Chromatin-bound RNAs are the primary product of transcription that undergo on-chromatin processing such as capping, splicing, and polyadenylation. These processing steps then determine the fate of RNAs. Albeit its vital importance, a simple and robust method for isolating different fractions of chromatin-bound RNAs is missing in plants.A. thaliana. The chromatin-bound RNAs isolation is based on the 1 M UREA wash that removes the majority of non-chromatin-associated proteins from the nucleus, as previously developed in mammalian cells. On-demand, the isolated chromatin-bound RNAs can be either used directly for gene-specific analysis or subject to further rRNA removal and also the optional polyadenylated RNA removal, followed by high-throughput sequencing. Detailed protocols for these procedures are also provided. Comparison of sequencing results of chromatin-bound RNAs with and without polyadenylated RNA removal revealed that a small fraction of CB-RNAs is polyadenylated but not yet fully spliced, representing RNA-processing intermediate on-chromatin.Here, we describe our updated method and the associated step-by-step protocol for chromatin-bound RNAs isolation in This optimized chromatin-bound RNAs purification method is simple and robust and can be used to study transcription and its-coupled RNA processing in plants.The online version contains supplementary material available at 10.1186/s13007-022-00967-y. As sessile organisms, plants perceive the fluctuant environmental stimuli and adjust their internal gene expression constantly to be able to adapt to the local conditions. To a certain extent, plants’ phenotypic plasticity is determined at the transcriptional level. However, transcription is a rather dynamic process; the steady-state RNA levels are an outcome of transcription initiation, productive elongation, and termination, followed by RNA processing and decay. Although conventional mRNA-seq provided a robust way to convey transcription control, it is only by directly tracking newly synthesized RNA that the instant changes in plant responses to developmental, environmental stimuli, and disease can be revealed.Over the past decades, a series of high-throughput sequencing methods have been developed for investigating nascent RNA molecules from the pool of cellular RNA . AccordiSince 2015, the GRO-seq method has been modified and adapted to profile the nascent transcripts from Pol II and Pol V in different plant species –15. Pol FLC in Arabidopsis [FLC intron1 revealed autonomous pathway components such as FCA and FLD functions to coordinately repress transcription initiation and elongation [A. thaliana [We have initially adapted the CB-RNAs isolation method for the study of bidopsis . Combineongation . Recentlthaliana , 19. Sinthaliana –22. Compthaliana . Albeit Arabidopsis thaliana seedlings. Combined with quantitative PCR or next-generation sequencing, the isolated CB-RNAs is suitable for analyzing the co-transcriptional intron splicing status both at the single gene and genome-wide level. For a given locus, the CB-RNA reflects the sum of elongating RNA (RNAe: RNAs being transcribed at the locus) and full-length polyadenylated RNA (RNAf: RNAs have been fully transcribed and polyadenylated but not yet released from chromatin). Hence, we also provided a step-by-step method to separate those two CB-RNA fractions and analyzed the corresponding distinct features. Our data showed that polyadenylated RNA likely represents the intermediate of on-chromatin RNA processing and accounts for only a small fraction of CB-RNAs. We thus propose a guideline on when the rRNA and or polyadenylated RNA needs to be removed before downstream analysis. Our work provided a simple and robust CB-RNAs isolation method in Arabidopsis, which in principle can be easily adapted to be used in a wide range of plant species.Here, we describe our up-to-date method for chromatin-bound RNAs isolation from After transcription initiation, RNA polymerase II (Pol II) forms a stable complex with DNA template which resists nonionic detergent and urea. Taking advantage of this feature, Pol II associated nascent RNAs were successfully isolated from the free cytoplasmic and nucleoplasmic RNAs in different model systems, including mammals, Drosophila, and yeast. Based on these pre-existing knowledge, we describe a method suitable for CB-RNAs extraction in Arabidopsis and likely other plant species such as soybean. The extraction procedures mainly involve a nuclei extraction step followed by two stringent wash steps with a buffer containing urea Fig. A. The reCB-RNAs isolation captures all the chromatin-associated RNA species, including the nascent transcripts containing introns that are going to be, but not yet spliced out. This allows for studying co-transcriptional processes, such as co-transcriptional intron splicing (CTS) at a single gene or in the whole genome. Here, to evaluate the enrichment of nascent RNA in different extraction fraction, the ratio of unspliced RNA to spliced RNA of a specific intron at 5 randomly selected genes were estimated Fig. . Primer To investigate the intron splicing status on a genome-wide scale, we performed CB-RNA-seq. As shown in Fig. We first analyzed the sequencing reads distribution along genes of CB-RNA-seq data with only the rRNA depleted but with polyadenylated RNA kept (RNAe + RNAf). Compared with mRNA-seq data, two distinct features were observed in CB-RNA-seq data. Firstly, the read density of CB-RNA-seq declined gradually from 5ʹ to 3ʹ end, while no such pattern was observed in mRNA-seq Fig. A and B. Next, we wondered about the relationship between RNAe and RNAf in the isolated CB-RNAs. Thus, we analyzed and compared the sequencing results obtained from RNAe + RNAf, RNAe, and RNAf Figs.  and 5A. Since multiple factors could also affect CTS efficiency, such as the intron positions at a given gene and intron numbers, we looked at how CTS efficiency correlates with the order of intron within genes. We extracted genes with the same intron numbers and analyzed the corresponding 3ʹSS and 5ʹSS at each individual intron from RNAe, RNAf, and RNAe + RNAf Fig. : Fig. S1Next, as intron numbers affect splicing efficiency , we plotA. thaliana seedlings. Given the unique advantages of this approach, the CB-RNA extraction method is considered the most straightforward way for nascent RNA isolation. Firstly, the procedure is relatively simple, time-saving, and can be done with limited materials (One gram of seedlings are sufficient for most of applications). The entire CB-RNA isolation procedure can be done within 2–3 h from raw materials to purified RNAs. Secondly, the method is more economical compared with other parallel methods such as pNET-seq and GRO-seq given that CB-RNAs isolation does not involve immunoprecipitation steps that are time-consuming and expensive. Thirdly, once the CB-RNAs is obtained, it can be handled the same way as the total RNA, which is compatible with all sorts of downstream applications or sequencing library construction strategies. For example, the isolated CB-RNAs can be combined with specific downstream methods to assay 3’ end processing, RNA methylation, RNA editing, anti-sense RNA, circular RNA, and so on at the chromatin level. CB-RNA isolation method should be applicable in a wide range of plant species as illustrated successfully in Soybean [Nascent RNA sequencing has been widely used to study the co-transcriptional regulation in many species. However, isolating the high-quality nascent RNAs from plant tissue is still challenging due to the presence of cell walls and other reasons. Several methods, such as CB-RNA-seq, pNET-seq, and GRO-seq, have been developed recently to detect nascent RNA and reveal plant-specific transcriptional features. In this study, we provided a detailed protocol for isolation of the chromatin-bound RNA from  Soybean . HoweverThere are also limitations for CB-RNAs isolation and sequencing. Firstly, our current protocol only recovers CB-RNAs above 200nt. This is due to the column purification of CB-RNA (with 200nt cut-off for the Qiagen RNA miniprep kit) and also the size selection in the Illumina RNA-seq library generation step. Thus, caution and adjustment of the protocol will be needed when the nascent RNAs below 200nt are of interest. Secondly, in association with the first point, the CB-RNAs is suitable for studying nascent RNA and its associated RNA-processing steps but less suitable for looking at the Pol II occupancy. For example, Pol II pauses at both 5ʹ and 3ʹ end of the gene in Arabidopsis , while tOur data showed that a small fraction of full-length polyadenylated RNA (RNAf) is included in the isolated CB-RNAs Fig. D. The inWe described a simple and robust method to enrich chromatin-bound RNAs that include nascent RNA undergone elongation by Pol II and polyadenylated RNA that has undergone RNA processing on chromatin. The isolated chromatin-bound RNAs are compatible with all sorts of downstream applications, such as the studying of co-transcriptional RNA-processing combined with next-generation sequencing.Arabidopsis thaliana (Col-0) seeds were sterilized in 10% sodium hypochlorite for 10 min and rinsed by sterile distilled water 3 to 4 times before being sown on 1/2 MS media. Seeds were stratified at 4 °C for 3 days and transferred to a growth chamber , 16 h light/8 h dark, 22 °C. 10-day-old seedlings were collected and snap-frozen in liquid N2 for the following chromatin-bound RNAs (CB-RNAs) extraction.Note that RNase-free chemicals and solution are critical for CB-RNA isolation.2 (Invitrogen AM9530G), Triton X-100 (Sigma T8787), DTT , Proteinase inhibitor cocktail (Roche 11873580001), Glycerol (Invitrogen 15514-011), 1 M EDTA (Invitrogen 15575-038), 1 M Tris–HCl pH 7.5 (Invitrogen 15567-027), 5 M NaCl (Invitrogen AM9759), Urea (Sigma U5378), Tween-20 (Sigma P7949), Chloroform , Ethanol , Isopropyl alcohol , Nuclease-free H2O (Invitrogen 10977-015).Sucrose (Sigma S0389), Ficoll (Sigma F4375), Dextran T40 (Sigma 31389), HEPES (Sigma 54457), MgClYeast-tRNA (Roche 10109525001), RNase inhibitor (Promega N251A), RNaseZap (Invitrogen AM9780), TRIzol Reagent (Invitrogen 15596018), 3 M Sodium Acetate pH5.5 (Invitrogen AM9740), Perfect Start Green qPCR SuperMix (TransGene AQ601-04), RNeasy RNA purification Kit (Qiagen 74104), mRNA Capture Beads (Vazyme N401), Qubit RNA Assay Kit (Qubit Q32852), RNA secure Reagent (Ambion AM7006), Turbo DNase (Invitrogen AM2238), riboPOOLs  (siTOOLs Biotech), Streptavidin magpoly beads (SMART lifesciences SM01710), Low Melting Point Agarose (Invitrogen 16520-050), 10 × TBE Buffer (Invitrogen 15581-044), DNA size Marker , NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB E7760).Phase-lock tube (Invitrogen A33248), 0.45 μm sterile filter ; 0.45 μm nylon membrane , Miracloth (Millipore 475855), 50 mL Plastic Funnel, Nuclease-free 1.5 mL, 2 mL and 0.2 mL microcentrifuge tubes; Nuclease-free 15 mL and 50 mL falcon tubes; Nuclease-free pipette tips.anti-H3 (Abcam 1791), anti-Ser2P CTD Pol II (Active Motif 61083), anti-UBC .For CB-RNA isolation:2, 0.5% Triton X-100, 2 mM DTT freshly added, 1 × cocktail freshly added (100 × cocktail stock solution: 2 tablet/ml Nuclease-free H2O), 1 × tRNA freshly added (100 × tRNA stock solution: 10 μg/μL), 1 μl RNase inhibitor was added in every 100 μL buffer. Adjust pH with 1 M KOH to 7.5. The solution is filtered through 0.45 μm PES membrane and can be kept at − 20 °C for up to 3 months.0.44 M sucrose, 1.25% (wt/vol) Ficoll, 2.5% (wt/vol) Dextran, 20 mM HEPES pH 7.4, 10 mM MgCl50% Glycerol, 0.5 mM EDTA, 25 mM Tris–HCl pH 7.5, 100 mM NaCl, 2 mM DTT freshly added, 5 × tRNA freshly added, 2 × cocktail freshly added, 2 μL/100 μL RNase inhibitor freshly added. Solution is filtered through 0.45 μm nylon membrane and can be kept at − 20 °C for up to 3 months.25 mM Tris–HCl pH 7.5, 300 mM NaCl, 1 M Urea, 0.5 mM EDTA, 1% Tween 20 Solution is filtered through 0.45 μm nylon membrane and can be kept at − 20 °C for up to 3 months.For rRNA removal:10 mM Tris–HCl (pH 7.5); 1 mM EDTA; 2 M NaCl.0.1 M NaOH; 0.05 M NaCl.10 mM Tris–HCl (pH 7.5); 1 mM EDTA; 1 M NaCl.Use RNasezap to clean the bench and pipette before starting.Pre-cool the centrifuge to 4 °C.Prepare working solutions  freshly before use.Given that endogenous RNAase can’t be inactivated thoroughly during the procedure, handling one sample each time is recommended.Take 1 g of Arabidopsis seedlings and grind them in liquid nitrogen to a fine powder.Resuspend the ground powder with 8 mL pre-cooled Honda buffer and homogenize quickly by stirring with a 1 mL tip. Filter the solution through two layers of Miracloth into a new ice-cold 50 mL falcon tube. .g for 5 min.Centrifuge the filtrate immediately at 4 °C, 3500Remove the supernatant, resuspend the pellet immediately with 1 mL pre-cooled Honda buffer, and transfer to a new ice-cold 2 mL tube. .g for 1 min.Centrifuge immediately at 4 °C, 8000Remove the supernatant thoroughly and weigh the pellet.Resuspend the pellet with 1 volume (w/v) of nuclei resuspension buffer. . Stir to mix the pellet and pipet up and down several times with an end-cut 200μL tip. .After homogenization, add 2 volumes of wash buffer into the above mixture, and pipette gently 20 times with an end-cut 1 mL tip to ensure samples are thoroughly mixed. Store the mixture on ice for 1 min.g for 1 min. Remove the supernatant thoroughly. Centrifuge immediately at 4 °C, 8000Resuspend the pellet again with 1 volume of nuclei resuspension buffer. Stir to mix the pellet and pipette gently several times with an end-cut 200 μL tip.After homogenization, immediately add 1 volume of wash buffer to the mixture and pipette up and down 20 times with an end-cut 1 mL tip to ensure samples are thoroughly mixed.Store the mixture on ice for 1 min.g for 2 min. Remove the supernatant thoroughly. Centrifuge immediately at 4 °C, 12,000Resulting chromatin can be snap frozen and stored at − 80 °C.16.Resuspend the pellet from step 15 (Chromatin-Bound fraction), step 8 , step 10 , and step 14  with 1 mL Trizol solution.17.Vortex to mix thoroughly and stay at room temperature for 10 min.18.Add 0.2 mL chloroform, vortex for 10 s, and leave at room temperature for 5 min.19.g for 2 min at room temperature just before use. Transfer the solution from step 18 to a phase-lock tube. Centrifuge at 14,000g, 10 min at 4 °C.Spin the phase-lock tube at 14,00020.Carefully transfer the supernatant into a new tube and add a 0.8 × volume of 100% ethanol.21.g room temperature for 1 min until all the solutions pass through.Rotate to mix and centrifuge briefly at 600 g for a few seconds. Transfer the solution (including precipitation if any) to a Qiagen RNA Column. Centrifuge multiple times at 800022.g for 1 min.Wash the column with 700 μL RW1 buffer, centrifuge at room temperature, 12,00023.g for 1 min. Repeat once.Wash the column with 500 μL RPE buffer, centrifuge at room temperature, 12,00024.g for 2 min.Change to a new 2 mL tube. Centrifuge at 12,00025.2O, stay on bench for 2–5 min, centrifuge 12,000g for 1 min to elute. Repeat once and obtain 100 μL eluted RNAs in total.Change the column to a new 1.5 mL tube. Add 50 μL Nuclease-free H26.Measure the RNA concentration with a Nano-Drop machine.27.RNA sample (2 to 5 μg): 100 μL10 × DNase I buffer: 12 μLRNase Inhibitor (40U/μL): 2 μLTurbo DNase I (2U/μL): 2 μL2O: 4 μL25 × RNA secure HSet up the following Turbo DNase I digestion reaction:28.Incubate at 37 °C for 1 h.29.Add 3.5 volumes of RLT buffer and 2.5 volumes of Ethanol to the sample from the last step and mix well. (Qiagen RNeasy Mini Kit)30.g for 30 s at room temperature. Perform multiple times until all the solutions pass through.Transfer the mixture to a Qiagen RNA Column and centrifuge at 10,00031.g for 30 s at room temp. Repeat once and centrifuge at 14,000g for 2 min.Discard the flowthrough. Add 500 μL RPE buffer to the column and centrifuge at 12,00032.Transfer the column to a new 1.5 mL Tube.33.g for 1 min. Repeat once.Add 50 μL RNase-free water to column and still for 5 min. Then centrifuge at the 14,00034.Transfer 100 μL eluted RNA sample to a fresh 1.5 mL tube.35.Set up the precipitation reaction as follow: RNA sample: 100 μL 3M NaOAc: 10 μL Isopropanol: 110 μLGlycoblue: 1 μL36.g 4 °C for 15 min.Incubate at − 80 °C for 30 min, and spin at 14,00037.Wash the RNA pellet with 80% ethanol twice.38.2O.Remove any solution thoroughly with a pipette and dissolve the RNA pellet with 15 μL H39.Check the RNA concentration with Nanodrop and take 1 μL (≥ 10 ng) for RNA quality check by running it on a 2% (wt/vol) low melting agarose gel (1 × TBE gel with 1 × SYBR gold fluorescent dye added).40.Take 500 ng RNA sample for qPCR analysis. Alternatively, the RNA sample can be subjected to poly(A) RNA removal and rRNA-depletion steps, followed by RNA-seq library construction. The isolated chromatin-bound RNAs can be stored at − 80 °C for several months.Set up the following reverse transcription reaction mix (10 μL):RNA sample (500 ng): 5.5 μL10 mM dNTPs: 0.5 μLPrimer mix: 0.5 μLHeat the mixture at 65 °C for 5 min and place the tube on ice for at least 2 min. (The primer mix needs to be prepared according to the number of gene-specific primers used. 1 pmol of each primer is included for each reaction. A list of primers used in this study can be found in Table Prepare the following as mix and add 3.5 μL into each reaction.5x M-MLV buffer: 2 μLM-MLV Enzyme (200U/μL): 0.5 μLRNase Inhibitor (40U/μL): 0.5 μLRNase-free water: 0.5 μLPlace the tube on a PCR thermocycler with the 105 °C heated lid and perform the PCR cycles as follows:42 °C 1 h, 80 °C 5 min, and hold at 4 °C.Dilute the cDNA sample 40 times with nuclease-free water.Set the following qPCR reaction mix (10 μL):cDNA (Diluted): 4 μLqPCR SuperMix: 5 μL10 μM F Primer: 0.5 μL10 μM R Primer: 0.5 μL from 4 °C and warm them to room temperature.Take 8 μg chromatin-bound RNA sample. Dilute it to 50 μL with nuclease-free water in a nuclease-free 1.5 mL tube.Gently mix mRNA Capture Beads and transfer 50 μL beads into the RNA sample. Mix with a pipette.Denature the RNA by incubation at 65 °C for 5 min and place the tube on ice immediately.Leave the sample at room temperature for 5 min.Place on the magnet for 5 min to separate poly-A RNA from the rest of the CB-RNAs.Take out the supernatant to a new 1.5 mL tube and set it aside.Add 200 μL Beads Wash buffer to beads and mix with pipette thoroughly.Place the tube on a magnet for 5 min and discard the supernatant.Add 50 μL Tris Buffer to resuspend beads and mix with pipette thoroughly.Incubate the tube at 80 °C for 2 min and set the tube at room temperature for 2 min.Add 50 μL Beads Binding buffer and mix with pipette thoroughly. Incubate the tube at room temperature for 5 min.Place the tube on a magnet for 5 min, transfer the supernatant to a new 1.5 mL tube, and mix with the sample in step 7.Add 1/10 volume of 3 M NaOAc and 1 μL Glycoblue, and mix thoroughly.Add 1 volume of isopropanol and mix thoroughly.Incubate at − 80 °C for 20 min.g 4 °C for 10 min.Centrifuge at 14,000Remove supernatant and wash the pellet twice with 80% ethanol.g 4 °C for 5 min and discard the supernatant.Centrifuge at 14,000Dissolve the pellet in 13 μL of RNase-free water.Hybridization of riboPOOL probe to RNASet up the following reaction mix:RNA sample (2 μg CB-RNAs): 13 μL50 μm riboPOOL probe: 1 μLHybridization buffer: 5 μLRNase inhibitor (40U/μL): 1 μLVortex to mix and spin down the droplet.Incubate at 68 °C for 10 min to denature RNA.Allow cooling slowly to 37 °C for optimal hybridization. (By setting ramp rate at 3 °C/min)Preparation of beads (Two steps of depletion)5.Resuspend the Streptavidin magpoly beads by carefully vertexing the tube at medium speed.6.Transfer 60 μL of beads suspension per sample to a fresh tube.  in a single tube).7.Place the tube on a magnetic rack and wait for 1 min.8.Aspirate and discard all supernatant.9.Add 100 μL per sample of beads Resuspension buffer, and agitate the tube well to resuspend beads. Place on magnetic rack for 1 min, aspirate and discard supernatant.10.Repeat step 9.11.Resuspend beads in 80 μL depletion buffer per sample and divide the beads into 2 tubes.Ribosomal RNA depletion12.Briefly centrifuge the tube containing ~ 20 μL hybridized riboPOOL and CB-RNAs.13.Transfer 40 μL of the prepared beads  into the tube containing hybridized riboPOOL-RNA solution. Agitate the tube to resuspend well.14.Incubate the tube at 37 °C for 15 min (mix every 5 min), followed by a 50 °C incubation for 5 min.15.Spin briefly and place the tube on the magnet for 2 min then carefully transfer the supernatant to the other tube containing beads (without depletion buffer from step 13), repeat step 14.16.Place the tube on the magnet for 2 min and carefully transfer the supernatant to a new tube.RNA clean-up17.Add nuclease-free water to the rRNA-depleted RNA sample from the last step to bring the volume to 100 μL.18.Set up the following precipitation reaction:RNA sample: 100 μL10 mg/mL Glycoblue: 1 μL3M NaOAc: 11 μL100% ethanol: 280 μL19.Mix well, and incubate at − 20 °C for at least 1 h.20.g 4 °C for 10 min.Centrifuge at 14,00021.Remove supernatant and wash twice with 80% ethanol.22.g 4 °C for 5 min and discard the supernatant.Centrifuge at 14,00023.Air dry and dissolve the pellet in 12 μL of RNase-free water.24.Check the RNA concentration with Qubit RNA Assay Kit and take 50 ng for CB-RNAs library construction.Paired-end sequencing was performed on the bar-coded library using an Nova Seq 6000. The adapters, Ns and low-quality bases were removed from raw data using the Trimmomatic  package Additional file 1: Bioinformatic pipeline.Additional file 2: Fig. S1. The relationship between co-transcriptional splicing (CTS) efficiency and intron order in RNAe,RNAf, RNAe+RNAf and mRNA.


Drosophila melanogaster has become a powerful model in genetic research thanks to its genetic similarity to mammals. This project mainly aimed to generate new evidence for the toxic/genotoxic properties of different nanopesticides , an inorganic nanoparticle as an active ingredient (copper(II) hydroxide [Cu(OH)2] nanopesticides, 15 ± 6 nm), a polymer-based nanopesticide , and an inorganic nanoparticle associated with an organic active ingredient ) and their microparticulate forms  sulfate pentahydrate (CuSO4·5H2O), acephate, and validamycin) widely used against agricultural pests, while also showing the merits of using Drosophila—a non-target in vivo eukaryotic model organism—in nanogenotoxicology studies. Significant biological effects were noted at the highest doses of permethrin (0.06 and 0.1 mM), permethrin nanopesticides (1 and 2.5 mM), CuSO4·5H2O (1 and 5 mM), acephate and acephate nanopesticides , and validamycin and validamycin nanopesticides . The results demonstrating the toxic/genotoxic potential of these nanopesticides through their impact on cellular internalization and gene expression represent significant contributions to future nanogenotoxicology studies.New insights into the interactions between nanopesticides and edible plants are required in order to elucidate their impacts on human health and agriculture. Nanopesticides include formulations consisting of organic/inorganic nanoparticles.  Scientists and researchers are desperately working to come up with new ways to increase food production, as the global population is projected to grow by about 48% by 2050 . ShrinkiThe key concept in nanopesticide formulations involves improving the delivery and efficacy of the active ingredient(s) through nanoscale particles . HoweverResearch into certain applications of nanotechnology in agriculture has become increasingly popular over the last decade, with a particular interest in nanopesticides ,16,17,1850  decreased with droplet size, pointing to an increase in the uptake of smaller droplets  nanopesticide as an inorganic NP, an acephate nanopesticide as a polymer-based nanopesticide, a and validamycin nanopesticide as an inorganic nanoparticle associated with an organic active ingredient), as well as microparticle forms of these nanopesticides  sulfate pentahydrate [CuSO4·5H2O], acephate, and validamycin) on D. melanogaster—a non-target eukaryotic model organism. The experiments included a Drosophila comet assay (for primary DNA damage and oxidative DNA damage in hemocytes and midgut cells), Drosophila SMART to detect any somatic mutation and/or recombination activity in wing imaginal disc cells, and measurements of intracellular reactive oxygen species (ROS) in the hemocytes and midgut cells of Drosophila larvae, along with assessments of oxidative stress effects (glutathione (GSH) amount), lipid peroxidation product formation , levels of nanopesticides crossing the intestinal barrier , changes in the expression of genes controlling the integrity of the intestinal barrier and general stress genes, phenotypic changes in different generations , locomotor behavior , and morphological abnormalities. The selected nanopesticides had not been used in any previous research for this purpose; hence, the determination of the nanotoxic and nanogenotoxic potential of these substances through in vivo and in vitro testing was a rather important step towards establishing the biosafety profiles of these substances. In addition, we also evaluated whether there was a possible correlation between the bulk  and nanoscale  forms of these compounds that may influence the extent of their toxicity and genotoxicity. In this respect, this research project successfully fills a knowledge gap in the literature regarding these nanopesticides. The findings derived from our experiments may provide valuable data concerning the cytotoxic and genotoxic potential of nanopesticides widely used in the agricultural sector.This study will hopefully encourage scientific efforts towards developing nanopesticides that are less harmful to nature than conventional pesticides. n humans ,65, respn humans ,67. Evenn humans ,69,70, i2 nanopesticides, acephate nanopesticides, and validamycin nanopesticides, respectively  [2 nanopesticides, respectively. Greater hydrodynamic diameter and lower zeta potential values indicate the existence of a tendency for nanoparticles to aggregate. To prevent such aggregation, we carried out all experiments by utilizing freshly prepared dispersions through the sonication process. The amount of active permethrin in the synthesized permethrin nanopesticides was determined using the Shimadzu Prominence HPLC System (Japan) and Inertsil ODS-3 C18 column. The measurements were carried out at 40 °C with a 20 µL injection, a 1 mL/min flow rate, and a 90:10 acetonitrile:water mobile phase; the amount of permethrin was determined with a UV detector at 225 nm. To that end, firstly, a calibration chart was created with 20, 40, 60, 80, and 100 ppm standard permethrin solutions  . The aveolutions .2 nanopesticides was characterized by XRD measurements performed according to the powder diffraction technique using the Empyrean X-ray diffractometer   [The crystal structure of Cu(OH)lytical) . Measureandards) . In the 3 particles during the reaction  [3 in the crystal phase correspond to reflections from the planes (004), (110), (112), (114), (300), (304), (118), and (224), respectively.X-ray diffraction measurements were taken from powder samples taken at different reaction times in order to understand whether there was a change in the crystal structure of the CaCOreaction . Figure  (JCPDS) ,74. In tLAL assays were utilized to measure the endotoxin levels . Endotox50 values. The LC50 value for permethrin was 0.1 mM, while it was 2.5 mM for permethrin nanopesticides, validamycin, and validamycin nanopesticides, and 5 mM for CuSO4·5H2O, Cu(OH)2 nanopesticides, acephate, acephate nanopesticides, and CaCO3 (4·5H2O at the highest dose (10 mM) significantly increased the lethality in the larval stages  was assessed. It had a viability  of 94 and 93% for the tested concentrations of ethanol and nanocapsules, respectively. The viability of clear distilled water was 100%. The toxic effects of the exposure to the compounds, applied throughout the entire larval development, were specified as variations in the ability to reach the adult stage. When applied at different concentrations from 0.01 to 10 mM throughout the entire larval development, a toxic effect was observed at the applied concentrations of 0.5, 1, 2.5, 5, 7.5, and 10 mM on the ability of permethrin-exposed larvae to reach the adult stage, while no toxic effects were observed at other concentrations. On the other hand, a toxic effect was observed at the applied concentrations of 5, 7.5, and 10 mM upon exposure to permethrin nanopesticides, whereas no toxic effect was observed at other concentrations  and nanocapsules used during the preparation of nanopermethrin (permethrin nanopesticide LCtrations A. As a rtrations A.4·5H2O and Cu(OH)2 nanopesticides, impairing the ability to reach the adult stage, no toxic effect was observed at other concentrations. After the toxicity study for both CuSO4·5H2O and Cu(OH)2 nanopesticides, the concentration range was determined to be 0.01, 0.1, 1, and 5 mM in the tests used within the scope of this project , permethrin nanopesticides (2.5 mM), acephate and acephate nanopesticides (5 mM), and validamycin and validamycin nanopesticides (2.5 mM). As shown in Drosophila adults exposed to permethrin (0.1 mM), permethrin nanopesticides (2.5 mM), acephate and acephate nanopesticides (5 mM), and validamycin and validamycin nanopesticides (2.5 mM) were found to have anomalies in the mouth and leg regions. No morphological changes were observed in the groups treated with CuSO4·5H2O and Cu(OH)2 nanopesticides.Drosophila individuals belonging to the F1, F2, and F3 generations were obtained from those F0 individuals. The F1, F2, and F3 generations contained both normal and defective flies. In the F0, F1, F2, and F3 generations, the highest doses of permethrin (0.1 mM), permethrin nanopesticides (2.5 mM), acephate and acephate nanopesticides (5 mM), and validamycin and validamycin nanopesticides (2.5 mM) caused phenotypic variations in the mouth and leg regions (4·5H2O and Cu(OH)2 nanopesticides.After exposure to different concentrations of compounds, phenotypic variations were examined in F0 . Then, adult  regions . On the Each application concentration was performed in three replicates for each generation. When a total of 1500 flies was observed, phenotypic variations observed in the mouths of individuals treated with permethrin (0.1 mM) amounted to 28 ± 1.8%  of the total population. However, the rates of phenotypic variation observed in the mouths of individuals treated with permethrin nanopesticides (2.5 mM), acephate and acephate nanopesticides (5 mM), and validamycin and validamycin nanopesticides (2.5 mM) were 30 ± 0.4% (450 adults), 25 ± 1.3% (375 adults), 29 ± 1.5% (435 adults), 26 ± 1.8% (390 adults), and 27 ± 1.6% (405 adults) of the total population, respectively B–F. FiguAs for the phenotypic variations observed in the legs, the rate of those observed in the legs of flies exposed to permethrin (0.1 mM) was 26 ± 0.3% of the total population (390 adults); the other nanopesticides caused variations at the following rates: 29 ± 0.6% (435 adults), 22 ± 1.8% (330 adults), 27 ± 1.3% (405 adults), 25 ± 1.4% (375 adults), and 26 ± 1.7% (390 adults) of the total population, respectively B–F. FiguImpaired climbing ability is often considered to be an indicator of seriously damaged locomotor behavior in flies. Significant differences were detected in climbing behavior between the study and control flies after 7-day exposure to the nanopesticides.The locomotor activity of 2% ethanol used as a permethrin solvent and the nanocapsules (2.5 mM) used during the preparation of permethrin nanopesticides was determined to be 94 ± 3.2% and 91 ± 3.6%, respectively. The concentrations in permethrin application  were determined to be 90 ± 4.6%, 86 ± 5.2%, 81 ± 6.3%, and 67 ± 7.4%, respectively, when compared with the control group, while the concentrations used in permethrin nanopesticide application  were determined to be 88 ± 3.8%, 84 ± 4.7%, 80 ± 5.1%, and 69 ± 5.9%, respectively, when compared with the control group A,B.4·5H2O and Cu(OH)2 nanopesticides were 89 ± 4.2%, 84 ± 5.4%, 80 ± 5.1%, and 60 ± 6.3%, respectively. They were determined to be 90 ± 3.5%, 86 ± 5.2%, 82 ± 4.7%, and 79 ± 5.8% in Cu(OH)2 nanopesticide application  of CuSOlication C.The locomotor activity upon exposure to the nanocapsules (5 mM PEG-400) used during the preparation of the acephate nanopesticide was determined to be 90 ± 3.6%. The concentrations in acephate  were determined to be 88 ± 4.2%, 85 ± 4.7%, 80 ± 5.4%, and 52 ± 6.7%, respectively, while the concentrations in acephate nanopesticide application  were determined to be 91 ± 3.3%, 87 ± 4.2%, 83 ± 4.5%, and 59 ± 5.3%, respectively, as compared to the control group D.3 application  were determined as to be ± 4.4%, 90 ± 5.1%, 88 ± 4.8%, and 86 ± 5.7%, respectively, as compared to controls (3) used during the preparation of the validamycin nanopesticide was determined to be 89 ± 3.9%. When the concentrations  in validamycin and validamycin nanopesticide applications were compared with those of the control group, they were determined to be 87 ± 4.4%, 84 ± 5.1%, 79 ± 5.3%, and 53 ± 5.9% respectively, while in validamycin nanopesticide applications, they were determined to be 89 ± 3.2%, 86 ± 4.6%, 81 ± 4.9%, and 63 ± 5.3%, respectively (The concentrations in CaCOcontrols E. On theectively F.4·5H2O (5 mM), acephate and acephate nanopesticides (5 mM), and validamycin and validamycin nanopesticide (2.5 mM) were found to impair locomotor activity at statistically significant levels.The highest doses of permethrin (0.1 mM), permethrin nanopesticides (2.5 mM), CuSODrosophila larvae. On the other hand, there was no significant difference in glutathione levels upon exposure to Cu(OH)2 nanopesticides and CaCO3 as compared to the control substances controls A–F.Drosophila intestinal barrier, effects on the cytoplasmic matrix of intestinal enterocytes of larvae , effects on microvilli, internalization into the intestinal lumen, and effects on the lumen, as well as their interactions with cellular components  in the lumen, are shown in The interactions of the lowest and highest concentrations of nanopesticides and control substances with the In the midgut lumen, nanopesticide particles not yet taken into the enterocytes were located in the vicinity of microvilli A, while 2 nanopesticide or CaCO3 applications . In other words, we determined that nanopesticides and their microparticle forms did not cause mutagenic and/or recombinogenic effects in Drosophila SMART assays (3mwh/flr) showed mutation and recombination, only clone types forming due to mutation were observed in serrate-wing flies (mwh/TM3) thanks to suppression of recombination by their balancer chromosomes. Therefore, no evaluation was conducted in that regard in serrate-wing flies [Comparison of study groups with controls revealed no significant changes for all clone types  was determined by trypan blue assay, and the concentration range selection was performed. In order to study non-toxic concentrations via the comet method, values of 70% and above were taken into account for cell viability in the test groups [4·5H2O, acephate, acephate nanopesticides, validamycin, and validamycin nanopesticides caused significant single-stranded DNA damage in Drosophila hemocytes and midgut cells. On the other hand, there were no significant differences in the levels of single-stranded DNA damage at the concentrations of Cu(OH)2 nanopesticides and CaCO3 as compared to the control group 2 nanopesticides, acephate, acephate nanopesticides, validamycin, and validamycin nanopesticides. There was a significant increase in the expression of the Hsp70, Hsp83, CAT, SOD2, and p53 genes in larvae after exposure to permethrin  and permethrin nanopesticides . However, a statistically significant increase (upregulation) for the Hsp70, Hsp83, CAT, SOD2, and p53 genes was observed in larvae exposed to 0.06 and 0.1 mM concentrations of permethrin and 1 and 2.5 mM concentrations of permethrin nanopesticides. The greatest increase for these genes was detected at the highest concentrations of both permethrin (0.1 mM) and permethrin nanopesticides (2.5 mM) (p ≤ 0.5 and p ≤ 0.01. The data represent the mean ± standard error from three independent experiments.The percentages of mRNA expression of the (2.5 mM) A,B. The 4·5H2O, Cu(OH)2 nanopesticides, acephate, acephate nanopesticides, validamycin, and validamycin nanopesticides, the genes  and some gene markers  associated with the gut barrier response to xenobiotics were used for the first time. After exposure to permethrin , no statistically significant differences were observed in the expression of Duox, Hml, Muc68D, and/or PPO2—gene markers associated with midgut and hemocyte interactions; however, a statistically significant downregulation was observed in larvae exposed to permethrin nanopesticides . On the other hand, a significant decrease in mRNA expression of Ogg1—a gene associated with DNA repair—was observed at 0.06 and 0.1 mM doses of permethrin, as well as 1 and 2.5 mM concentrations of permethrin nanopesticides. No statistical differences were observed in the expression of Hsp70, Hsp83, CAT, SOD2, p53, Ogg1, Duox, Hml, Muc68D, or PPO2 for the nanocapsules (2.5 mM) used during the preparation of the permethrin nanopesticides. The level of statistical significance was taken as p ≤ 0.01. The data represent the mean ± standard error from three independent experiments  and Cu(OH)2 nanopesticides . However, a significant decrease in Hsp70 expression occurred after exposure to 1 and 5 mM concentrations of both CuSO4·5H2O and Cu(OH)2 nanopesticides, in SOD2 after 0.01–5 mM CuSO4·5H2O, and in Hsp831 only after the 5 mM doses of CuSO4·5H2O and Cu(OH)2 nanopesticides , and statistically significant results were obtained at 1 and 5 mM concentrations 2 nanopesticides, while significant decreases were found in the expression of all genes in question at the highest concentration (5 mM) of Cu(OH)2 nanopesticides , no statistical changes were observed in the expression of the Duox, Hml, Muc68D, and PPO2 genes, associated with midgut–hemocyte interaction. In the larvae exposed to 0.1, 1, and 5 mM concentrations of CuSO4·5H2O, concentration-dependent decreases were observed in the mRNA expression of the Ogg1 gene when compared to the larvae of the control group, and statistically significant results were found at 1 and 5 mM concentrations of CuSO4·5H2O , the expression of Duox, Hml, Muc68D, and PPO2 generally decreased in a concentration-dependent manner. Statistically significant decreases were observed in the mRNA expression of Duox and Hml in larvae exposed to 0.01, 0.1, 1, and 5 mM concentrations of Cu(OH)2 nanopesticides, as compared to the larvae of the control group. Significant decreases were observed in the expression of Muc68D and PPO2 in larvae after the highest dose of Cu(OH)2 nanopesticides (5 mM). Compared to the control group, in larvae exposed to CuSO4·5H2O, a concentration-dependent decrease was observed in the mRNA expression of the Ogg1 gene, while a statistically significant decrease was observed at 1 and 5 mM concentrations. Compared to the control group, an increase was observed in the mRNA expression of the Ogg1 gene in larvae exposed to 0.01, 0.1, and 1 mM concentrations of Cu(OH)2 nanopesticides, while a decrease was observed at the 5 mM concentration. However, these results were not statistically significant  and acephate nanopesticides  caused concentration-dependent increases in the expression of the sticides E,F. The Ogg1 gene when compared to the larvae of the control group, and statistically significant results were found at 1 and 5 mM concentrations of acephate . However, in larvae exposed to 0.01, 0.1, 1, and 5 mM concentrations of acephate, dose-dependent decreases were observed in the mRNA expression of the acephate E. On the results E,F. No sp ≤ 0.01 E,F.3  in the nanocapsules used during the preparation of the validamycin nanopesticides caused no significant changes in the expression of the Hsp70, Hsp83, CAT, SOD2, and p53 genes. However, some dose-dependent changes were observed for the Hsp70 gene in all doses of CaCO3, with a statistically significant decrease occurring only at the highest dose (5 mM) of CaCO3. In addition, a statistically significant increase in the expression of the p53 gene was observed at the highest concentration of CaCO3 (5 mM) . The level of statistical significance was taken as p ≤ 0.5 (Exposure to CaCO3 (5 mM) I. On the p ≤ 0.5 I.Hsp70, Hsp83, CAT, SOD2, and p53. A significant increase in the expression of these genes was noted in larvae exposed to 1 and 2.5 mM concentrations of validamycin and validamycin nanopesticides . However, in larvae exposed to 0.01, 0.1, 1, and 2.5 mM concentrations of validamycin, dose-dependent decreases were observed in the mRNA expression of the Ogg1 gene when compared to the larvae of the control group, and statistically significant results were found at 0.1, 1, and 2.5 mM doses of validamycin . Moreover, significant decreases occurred in the expression of the Muc68D gene at the highest concentration of validamycin nanopesticides (2.5 mM). Compared to the control group, in larvae exposed to 0.01, 0.1, 1, and 2.5 mM concentrations of validamycin and validamycin nanopesticides, a decrease in the mRNA expression of the Ogg1 gene was observed in general, in a concentration-dependent manner, while statistically significant results were found at doses of 0.1, 1, and 2.5 mM  and validamycin nanopesticides  led to dose-dependent changes in the expression of sticides G,H. The idamycin G. In thed 2.5 mM G,H. No sp ≤ 0.01 G,H.4·5H2O, acephate, and validamycin) using a non-target in vivo eukaryotic model organism. To this end, we carried out detailed comparative investigations of potential toxic/genotoxic properties, along with their effects on the gene expression and cellular uptake properties of conventional- and nanoformulations of four different pesticides that are widely used in the agricultural sector. The results obtained in this research endeavor support the advantages of using D. melanogaster as a reliable model organism to evaluate the biological properties of different nanopesticide formulations and their microparticle forms.In this study, we explored the potential risks associated with various nanopesticide formulations and their microparticle forms  in D. melanogaster is 46.47 ppm, while its LC25 is 22.5 ppm [25 value obtained with permethrin alone and with PBO–permethrin combinations at different rates, no genotoxic effects were detected in either application group in the Drosophila SMART assays [A. aegypti, C. tritaeniorhynchus, E. coli, E. ludwigii, B. subtilis, C. sativus, Z. mays, L. esculentum, and A. cepa), and these effects may vary depending on the cell, organism, and particle size [Previous research involving permethrin indicates that its median lethal concentration  NPs after exposure through ingestion in D. melanogaster were evaluated using SMART, comet, and lipid peroxidation assays, reporting that such materials induced single-stranded DNA damage, somatic mutation and recombination, ROS formation, and lipid peroxidation product formation [4·5H2O and CuO NPs [2 nanopesticides did not cause any negative biological effects on D. melanogaster. In addition, considering the results of studies with CuSO4·5H2O similar to previous findings in the literature, it was observed that CuSO4·5H2O induced oxidative damage and genotoxicity at high doses. Morgado et al. [T. molitor and P. pruinosus. This nanopesticide generally caused milder effects on both species, and although its toxic impact was highly species-specific, the authors concluded that such nanopesticides could be a good alternative for mitigating the environmental impact, due to their lower toxicity to soil organisms as compared to conventional pesticides.The literature review showed that the in vivo toxicity and genotoxicity of CuSOormation ,45,47. M CuO NPs . Our stuo et al.  found thD. melanogaster [Choudhury et al.  noted thnogaster ,53. In t3 nanoparticles can be attributed to the prolonged release of the active ingredient spanning over 14 days [A. aegypti [D. melanogaster [3 alone in this project determined that it caused no negative biological effects . However, exposure to validamycin and validamycin nanopesticides caused statistically significant increases in single-stranded DNA damage, ROS formation, lipid peroxidation product formation, and glutathione levels at high concentrations, but no genotoxic effects were detected by SMART assays.The long-term effectiveness of validamycin formulated with CaCO 14 days . Validam aegypti , and hadnogaster . The appD. melanogaster’s intestinal epithelial cells and cellular components such as the cytoplasm, vacuoles, and/or microvilli in the intestinal lumen [2 nanopesticides migrated into the intestinal epithelial cells of D. melanogaster. Decreases in locomotor activity [D. melanogaster as a result of acute sublethal acephate exposure. In line with these findings, we found that the locomotor activity decreased at a statistically significant rate upon exposure to the highest doses (5 mM) of acephate and acephate nanopesticides. Moreover, significant biological effects on adult Drosophila flies were observed once they were exposed to permethrin, permethrin nanopesticides, CuSO4.5H2O, acephate, acephate nanopesticides, validamycin, and validamycin nanopesticides, including the formation of intracellular ROS in the blood cells, hemocytes, and midgut cells; oxidative stress effects (GSH amount); lipid peroxidation product formation ; single-stranded DNA damage; oxidative DNA damage; phenotypic changes in different generations; changes in locomotor activity ; and morphological disorders. In addition, interactions of nanopesticides with the Drosophila intestinal barrier; their effects on the cytoplasmic matrix of the intestinal enterocytes , microvilli, and intestinal lumen; and their interactions with the cellular components  in the lumen are described in this study.The relevant literature notes that CuO NPs can transfer into al lumen ,47. Likeactivity  and fligactivity  have beeHsp gene family. Changes in the expression of Hsp genes in D. melanogaster are quite sensitive indicators for any biological stress [CAT and SOD2—play a role in cellular responses to any change in free radical balance. The Ogg1 and p53 genes, which are involved in DNA repair and genomic integrity, are safe factors for cell health . The cell-protective p53 gene appears to be a crucial center in the cellular genotoxic stress response, acting as a transcription factor to elicit cellular functions, including DNA damage and repair, cell-cycle arrest, and apoptosis. The p53 gene normally accumulates in the nucleus, and is converted into an active form of DNA binding to control several gene sets to prevent proliferation of DNA-damaged cells [Hsp70 and Hsp83), those responsible for antioxidant defense (CAT and SOD2), DNA repair (Ogg1), and p53 (responsible for genomic integrity), as well as some gene markers  associated with the intestinal barrier response to xenobiotics—were used for the first time in this study.Exposure to pesticides, nanopesticides, or other xenobiotic compounds can activate different metabolic or physiological pathways according to their mode of action. Therefore, the expression of several gene markers associated with cellular uptake, along with biological effects and changes, may be affected after pesticide/nanopesticide exposure. Cells respond to any external influence in a systematic and coordinated manner at the molecular level via a cascading effect. One of the earliest steps involved in the first response is related to the l stress ,86. The l stress . Furthered cells . The chaSOD2, p53, Hsp70, Hsp83, and CAT genes of the larvae after exposure to permethrin (0.06 and 0.1 mM), permethrin nanopesticides (1 and 2.5 mM), acephate and acephate nanopesticides (1 and 5 mM), and validamycin and validamycin nanopesticides (1 and 2.5 mM). Previous works in the literature show that acute sublethal acephate exposure in D. melanogaster could induce oxidative stress  in hemocytes [A significant increase was observed in the expression of the emocytes ,53. Thes4·5H2O and Cu(OH)2 nanopesticides led to significant decreases in the expression of the Hsp70, Hsp83, and SOD2 genes after larval exposure to 0.01, 0.1, 1, and/or 5 mM doses of CuSO4·5H2O and Cu(OH)2 nanopesticides. In addition, CuSO4·5H2O exposure (1 and 5 mM) caused an increase in the expression of the CAT and p53 genes, while Cu(OH)2 nanopesticide exposure resulted in significant decreases in the expression of the Hsp70, Hsp83, CAT, SOD2, and p53 genes.Exposure to CuSO3 exposure in the nanocapsules used during the preparation of the validamycin nanopesticides, no statistical differences were observed for the Hsp83, CAT, and SOD2 genes at 01, 0.1, 1, and 5 mM doses of CaCO3, as compared to the controls. In addition, a statistically significant decrease was observed for the Hsp70 gene at a 5 mM dose of CaCO3, while a significant increase was observed for the p53 gene.As a result of the CaCO3 and validamycin in the nanocapsules, significant decreases were detected after exposure to permethrin nanopesticides, Cu(OH)2 nanopesticides, acephate nanopesticides, and validamycin nanopesticides.The level of penetration of pesticides and nanopesticides through the intestinal barrier, along with their effects on the intestinal lumen in the larval stage, was evaluated by looking at their effects on the genes and gene markers  that control the integrity of the intestinal barrier. The expression of Duox, Hml, Muc68D, and/or PPO2, which was analyzed in larvae after their exposure to pesticides and nanopesticides, was compared to that of the control group larvae. While no statistical differences were noted for CaCOOgg1 gene—a gene associated with DNA repair. An increase, with no statistical significance, in the mRNA expression of the Ogg1 gene was observed in larvae exposed to 0.01, 0.1, and 1 mM concentrations of Cu(OH)2 nanopesticides as compared to the control group, whereas a decrease was observed at 5 mM. No significant changes were observed in the expression of Duox, Hml, Muc68D, PPO2, and Ogg1 analyzed in larvae after exposure to CaCO3 . The nanocapsules used for the preparation of the nanopesticides caused no significant changes in the expression of Hsp70, Hsp83, CAT, SOD2, p53, Ogg1, Duox, Hml, Muc68D, or PPO2.On the other hand, permethrin at doses of 0.06 and 0.1 mM, permethrin nanopesticides at 1 and 2.5 mM, CuSO4.5H2O at 1 and 5 mM, acephate at 1 and 5 mM, acephate nanopesticides at 0.1, 1 and 5 mM, and validamycin nanopesticides at doses of 0.1, 1 and 2.5 mM all caused a significant decrease in the mRNA expression of the Drosophila is partially controlled by crosslinking of proteins through transglutaminases [Muc68D) is expressed only in larvae [Muc68D gene suggests a lower protective role of the peritrophic membrane.The first physical barrier to protect hosts from xenobiotics and toxins is the peritrophic membrane and its epithelial integrity ,89. Its aminases . Mucin 6n larvae . DecreasDrosophila [Anopheles mosquitoes with decreased DUOX expression, due to decreased dityrosine crosslinking of the peritrophic membranes [Peroxidase, or dual oxidase (DUOX), is an enzyme that catalyzes ROS generation by localizing in the plasma membrane and phagosome membrane of the NADPH oxidase family proteins in osophila , and it osophila . In addiosophila . Intestiembranes . This isDrosophila larvae [Hemolectin (Hml) is a clotting factor in a larvae , requirea larvae . Our resa larvae . In this4 treatment. In both CuO NPs and CuSO4, copper caused a significant reduction in SOD at almost all doses. CuSO4 exposure caused a significant increase in the expression of the CAT gene at all tested doses, while the expression of the CAT gene following CuO NP exposure caused a statistically significant decrease—especially at 10 mM. In addition, a significant decrease and increase in expression of the p53 gene was observed after exposure of Drosophila larvae to both CuO NPs (10 mM) and CuSO4 (2 mM) [Hsp70 and SOD2 genes, while a significant increase in the expression of the CAT and p53 genes was detected at 0.01, 0.1, 1, and 5 mM concentrations of CuSO4·5H2O, in accordance with the studies in the literature. Dose-dependent decreases in the expression of Duox, Hml, Muc68D, PPO2, and upd3—the genes associated with midgut–hemocyte interaction—were detected in Drosophila larvae exposed to different doses of CuO NPs . Although no changes occurred in the expression of the Muc68D gene, a significant decrease was observed in the expression of Duox, Hml, and Upd3. Furthermore, a statistically significant effect was recorded in the expression of the PPO2 gene only at the high concentration (10 mM). However, no significant change was noted in the expression of the Ogg1 gene [When other studies in the literature were reviewed, we saw that a significant decrease was noted for Hsp70 upon exposure to CuO NPs at doses of 0.08, 0.4, 2, and 10 mM, while significant effects were observed only at 0.08 mM after CuSO4 (2 mM) . In thisgg1 gene .Despite there having been plenty of research into the effectiveness of nanopesticides on target organisms ,69,98, tLycopersicum esculentum, Cucumis sativus, and Zea mays. Comparable results were reported for an atrazine polymeric nanopesticide (particle size: about 250 nm in diameter) after being tested on non-target maize plants [2 NPs, Cu(OH)2 nanowires, Cu NPs, and atrazine-loaded polymeric NPs—on different plant species.Some previous works have dealt with the toxic effects of nanopesticides on non-target plant species. For instance, Kumar et al.  showed te plants . Certaine plants ,101. Grie plants  summarizEnchytraeus crypticus [Eisenia fetida [Caenorhabditis elegans [Acanthoscelides obtectus [Folsomia candida [Hypoaspis aculeifer). Jacques et al. [Caenorhabditis elegans. Firdaus et al. [E. fetida and Lumbricus terrestris), discovering that nanoforms caused approximately 50% more bifenthrin accumulation. From a similar perspective, Neves et al. (2019) showed that commercial inorganic nanopesticides (diameter: >1000 nm) had a higher toxicity to Folsomia candida as compared to conventional formulations (Cu(OH)2 solution). On the other hand, Pascoli et al. [C. elegans, whereas the bulk formulation impaired pharyngeal pumping and GST-4 protein expression. Phanse et al. [Risk assessment of engineered nanomaterials for terrestrial organisms has been carried out over the last several decades, and nanopesticide research has recently begun to gain momentum. The use of several soil organisms is recommended for ecotoxicological models for the risk assessment of chemicals. While adaptations have been proposed for engineered nanomaterials, including some specific applications such as nanobiomaterials ,104,105,rypticus  and Eisea fetida ), nemato elegans ), and arobtectus , Folsomi candida , and Hyps et al.  showed ts et al.  tested ti et al.  found the et al.  developeOncorhynchus mykiss [Channa punctatus [Prochilodus lineatus [Danio rerio [Daphnia magna [Daphnia similis [Leptocheirus plumulosus [Ceriodaphnia dubia [Pseudokirchneriella subcapitata [Chlorella vulgaris [Closterium sp. [Lemna valdiviana [Lithobates catesbeianus [2 nanopesticides [D. magna, the authors detected a decrease in the expression of detoxification-related genes following short-term (24 h) exposure, although the expression of these genes increased significantly after long-term exposure (48 h). On the other hand, it has been determined that the expression of genes related to the reproductive system varies depending on the dose and exposure time. Vignardi et al. [2 (< 1000 nm) and conventional pesticides  caused similar levels of toxicity in Leptocheirus plumulosus.Risk assessment of engineered nanomaterials for aquatic organisms has been undertaken for quite some time ,113. Jens mykiss , Channa unctatus , Prochillineatus , Danio rio rerio ,119), cria magna , Daphnia similis , Leptochumulosus , and Ceria dubia ), algae capitata , Chlorelvulgaris , Closterrium sp. , Lemna vldiviana ), and amsbeianus ,126). Amsticides . When thi et al.  reportedA. aegypti [C. tritaeniorhynchus [C. pipiens [C. quinquefasciatus [Spodoptera littoralis [Blattella germanica (German cockroach) [Various studies have looked into the toxicity or insect-repellent effects of nanopesticides on flying target organisms such as  aegypti , C. tritrhynchus , C. pipi pipiens , C. quinasciatus , Spodoptttoralis , and Blackroach) . Howeverckroach) . A signi2, 99.9%), sodium carbonate , N-butanol , cyclohexane , methanol , ethanol , acetonitrile , validamycin , dichloromethane , polyethylene glycol (PEG) (H(OCH2CH2)nOH, ≥98), n-butyl acetate , sec-butyl alcohol , copper(II) sulfate pentahydrate (CuSO4·5H2O), and calcium carbonate (CaCO3) were purchased from Merck . Ethyl methanesulfonate , hydrogen peroxide , acephate , permethrin , hexadecyl trimethyl ammonium bromide , phosphatidylcholine (from soybean lecithin) , and ammonium glycyrrhizate  were purchased from Sigma Chemical Co. . Copper hydroxide (Cu(OH)2) particles were purchased from DuPont as a commercial biocide (Kocide® 3000). For negative control groups, we used sterile distilled water, ethanol (2%), nanocapsules (2.5 mM), nanocapsules (5 mM PEG-400), and nanocapsules (2.5 mM CaCO3) for the preparation of the CuSO4·5H2O and Cu(OH)2 nanopesticides, permethrin, permethrin nanopesticides, acephate nanopesticides, and validamycin nanopesticides, respectively.Calcium chloride (CaCl21H20Cl2O3) is a chemical compound characterized by rather low water solubility. Therefore, we used the method recommended in the literature to prepare nanopermethrin in a nanometric powder form that could be easily dispersed in water [Permethrin  was purchased and used for the Cu(OH)2 nanopesticide whose toxicity was tested in this study [® 3000 Cu(OH)2 nanopesticide containing 35% Cu2+ equivalent was optimized. To that end, 1 mg/mL dispersions were prepared after the greenish-blue-colored Cu(OH)2 nanopesticide was thoroughly ground in a mortar. As dispersion preparation methods, mixing with a magnetic stirrer at 1200 rpm, holding in an ultrasonic bath, and combining these two methods were systematically tried. The particle sizes and size distributions of the dispersions obtained for each trial were characterized by dynamic light scattering (DLS) measurements on a Malvern Zetasizer ZS device.A commercial biocide  with the hydrophilic polymer PEG [Nanoacephate was prepared by encapsulating acephate  was performed by utilizing the reaction of calcium chloride and sodium carbonate via the reverse-phase microemulsion method in a solution medium containing validamycin [3 particles were precipitated. The synthesized CaCO3 particles were dried at 6000 rpm for 15 min. After separation by centrifugation, they were washed twice with 2 mL of deionized water and methanol, and then dried in a vacuum oven at 313 K for 18 h. The hydrodynamic diameters of the synthesized CaCO3 particles were determined by DLS measurements, while their size and morphology were characterized by transmission electron microscopy (TEM) and scanning electron microscopy (SEM) measurements. The changes in the crystal structure of the CaCO3 particles were investigated by XRD measurements performed both during the synthesis stages and after validamycin loading.Synthesis of nano-CaCOidamycin . The exp3 particles synthesized during the preparation of the Cu(OH)2 and validamycin nanopesticides was characterized by X-ray diffraction (XRD) measurements performed on powder samples using the Empyrean X-ray diffractometer from PANalytical . Measurements were performed through Cu Kα irradiation at 2θ in the angle range of 10–90° and at a scanning speed of 0.01°. The permethrin content in the prepared permethrin nanopesticide was characterized using a Shimadzu Prominence High-Performance Liquid Chromatography (HPLC) system with a UV detector at 225 nm and an Inertsil ODS-3 C18 column. Prior to dispersion, a pre-wetting procedure was applied to the permethrin nanopesticides, Cu(OH)2 nanopesticides, acephate nanopesticides, and validamycin nanopesticides in 0.5% ethanol, after which the nanopesticides were dispersed in 0.05% BSA in Milli-Q water in order to enhance their workability. Then, the permethrin nanopesticides, Cu(OH)2 nanopesticides, acephate nanopesticides, and validamycin nanopesticides were exposed to ultrasonic vibration at 20 kHz for 30 min using a Branson Digital Sonifier system (S-250D)  to obtain a stock dispersion of 2.56 mg/mL, in line with previously described protocols [The characterization of the nanopesticides was achieved through analyses TEM (LEO 906 E TEM by ZEISS) , SEM (LEO 1430 by ZEISS) , DLS , and laser Doppler ve-locimetry (LDV)  . A HyperCOOL HC3110 lyophilizer was used in the lyophilization processes. The crystal structure of the CaCOrotocols ,136.Escherichia coli E50-640) for each assay. Endotoxin standards and diluted samples were assessed for endotoxin testing in pyrogen-free microplates  using a BioTek Synergy 2 microplate reader  at 37 °C. Absorbance was measured at 405–410 nm. We utilized commercially available control endotoxins (lipopolysaccharide (LPS); 0.5 EU/mL) and LAL water as positive and negative controls, respectively. The percentage of recovery spike values was calculated as follows:2 nanopesticides ; 100.6, 107.4, 105.4, and 108.2% for acephate nanopesticides ; and 105.2, 101.8, 108.4, and 113.2% for validamycin nanopesticides .The endotoxin content was measured by chromogenic Limulus amebocyte lysate (LAL) assay (Lonza (QCL-1000TM), Inc., Walkersville, MD, USA), as per the protocol described in the manual and in previous studies ,140,141.50 values, a total of 25 third-instar Drosophila larvae at 72 h were randomly chosen and exposed to different doses of permethrin, permethrin nanopesticides, CuSO4·5H2O, Cu(OH)2 nanopesticides, acephate, acephate nanopesticides, CaCO3, validamycin, and validamycin nanopesticides through ingestion  hydrated with 10 mL of the freshly prepared test solutions). We then recorded the numbers of emerging flies 10 days after exposure, and the larva-killing capacity of the materials was recorded as the percentage of larvae unable to grow into adults. Four replications with 25 larvae were conducted for each treatment. This testing was repeated 3 times on the subsequent days.To calculate the LCDrosophila flies were anesthetized with diethyl ether before testing. Then, they were placed into the pots, where they were exposed to insecticides. To allow air transfer, the pots were covered with clean muslin and rubber at the top. After 15 min, the flies were transferred to clean pots. Wet cotton was put on the muslins to maintain humidity. 24 h later, the numbers and rates of the dead flies were recorded.The nanopesticides were exposed to the pots 24 h before the testing, and the experiments were performed at room temperature with 50% relative humidity ,143,144.Drosophila larvae and adults were cultured at 25 ± 1 °C (in a 12:12 h light:dark cycle) with 60% humidity on a food culture consisting of cornmeal, sugar, yeast, agar, propionic acid, and nipagin. Three different mutant strains were used in the study: wild Canton-S, flare-3, and multiple wing hairs. In the wing SMART assay, two distinct fly strains were used: flare-3 with genetic constitution 3/In (3LR) TM3flr, sBd and multiple wing hairs with mwh/mwh genetic constitution. The study by Lindsley and Zimm [4·5H2O, Cu(OH)2 nanopesticides, acephate, acephate nanopesticides, CaCO3, validamycin, and validamycin nanopesticides, we measured the egg-to-adult survival rates in flies. The adult Canton-S flies were placed in darkened bottles with food culture, facilitating egg collection, which was performed every 8 h. Each test sample containing 50 eggs was transferred to plastic vials, into which we placed 4 g of instant food culture specifically designed for Drosophila . The food culture had been saturated with 10 mL of various concentrations of permethrin nanopesticides , permethrin , Cu(OH)2 nanopesticides and CuSO4·5H2O , acephate nanopesticides and acephate  and validamycin nanopesticides and validamycin  dispersions, and the final concentrations of food culture for permethrin nanopesticides , permethrin , Cu(OH)2 nanopesticides , CuSO4·5H2O , acephate nanopesticides , acephate , validamycin nanopesticides , validamycin , and CaCO3  are indicated as mg/g.and Zimm  providesThe pesticide doses are expressed as mM throughout the text. Such doses were designated in accordance with previous works in the relevant literature ,146,147.2 nanopesticides, acephate nanopesticides, and validamycin nanopesticides were designated so that the highest dose should not exceed 5 mM. For each dose, we utilized a total of five different replicate samples. The adult flies that survived the pesticide exposure were then collected to determine their counts in order that the survival rate could be calculated. In total, 100 surviving adult flies per treatment were examined for any developmental changes under stereomicroscopy (SLX-2 STEREOZOOM) to assess the impact of exposure during the egg-to-larva phase, and then the morphological changes observed across various body parts—such as the head, thorax, wings, legs, and abdominal area—were recorded in detail [The ideal EMS dose (1 mM) to cause effective mutagenesis was based on the evidence from our previous works ,148. We n detail .The F0 (parent), F1, F2, and F3 generation flies in the control groups, along with those in the study groups exposed to test chemicals, were meticulously observed under a stereomicroscope to record any possible phenotypic variations in the head, thorax, eyes, mouth, wings, legs, or abdominal region. In this process, we followed previously published protocols in the literature ,150,151.In accordance with the procedures proposed in the previous work in the literature, the locomotor behavior of the flies was measured through climbing assays ,152,153.g. The GSH levels in the exposed larvae were measured using Ellman’s reagent [Oxidative stress assays were performed by measuring glutathione (GSH) levels. The methods applied were mainly as follows: 40 larvae were used for each concentration in the control and study groups, and 5 repetitive tests were carried out in each concentration group. After the cuticle was removed, it was homogenized in 1 mL of cold homogenizing solution  in order to obtain 10% tissue homogenate from the internal tissues of the larvae belonging to the control and nanopesticide-exposed groups. Then, the supernatant to be used in the experiment was obtained by centrifuging at 9000–10,000×  reagent ,155. TheDrosophila larvae (72 ± 4 h) were exposed to the compounds used as negative controls, nanopesticides, and different concentrations of microparticle forms of the nanopesticides for approximately 24 h. The lipid peroxidation or thiobarbituric-acid-reactive species (TBARS) assay was performed in accordance with the method modified by Carmona et al. [Drosophila larvae (each weighing about 0.5 g) were chosen and separately exposed to the control substances, nanopesticides, and microparticle forms of the nanopesticides. The experiments were carried out with two independent tests and three replications in each independent test. The larvae in the control and exposed groups were homogenized with 0.5% w/v TCA (trichloroacetic acid). The larval homogenates were kept on ice for 30 min and then filtered. Then, 0.5 mL of the filtered tissue homogenates and 0.5 mL of 0.5% w/v TBA (2-thiobarbituric acid) solution were mixed and incubated at 70 °C for 30 min. Then, the absorbance values were measured at 532 nm on a spectrophotometer. The TBARS levels were converted to MDA, and the molar consumption coefficient (1.56 × 105 M−1) was used to achieve results displayed as mg of MDA/kg of sample.The lipid peroxidation tests were carried out by measuring the amounts of malondialdehyde in the control and study groups. Third-instar a et al.  and Tiroa et al. . The appDrosophila larvae (exposed for four days), which were collected, washed carefully, dissected in phosphate buffer , and manipulated in line with our previously described protocol [2HPO4 (disodium hydrogen phosphate), 1.4 mM KH2PO4 (potassium dihydrogen phosphate)) was prepared, and then 0.15 M PBS was diluted with distilled water at a ratio of 2:1 to obtain 0.1 M PBS. Subsequently, 72 ± 4 h old Drosophila larvae were exposed to the lowest and highest concentrations of nanopesticides 2 nanopesticides, 0.01 mM and 5 mM for acephate nanopesticides, and 0.01 mM and 2.5 mM for validamycin nanopesticides, respectively). After 24 h of exposure, midguts from 96 ± 4 h old larvae from the control and treatment groups were dissected in 0.1 M phosphate-buffered saline . Then, a fixative solution containing 4% paraformaldehyde and 1% glutaraldehyde was obtained by adding 4 g of paraformaldehyde and 0.1 mL of glutaraldehyde to 100 mL of 0.15 M PBS. Midguts isolated from the larvae were fixed in a fixative solution for 2 h at +4 °C. Midgut dissection performed in Drosophila specimens is shown in the literature [4) fixative prepared in 1% Sorensen phosphate buffer (prepared with 1% OsO4 and 0.8% potassium hexacyanoferrate II) was fixed on the rotator at room temperature for 2 h. Washing was performed with 0.1 M Sorensen phosphate buffer at room temperature in 3 repetitions for 10 min on the rotator. The bottles were changed in the last wash. In the dehydration process, they were kept in 50% and 70% ethanol through 3 repetitions at +4 °C for 2 × 15 min. En bloc staining, which is an electron microscopy staining technique, was performed at +4 °C for 1 h in a 1% uranyl acetate solution prepared with 75% ethanol. As a continuation of the dehydration process, it was kept in 80%, 90%, and 100% ethanol for 10 min at +4 °C for 3 repetitions. Before the embedding of the samples, the tissues were kept on the rotator with propylene oxide for 10 min and 4 h at room temperature with propylene oxide + araldite master mix + 2% accelerator benzyldimethyl amine (BDMA) (1:1) as an intermediate solution for 2 repetitions. Before the embedding process, the pieces were removed from the bottles with the help of toothpicks, and the excess araldite was removed by rolling on the filter paper. Pieces were taken from this mixture in a separate bottle, rotated at room temperature for 1 night, and embedded in the mixture . After embedding, it was kept at 60 °C for 48 h. Once it had become a block, Cu was placed on grids, and thick and thin sections were obtained. After the samples were examined via TEM, the cellular uptake of nanopesticides through the intestinal barrier into the intestinal lumen of Drosophila larvae was evaluated using the images.Internalization of nanopesticides within the intestinal barrier was assessed in 3rd-instar protocol ,158,159.terature . The folDrosophila larvae once they had been exposed to the chemicals [Intracellular ROS generation was assessed by dichloro-dihydro-fluorescein diacetate (DCFH-DA) screening in hemocytes and midgut cells from hemicals ; the prohemicals ,158,159.hemicals .Drosophila larvae were used as target cells. The procedures described in previously published works in the literature were followed at every stage [Drosophila instant food culture that was pre-wetted with the following concentrations: control (distilled water) at 0, and study groups at different concentrations of permethrin nanopesticides , permethrin , Cu(OH)2 nanopesticides and CuSO4·5H2O , acephate nanopesticides and acephate , and validamycin nanopesticides and validamycin  dispersions, for 24 ± 2 h. Clean distilled water was used as a negative control [DNA breakage was assessed by comet assays, where the hemocytes collected from the ry stage ,163,164. control , while E control ,161 was Escherichia coli bacteria, the levels of oxidized purine and pyrimidine bases can be determined by modifying the comet assay. Endo III shows the number of specifically oxidized pyrimidine bases, while Fpg shows the number of specifically oxidized purine bases [Drosophila larvae under in vivo conditions. Enzyme application was performed by selecting certain enzymes to assess whether the observed DNA damage was caused by purine bases or pyrimidine bases.The comet assay can assess single-stranded DNA damage as well as oxidative damage to DNA using some bacterial enzymes. Therefore, using the bacterial enzymes endonuclease III (Endo III) and formamidopyrimidine DNA glycosylase (Fpg) isolated from ne bases ,82,83,84Hsp70, NM_169441.2), heat-shock protein 83 , catalase , superoxide dismutase , p53 (NM_206544.2), and Ogg1 (NM_132271.5). The second set of genes was associated with the intestinal barrier’s response to physical stress: dual oxidase , hemolectin , mucin 68D , and prophenoloxidase 2 . The expression of the selected genes in treated and untreated larvae was detected through homogenized groups of 30 third-instar larvae (50 mg) in TRIzol® Reagent . RNase-free DNase I  was used to remove DNA contamination. The quantity of mRNA in each sample was determined by means of a NanoDrop device. cDNA was synthesized through the use of 1 mg of total RNA with the Transcriptor First-Strand cDNA Synthesis Kit , and stored at 20 °C. The resulting cDNA was subjected to real-time RT-PCR analysis on a Light Cycler 480  to determine the relative expression of the selected genes by using β-actin as the housekeeping control. The reaction conditions for all genes were as follows: one cycle of pre-incubation for 5 min at 95 °C, and amplification was repeated 45 times . The data were analyzed using the StepOneTM software v 2.2.2. The mean cycle threshold (Ct) value for the targeted gene was used to calculate the relative expression with the relative quantification (RQ) value and formula: RQ = 2−ΔCT × 100, where ΔCT = CT of target gene − CT of an endogenous housekeeping gene. Evaluation of 2−ΔCT reveals the fold change in gene expression, normalized to the internal control (β-actin), enabling the comparison of the larvae treated with different substances [We also examined whether there were changes in the expression of certain genes. The first set of genes was associated with general stress: heat-shock protein 70  assay, comet assay, and gene expression changes) and one-way ANOVA (climbing assay and lipid peroxidation assay) using SigmaPlot version 11.0 . Mean mortality values for 24 h were calculated and compared through Duncan’s multiple range test (p ≤ 0.05), and the LC50 values were calculated using a statistical analysis tool described in [U-test. All data from the experiments are presented as the means of two independent experiments, including duplicates of each one, unless stated otherwise. The values are presented as arithmetic means ± standard error.The normality of variance was analyzed using the Kolmogorov–Smirnov and Shapiro–Wilk tests, and homogeneity of variance was evaluated by Levene’s test. Data with normal distribution and equal variance were analyzed using Student’s ribed in . Data wiD. melanogaster—a eukaryotic in vivo model organism. This completed project provides a useful framework in determining the priorities for future in vivo research towards stronger risk analysis of nanopesticides used in agricultural areas all over the world. The main conclusions can be summarized as follows:(1)Characterization of nanoparticles for size and diameter should be specified in detail by TEM and SEM imaging techniques, along with XRD and HPLC.(2)Endotoxin levels at all different doses of test chemicals were below the detectable limit level (0.116667 EU/mL), and these nanopesticides were not contaminated with endotoxins.(3)Lower doses of the tested nanopesticides showed no hazardous effects on the ability of fruit flies to reach the adult stage.(4)All nanopesticides other than copper-based pesticides caused morphological changes in the abdomen, wing, mouth, and leg regions of adult flies.(5)Drosophila.All nanopesticides at the highest doses—except for copper-based ones—had significant pro-oxidant effects in (6)Drosophila larvae.High doses of pesticides caused significant changes in glutathione and lipid peroxidation formation in (7)Single-stranded and oxidative DNA damage was mediated by oxidative damage to the pyrimidine bases.(8)Nanopesticides and microparticle forms caused no mutagenic and/or recombinogenic effects.(9)All pesticides at the highest doses—except for copper-based ones—caused phenotypic variations across three generations of fruit flies.(10)Nanopesticides and microparticle forms significantly impaired the climbing and walking ability of adult flies.(11)Ingested nanopesticides found their way through the intestinal barrier into the intestinal lumen.(12)Hsp70 and Hsp83), antioxidant defense genes (CAT and SOD2), and a genomic integrity gene (p53).Pesticides/nanopesticides caused a significant increase in the expression of stress genes ((13)DUOX, Hml, Muc68D, and/or PPO2 genes, as well as in the mRNA expression of Ogg1—a gene associated with DNA repair.Nanopesticides and microparticle pesticides induced significant impairment in the expression of the The widespread use of nanopesticides in agricultural areas has recently triggered a scientific interest in determining the merits and drawbacks of nanopesticides. In that regard, this study stands out as an original research endeavor, as it explores the effects of different types of nanopesticides and microparticle forms of pesticides through assays for toxicity, genotoxicity, phenotypic and behavioral changes, biochemical impacts, cellular uptake, and gene expression in