Food contamination with fipronil alters gene expression associated with foraging in Africanized honey bees

Food contamination with fipronil alters gene expression associated with foraging in Africanized honey bees | 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 Food contamination with fipronil alters gene expression associated with foraging in Africanized honey bees Yan Souza Lima, Isabella Cristina de Castro Lippi, Jaine da Luz Scheffer, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4484576/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 15 Aug, 2024 Read the published version in Environmental Science and Pollution Research → Version 1 posted 6 You are reading this latest preprint version Abstract Taking into consideration that bees can be contaminated by pesticides through the ingestion of contaminated floral resources, we can utilize genetic techniques to assess effects that are scarcely observed in behavioral studies. This study aimed to investigate the genetic effects of ingesting lethal and sublethal doses of the insecticide fipronil in foraging honey bees during two periods of acute exposure. Bees were exposed to fipronil through contaminated honey syrup at two dosages (LD50 = 0.19 μg/bee; LD50/100 = 0.0019 μg/bee) and for two durations (one and four hours). Following exposure, we measured syrup consumption per bee, analyzed the transcriptome of bee brain tissue, and identified differentially expressed genes (DEGs), categorizing them functionally based on Gene Ontology (GO). The results revealed a significant genetic response in honey bees after exposure to fipronil, regardless of the dosage used. Fipronil affected various metabolic, transport, and cellular regulation pathways, as well as detoxification processes and xenobiotic substance detection. Additionally, downregulation of several DEGs belonging to the Olfactory Binding Protein (OBP) family was observed, suggesting potential physiological alterations in bees that may lead to disoriented behaviors and reduced foraging efficiency. Foraging bees Foraging efficiency Insecticide Transcriptome Gene expression Detoxification Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Due to changes in agricultural practices and the intensification of agriculture, the use of pesticides has significantly increased worldwide in recent decades (Tudi et al. 2021). This practice has sparked debates and concerns globally, as the indiscriminate and large-scale use of these products has significant negative impacts on both the environment and human health (Bonmatin et al. 2015; Sharma et al. 2019; García et al. 2022). Due to the widespread and incorrect use of pesticides, they can affect areas near application sites, resulting in contamination of natural and conservation areas (Zaller et al. 2022), water bodies (Stehle and Schulz 2015), as well as non-target plants and animals, as observed in bees (Cech et al. 2023). Regarding how bees are contaminated, exposures can occur through ingestion, when bees searching for floral resources are exposed to pesticides through consumption or contact with contaminated food (nectar, pollen, or water) (Rortais et al. 2017). This type of exposure can be continuous and prolonged, as various pesticides are absorbed by plants, reaching nectar and pollen through sap flow (Goulson 2013). Thus, when these floral resources are collected by bees, they can be stored and consumed by other individuals in the colony in sublethal doses, resulting in impacts on bee activities and survival (Rortais et al. 2017). Among these pesticides, fipronil is a broad-spectrum systemic insecticide responsible for contaminating floral resources and various bee products (Bonmatin et al. 2015; Singh et al. 2021). This insecticide acts as a non-competitive blocker of the gamma-aminobutyric acid receptor in the central nervous system of insects, affecting glutamate-dependent chloride channels, causing uncontrolled central nervous system activity, hyperexcitation, convulsions, paralysis, and death of insects (Gunasekara et al. 2007; Gonçalves et al. 2022). Several studies have highlighted the harmful effects of fipronil on Apis mellifera bees, resulting in changes in behavior, locomotion, cognitive abilities, learning, and memory of bees (Pisa et al. 2015; Zaluski et al. 2015; Roat et al. 2017; Bovi et al. 2018). Although studies evaluating the behavioral and physiological effects on bees exposed to pesticides are extremely relevant, various studies have highlighted that molecular genetics approaches are an important tool for detecting harmful effects that are often not identified in studies focused on bee physiology and behavior (Costa et al. 2022). Gene expression pattern analysis has been employed in various studies, investigating the effects of parasites on bees (Zanni et al. 2017), nutritional stress (Corby-Harris et al. 2014), mineral supplementation effects on gene expression (Camilli et al. 2022), as well as transcriptomic studies evaluating the effect of pesticides on the gene expression profile of contaminated bees (Li et al. 2019; Gao et al. 2022; Astolfi et al. 2022). Considering that bees' brains are the main organs affected by neurotoxic pesticides, these pesticides can easily disturb their nervous systems (Li et al. 2019). Therefore, advanced techniques, such as molecular genetics analyses, can be employed to more accurately assess the damage caused by these pesticides to bees (Azevedo and Nocelli 2022). Thus, the aim of this study was to investigate the influence of ingesting lethal and sublethal doses of fipronil insecticide after two periods of acute exposure on the gene expression profile of the brain tissue of Africanized A. mellifera bees in the foraging phase. Materials and methods Collection of forager honey bees Africanized honey bee hives ( A. mellifera ) housed at the experimental apiary of FMVZ - UNESP, Botucatu, São Paulo State, Brazil (22° 50' 28" S, 48° 25' 43" W, elevation 726 m) were used. The climate in the study area is classified as Aw according to the Köppen method. It features well-defined seasons, with hot and rainy summers, and cold and dry winters. The average relative humidity is 70%, with an annual average temperature around 21°C and an accumulated annual precipitation of approximately 1,500 mm (Franco et al. 2023). For the collection of forager bees, ten colonies of Africanized bees ( A. mellifera ) housed in Langstroth hives, standardized in terms of the number of frames containing brood and food, were used. Initially, two frames containing sealed brood from each hive were removed. These frames were protected with bags made of tulle fabric and returned to their respective hives. After a period of 24 hours, the protected frames were again removed from the hives, and the newly emerged bees were marked with a non-toxic pen on the pronotum region (Astolfi et al. 2022; Camilli et al. 2022). Immediately after marking the bees, the tulle bags were removed, and the frames containing the marked bees were returned to their respective hives. After the corresponding period for the development of worker bees into the forager phase (21 days), 180 previously marked bees were collected and placed in perforated Falcon tubes. The tubes containing the marked bees were placed in a freezer (-18°C) for approximately two minutes for anesthesia, allowing for the manipulation of the insects (Zaluski et al. 2015). The forager bees were then distributed on perforated Petri dishes and kept fasting for three hours to empty their honey crop. Exposure of bees to fipronil-contaminated food Both fipronil-contaminated and uncontaminated honey syrup in a 1:1 ratio were provided to bees housed in plates. Dilutions were made with the active ingredient Fipronil (Sigma-Aldrich®; 98.8% purity) to obtain honey syrup solutions with the lethal dose (LD 50 = 0.19 µg/bee) and sublethal dose (LD 50/100 = 0.0019 µg/bee) of the insecticide (Zaluski et al. 2015, Lunardi et al. 2017, Bovi et al. 2018), considering the average consumption of 50 µL of syrup per bee, corresponding to the average volume of the honey crop (Thompson and Hunt 1999). The honey syrup feeders remained available for bee consumption during two exposure periods, one and four hours. In each treatment, 30 bees were used, with an experimental design conducted in sextuplicate, containing five bees per repetition (three doses: LD 50 , LD 50/100 , and control; two exposure periods: one and four hours). The syrup feeders were weighed on an analytical balance before and after the exposure period. Bee syrup consumption was statistically evaluated by ANOVA, and for mean comparison, the Tukey test (p < 0.05) was applied using the R program version 4.4. After exposure, bees were collected and immediately frozen, remaining stored in an ultrafreezer (-80°C) until the time of transcriptomic analysis. RNA-Seq sequencing and analysis of differentially expressed genes For the preparation and sequencing of the RNA-Seq library, honey bees were decapitated, and the compound eyes were removed from each head to prevent potential pigment contamination (Astolfi et al. 2022). Subsequently, the heads were macerated to expose the brain material, which was then used for total RNA extraction. The RNA-Seq sequencing and analysis of differentially expressed genes followed the methodology described by Camilli et al. (2022) with some adaptations. Total RNA was isolated from the brains of A. mellifera foragers, with three brains pooled per sample. The extraction was performed using Trizol reagents following the manufacturer's guidelines (Chomczynski 1993). RNA quality and quantity were assessed using a Qubit fluorometer and an Agilent 2100 Bioanalyzer. RNA degradation was evaluated using a 1% agarose gel. For library preparation, 200 ng of total RNA served as the starting material for Illumina RNA sequencing (RNA-Seq), utilizing the SureSelect Strand Specific RNA Library Preparation Kit. Library quantification was performed via qPCR, followed by dilution to 12 pM. The libraries were then loaded onto the NextSeq flow cell and sequenced using the sequence by synthesis (SBS) method for 50 cycles, resulting in the generation of 150 bp fragments. The FASTQC program (Andrews et al. 2010) assessed adapter content and raw read quality. Trimmomatic v0.39 (Bolger et al. 2014) was used for sequencing quality control. Data alignment utilized the Burrows-Wheeler Aligner (BWA) v0.7.12 with Amel_HAv3.1 as the reference (Andrews et al. 2015). Analysis and visualization were performed using RStudio with ggplot2 v3.3.2 (Wickham et al. 2016). Differential expression analysis employed edgeR v3.30.3 (Robinson et al. 2010). Low-expression genes were filtered based on counts per million, normalized using the TMM method, and analyzed for significant regulation with an adjusted p-value < 0.05 and |log2 FC| ≥ 2 (Benjamini and Hochberg 1995). Classification of differentially expressed genes into functional categories We conducted a Gene Ontology (GO) analysis to categorize genes into functional groups and understand their biological implications in bees' response to insecticide exposure. The Differentially Expressed Genes (DEGs) were grouped into terms describing functions, processes, or cellular components associated with specific genes. This aimed to categorize and detail their functions, and they were then classified into three GO categories: Biological Processes (BP), Molecular Functions (MF), and Cellular Components (CC). The gene ontology analysis was performed on sets of genes found with differential expression, using the R software with the gprofiler2 package (Kolberg et al. 2020). We adjusted p-values in the Fisher's test using the FDR method to correct for multiple comparisons (Benjamini and Hochberg 1995), setting statistical significance for adjusted p-values < 0.05 and |log2 FC| ≥ 2. Results Consumption of food contaminated with fipronil Regarding the dosages of the insecticide provided in the food, significant differences were observed only during one hour of exposure (GL = 2; F = 15.17; p < 0.001). The highest consumption among treatments occurred with bees from the control treatment, followed by the lethal and sublethal doses, respectively (Table 1). Table 1 Mean consumption of fipronil-contaminated honey syrup (μL/bee) with lethal dose (LD 50 = 0.19 μg/bee) and sublethal dose (LD 50/100 = 0.0019 μg/bee) of the insecticide by Africanized honey bees after one and four hours of exposure Exposure time Control Lethal Dose (LD 50 ) Sublethal Dose (LD 50/100 ) 1 hour* 38,49±3,24a 29,77±5,89b 21,41±6,43c 4 hours 39,29±6,07 53,31±9,21 44,37±13,10 *Different letters in the row indicate significant differences by Tukey's test (p > 0.05). Differentially expressed genes (DEGs) altered after acute exposure to the fipronil insecticide in Africanized honey bees Following ingestion of syrup contaminated with the fipronil insecticide during one hour of exposure, there was a change in the expression of a total of 2,391 genes (FC > 2.0 and p < 0.05). Of these, 2,026 genes were differentially expressed at the lethal dosage and 1,829 at the sublethal dosage. A total of 1,464 genes showed altered expression in common for the two tested dosages. Genes exclusively altered at each dosage were also identified, with 562 DEGs for the lethal dose and 365 DEGs for the sublethal dose (Fig 1a). As bees were exposed to the insecticide for a longer period, there was a reduction in the number of differentially expressed genes. Bees contaminated after four hours of exposure showed a total of 147 altered genes (FC > 2.0 and p < 0.05). In the treatment with the lethal dose, 96 DEGs were recorded, while 102 DEGs were observed for the sublethal dose. In both treatments, 51 genes were altered in common (Fig 1b). Additionally, a total of 45 DEGs were exclusively observed in the treatment with the lethal dose, and 51 in bees treated with the sublethal dose. When analyzing the DEGs in bees contaminated with the insecticide, it is possible to distinguish a pattern in gene expression concerning the exposure periods and fipronil dosages used. After one hour of exposure, the majority of DEGs were downregulated, regardless of the dosage used (LD 50 = 1,118 DEGs; LD 50/100 = 1,010 DEGs). However, a significant number of genes were upregulated, 908 and 819, in the lethal and sublethal dosages, respectively (Fig 2a). As bees were exposed to fipronil for a longer period (4 hours), the opposite occurred in the gene expression pattern compared to the one hour exposure, with a greater number of upregulated genes in both tested dosages (LD 50 = 55 DEGs; LD 50/100 = 57 DEGs). However, dozens of genes had their regulation reduced after four hours of exposure, with 41 and 45 downregulated DEGs in the lethal and sublethal dosages, respectively (Fig 2b). The differentially expressed genes, analyzed through Gene Ontology (GO) enrichment analysis and grouped according to their functional categories [Biological Processes (BP), Molecular Functions (MF), and Cellular Components (CC)], revealed a distinct pattern. After one hour of exposure to the insecticide, the majority of DEGs in bees that ingested the lethal dose of fipronil showed higher enrichment in the cellular component category, with 21 terms. Biological Processes and Molecular Functions were enriched with 14 and 10 terms, respectively. Conversely, in the treatment where bees received the sublethal dose of the insecticide, a greater number of terms were enriched in the functional categories, especially in Biological Processes (33 terms), followed by Molecular Functions (24 terms) and Cellular Components (12 terms). All Gene Ontology groupings for all treatments are available in Supplementary Information (SI). The significantly enriched terms in Biological Processes in bees that consumed the lethal and sublethal doses of fipronil after one hour of exposure are mainly associated with biosynthetic and metabolic processes, translation, and transport of macro and micromolecules. In the cellular component category, the enriched terms are predominantly related to ribosomes and their subunits. Many DEGs involved in pathways of the cationic channel complex were also enriched, such as channels of metal ions, voltage-dependent calcium, and potassium channels. The corresponding DEGs to Molecular Functions are involved in pathways associated with ribosomes and voltage-dependent channel activity, being the main enriched terms. GO analysis for the DEGs at the intersection of treatments, i.e., those that occurred in both treatments, showed that in the treatment after one hour of exposure, the biological process category was enriched with 20 terms, followed by Molecular Functions (16 terms) and Cellular Components (10 terms), respectively (Fig 3). The main enriched pathways in the biological process category from GO include translation processes, biosynthetic, and metabolic processes. Molecular Functions were mainly enriched regarding ribosomal structures, activities of molecular structures, and transport channel activities. Similarly, the functional category of Cellular Components had greater significance in DEGs involved with ribosomal structures and transport channels. After four hours of exposure, Molecular Functions exhibited higher enrichment, with eight terms; whereas both Biological Processes and Cellular Components were enriched with only one term each. The DEGs associated with Molecular Functions are mainly linked to monooxygenase activity, oxidoreductase activities, and iron ion and tetrapyrrole bindings. Biological Processes mainly indicate DEGs associated with carbohydrate metabolic processes, while Cellular Components are primarily associated with the extracellular region. On the other hand, bees exposed to the sublethal dose of fipronil for four hours showed enrichment in only two functional categories, with five terms enriching Molecular Functions and only one term enriching Biological Processes. The enriched pathways in Molecular Functions were monooxygenase activity, iron ion and tetrapyrrole bindings, oxidoreductase, and heme; while in Biological Processes, the only enriched pathway was related to the extracellular region. Regarding the intersection performed for the DEGs of the four hour exposure treatment, the Molecular Functions category showed a high number of enriched terms (n = 13 terms), compared to Biological Processes and Cellular Components, with only one term enriching each respective functional category of GO (Fig 4). Similarly to the GO analysis of the DEGs that received the lethal dose of fipronil, the intersection between the DEGs (lethal and sublethal doses) indicates the carbohydrate metabolic process as the only enriched pathway in Biological Processes; enrichment of the pathway associated with the extracellular region in Cellular Components; and in Molecular Functions, monooxygenase and oxidoreductase activity, as well as genes attributed to iron ion, tetrapyrrole, and heme bindings. Considering the large number of DEGs in the respective treatments, Table 2 presents the top ten most significant differentially expressed genes in terms of logFC value for each treatment (FC > 2.0 and p < 0.05); five upregulated and downregulated genes already characterized were selected for each treatment. The complete clustering analysis of all common DEGs after one and four hours of exposure to fipronil insecticide is available in the Supplementary Information (SI). Bees exposed for one hour to the lethal dose of fipronil showed downregulation of genes involved in DNA molecule repair (LOC100577751), immune response (LOC408917), protein synthesis, and cell adhesion (LOC102655710, LOC412791); there was upregulation of genes associated with transport and membrane components (LOC100576458, LOC107965279), odorant detection proteins ( Obp14 ), as well as structural cuticle genes (LOC724199). During the same exposure period (one hour), but with the sublethal dose ingested by bees, genes associated with ATP molecule production (LOC726367), locomotion (LOC551706), and enzymes responsible for protein metabolism (LOC410583) decreased their expression (downregulated). In the same treatment, there was an increase in the regulation of genes mainly associated with protein digestion (LOC551180), receptors and membrane components (LOC100576135, LOC107965279), and muscle contraction (LOC102656116). In treatments where bees were subjected to the insecticide for four hours, there was a similarity in the DEGs, regardless of the doses tested. There was a significant decrease in the regulation of genes encoding odorant-binding proteins ( Obp2 , Obp1 , Obp6 , Obp5 ); whereas the increased expression (upregulation) was represented by genes associated mainly with ribosomal structural components (LD 50 = LOC113219340, LOC113219383, LOC113219387; LD 50/100 = LOC113219340, LOC113219387), phospholipid degradation (LOC107965279), and immune response (LOC113218873). Table 2 Main differentially expressed genes (DEGs) in Africanized honeybees exposed orally to lethal and sublethal doses of the insecticide fipronil for periods of one and four hours Gene Gene description Role logFC Expression 1 hour – Lethal Dose LOC100577751 LIM/homeobox protein Lhx5 DNA repair -6,15 Downregulated LOC408917 Mitogen-activated protein kinase 15 Cellular response to stress -5,98 LOC102655710 Basic proline-rich protein Protein synthesis -5,85 LOC410809 Protein pygopus Metal ion binding -5,72 LOC412791 Flocculation protein FLO11 Cellular adhesion -5,58 LOC100576458 Urea transporter 2 Transmembrane transport activity 4,00 Upregulated Obp14 Odorant binding protein 14 Olfactory receptors 4,09 LOC724199 Early nodulin-75 Structural component of cuticle 4,16 LOC107965279 Pancreatic lipase-related protein 2 Phospholipid degradation 5,46 LOC100576677 DNA repair protein RAD51 homolog 4 DNA repair 5,55 1 hour – Sublethal Dose LOC102655710 Basic proline-rich protein Protein synthesis -6,16 Downregulated LOC726367 Glycerate kinase-like Oxidative phosphorylation -5,98 LOC100577751 LIM/homeobox protein Lhx5 DNA repair -5,97 LOC551706 Unconventional myosin-Ixb Motor activity -5,91 LOC410583 Ornithine aminotransferase, mitochondrial Aminotransferase -5,74 LOC551180 Aminopeptidase N Intermediate protein digestion 3,82 Upregulated LOC100576135 Glutamate receptor 1-like Membrane receptors 3,95 LOC107965279 Pancreatic lipase-related protein 2 Phospholipid degradation 4,37 LOC102656116 Allatotropin-like Muscle contraction 4,93 LOC102654608 Homeobox protein ceh-19 DNA binding 5,55 4 hours – Lethal Dose Obp2 Odorant binding protein 2 Olfactory receptors -11,13 Downregulated Obp1 Odorant binding protein 1 Olfactory receptors -10,65 Obp6 Odorant binding protein 6 Olfactory receptors -9,09 Obp5 Odorant binding protein 5 Olfactory receptors -5,80 LOC726362 4-coumarate--CoA ligase 1 Ligase activity -5,10 LOC113219340 large subunit ribosomal RNA Structural component ribosomes 4,96 Upregulated LOC113218873 Putative leucine-rich repeat-containing protein Immune regulation 4,96 LOC113219383 Large subunit ribosomal RNA Structural component ribosomes 4,96 LOC113219387 Large subunit ribosomal RNA Structural component ribosomes 4,96 LOC107965279 Pancreatic lipase-related protein 2 Phospholipid degradation 4,96 4 hours – Sublethal Dose Obp2 Odorant binding protein 2 Olfactory receptors -10,63 Downregulated Obp1 Odorant binding protein 1 Olfactory receptors -9,78 Obp6 Odorant binding protein 6 Olfactory receptors -9,18 LOC552229 Esterase B1 Hydrolysis activity -6,89 Obp5 Odorant binding protein 5 Olfactory receptors -6,18 LOC112935903 Cytochrome P450 6a14-like Xenobiotic detoxification 3,91 Upregulated LOC113219340 Large subunit ribosomal RNA Structural component ribosomes 4,30 LOC113219387 Large subunit ribosomal RNA Structural component ribosomes 4,91 LOC107965279 Pancreatic lipase-related protein 2 Phospholipid degradation 5,03 LOC113218873 Putative leucine-rich repeat-containing protein Immune regulation 5,11 Discussion Various studies have demonstrated the harmful effect of the insecticide fipronil on honey bees, stingless bees, and wild bees (Zaluski et al. 2015; Roat et al. 2017; Bovi et al. 2018). However, many of these studies have focused on assessing dosage-related mortality or behavioral effects (Renzi et al. 2016; Lunardi et al. 2017; Bovi et al. 2018; Mulvey and Cresswell 2020). Recently, the application of advanced genetic analysis techniques, such as transcriptomics in bees exposed to pesticides, has proven to be a promising approach for comprehensively understanding the effects of these chemicals (Dai 2021; Gao 2020, 2022; Astolfi et al. 2022). The genetic approach has contributed to a greater understanding of the overall effects of pesticides, filling important gaps in understanding their impacts on non-target organisms, such as bees (Fent et al. 2020; Astolfi et al. 2022; Tosi et al. 2022). It was observed that bees showed a significantly higher consumption rate of uncontaminated syrup compared to syrup containing lethal and sublethal doses of fipronil during the one hour period. There is substantial scientific evidence indicating that insecticides can have repellent effects, potentially causing bees to avoid treated areas due to their direct contact or residual presence in flower nectar (Sgolastra et al. 2018; Wu et al. 2021; Cullen et al. 2023). The marked number of DEGs in the one hour exposure treatment (n = 2,391 genes) compared to the four hour period (n = 147 genes) may be associated with a greater immune system response immediately after exposure to fipronil (James and Xu 2012; Decio et al. 2021; Orčić et al. 2022). Insecticide intoxication can induce immune reactions that occur rapidly, requiring the synthesis of new molecules with signaling functions, cellular tasks, and proliferation of additional immune cells (James and Xu 2012; Bajgar et al. 2015; Dolezal et al. 2019). During the first hour of exposure, a more intense stress response likely occurred, which returned to levels closer to normal expression after four hours, leading to a reduction in the number of DEGs over the exposure period (El-Seedi et al. 2022). Regarding the gene ontology analysis, it was possible to identify the main pathways affected in each treatment, associated with their respective enriched functional categories. In the treatment after one hour of exposure, both dosages showed similarity in enriched pathways, with biosynthetic and metabolic processes, translation, and macro and micromolecule transport being the main pathways grouped under BP (Biological Processes). Insecticide exposure can affect a variety of biosynthetic and metabolic processes in insects, including xenobiotic metabolism and monooxygenase activity, which play roles in pesticide detoxification and elimination (Pisa et al. 2015; Nauen et al. 2022; Ranganathan et al. 2022). Additionally, fipronil can interfere with bee hormonal regulation, affecting energy production, altering protein synthesis, and causing disturbances in cellular development and function (Holder et al. 2018; Gonçalves et al. 2022). By acting as an antagonist of the GABA receptor, fipronil disrupts the normal functionality of the insect nervous system, resulting in neuronal hyperexcitation that can affect gene expression in various metabolic, biosynthetic, and transport pathways, including genes involved in protein, carbohydrate, lipid synthesis, and macro and micromolecule transport (Pisa et al. 2015; Gonçalves et al. 2022). Exposure to the insecticide altered gene expression related to ribosomes and their subunits in bees, potentially causing changes in ribosomal RNA synthesis, ribosome assembly, and protein synthesis control (Shi et al. 2017). The alteration of several pathways involved in ribosomes due to pesticide contamination has been reported in multiple studies with other insecticides (Shi et al. 2017; Wu et al. 2017; Gao et al. 2020; Flores et al. 2021), with significant consequences for the metabolism, development, stress response, and cellular function of contaminated bees (Mao and Jing 2007; Shi et al. 2017; Wu et al. 2017). The results obtained from enriched pathways in GO reveal that exposure to fipronil impacts the functions of ion channels, which can lead to disruptions in both ionic balance and electrical signal transmission in cells. By directly blocking these channels, fipronil interferes with the normal flow of ions and modulates the activity of ion channels, altering their opening, closing, or conductance, leading to an imbalance in nerve signal transmission and alteration of physiological functions in these insects (Dong 2007; Murillo et al. 2011). One piece of evidence of fipronil's negative influence on ion channels is its damaging action on insects' Ca 2+ channels, which can have detrimental effects on neuronal signaling, muscle contraction, and energy metabolism, resulting in neuronal dysfunction, behavioral disorders, reduced locomotion, and flight (Dong 2007; Wu et al. 2021). Ca 2+ signals also play a crucial role in bees' learning and memory, as Ca 2+ is used in all stages of odorant information processing, from detection by the antenna to integration, learning, and memorization (Wu et al. 2021; Paten et al. 2022). Regarding the DEGs after one hour of exposure to the lethal dose of the fipronil insecticide, bees showed downregulation in the expression of genes involved in developmental functions and cell adhesion (LIM/homeobox protein Lhx5, Mitogen-activated protein kinase 15, Basic proline-rich protein, Flocculation protein FLO11). The decrease in the expression of these genes may compromise bees' ability to respond adequately to the stress caused by the insecticide and other external stressors, affecting their metabolic adaptation and survival (Zhao et al. 2007; Krizsan et al. 2014; Liu et al. 2020; Farhadi et al. 2023). Additionally, the Protein pygopus gene, associated with the Wnt signaling pathway and essential for biological process development and regulation, also showed downregulation (Parker et al. 2002). According to Martin and Kimelman (2009), the Wnt signaling pathway is involved in the development of the central nervous system of bees, cell fate determination during metamorphosis, and reproductive organ formation. On the other hand, exposure to the lethal dose of fipronil for one hour also showed upregulation of genes mainly involved in detoxification processes and xenobiotic substance detection (Urea transporter 2, Odorant binding protein 14, Early nodulin-75) (Schwaighofer et al. 2014; Nie et al. 2018), as well as genes contributing to lipid metabolism and defense against DNA damage (Pancreatic lipase-related protein 2, DNA repair protein RAD51 homolog 4) (Lee et al. 2010; Collins et al. 2021). When exposed to a sublethal dose of fipronil, after one hour of exposure, downregulation of genes involved in metabolic, developmental, and cellular transport processes (Glycerate kinase-like, LIM/homeobox protein Lhx5, Unconventional myosin-Ixb, Ornithine aminotransferase) was observed, as well as downregulation of the Basic proline-rich protein gene, responsible for encoding the proline-rich protein. Proline-rich proteins are predominantly found in insect hemolymph and may be involved in responding and adapting to different types of stressors, such as environmental (e.g., temperature, humidity, and ultraviolet radiation) and physiological (e.g., dehydration, injuries, and toxins) stressors (Micheu et al. 2000). According to Teulier et al. (2016), prolines may also be involved in the flight of hymenopterans. Although bees primarily use carbohydrates as an energy source, proline can be used as an alternative fuel to support muscle metabolism from resting conditions to the high energy production rate required during flight (Arrese and Soulages 2010; Teulier et al. 2016). In the same treatment (sublethal dose, one hour), upregulation of genes associated with protein and lipid digestion (Aminopeptidase N, Pancreatic lipase-related protein 2), neurotransmitters (Glutamate receptor 1-like), hormonal regulation, and gene expression (Allatotropin-like, Homeobox protein ceh-19) was observed. This suggests a compensatory response of contaminated bees to decrease the negative effects of the insecticide, involving a series of processes such as digestion and nutrient acquisition, synaptic transmission, and cellular development and differentiation (Belzunces et al. 2012; Pashte and Patil 2017). After four hours of exposure to fipronil, the GO showed that the terms of MF (Molecular Functions) were mainly associated with monooxygenase activity, oxidoreductase activities, and iron ion and tetrapyrrole binding. Monooxygenases play an essential role in insects exposed to insecticides, being involved in the metabolism and detoxification of these substances (Haas and Nauem 2021; Nauen et al. 2022). These enzymes are mainly represented by the cytochrome P450 family and are responsible for the chemical modification of insecticides, making them more soluble in water and facilitating their elimination in the insects' bodies (Nauen et al. 2022); overall, the increased activity of monooxygenases may be involved in the process of metabolizing and neutralizing fipronil's toxic compounds to reduce its concentration and minimize its adverse effects on bees (Feyereisen 2012; Haas and Nauem 2021). Bees exposed to fipronil for four hours show similar downregulation of genes, regardless of the tested dosage. With the exception of the Esterase B1 and 4-coumarate--CoA ligase 1 genes, related to antioxidant responses and detoxification in insects (Fent et al. 2020; Zhang et al. 2023); the other downregulated genes belong to the odorant binding protein family – OBPs ( Obp1 , Obp2 , Obp5 , Obp6 ). OBPs are predominantly expressed in chemosensory tissues such as antennae and maxillary palps, being responsible for detecting and discriminating odors by bees (Schwaighofer et al. 2014). These small, water-soluble proteins bind to and transport hydrophobic odor molecules, increasing the sensitivity and specificity of bees' olfactory system (Forêt and Maleszka 2006). Downregulation of Obp genes may have detrimental effects on odor detection and recognition, potentially affecting bees' foraging ability, reducing communication efficiency, hindering pheromone and floral resource identification (Li et al. 2015; Mayack 2023); impacting colony social structure, such as queen recognition, task organization, and defense against invaders (Forêt and Maleszka 2006; Schwaighofer et al. 2014). Genes upregulated after four hours of exposure, regardless of the dosage, were Large subunit ribosomal RNA (LOC113219340, LOC113219383, LOC113219387), Pancreatic lipase-related protein 2, and Putative leucine-rich repeat-containing protein, mainly involved in protein synthesis, lipid digestion, and immune response of insects (Shi et al. 2017; Wu et al. 2017; Gao et al. 2020; Collins et al. 2021). Additionally, upregulation was observed in the expression of the gene related to xenobiotic detoxification (Cytochrome P450 6a14-like). This is a gene responsible for encoding enzymes belonging to the cytochrome P450 family, which play an important role in the metabolism and detoxification of foreign substances, including insecticides (Nauen et al. 2022). Thus, after a stress response in bees exposed to fipronil, the increased expression of the Cytochrome P450 6a14-like gene can be understood as an attempt to neutralize the insecticide's toxic compounds (Fent et al. 2020). In addition to playing a significant role in xenobiotic detoxification, these enzymes are important for the biosynthesis and degradation pathways of endogenous compounds, such as pheromones, ecdysone, and juvenile hormone, which play crucial roles in insect growth and development (Zhao et al. 2018; Nauen et al. 2022). Conclusion This study unveils a robust genetic response in Africanized honey bees following acute exposure to the insecticide fipronil, regardless of the dosage ingested (lethal or sublethal). This is evidenced by the significant number of DEGs within the first hour of fipronil exposure, possibly due to rapid immune system activation. Gene expression analysis demonstrated that the insecticide impacts various Biological Processes, Molecular Functions, and Cellular Components in bees. Furthermore, alterations in the expression of several genes associated with detoxification and substance detection were observed, particularly genes from the Olfactory Binding Protein (OBP) family, indicating physiological changes in odor detection that may lead foraging honey bees to disoriented behaviors and reduced foraging and pollination efficiency. Declarations Author contributions The preparation of the study, data collection, and analysis were carried out by Yan Souza Lima, Isabella Cristina de Castro Lippi, Jaine da Luz Scheffer, and Juliana Sartori Lunardi. Marcus Vinícius Niz Alvarez and Samir Moura Kadri contributed to the genetic and bioinformatics analyses. Ricardo de Oliveira Orsi coordinated the study's development, methodology, analyzed the data, and corrected the manuscript. All authors read and approved the final manuscript text. Funding This work was supported by the Coordination for the Improvement of Higher Education Personnel (CAPES) - Financing Code 001, and by the State of São Paulo Research Foundation (FAPESP), process: 2020/10524-0. Data availability The data supporting this study's fndings are available from the corresponding author upon reasonable request. Ethics approval The use of bees in the study was previously approved by the Ethics Committee on Animal Use CEUA/FMVZ/UNESP (Botucatu, São Paulo State, Brazil), registered under protocol number 0006/2021. Consent to participate Not applicable. Consent to publish All authors have studied the manuscript thoroughly and consented to the publication. Competing interests The authors declare no competing interests. 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Orsi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3ElEQVRIiWNgGAWjYDACZhDBBmEf+ACh2YjXcnAGUVqQ1TDzEKPFvJ354eeKMhsGfunjDw/btt2Rk5/dwPa4Ao8WmcNsxpJnzqUxSPblGBzObXtmbHDnALvhGTxaJICOkWxsO8xgcIaHAajlcOIGiQQ2yQb8Wph/QrSwPzhs2Xa4fv4MwlrYoLYwGBxmbDucwHCDoBY2M8uGc2k8kj08Bgd7zh023HDnYLshXi38hx/fbCizkePnYX/84UfZYXn52c3HHuLTAgM8SKYwEqMB1WJSNYyCUTAKRsFwBwDQbUcgkvGmcwAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0001-8099-8277","institution":"UNESP: Universidade Estadual Paulista Julio de Mesquita Filho","correspondingAuthor":true,"prefix":"","firstName":"Ricardo","middleName":"de Oliveira","lastName":"Orsi","suffix":""}],"badges":[],"createdAt":"2024-05-27 11:13:07","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4484576/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4484576/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11356-024-34695-8","type":"published","date":"2024-08-15T15:58:26+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":59400452,"identity":"50a27234-279c-4d0b-bea7-373adabdd073","added_by":"auto","created_at":"2024-07-01 10:06:47","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":55444,"visible":true,"origin":"","legend":"\u003cp\u003eVenn diagram showing the DEGs in Africanized honeybees exposed orally to fipronil insecticide at lethal (LD\u003csub\u003e50\u003c/sub\u003e) and sublethal (LD\u003csub\u003e50/100\u003c/sub\u003e) doses after one (a) and four hours (b)\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4484576/v1/5a8aed60637fc88ca61844fe.png"},{"id":59400456,"identity":"393f4abe-d409-41b3-8e87-3f1df33dd8c1","added_by":"auto","created_at":"2024-07-01 10:06:48","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":43069,"visible":true,"origin":"","legend":"\u003cp\u003eHistogram depicting the downregulated and upregulated DEGs in Africanized honeybees exposed orally to fipronil insecticide at lethal (LD\u003csub\u003e50\u003c/sub\u003e) and sublethal (LD\u003csub\u003e50/100\u003c/sub\u003e) doses after one (a) and four hours (b)\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4484576/v1/0a66298197a52e6809650a4a.png"},{"id":59400455,"identity":"035dc98b-5b93-4b18-85a9-2dd0d57cc57e","added_by":"auto","created_at":"2024-07-01 10:06:48","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":140258,"visible":true,"origin":"","legend":"\u003cp\u003eGene Ontology (GO) analysis of the intersection between Africanized honey bees that ingested food contaminated with lethal and sublethal doses of fipronil after one hour of exposure [Red indicates Biological Processes (BP), green indicates Cellular Components (CC), and blue indicates Molecular Functions (MF)]\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4484576/v1/414d5d23c35831bd16908c31.png"},{"id":59400453,"identity":"97fb1d36-ac0b-4af7-8285-21e6766c0560","added_by":"auto","created_at":"2024-07-01 10:06:48","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":53745,"visible":true,"origin":"","legend":"\u003cp\u003eGene Ontology (GO) analysis of the intersection between Africanized honey bees that ingested food contaminated with lethal and sublethal doses of fipronil after four hours of exposure [Red indicates Biological Processes (BP), green indicates Cellular Components (CC), and blue indicates Molecular Functions (MF)]\u003c/p\u003e","description":"","filename":"42.png","url":"https://assets-eu.researchsquare.com/files/rs-4484576/v1/99b1b42e9c23a17bf3a014cd.png"},{"id":63071381,"identity":"5a8dc3f8-e68e-4b7e-b78f-832595a2f7af","added_by":"auto","created_at":"2024-08-22 20:06:43","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":920566,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4484576/v1/1e8556e5-0346-40ce-8917-6422681b657a.pdf"}],"financialInterests":"","formattedTitle":"Food contamination with fipronil alters gene expression associated with foraging in Africanized honey bees","fulltext":[{"header":"Introduction","content":"\u003cp\u003eDue to changes in agricultural practices and the intensification of agriculture, the use of pesticides has significantly increased worldwide in recent decades (Tudi et al. 2021). This practice has sparked debates and concerns globally, as the indiscriminate and large-scale use of these products has significant negative impacts on both the environment and human health (Bonmatin et al. 2015; Sharma et al. 2019; Garc\u0026iacute;a et al. 2022). Due to the widespread and incorrect use of pesticides, they can affect areas near application sites, resulting in contamination of natural and conservation areas (Zaller et al. 2022), water bodies (Stehle and Schulz 2015), as well as non-target plants and animals, as observed in bees (Cech et al. 2023).\u003c/p\u003e\n\u003cp\u003eRegarding how bees are contaminated, exposures can occur through ingestion, when bees searching for floral resources are exposed to pesticides through consumption or contact with contaminated food (nectar, pollen, or water) (Rortais et al. 2017). This type of exposure can be continuous and prolonged, as various pesticides are absorbed by plants, reaching nectar and pollen through sap flow (Goulson 2013). Thus, when these floral resources are collected by bees, they can be stored and consumed by other individuals in the colony in sublethal doses, resulting in impacts on bee activities and survival (Rortais et al. 2017).\u003c/p\u003e\n\u003cp\u003eAmong these pesticides, fipronil is a broad-spectrum systemic insecticide responsible for contaminating floral resources and various bee products (Bonmatin et al. 2015; Singh et al. 2021). This insecticide acts as a non-competitive blocker of the gamma-aminobutyric acid receptor in the central nervous system of insects, affecting glutamate-dependent chloride channels, causing uncontrolled central nervous system activity, hyperexcitation, convulsions, paralysis, and death of insects (Gunasekara et al. 2007; Gon\u0026ccedil;alves et al. 2022).\u003c/p\u003e\n\u003cp\u003eSeveral studies have highlighted the harmful effects of fipronil on \u003cem\u003eApis mellifera\u003c/em\u003e bees, resulting in changes in behavior, locomotion, cognitive abilities, learning, and memory of bees (Pisa et al. 2015; Zaluski et al. 2015; Roat et al. 2017; Bovi et al. 2018). Although studies evaluating the behavioral and physiological effects on bees exposed to pesticides are extremely relevant, various studies have highlighted that molecular genetics approaches are an important tool for detecting harmful effects that are often not identified in studies focused on bee physiology and behavior (Costa et al. 2022).\u003c/p\u003e\n\u003cp\u003eGene expression pattern analysis has been employed in various studies, investigating the effects of parasites on bees (Zanni et al. 2017), nutritional stress (Corby-Harris et al. 2014), mineral supplementation effects on gene expression (Camilli et al. 2022), as well as transcriptomic studies evaluating the effect of pesticides on the gene expression profile of contaminated bees (Li et al. 2019; Gao et al. 2022; Astolfi et al. 2022). Considering that bees\u0026apos; brains are the main organs affected by neurotoxic pesticides, these pesticides can easily disturb their nervous systems (Li et al. 2019). Therefore, advanced techniques, such as molecular genetics analyses, can be employed to more accurately assess the damage caused by these pesticides to bees (Azevedo and Nocelli 2022).\u003c/p\u003e\n\u003cp\u003eThus, the aim of this study was to investigate the influence of ingesting lethal and sublethal doses of fipronil insecticide after two periods of acute exposure on the gene expression profile of the brain tissue of Africanized \u003cem\u003eA. mellifera\u003c/em\u003e bees in the foraging phase.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cstrong\u003eCollection of forager honey bees\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfricanized honey bee hives (\u003cem\u003eA. mellifera\u003c/em\u003e) housed at the experimental apiary of FMVZ - UNESP, Botucatu, S\u0026atilde;o Paulo State, Brazil (22\u0026deg; 50\u0026apos; 28\u0026quot; S, 48\u0026deg; 25\u0026apos; 43\u0026quot; W, elevation 726 m) were used. The climate in the study area is classified as Aw according to the K\u0026ouml;ppen method. It features well-defined seasons, with hot and rainy summers, and cold and dry winters. The average relative humidity is 70%, with an annual average temperature around 21\u0026deg;C and an accumulated annual precipitation of approximately 1,500 mm (Franco et al. 2023).\u003c/p\u003e\n\u003cp\u003eFor the collection of forager bees, ten colonies of Africanized bees (\u003cem\u003eA. mellifera\u003c/em\u003e) housed in Langstroth hives, standardized in terms of the number of frames containing brood and food, were used. Initially, two frames containing sealed brood from each hive were removed. These frames were protected with bags made of tulle fabric and returned to their respective hives. After a period of 24 hours, the protected frames were again removed from the hives, and the newly emerged bees were marked with a non-toxic pen on the pronotum region (Astolfi et al. 2022; Camilli et al. 2022). Immediately after marking the bees, the tulle bags were removed, and the frames containing the marked bees were returned to their respective hives. After the corresponding period for the development of worker bees into the forager phase (21 days), 180 previously marked bees were collected and placed in perforated Falcon tubes.\u003c/p\u003e\n\u003cp\u003eThe tubes containing the marked bees were placed in a freezer (-18\u0026deg;C) for approximately two minutes for anesthesia, allowing for the manipulation of the insects (Zaluski et al. 2015). The forager bees were then distributed on perforated Petri dishes and kept fasting for three hours to empty their honey crop.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExposure of bees to fipronil-contaminated food\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBoth fipronil-contaminated and uncontaminated honey syrup in a 1:1 ratio were provided to bees housed in plates. Dilutions were made with the active ingredient Fipronil (Sigma-Aldrich\u0026reg;; 98.8% purity) to obtain honey syrup solutions with the lethal dose (LD\u003csub\u003e50\u003c/sub\u003e = 0.19 \u0026micro;g/bee) and sublethal dose (LD\u003csub\u003e50/100\u0026nbsp;\u003c/sub\u003e= 0.0019 \u0026micro;g/bee) of the insecticide (Zaluski et al. 2015, Lunardi et al. 2017, Bovi et al. 2018), considering the average consumption of 50 \u0026micro;L of syrup per bee, corresponding to the average volume of the honey crop (Thompson and Hunt 1999).\u003c/p\u003e\n\u003cp\u003eThe honey syrup feeders remained available for bee consumption during two exposure periods, one and four hours. In each treatment, 30 bees were used, with an experimental design conducted in sextuplicate, containing five bees per repetition (three doses: LD\u003csub\u003e50\u003c/sub\u003e, LD\u003csub\u003e50/100\u003c/sub\u003e, and control; two exposure periods: one and four hours).\u003c/p\u003e\n\u003cp\u003eThe syrup feeders were weighed on an analytical balance before and after the exposure period. Bee syrup consumption was statistically evaluated by ANOVA, and for mean comparison, the Tukey test (p \u0026lt; 0.05) was applied using the R program version 4.4. After exposure, bees were collected and immediately frozen, remaining stored in an ultrafreezer (-80\u0026deg;C) until the time of transcriptomic analysis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRNA-Seq sequencing and analysis of differentially expressed genes\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor the preparation and sequencing of the RNA-Seq library, honey bees were decapitated, and the compound eyes were removed from each head to prevent potential pigment contamination (Astolfi et al. 2022). Subsequently, the heads were macerated to expose the brain material, which was then used for total RNA extraction.\u003c/p\u003e\n\u003cp\u003eThe RNA-Seq sequencing and analysis of differentially expressed genes followed the methodology described by Camilli et al. (2022) with some adaptations. Total RNA was isolated from the brains of A. mellifera foragers, with three brains pooled per sample. The extraction was performed using Trizol reagents following the manufacturer\u0026apos;s guidelines (Chomczynski 1993). RNA quality and quantity were assessed using a Qubit fluorometer and an Agilent 2100 Bioanalyzer. RNA degradation was evaluated using a 1% agarose gel. For library preparation, 200 ng of total RNA served as the starting material for Illumina RNA sequencing (RNA-Seq), utilizing the SureSelect Strand Specific RNA Library Preparation Kit. Library quantification was performed via qPCR, followed by dilution to 12 pM. The libraries were then loaded onto the NextSeq flow cell and sequenced using the sequence by synthesis (SBS) method for 50 cycles, resulting in the generation of 150 bp fragments.\u003c/p\u003e\n\u003cp\u003eThe FASTQC program (Andrews et al. 2010) assessed adapter content and raw read quality. Trimmomatic v0.39 (Bolger et al. 2014) was used for sequencing quality control. Data alignment utilized the Burrows-Wheeler Aligner (BWA) v0.7.12 with Amel_HAv3.1 as the reference (Andrews et al. 2015). Analysis and visualization were performed using RStudio with ggplot2 v3.3.2 (Wickham et al. 2016). Differential expression analysis employed edgeR v3.30.3 (Robinson et al. 2010). Low-expression genes were filtered based on counts per million, normalized using the TMM method, and analyzed for significant regulation with an adjusted p-value \u0026lt; 0.05 and |log2 FC| \u0026ge; 2 (Benjamini and Hochberg 1995).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClassification of differentially expressed genes into functional categories\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe conducted a Gene Ontology (GO) analysis to categorize genes into functional groups and understand their biological implications in bees\u0026apos; response to insecticide exposure. The Differentially Expressed Genes (DEGs) were grouped into terms describing functions, processes, or cellular components associated with specific genes. This aimed to categorize and detail their functions, and they were then classified into three GO categories: Biological Processes (BP), Molecular Functions (MF), and Cellular Components (CC). The gene ontology analysis was performed on sets of genes found with differential expression, using the R software with the gprofiler2 package (Kolberg et al. 2020). We adjusted p-values in the Fisher\u0026apos;s test using the FDR method to correct for multiple comparisons (Benjamini and Hochberg 1995), setting statistical significance for adjusted p-values \u0026lt; 0.05 and |log2 FC| \u0026ge; 2.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eConsumption of food contaminated with fipronil\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRegarding the dosages of the insecticide provided in the food, significant differences were observed only during one hour of exposure (GL = 2; F = 15.17; p \u0026lt; 0.001). The highest consumption among treatments occurred with bees from the control treatment, followed by the lethal and sublethal doses, respectively (Table 1).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1\u003c/strong\u003e Mean consumption of fipronil-contaminated honey syrup (\u0026mu;L/bee) with lethal dose (LD\u003csub\u003e50\u0026nbsp;\u003c/sub\u003e=\u0026nbsp;0.19 \u0026mu;g/bee) and sublethal dose (LD\u003csub\u003e50/100\u0026nbsp;\u003c/sub\u003e=\u0026nbsp;0.0019 \u0026mu;g/bee) of the insecticide by Africanized honey bees after one and four hours of exposure\u003c/p\u003e\n\u003cdiv align=\"\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"568\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"25%\"\u003e\n \u003cp\u003eExposure time\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\"\u003e\n \u003cp\u003eControl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\"\u003e\n \u003cp\u003eLethal Dose\u003c/p\u003e\n \u003cp\u003e(LD\u003csub\u003e50\u003c/sub\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\"\u003e\n \u003cp\u003eSublethal Dose\u003c/p\u003e\n \u003cp\u003e(LD\u003csub\u003e50/100\u003c/sub\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25%\"\u003e\n \u003cp\u003e1 hour*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\"\u003e\n \u003cp\u003e38,49\u0026plusmn;3,24a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\"\u003e\n \u003cp\u003e29,77\u0026plusmn;5,89b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\"\u003e\n \u003cp\u003e21,41\u0026plusmn;6,43c\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25%\"\u003e\n \u003cp\u003e4 hours\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\"\u003e\n \u003cp\u003e39,29\u0026plusmn;6,07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\"\u003e\n \u003cp\u003e53,31\u0026plusmn;9,21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\"\u003e\n \u003cp\u003e44,37\u0026plusmn;13,10\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u0026nbsp;*Different letters in the row indicate significant differences by Tukey\u0026apos;s test (p \u0026gt; 0.05).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDifferentially expressed genes (DEGs) altered after acute exposure to the fipronil insecticide in Africanized honey bees\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFollowing ingestion of syrup contaminated with the fipronil insecticide during one hour of exposure, there was a change in the expression of a total of 2,391 genes (FC \u0026gt; 2.0 and p \u0026lt; 0.05). Of these, 2,026 genes were differentially expressed at the lethal dosage and 1,829 at the sublethal dosage. A total of 1,464 genes showed altered expression in common for the two tested dosages. Genes exclusively altered at each dosage were also identified, with 562 DEGs for the lethal dose and 365 DEGs for the sublethal dose (Fig 1a).\u003c/p\u003e\n\u003cp\u003eAs bees were exposed to the insecticide for a longer period, there was a reduction in the number of differentially expressed genes. Bees contaminated after four hours of exposure showed a total of 147 altered genes (FC \u0026gt; 2.0 and p \u0026lt; 0.05). In the treatment with the lethal dose, 96 DEGs were recorded, while 102 DEGs were observed for the sublethal dose. In both treatments, 51 genes were altered in common (Fig 1b). Additionally, a total of 45 DEGs were exclusively observed in the treatment with the lethal dose, and 51 in bees treated with the sublethal dose.\u003c/p\u003e\n\u003cp\u003eWhen analyzing the DEGs in bees contaminated with the insecticide, it is possible to distinguish a pattern in gene expression concerning the exposure periods and fipronil dosages used. After one hour of exposure, the majority of DEGs were downregulated, regardless of the dosage used (LD\u003csub\u003e50\u003c/sub\u003e = 1,118 DEGs; LD\u003csub\u003e50/100\u0026nbsp;\u003c/sub\u003e= 1,010 DEGs). However, a significant number of genes were upregulated, 908 and 819, in the lethal and sublethal dosages, respectively (Fig 2a). As bees were exposed to fipronil for a longer period (4 hours), the opposite occurred in the gene expression pattern compared to the one hour exposure, with a greater number of upregulated genes in both tested dosages (LD\u003csub\u003e50\u003c/sub\u003e = 55 DEGs; LD\u003csub\u003e50/100\u0026nbsp;\u003c/sub\u003e= 57 DEGs). However, dozens of genes had their regulation reduced after four hours of exposure, with 41 and 45 downregulated DEGs in the lethal and sublethal dosages, respectively (Fig 2b).\u003c/p\u003e\n\u003cp\u003eThe differentially expressed genes, analyzed through Gene Ontology (GO) enrichment analysis and grouped according to their functional categories [Biological Processes (BP), Molecular Functions (MF), and Cellular Components (CC)], revealed a distinct pattern. After one hour of exposure to the insecticide, the majority of DEGs in bees that ingested the lethal dose of fipronil showed higher enrichment in the cellular component category, with 21 terms. Biological Processes and Molecular Functions were enriched with 14 and 10 terms, respectively. Conversely, in the treatment where bees received the sublethal dose of the insecticide, a greater number of terms were enriched in the functional categories, especially in Biological Processes (33 terms), followed by Molecular Functions (24 terms) and Cellular Components (12 terms). All Gene Ontology groupings for all treatments are available in Supplementary Information (SI).\u003c/p\u003e\n\u003cp\u003eThe significantly enriched terms in Biological Processes in bees that consumed the lethal and sublethal doses of fipronil after one hour of exposure are mainly associated with biosynthetic and metabolic processes, translation, and transport of macro and micromolecules. In the cellular component category, the enriched terms are predominantly related to ribosomes and their subunits. Many DEGs involved in pathways of the cationic channel complex were also enriched, such as channels of metal ions, voltage-dependent calcium, and potassium channels. The corresponding DEGs to Molecular Functions are involved in pathways associated with ribosomes and voltage-dependent channel activity, being the main enriched terms.\u003c/p\u003e\n\u003cp\u003eGO analysis for the DEGs at the intersection of treatments, i.e., those that occurred in both treatments, showed that in the treatment after one hour of exposure, the biological process category was enriched with 20 terms, followed by Molecular Functions (16 terms) and Cellular Components (10 terms), respectively (Fig 3). The main enriched pathways in the biological process category from GO include translation processes, biosynthetic, and metabolic processes. Molecular Functions were mainly enriched regarding ribosomal structures, activities of molecular structures, and transport channel activities. Similarly, the functional category of Cellular Components had greater significance in DEGs involved with ribosomal structures and transport channels.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAfter four hours of exposure, Molecular Functions exhibited higher enrichment, with eight terms; whereas both Biological Processes and Cellular Components were enriched with only one term each. The DEGs associated with Molecular Functions are mainly linked to monooxygenase activity, oxidoreductase activities, and iron ion and tetrapyrrole bindings. Biological Processes mainly indicate DEGs associated with carbohydrate metabolic processes, while Cellular Components are primarily associated with the extracellular region.\u003c/p\u003e\n\u003cp\u003eOn the other hand, bees exposed to the sublethal dose of fipronil for four hours showed enrichment in only two functional categories, with five terms enriching Molecular Functions and only one term enriching Biological Processes. The enriched pathways in Molecular Functions were monooxygenase activity, iron ion and tetrapyrrole bindings, oxidoreductase, and heme; while in Biological Processes, the only enriched pathway was related to the extracellular region.\u003c/p\u003e\n\u003cp\u003eRegarding the intersection performed for the DEGs of the four hour exposure treatment, the Molecular Functions category showed a high number of enriched terms (n = 13 terms), compared to Biological Processes and Cellular Components, with only one term enriching each respective functional category of GO (Fig 4).\u003c/p\u003e\n\u003cp\u003eSimilarly to the GO analysis of the DEGs that received the lethal dose of fipronil, the intersection between the DEGs (lethal and sublethal doses) indicates the carbohydrate metabolic process as the only enriched pathway in Biological Processes; enrichment of the pathway associated with the extracellular region in Cellular Components; and in Molecular Functions, monooxygenase and oxidoreductase activity, as well as genes attributed to iron ion, tetrapyrrole, and heme bindings.\u003c/p\u003e\n\u003cp\u003eConsidering the large number of DEGs in the respective treatments, Table 2 presents the top ten most significant differentially expressed genes in terms of logFC value for each treatment (FC \u0026gt; 2.0 and p \u0026lt; 0.05); five upregulated and downregulated genes already characterized were selected for each treatment. The complete clustering analysis of all common DEGs after one and four hours of exposure to fipronil insecticide is available in the Supplementary Information (SI).\u003c/p\u003e\n\u003cp\u003eBees exposed for one hour to the lethal dose of fipronil showed downregulation of genes involved in DNA molecule repair (LOC100577751), immune response (LOC408917), protein synthesis, and cell adhesion (LOC102655710, LOC412791); there was upregulation of genes associated with transport and membrane components (LOC100576458, LOC107965279), odorant detection proteins (\u003cem\u003eObp14\u003c/em\u003e), as well as structural cuticle genes (LOC724199).\u003c/p\u003e\n\u003cp\u003eDuring the same exposure period (one hour), but with the sublethal dose ingested by bees, genes associated with ATP molecule production (LOC726367), locomotion (LOC551706), and enzymes responsible for protein metabolism (LOC410583) decreased their expression (downregulated). In the same treatment, there was an increase in the regulation of genes mainly associated with protein digestion (LOC551180), receptors and membrane components (LOC100576135, LOC107965279), and muscle contraction (LOC102656116).\u003c/p\u003e\n\u003cp\u003eIn treatments where bees were subjected to the insecticide for four hours, there was a similarity in the DEGs, regardless of the doses tested. There was a significant decrease in the regulation of genes encoding odorant-binding proteins (\u003cem\u003eObp2\u003c/em\u003e,\u003cem\u003e\u0026nbsp;Obp1\u003c/em\u003e,\u003cem\u003e\u0026nbsp;Obp6\u003c/em\u003e,\u003cem\u003e\u0026nbsp;Obp5\u003c/em\u003e); whereas the increased expression (upregulation) was represented by genes associated mainly with ribosomal structural components (LD\u003csub\u003e50\u003c/sub\u003e = LOC113219340, LOC113219383, LOC113219387; LD\u003csub\u003e50/100\u0026nbsp;\u003c/sub\u003e= LOC113219340, LOC113219387), phospholipid degradation (LOC107965279), and immune response (LOC113218873).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2\u003c/strong\u003e Main differentially expressed genes (DEGs) in Africanized honeybees exposed orally to lethal and sublethal doses of the insecticide fipronil for periods of one and four hours\u003c/p\u003e\n\u003cdiv align=\"center\"\u003e\n \u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"904\"\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003ctd width=\"13.067552602436324%\"\u003e\n \u003cp\u003eGene\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"34.21926910299003%\"\u003e\n \u003cp\u003eGene description\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"31.561461794019934%\"\u003e\n \u003cp\u003eRole\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.862679955703212%\"\u003e\n \u003cp\u003elogFC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.289036544850498%\" valign=\"top\"\u003e\n \u003cp\u003eExpression\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"100%\" colspan=\"5\"\u003e\n \u003cp\u003e1 hour \u0026ndash; Lethal Dose\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"13.067552602436324%\"\u003e\n \u003cp\u003eLOC100577751\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"34.21926910299003%\"\u003e\n \u003cp\u003eLIM/homeobox protein Lhx5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"31.561461794019934%\"\u003e\n \u003cp\u003eDNA repair\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.862679955703212%\"\u003e\n \u003cp\u003e-6,15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.289036544850498%\" rowspan=\"5\"\u003e\n \u003cp\u003e\u003cem\u003eDownregulated\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.070242656449553%\"\u003e\n \u003cp\u003eLOC408917\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"39.46360153256705%\"\u003e\n \u003cp\u003eMitogen-activated protein kinase 15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"36.39846743295019%\"\u003e\n \u003cp\u003eCellular response to stress\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.067688378033205%\"\u003e\n \u003cp\u003e-5,98\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.070242656449553%\"\u003e\n \u003cp\u003eLOC102655710\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"39.46360153256705%\"\u003e\n \u003cp\u003eBasic proline-rich protein\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"36.39846743295019%\"\u003e\n \u003cp\u003eProtein synthesis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.067688378033205%\"\u003e\n \u003cp\u003e-5,85\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.070242656449553%\"\u003e\n \u003cp\u003eLOC410809\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"39.46360153256705%\"\u003e\n \u003cp\u003eProtein pygopus\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"36.39846743295019%\"\u003e\n \u003cp\u003eMetal ion binding\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.067688378033205%\"\u003e\n \u003cp\u003e-5,72\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.070242656449553%\"\u003e\n \u003cp\u003eLOC412791\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"39.46360153256705%\"\u003e\n \u003cp\u003eFlocculation protein FLO11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"36.39846743295019%\"\u003e\n \u003cp\u003eCellular adhesion\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.067688378033205%\"\u003e\n \u003cp\u003e-5,58\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"13.067552602436324%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"34.21926910299003%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"31.561461794019934%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.862679955703212%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.289036544850498%\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"13.067552602436324%\"\u003e\n \u003cp\u003eLOC100576458\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"34.21926910299003%\"\u003e\n \u003cp\u003eUrea transporter 2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"31.561461794019934%\"\u003e\n \u003cp\u003eTransmembrane transport activity\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.862679955703212%\"\u003e\n \u003cp\u003e4,00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.289036544850498%\" rowspan=\"5\"\u003e\n \u003cp\u003e\u003cem\u003eUpregulated\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.070242656449553%\"\u003e\n \u003cp\u003e\u003cem\u003eObp14\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"39.46360153256705%\"\u003e\n \u003cp\u003eOdorant binding protein 14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"36.39846743295019%\"\u003e\n \u003cp\u003eOlfactory receptors\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.067688378033205%\"\u003e\n \u003cp\u003e4,09\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.070242656449553%\"\u003e\n \u003cp\u003eLOC724199\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"39.46360153256705%\"\u003e\n \u003cp\u003eEarly nodulin-75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"36.39846743295019%\"\u003e\n \u003cp\u003eStructural component of cuticle\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.067688378033205%\"\u003e\n \u003cp\u003e4,16\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.070242656449553%\"\u003e\n \u003cp\u003eLOC107965279\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"39.46360153256705%\"\u003e\n \u003cp\u003ePancreatic lipase-related protein 2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"36.39846743295019%\"\u003e\n \u003cp\u003ePhospholipid degradation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.067688378033205%\"\u003e\n \u003cp\u003e5,46\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.070242656449553%\"\u003e\n \u003cp\u003eLOC100576677\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"39.46360153256705%\"\u003e\n \u003cp\u003eDNA repair protein RAD51 homolog 4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"36.39846743295019%\"\u003e\n \u003cp\u003eDNA repair\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.067688378033205%\"\u003e\n \u003cp\u003e5,55\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"100%\" colspan=\"5\"\u003e\n \u003cp\u003e1 hour \u0026ndash; Sublethal Dose\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"13.067552602436324%\"\u003e\n \u003cp\u003eLOC102655710\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"34.21926910299003%\"\u003e\n \u003cp\u003eBasic proline-rich protein\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"31.561461794019934%\"\u003e\n \u003cp\u003eProtein synthesis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.862679955703212%\"\u003e\n \u003cp\u003e-6,16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.289036544850498%\" rowspan=\"5\"\u003e\n \u003cp\u003e\u003cem\u003eDownregulated\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.070242656449553%\"\u003e\n \u003cp\u003eLOC726367\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"39.46360153256705%\"\u003e\n \u003cp\u003eGlycerate kinase-like\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"36.39846743295019%\"\u003e\n \u003cp\u003eOxidative phosphorylation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.067688378033205%\"\u003e\n \u003cp\u003e-5,98\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.070242656449553%\"\u003e\n \u003cp\u003eLOC100577751\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"39.46360153256705%\"\u003e\n \u003cp\u003eLIM/homeobox protein Lhx5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"36.39846743295019%\"\u003e\n \u003cp\u003eDNA repair\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.067688378033205%\"\u003e\n \u003cp\u003e-5,97\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.070242656449553%\"\u003e\n \u003cp\u003eLOC551706\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"39.46360153256705%\"\u003e\n \u003cp\u003eUnconventional myosin-Ixb\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"36.39846743295019%\"\u003e\n \u003cp\u003eMotor activity\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.067688378033205%\"\u003e\n \u003cp\u003e-5,91\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.070242656449553%\"\u003e\n \u003cp\u003eLOC410583\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"39.46360153256705%\"\u003e\n \u003cp\u003eOrnithine aminotransferase, mitochondrial\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"36.39846743295019%\"\u003e\n \u003cp\u003eAminotransferase\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.067688378033205%\"\u003e\n \u003cp\u003e-5,74\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"13.067552602436324%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"34.21926910299003%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"31.561461794019934%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.862679955703212%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.289036544850498%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"13.067552602436324%\"\u003e\n \u003cp\u003eLOC551180\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"34.21926910299003%\"\u003e\n \u003cp\u003eAminopeptidase N\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"31.561461794019934%\"\u003e\n \u003cp\u003eIntermediate protein digestion\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.862679955703212%\"\u003e\n \u003cp\u003e3,82\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.289036544850498%\" rowspan=\"5\"\u003e\n \u003cp\u003e\u003cem\u003eUpregulated\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.070242656449553%\"\u003e\n \u003cp\u003eLOC100576135\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"39.46360153256705%\"\u003e\n \u003cp\u003eGlutamate receptor 1-like\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"36.39846743295019%\"\u003e\n \u003cp\u003eMembrane receptors\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.067688378033205%\"\u003e\n \u003cp\u003e3,95\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.070242656449553%\"\u003e\n \u003cp\u003eLOC107965279\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"39.46360153256705%\"\u003e\n \u003cp\u003ePancreatic lipase-related protein 2\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"36.39846743295019%\"\u003e\n \u003cp\u003ePhospholipid degradation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.067688378033205%\"\u003e\n \u003cp\u003e4,37\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.070242656449553%\"\u003e\n \u003cp\u003eLOC102656116\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"39.46360153256705%\"\u003e\n \u003cp\u003eAllatotropin-like\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"36.39846743295019%\"\u003e\n \u003cp\u003eMuscle contraction\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.067688378033205%\"\u003e\n \u003cp\u003e4,93\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.070242656449553%\"\u003e\n \u003cp\u003eLOC102654608\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"39.46360153256705%\"\u003e\n \u003cp\u003eHomeobox protein ceh-19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"36.39846743295019%\"\u003e\n \u003cp\u003eDNA binding\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.067688378033205%\"\u003e\n \u003cp\u003e5,55\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"100%\" colspan=\"5\"\u003e\n \u003cp\u003e4 hours \u0026ndash; Lethal Dose\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"13.067552602436324%\"\u003e\n \u003cp\u003e\u003cem\u003eObp2\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"34.21926910299003%\"\u003e\n \u003cp\u003eOdorant binding protein 2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"31.561461794019934%\"\u003e\n \u003cp\u003eOlfactory receptors\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.862679955703212%\"\u003e\n \u003cp\u003e-11,13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.289036544850498%\" rowspan=\"5\"\u003e\n \u003cp\u003e\u003cem\u003eDownregulated\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.070242656449553%\"\u003e\n \u003cp\u003e\u003cem\u003eObp1\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"39.46360153256705%\"\u003e\n \u003cp\u003eOdorant binding protein 1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"36.39846743295019%\"\u003e\n \u003cp\u003eOlfactory receptors\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.067688378033205%\"\u003e\n \u003cp\u003e-10,65\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.070242656449553%\"\u003e\n \u003cp\u003e\u003cem\u003eObp6\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"39.46360153256705%\"\u003e\n \u003cp\u003eOdorant binding protein 6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"36.39846743295019%\"\u003e\n \u003cp\u003eOlfactory receptors\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.067688378033205%\"\u003e\n \u003cp\u003e-9,09\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.070242656449553%\"\u003e\n \u003cp\u003e\u003cem\u003eObp5\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"39.46360153256705%\"\u003e\n \u003cp\u003eOdorant binding protein 5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"36.39846743295019%\"\u003e\n \u003cp\u003eOlfactory receptors\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.067688378033205%\"\u003e\n \u003cp\u003e-5,80\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.070242656449553%\"\u003e\n \u003cp\u003eLOC726362\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"39.46360153256705%\"\u003e\n \u003cp\u003e4-coumarate--CoA ligase 1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"36.39846743295019%\"\u003e\n \u003cp\u003eLigase activity\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.067688378033205%\"\u003e\n \u003cp\u003e-5,10\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"13.067552602436324%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"34.21926910299003%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"31.561461794019934%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.862679955703212%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.289036544850498%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"13.067552602436324%\"\u003e\n \u003cp\u003eLOC113219340\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"34.21926910299003%\"\u003e\n \u003cp\u003elarge subunit ribosomal RNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"31.561461794019934%\"\u003e\n \u003cp\u003eStructural component ribosomes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.862679955703212%\"\u003e\n \u003cp\u003e4,96\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.289036544850498%\" rowspan=\"5\"\u003e\n \u003cp\u003e\u003cem\u003eUpregulated\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.070242656449553%\"\u003e\n \u003cp\u003eLOC113218873\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"39.46360153256705%\"\u003e\n \u003cp\u003ePutative leucine-rich repeat-containing protein\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"36.39846743295019%\"\u003e\n \u003cp\u003eImmune regulation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.067688378033205%\"\u003e\n \u003cp\u003e4,96\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.070242656449553%\"\u003e\n \u003cp\u003eLOC113219383\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"39.46360153256705%\"\u003e\n \u003cp\u003eLarge subunit ribosomal RNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"36.39846743295019%\"\u003e\n \u003cp\u003eStructural component ribosomes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.067688378033205%\"\u003e\n \u003cp\u003e4,96\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.070242656449553%\"\u003e\n \u003cp\u003eLOC113219387\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"39.46360153256705%\"\u003e\n \u003cp\u003eLarge subunit ribosomal RNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"36.39846743295019%\"\u003e\n \u003cp\u003eStructural component ribosomes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.067688378033205%\"\u003e\n \u003cp\u003e4,96\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.070242656449553%\"\u003e\n \u003cp\u003eLOC107965279\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"39.46360153256705%\"\u003e\n \u003cp\u003ePancreatic lipase-related protein 2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"36.39846743295019%\"\u003e\n \u003cp\u003ePhospholipid degradation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.067688378033205%\"\u003e\n \u003cp\u003e4,96\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"100%\" colspan=\"5\"\u003e\n \u003cp\u003e4 hours \u0026ndash; Sublethal Dose\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"13.067552602436324%\"\u003e\n \u003cp\u003e\u003cem\u003eObp2\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"34.21926910299003%\"\u003e\n \u003cp\u003eOdorant binding protein 2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"31.561461794019934%\"\u003e\n \u003cp\u003eOlfactory receptors\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.862679955703212%\"\u003e\n \u003cp\u003e-10,63\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.289036544850498%\" rowspan=\"5\"\u003e\n \u003cp\u003e\u003cem\u003eDownregulated\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.070242656449553%\"\u003e\n \u003cp\u003e\u003cem\u003eObp1\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"39.46360153256705%\"\u003e\n \u003cp\u003eOdorant binding protein 1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"36.39846743295019%\"\u003e\n \u003cp\u003eOlfactory receptors\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.067688378033205%\"\u003e\n \u003cp\u003e-9,78\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.070242656449553%\"\u003e\n \u003cp\u003e\u003cem\u003eObp6\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"39.46360153256705%\"\u003e\n \u003cp\u003eOdorant binding protein 6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"36.39846743295019%\"\u003e\n \u003cp\u003eOlfactory receptors\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.067688378033205%\"\u003e\n \u003cp\u003e-9,18\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.070242656449553%\"\u003e\n \u003cp\u003eLOC552229\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"39.46360153256705%\"\u003e\n \u003cp\u003eEsterase B1\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"36.39846743295019%\"\u003e\n \u003cp\u003eHydrolysis activity\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.067688378033205%\"\u003e\n \u003cp\u003e-6,89\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.070242656449553%\"\u003e\n \u003cp\u003e\u003cem\u003eObp5\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"39.46360153256705%\"\u003e\n \u003cp\u003eOdorant binding protein 5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"36.39846743295019%\"\u003e\n \u003cp\u003eOlfactory receptors\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.067688378033205%\"\u003e\n \u003cp\u003e-6,18\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"13.067552602436324%\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"34.21926910299003%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"31.561461794019934%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.862679955703212%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.289036544850498%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"13.067552602436324%\"\u003e\n \u003cp\u003eLOC112935903\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"34.21926910299003%\"\u003e\n \u003cp\u003eCytochrome P450 6a14-like\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"31.561461794019934%\"\u003e\n \u003cp\u003eXenobiotic detoxification\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.862679955703212%\"\u003e\n \u003cp\u003e3,91\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.289036544850498%\" rowspan=\"5\"\u003e\n \u003cp\u003e\u003cem\u003eUpregulated\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.070242656449553%\"\u003e\n \u003cp\u003eLOC113219340\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"39.46360153256705%\"\u003e\n \u003cp\u003eLarge subunit ribosomal RNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"36.39846743295019%\"\u003e\n \u003cp\u003eStructural component ribosomes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.067688378033205%\"\u003e\n \u003cp\u003e4,30\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.070242656449553%\"\u003e\n \u003cp\u003eLOC113219387\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"39.46360153256705%\"\u003e\n \u003cp\u003eLarge subunit ribosomal RNA\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"36.39846743295019%\"\u003e\n \u003cp\u003eStructural component ribosomes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.067688378033205%\"\u003e\n \u003cp\u003e4,91\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.070242656449553%\"\u003e\n \u003cp\u003eLOC107965279\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"39.46360153256705%\"\u003e\n \u003cp\u003ePancreatic lipase-related protein 2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"36.39846743295019%\"\u003e\n \u003cp\u003ePhospholipid degradation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.067688378033205%\"\u003e\n \u003cp\u003e5,03\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.070242656449553%\"\u003e\n \u003cp\u003eLOC113218873\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"39.46360153256705%\"\u003e\n \u003cp\u003ePutative leucine-rich repeat-containing protein\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"36.39846743295019%\"\u003e\n \u003cp\u003eImmune regulation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.067688378033205%\"\u003e\n \u003cp\u003e5,11\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eVarious studies have demonstrated the harmful effect of the insecticide fipronil on honey bees, stingless bees, and wild bees (Zaluski et al. 2015; Roat et al. 2017; Bovi et al. 2018). However, many of these studies have focused on assessing dosage-related mortality or behavioral effects (Renzi et al. 2016; Lunardi et al. 2017; Bovi et al. 2018; Mulvey and Cresswell 2020). Recently, the application of advanced genetic analysis techniques, such as transcriptomics in bees exposed to pesticides, has proven to be a promising approach for comprehensively understanding the effects of these chemicals (Dai 2021; Gao 2020, 2022; Astolfi et al. 2022). The genetic approach has contributed to a greater understanding of the overall effects of pesticides, filling important gaps in understanding their impacts on non-target organisms, such as bees (Fent et al. 2020; Astolfi et al. 2022; Tosi et al. 2022).\u003c/p\u003e\n\u003cp\u003eIt was observed that bees showed a significantly higher consumption rate of uncontaminated syrup compared to syrup containing lethal and sublethal doses of fipronil during the one hour period. There is substantial scientific evidence indicating that insecticides can have repellent effects, potentially causing bees to avoid treated areas due to their direct contact or residual presence in flower nectar (Sgolastra et al. 2018; Wu et al. 2021; Cullen et al. 2023).\u003c/p\u003e\n\u003cp\u003eThe marked number of DEGs in the one hour exposure treatment (n = 2,391 genes) compared to the four hour period (n = 147 genes) may be associated with a greater immune system response immediately after exposure to fipronil (James and Xu 2012; Decio et al. 2021; Orčić et al. 2022). Insecticide intoxication can induce immune reactions that occur rapidly, requiring the synthesis of new molecules with signaling functions, cellular tasks, and proliferation of additional immune cells (James and Xu 2012; Bajgar et al. 2015; Dolezal et al. 2019). During the first hour of exposure, a more intense stress response likely occurred, which returned to levels closer to normal expression after four hours, leading to a reduction in the number of DEGs over the exposure period (El-Seedi et al. 2022).\u003c/p\u003e\n\u003cp\u003eRegarding the gene ontology analysis, it was possible to identify the main pathways affected in each treatment, associated with their respective enriched functional categories. In the treatment after one hour of exposure, both dosages showed similarity in enriched pathways, with biosynthetic and metabolic processes, translation, and macro and micromolecule transport being the main pathways grouped under BP (Biological Processes). Insecticide exposure can affect a variety of biosynthetic and metabolic processes in insects, including xenobiotic metabolism and monooxygenase activity, which play roles in pesticide detoxification and elimination (Pisa et al. 2015; Nauen et al. 2022; Ranganathan et al. 2022). Additionally, fipronil can interfere with bee hormonal regulation, affecting energy production, altering protein synthesis, and causing disturbances in cellular development and function (Holder et al. 2018; Gon\u0026ccedil;alves et al. 2022). By acting as an antagonist of the GABA receptor, fipronil disrupts the normal functionality of the insect nervous system, resulting in neuronal hyperexcitation that can affect gene expression in various metabolic, biosynthetic, and transport pathways, including genes involved in protein, carbohydrate, lipid synthesis, and macro and micromolecule transport (Pisa et al. 2015; Gon\u0026ccedil;alves et al. 2022).\u003c/p\u003e\n\u003cp\u003eExposure to the insecticide altered gene expression related to ribosomes and their subunits in bees, potentially causing changes in ribosomal RNA synthesis, ribosome assembly, and protein synthesis control (Shi et al. 2017). The alteration of several pathways involved in ribosomes due to pesticide contamination has been reported in multiple studies with other insecticides (Shi et al. 2017; Wu et al. 2017; Gao et al. 2020; Flores et al. 2021), with significant consequences for the metabolism, development, stress response, and cellular function of contaminated bees (Mao and Jing 2007; Shi et al. 2017; Wu et al. 2017).\u003c/p\u003e\n\u003cp\u003eThe results obtained from enriched pathways in GO reveal that exposure to fipronil impacts the functions of ion channels, which can lead to disruptions in both ionic balance and electrical signal transmission in cells. By directly blocking these channels, fipronil interferes with the normal flow of ions and modulates the activity of ion channels, altering their opening, closing, or conductance, leading to an imbalance in nerve signal transmission and alteration of physiological functions in these insects (Dong 2007; Murillo et al. 2011). One piece of evidence of fipronil\u0026apos;s negative influence on ion channels is its damaging action on insects\u0026apos; Ca\u003csup\u003e2+\u003c/sup\u003e channels, which can have detrimental effects on neuronal signaling, muscle contraction, and energy metabolism, resulting in neuronal dysfunction, behavioral disorders, reduced locomotion, and flight (Dong 2007; Wu et al. 2021). Ca\u003csup\u003e2+\u003c/sup\u003e signals also play a crucial role in bees\u0026apos; learning and memory, as Ca\u003csup\u003e2+\u003c/sup\u003e is used in all stages of odorant information processing, from detection by the antenna to integration, learning, and memorization (Wu et al. 2021; Paten et al. 2022).\u003c/p\u003e\n\u003cp\u003eRegarding the DEGs after one hour of exposure to the lethal dose of the fipronil insecticide, bees showed downregulation in the expression of genes involved in developmental functions and cell adhesion (LIM/homeobox protein Lhx5, Mitogen-activated protein kinase 15, Basic proline-rich protein, Flocculation protein FLO11). The decrease in the expression of these genes may compromise bees\u0026apos; ability to respond adequately to the stress caused by the insecticide and other external stressors, affecting their metabolic adaptation and survival (Zhao et al. 2007; Krizsan et al. 2014; Liu et al. 2020; Farhadi et al. 2023). Additionally, the Protein pygopus gene, associated with the Wnt signaling pathway and essential for biological process development and regulation, also showed downregulation (Parker et al. 2002). According to Martin and Kimelman (2009), the Wnt signaling pathway is involved in the development of the central nervous system of bees, cell fate determination during metamorphosis, and reproductive organ formation. On the other hand, exposure to the lethal dose of fipronil for one hour also showed upregulation of genes mainly involved in detoxification processes and xenobiotic substance detection (Urea transporter 2, Odorant binding protein 14, Early nodulin-75) (Schwaighofer et al. 2014; Nie et al. 2018), as well as genes contributing to lipid metabolism and defense against DNA damage (Pancreatic lipase-related protein 2, DNA repair protein RAD51 homolog 4) (Lee et al. 2010; Collins et al. 2021).\u003c/p\u003e\n\u003cp\u003eWhen exposed to a sublethal dose of fipronil, after one hour of exposure, downregulation of genes involved in metabolic, developmental, and cellular transport processes (Glycerate kinase-like, LIM/homeobox protein Lhx5, Unconventional myosin-Ixb, Ornithine aminotransferase) was observed, as well as downregulation of the Basic proline-rich protein gene, responsible for encoding the proline-rich protein. Proline-rich proteins are predominantly found in insect hemolymph and may be involved in responding and adapting to different types of stressors, such as environmental (e.g., temperature, humidity, and ultraviolet radiation) and physiological (e.g., dehydration, injuries, and toxins) stressors (Micheu et al. 2000). According to Teulier et al. (2016), prolines may also be involved in the flight of hymenopterans. Although bees primarily use carbohydrates as an energy source, proline can be used as an alternative fuel to support muscle metabolism from resting conditions to the high energy production rate required during flight (Arrese and Soulages 2010; Teulier et al. 2016). In the same treatment (sublethal dose, one hour), upregulation of genes associated with protein and lipid digestion (Aminopeptidase N, Pancreatic lipase-related protein 2), neurotransmitters (Glutamate receptor 1-like), hormonal regulation, and gene expression (Allatotropin-like, Homeobox protein ceh-19) was observed. This suggests a compensatory response of contaminated bees to decrease the negative effects of the insecticide, involving a series of processes such as digestion and nutrient acquisition, synaptic transmission, and cellular development and differentiation (Belzunces et al. 2012; Pashte and Patil 2017).\u003c/p\u003e\n\u003cp\u003eAfter four hours of exposure to fipronil, the GO showed that the terms of MF (Molecular Functions) were mainly associated with monooxygenase activity, oxidoreductase activities, and iron ion and tetrapyrrole binding. Monooxygenases play an essential role in insects exposed to insecticides, being involved in the metabolism and detoxification of these substances (Haas and Nauem 2021; Nauen et al. 2022). These enzymes are mainly represented by the cytochrome P450 family and are responsible for the chemical modification of insecticides, making them more soluble in water and facilitating their elimination in the insects\u0026apos; bodies (Nauen et al. 2022); overall, the increased activity of monooxygenases may be involved in the process of metabolizing and neutralizing fipronil\u0026apos;s toxic compounds to reduce its concentration and minimize its adverse effects on bees (Feyereisen 2012; Haas and Nauem 2021).\u003c/p\u003e\n\u003cp\u003eBees exposed to fipronil for four hours show similar downregulation of genes, regardless of the tested dosage. With the exception of the Esterase B1 and 4-coumarate--CoA ligase 1 genes, related to antioxidant responses and detoxification in insects (Fent et al. 2020; Zhang et al. 2023); the other downregulated genes belong to the odorant binding protein family \u0026ndash; OBPs (\u003cem\u003eObp1\u003c/em\u003e,\u003cem\u003e\u0026nbsp;Obp2\u003c/em\u003e,\u003cem\u003e\u0026nbsp;Obp5\u003c/em\u003e,\u003cem\u003e\u0026nbsp;Obp6\u003c/em\u003e). OBPs are predominantly expressed in chemosensory tissues such as antennae and maxillary palps, being responsible for detecting and discriminating odors by bees (Schwaighofer et al. 2014). These small, water-soluble proteins bind to and transport hydrophobic odor molecules, increasing the sensitivity and specificity of bees\u0026apos; olfactory system (For\u0026ecirc;t and Maleszka 2006). Downregulation of \u003cem\u003eObp\u003c/em\u003e genes may have detrimental effects on odor detection and recognition, potentially affecting bees\u0026apos; foraging ability, reducing communication efficiency, hindering pheromone and floral resource identification (Li et al. 2015; Mayack 2023); impacting colony social structure, such as queen recognition, task organization, and defense against invaders (For\u0026ecirc;t and Maleszka 2006; Schwaighofer et al. 2014).\u003c/p\u003e\n\u003cp\u003eGenes upregulated after four hours of exposure, regardless of the dosage, were Large subunit ribosomal RNA (LOC113219340, LOC113219383, LOC113219387), Pancreatic lipase-related protein 2, and Putative leucine-rich repeat-containing protein, mainly involved in protein synthesis, lipid digestion, and immune response of insects (Shi et al. 2017; Wu et al. 2017; Gao et al. 2020; Collins et al. 2021). Additionally, upregulation was observed in the expression of the gene related to xenobiotic detoxification (Cytochrome P450 6a14-like). This is a gene responsible for encoding enzymes belonging to the cytochrome P450 family, which play an important role in the metabolism and detoxification of foreign substances, including insecticides (Nauen et al. 2022). Thus, after a stress response in bees exposed to fipronil, the increased expression of the Cytochrome P450 6a14-like gene can be understood as an attempt to neutralize the insecticide\u0026apos;s toxic compounds (Fent et al. 2020). In addition to playing a significant role in xenobiotic detoxification, these enzymes are important for the biosynthesis and degradation pathways of endogenous compounds, such as pheromones, ecdysone, and juvenile hormone, which play crucial roles in insect growth and development (Zhao et al. 2018; Nauen et al. 2022).\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study unveils a robust genetic response in Africanized honey bees following acute exposure to the insecticide fipronil, regardless of the dosage ingested (lethal or sublethal). This is evidenced by the significant number of DEGs within the first hour of fipronil exposure, possibly due to rapid immune system activation. Gene expression analysis demonstrated that the insecticide impacts various Biological Processes, Molecular Functions, and Cellular Components in bees. Furthermore, alterations in the expression of several genes associated with detoxification and substance detection were observed, particularly genes from the Olfactory Binding Protein (OBP) family, indicating physiological changes in odor detection that may lead foraging honey bees to disoriented behaviors and reduced foraging and pollination efficiency.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe preparation of the study, data collection, and analysis were carried out by Yan Souza Lima, Isabella Cristina de Castro Lippi, Jaine da Luz Scheffer, and Juliana Sartori Lunardi. Marcus Vin\u0026iacute;cius Niz Alvarez and Samir Moura Kadri contributed to the genetic and bioinformatics analyses. Ricardo de Oliveira Orsi coordinated the study\u0026apos;s development, methodology, analyzed the data, and corrected the manuscript. All authors read and approved the final manuscript text.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Coordination for the Improvement of Higher Education Personnel (CAPES) - Financing Code 001, and by the State of S\u0026atilde;o Paulo Research Foundation (FAPESP), process: 2020/10524-0.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data supporting this study\u0026apos;s fndings are available from the corresponding author upon reasonable request.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe use of bees in the study was previously approved by the Ethics Committee on Animal Use CEUA/FMVZ/UNESP (Botucatu, S\u0026atilde;o Paulo State, Brazil), registered under protocol number 0006/2021.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to publish\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors have studied the manuscript thoroughly and consented to the publication.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAndrews S (2010) FastQC: A quality control tool for high throughput sequence data (2010) Babraham Bioinformatics. www.bioinformatics.babraham.ac.uk/projects/fastqc/\u003c/li\u003e\n\u003cli\u003eAndrews S, Pyl PT, Huber W (2015) HTSeq\u0026mdash;a Python framework to work with high-throughput sequencing data. 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PNAS 104(32):13182\u0026ndash;13186. https://doi.org/10.1073/pnas.0705464104\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"environmental-science-and-pollution-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"espr","sideBox":"Learn more about [Environmental Science and Pollution Research](https://www.springer.com/journal/11356)","snPcode":"11356","submissionUrl":"https://submission.nature.com/new-submission/11356/3","title":"Environmental Science and Pollution Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Foraging bees , Foraging efficiency , Insecticide , Transcriptome , Gene expression , Detoxification","lastPublishedDoi":"10.21203/rs.3.rs-4484576/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4484576/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Taking into consideration that bees can be contaminated by pesticides through the ingestion of contaminated floral resources, we can utilize genetic techniques to assess effects that are scarcely observed in behavioral studies. This study aimed to investigate the genetic effects of ingesting lethal and sublethal doses of the insecticide fipronil in foraging honey bees during two periods of acute exposure. Bees were exposed to fipronil through contaminated honey syrup at two dosages (LD50 = 0.19 μg/bee; LD50/100 = 0.0019 μg/bee) and for two durations (one and four hours). Following exposure, we measured syrup consumption per bee, analyzed the transcriptome of bee brain tissue, and identified differentially expressed genes (DEGs), categorizing them functionally based on Gene Ontology (GO). The results revealed a significant genetic response in honey bees after exposure to fipronil, regardless of the dosage used. Fipronil affected various metabolic, transport, and cellular regulation pathways, as well as detoxification processes and xenobiotic substance detection. Additionally, downregulation of several DEGs belonging to the Olfactory Binding Protein (OBP) family was observed, suggesting potential physiological alterations in bees that may lead to disoriented behaviors and reduced foraging efficiency.","manuscriptTitle":"Food contamination with fipronil alters gene expression associated with foraging in Africanized honey bees","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-01 10:06:43","doi":"10.21203/rs.3.rs-4484576/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Minor Revision","date":"2024-07-17T01:38:32+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2024-06-13T17:09:52+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-06-13T15:34:08+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Environmental Science and Pollution Research","date":"2024-06-13T15:30:30+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-05-30T04:23:45+00:00","index":"","fulltext":""},{"type":"submitted","content":"Environmental Science and Pollution Research","date":"2024-05-28T06:41:14+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"environmental-science-and-pollution-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"espr","sideBox":"Learn more about [Environmental Science and Pollution Research](https://www.springer.com/journal/11356)","snPcode":"11356","submissionUrl":"https://submission.nature.com/new-submission/11356/3","title":"Environmental Science and Pollution Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"ee9162fc-441d-44e0-aa22-2fc6e182fcdd","owner":[],"postedDate":"July 1st, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-08-22T19:35:39+00:00","versionOfRecord":{"articleIdentity":"rs-4484576","link":"https://doi.org/10.1007/s11356-024-34695-8","journal":{"identity":"environmental-science-and-pollution-research","isVorOnly":false,"title":"Environmental Science and Pollution Research"},"publishedOn":"2024-08-15 15:58:26","publishedOnDateReadable":"August 15th, 2024"},"versionCreatedAt":"2024-07-01 10:06:43","video":"","vorDoi":"10.1007/s11356-024-34695-8","vorDoiUrl":"https://doi.org/10.1007/s11356-024-34695-8","workflowStages":[]},"version":"v1","identity":"rs-4484576","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4484576","identity":"rs-4484576","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}