metabolic pathways, including ABC transporters, alanine, aspartate and glutamate metabolism. Differentially expressed genes (DEGs) were closely related to Toll and Imd signaling pathway and Longevity regulating pathway. A combined metabolomics and transcriptomics analysis revealed that PE and PS MPs co-exposure induced alterations in gene expression and metabolites related to the immune system and energy metabolism, thereby affecting \u003cem\u003eDrosophila\u003c/em\u003e lifespan. The findings provided a mechanistic understanding for the effects of PE and PS MPs co-exposure on \u003cem\u003eDrosophila\u0026rsquo;s\u003c/em\u003e lifespan.\u003c/p\u003e","manuscriptTitle":"Multi-omics Analysis Uncovers Lifespan Effects of Polyethylene and Polystyrene Microplastics Coexposure in Drosophila melanogaster","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-09 05:24:59","doi":"10.21203/rs.3.rs-7459263/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b0b98d84-fbbe-40a0-868c-67fc0f263ea1","owner":[],"postedDate":"September 9th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-09-10T13:46:14+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-09 05:24:59","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7459263","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7459263","identity":"rs-7459263","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}