Beyond the obvious: a realistic and holistic approach reveals multiple disorders caused by reduced-risk pesticides on individuals and colonies of bumble bees

Beyond the obvious: a realistic and holistic approach reveals multiple disorders caused by reduced-risk pesticides on individuals and colonies of bumble 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 Beyond the obvious: a realistic and holistic approach reveals multiple disorders caused by reduced-risk pesticides on individuals and colonies of bumble bees Lívia Maria Negrini Ferreira, Gaetana Mazzeo, Maria Augusta Pereira Lima This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8311248/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 6 You are reading this latest preprint version Abstract There is growing evidence of the negative impacts of prolonged exposure to sublethal concentrations of pesticides that are considered safe for bees, such as biopesticides, herbicides, and fungicides. In this study, we investigated the effects of four different types of pesticides on individuals and colonies of Bombus terrestris , using a holistic approach. Bees were orally and chronically exposed to either pure honey syrup (control, CTRL) or honey syrup contaminated with the neonicotinoid acetamiprid (ACE), the herbicide glyphosate (GLY), the fungicide metalaxyl-M (MET), or the biopesticide sweet orange essential oil (OEO). Sublethal effects were observed for all pesticide treatments at both individual and colony levels; however, lethal effects were only observed in colonies treated with OEO, likely due to the higher concentration tested for this product. Pesticide-exposed colonies experienced negative impacts on mass gain (ACE, OEO, GLY), food ingestion (all pesticide treatments), and number of eggs (all pesticide treatments). Pesticide-exposed individuals showed negative impacts on food ingestion (OEO). The walking behavior of workers was affected by pesticide exposure at the colony level, and by social isolation when treated individually. Overall, our results demonstrate that chronic exposure to pesticides considered safe for bees can cause detrimental sublethal effects on bumble bees, potentially contributing to the decline of pollinators. We also highlight the importance of considering the social context when assessing pesticide toxicity in social insects. Chronic exposure Biopesticide Herbicide Fungicide Neonicotinoid Bee Colony Health Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1 INTRODUCTION Pollination is one of the most significant ecosystem services provided by insects, with bees being the primary pollinators among animals (Khalifa et al., 2021 ). In the Mediterranean basin, bee diversity is particularly high (Michener, 2007 ; Mazzeo et al., 2019 ), and bees are associated with increased crop productivity, even for self-fertile cultivars (Bartual et al., 2018 ; Marqués et al., 2019 ; Kleftodimos et al., 2021 ). Despite the importance of bees for Mediterranean crop production, some agricultural practices in the region, such as the use of pesticides, negatively impact bee composition and local populations of various species (Turrisi et al., 2021 ). Although pesticides have long been recognized as a major threat to bees, their use remains widespread and requires increased regulation (Epstein, 2014 ; Nicholson et al., 2024 ). The susceptibility of wild bees to pesticides differs from that of honey bees ( Apis mellifera ), which is the model species used for testing pesticide toxicity in bees (Arena and Sgolastra, 2014 ; Cham et al., 2019 ; Schmolke et al., 2021 ; Varga-Szilay and Tóth, 2022 ). Bumble bees ( Bombus ), in particular, are important wild pollinators in temperate regions (Nicholson and Ricketts, 2019 ; McGrady et al., 2020 ), and studies have shown they can be more susceptible to pesticides than honey bees (Arena and Sgolastra, 2014 ; Gradish et al., 2019 ; Schmolke et al., 2021 ). Oral exposure, through the ingestion of contaminated pollen and nectar, is particularly concerning for bumble bees due to their life history. Bumble bee queens and larvae consume only unprocessed pollen and nectar, which may lead to higher oral pesticide doses relative to their body mass (Arena and Sgolastra, 2014 ; Gradish et al., 2019 ). Additionally, bumble bee larvae may consume up to 130 times more pollen per day than honey bee larvae, increasing the amount of contaminated food they ingest (Gradish et al., 2019 ). Residues of insecticides, fungicides, and herbicides have been found in the nectar and pollen of plants visited by bumble bees (Zioga et al., 2020 ), and residues detected in the colonies and bodies of these insects further confirm the collection, storage, and ingestion of pesticides (Botías et al., 2017 ; Main et al., 2020 ; Zioga et al., 2023 ; Nicholson et al., 2024 ). Oral exposure can occur even days after plants have been sprayed with pesticides, including wild plants that are not the target crops, and can persist for many days or even weeks (Zioga et al., 2023 ; Kuivila et al., 2021 ). Exposure to contaminated food can lead to the collapse of bee colonies, and even low-risk compounds or sublethal doses can cause colony morbidity due to bioaccumulation or interactions with chemical mixtures, nutritional stress, or diseases (Holder et al., 2018 ; Calatayud-Vernich et al., 2019 ; Magal et al., 2019 , 2020 ; Traynor et al., 2021 ). In addition to the death of individuals caused by lethal doses, exposure to sublethal doses of pesticides can also harm the survival of colonies and populations of bees (Stanley et al., 2015 ; Crall et al., 2019 ; Weidenmüller et al., 2022 ). These sublethal doses can impair the proper functioning of bee colonies due to multiple effects on individual workers (Marques et al., 2020 ; Costa et al., 2020 ; Miotelo et al., 2021 ). Although synthetic insecticides, particularly neonicotinoids, are commonly identified as the main pesticides harmful to bees, growing evidence suggests that herbicides, fungicides, and biopesticides can also pose risks to bees (Seide et al., 2018 ; Iwasaki and Hogendoorn, 2021 ; Battisti et al., 2022; Catania et al., 2023 ; Lima et al., 2024 ). In some cases, these compounds can be just as toxic to bees as synthetic insecticides (Barbosa et al., 2015 ; Tomé et al., 2015 ; Bernardes et al., 2018 ; Marques et al., 2020 ; Padilha et al., 2020 ; Piovesan et al., 2020 ). In this study, we investigated the lethal and sublethal effects of different types of pesticides on individuals and colonies of Bombus terrestris Linnaeus, 1758 (Apidae: Bombini) under laboratory conditions. We chronically exposed individuals and colonies to food contaminated with field-realistic doses of acetamiprid (a neonicotinoid insecticide), sweet orange essential oil (a biopesticide), glyphosate (an herbicide), or metalaxyl-M (a fungicide). Individuals were orally exposed for one week, and colonies for four weeks, with weekly assessments. Our study aimed to test the following hypotheses: (1) Pesticides from different groups decrease the survival of bumble bees; (2) The performance of bumble bee colonies is negatively affected by pesticides from different groups; (3) Pesticides from different groups alter the behavior of bumble bees; (4) The social level of exposure (individual or colony) affects the impact of pesticides on bumble bee behavior. 2 MATERIAL AND METHODS 2.1 Bees and pesticides We acquired large colonies of B. terrestris containing a queen, workers, brood, and sugar water (Natupol Excel line from Koppert Biological Systems, Netherlands). The sugar water was removed before the experiments. The colonies were housed in a BugDorm-4E4590DH specimen handling cage (W93.0 × D47.5 × H47.5 cm). The bees had free access to exit the nest and collect honey syrup (1:1 v/v honey and distilled water) from feeders placed inside the cage. Honey bee pollen (Koppert Biological Systems, Netherlands) was supplied once a week and placed inside the colony. In all experiments, we used commercial formulations of acetamiprid (Epik® SL; Sipcam, a.i.: 4.67%, 50 g/l), metalaxyl-M (Ridomil® Gold SL; Syngenta, a.i.: 43.88%, 465 g/l), glyphosate (Taifun® MK CL; Adama, a.i.: 30.8%, 360 g/l) and sweet orange essential oil (Prev-Am® Plus; Oroagri International Ltd, a.i.: 5.88%, 60 g/l). For synthetic pesticides, we used field-realistic concentrations found between the range of previously reported concentrations found in nectar residuals, as follows: 0.01 µg/ml acetamiprid (Pohorecka et al., 2012 ), 0.05 µg/ml of metalaxyl-M (Gong et al., 2020 ) and 30 µg/ml of glyphosate (Thompson et al., 2014 ). Acetamiprid, glyphosate, and metalaxyl-M are systemic pesticides previously found in plants visited by bees and in the bees themselves (Kiljanek et al., 2017 ; Thompson et al., 2019 ; El Agrebi et al., 2020 ; Zioga et al., 2020 , 2023 ; Rondeau and Raine, 2022 ). We used realistic concentrations based on studies that reported field doses of pesticides applied to the crop analyzed. Among the reported concentrations, we selected one that closely matched residue levels found in other studies for each active ingredient. Since no data are available on sweet orange essential oil residues in nectar, we used the recommended field concentration for tomato crops (476 µg/ml). This concentration simulates the deposition of the biopesticide on flowers when applied during flowering. Note that this commercial formulation is not recommended for use during flowering; therefore, we tested a worst-case scenario, assuming misuse of the product. 2.2 Pesticide oral exposure 2.2.1 Colony-level exposure Each colony was maintained in a translucent cage and provided with a single feeder containing uncontaminated honey syrup (control; CTRL) or honey syrup contaminated with acetamiprid (ACE), sweet orange essential oil (OEO), glyphosate (GLY), or metalaxyl-M (MET). Each colony represented one replicate, with three replicates per treatment, totaling 15 bumble bee colonies in the experiment. Each colony had access to its designated feeder for four weeks. Feeders were cleaned, and food was replaced every 24 hours to prevent syrup fermentation and pesticide degradation. Feeders were weighed daily before and after exposure to estimate honey syrup consumption. Colonies were weighed once a week (N = 4), when the dead bees outside the nest were counted and removed. At the end of the fourth week, colonies were opened to assess queen presence/absence and to count the number of eggs laid, estimating colony reproductive performance. Laboratory temperature and relative humidity (RH) were monitored using a data logger (EasyLog® EL-USB-2). 2.2.2 Individual-level exposure The individual-level bioassays were conducted using the same colonies as the colony-level bioassays, and both were performed simultaneously. Each colony assigned to one of the five treatments (CTRL, ACE, OEO, GLY, MET) served as a replicate. Five adult bumble bee workers were collected from each colony and assigned to the same pesticide treatment as their colony of origin. Individuals were cold-anesthetized and weighed, and only those within the size range of 0.13–0.36 g were included to standardize this variable, as bee size can influence pesticide sensitivity (Sgolastra et al., 2017 ; Linguadoca et al., 2022 ). The selected workers were placed in individual 650 mL plastic cages (15.0 × 15.0 × 7.0 cm) with a perforated, mesh-covered lid for ventilation. A 3 mL syringe, containing honey syrup (same treatments and concentrations previously described) was inserted horizontally into each cage. Individual bumble bees had access to the honey syrup for one week. After one week, the procedure was repeated with five new workers from each colony, continuing until the four-week colony-level exposure ended. Syringes were replaced daily to prevent syrup fermentation and pesticide degradation. Each syringe was weighed before and after daily exposure to measure individual food consumption. Bees´ survival was recorded every 24 hours. Bumble bees were kept in a dark room at 22 ± 2°C and 65% RH. 2.3 Assessment of behavioral effects on workers exposed at individual and colony-levels Surviving bees from the individual-level bioassay were filmed for behavioral analysis after one week of exposure. Bees were grouped according to pesticide treatment and filmed together, resulting in 15 videos (five pesticide treatments × three replicates). For the colony-level exposure, behavioral assessments were conducted weekly for four weeks. In each week, we recorded the behavior of five workers from each pesticide-treatment colony, totaling 60 videos (five treatments × three replicates × four weeks). Videos were recorded using a Logitech C922 Pro Stream Webcam and Logitech Capture software (version 2.08.11) at 30 fps and full HD resolution. For filming, each group was placed in a Petri dish (140 mm diameter × 20 mm height) in a dark room illuminated with a fluorescent light source and recorded for 10 minutes. 2.4 Data analysis To analyze the effects of different treatments on foraging behavior, we used generalized linear models (GLMs) with the “GAMLSS” package (Rigby & Stasinopoulos, 2005 ). Pesticide treatments were compared to the control using Dunnett’s tests with the “emmeans” package (Lenth, 2024 ). At the colony level, we performed three GLMs to assess how pesticide treatment (categorical variable: CTRL, ACE, OEO, GLY, MET) and weeks of exposure (continuous variable: 1–4) influenced (1) the number of dead bees outside the nest (“number of dead bees”), (2) colony mass variation (“colony mass variation”), and (3) honey syrup consumption (“food ingested”). Additionally, we performed two GLMs to evaluate the effect of pesticide treatment (categorical variable: CTRL, ACE, OEO, GLY, MET) on egg production (“number of eggs”) after four weeks of exposure. We calculated the correlation between “mass gain” and “food ingested” to ensure that pesticide effects were not confounded by a strong correlation between these variables. Finally, we performed a GLM to assess whether weekly honey syrup consumption (“food ingested per week”) was influenced by temperature (continuous variable) and pesticide treatment (categorical variable: CTRL, ACE, OEO, GLY, MET). At the individual level, we used Kaplan-Meier analysis to assess the effect of each pesticide treatment on bumble bee survival, estimating survival curves and median survival times (LT 50 ). For this analysis, we considered only survival status (dead or alive) during the first 96 hours of exposure. Curve similarity was tested using the log-rank test, with comparisons adjusted using Bonferroni correction (p < 0.05). Pesticide-contaminated treatments were not compared to each other; instead, ACE, OEO, GLY, and MET survival curves were compared only to the CTRL curve. Analyses were performed using the R packages “survival” (Therneau & Grambsch, 2000 ; Therneau, 2024 ), “survminer” (Kassambara et al., 2024 ), and “dplyr” (Wickham et al., 2023 ). Additionally, we performed two GLMs to assess how honey syrup consumption (“food ingested”) was influenced by: (1) pesticide treatment (categorical variable: CTRL, ACE, OEO, GLY, MET) and exposure duration (continuous variable: 24–96 h), and (2) pesticide treatment and bee mass (continuous variable: “bee mass”). The behaviors of bees across pesticide treatments and social levels (colony or individual) were analyzed using Ethoflow®, a software that employs computer vision and artificial intelligence (AI) to monitor behavioral parameters (Bernardes et al., 2021 ). The AI k-means algorithm and combinatorial optimization were used to track individual behavior while preserving identity within the group. The measured behavioral variables included: distance walked (cm), mean walking speed (cm/s), meandering (average turning angle in degrees), resting time (s), mean movement time (proportion of time spent in intermediate activity: distance walked > 0.07 and ≤ 0.4 cm/frame), mean fast time (proportion of time spent in high activity: distance walked > 0.4 cm/frame), and group density network (interactions between an individual and others in the group). Behavioral measurements obtained via Ethoflow® were analyzed using generalized linear models (GLMs) to assess the effects of pesticide treatment (categorical variable: CTRL, ACE, OEO, GLY, MET) on each behavioral parameter previously described, at both social levels (colony or individual). For colony-level exposure, we also examined how the number of weeks of exposure (continuous variable: 1 to 4) influenced behavioral parameters. Pesticide-contaminated treatments were not compared to each other; instead, ACE, OEO, GLY, and MET results were compared only to CTRL. The MET treatment was excluded from individual-level “group density network” analyses due to an insufficient number of surviving bees per group (minimum required = two). All analyses were conducted in R (version 4.3.2; R Core Team, 2023 ), and graphs were generated using the “ggplot2” package. 3 RESULTS 3.1 Colony-level pesticide exposure The number of dead bees outside the nest was influenced by pesticide treatment (χ² = 48.513, df = 4, p < 0.001) and week (χ² = 31.618, df = 1, p < 0.001), but there was no interaction between treatment and week (χ² = 2.957, df = 4, p = 0.565) (Fig. 1 ). Compared to the CTRL, the number of dead bees was higher in OEO-treated colonies (Z = 5.358, p < 0.001) (Fig. 1 A). In colonies treated with ACE, GLY, and MET, the number of dead bees did not differ from the CTRL (ACE: Z = 0.520, p = 0.925; GLY: Z = 0.965, p = 0.705; MET: Z = -2.164, p = 0.102) (Fig. 1 A). Over the weeks, the number of dead bees increased in all treatments except OEO (CTRL: Z = 2.686, p = 0.007; ACE: Z = 2.438, p = 0.015; OEO: Z = 1.369, p = 0.171; GLY: Z = 2.308, p = 0.021; MET: Z = 2.504, p = 0.012) (Fig. 1 B). The variation in colony mass relative to the beginning of exposure was influenced by the interaction between treatment and the number of weeks (χ² = 24.471, df = 4, p < 0.001) (Fig. 1 ). By the end of the four weeks of exposure, the total mass gain of MET colonies did not differ from CTRL colonies (t = -0.279, df = 49, p = 0.983) (Fig. 1 C). ACE and GLY colonies gained less mass than CTRL colonies, while OEO colonies lost mass (ACE: t = -2.793, df = 49, p = 0.026; OEO: t = -11.502, df = 49, p < 0.001; GLY: t = -3.541, df = 49, p = 0.003) (Fig. 1 C). Over the weeks, CTRL and MET colonies tended to gain mass (CTRL: t = 2.354, df = 49, p = 0.023; MET: t = 2.668, df = 49, p = 0.01), while OEO colonies tended to lose mass (t = -3.984, df = 49, p < 0.001) (Fig. 1 D). The number of weeks had no significant effect on mass gain or loss in ACE and GLY colonies (ACE: t = 1.913, df = 49, p = 0.062; GLY: t = 0.241, df = 49, p = 0.81) (Fig. 1 D). Figure 1 . Effects of pesticides on bumble bees’ mortality and colony mass variation over four weeks of colony exposure. (A) Mean number of dead bees per treatment after four weeks of exposure. (B) Generalized Linear Model regression of the number of dead bees per treatment as a function of weeks of exposure. (C) Total colony mass gain or loss per treatment after four weeks of exposure. (D) Generalized Linear Model regression of total mass gain or loss per treatment as a function of weeks of exposure. Honey syrup treatments were: (CTRL) uncontaminated (control); (ACE) contaminated with acetamiprid; (OEO) contaminated with sweet orange essential oil; (GLY) contaminated with glyphosate; and (MET) contaminated with metalaxyl-M. Each colony was considered a treatment replicate, with three replicates per treatment, totaling 15 observed colonies. The asterisk indicates a significant difference between pesticide-exposed colonies in comparison to control ( p < 0.05). The amount of food ingested per week was affected by the interaction between treatment and the number of weeks (χ² = 13.854, df = 4, p = 0.008) (Fig. 2 ). The amount of food ingested each week was higher in CTRL colonies compared to all pesticide-exposed treatments during the first, second, and third weeks ( p < 0.05) (Fig. 2 A). In the fourth week, food ingestion in CTRL colonies did not differ from that of ACE, GLY, and MET colonies but remained higher than in OEO colonies (ACE: t = -1.939, df = 48, p = 0.182; OEO: t = -7.306, df = 48, p < 0.001; GLY: t = -0.405, df = 48, p = 0.958; MET: t = -1.062, df = 48, p = 0.649) (Fig. 2 A). Over the weeks, the weekly amount of food ingested tended to decrease in CTRL, ACE, and OEO colonies (CTRL: t = -4.103, df = 48, p < 0.001; ACE: t = -2.070, df = 48, p = 0.044; OEO: t = -7.734, df = 48, p < 0.001) (Fig. 2 A). The number of weeks did not affect food ingestion in GLY and MET colonies (GLY: t = -1.585, df = 48, p = 0.12; MET: t = -0.598, df = 48, p = 0.553) (Fig. 2 B). Weekly colony mass loss or gain was moderately correlated with weekly food ingestion (r = 0.547; t = 4.977, df = 58, p < 0.001) (Fig. 2 C). Ambient temperature ranged from 18.62°C to 25.17°C, and its interaction with treatment significantly affected food ingestion by the colonies (χ² = 12.066, df = 4, p = 0.017) (Fig. 2 C). The amount of food ingested by CTRL colonies tended to increase with temperature (t = 4.095, df = 49, p < 0.001), whereas temperature had no significant effect on pesticide-exposed treatments (ACE: t = -1.148, df = 49, p = 0.257; OEO: t = 0.505, df = 49, p = 0.616; GLY: t = 1.165, df = 49, p = 0.25; MET: t = -1.097, df = 49, p = 0.278) (Fig. 2 C). Figure 2 . Food ingestion by Bombus terrestris colonies over four weeks of exposure to different pesticides. (A) Weekly amount of food ingested by each colony per treatment. (B) Generalized Linear Model (GLM) regression of the weekly amount of food ingested by each colony per treatment as a function of exposure duration. (C) Amount of food ingested by colonies submitted to different pesticides as a function of temperature. Honey syrup treatments were: (CTRL) uncontaminated (control); (ACE) contaminated with acetamiprid; (OEO) contaminated with sweet orange essential oil; (GLY) contaminated with glyphosate; and (MET) contaminated with metalaxyl-M. Each colony was considered a treatment replicate, with three replicates per treatment, totaling 15 observed colonies. The asterisk indicates a significant difference between pesticide-exposed colonies in comparison to control ( p < 0.05). The number of eggs in the colony after four weeks of exposure was significantly affected by treatment (χ² = 23.696, df = 4, p < 0.001) (Fig. 3 ). Egg numbers were significantly higher in the CTRL colonies compared to all pesticide-exposed colonies (ACE: Z = -3.9, p < 0.001; OEO: Z = -6.524, p < 0.001; GLY: Z = -5.885, p < 0.001; MET: Z = -2.824, p = 0.017) (Fig. 3 ). At the end of the four-week exposure period, the number of queens present in the pesticide-treated colonies (ACE, OEO, GLY, and MET) was insufficient to permit a statistical comparison of queen mass with the CTRL group. Queen presence was recorded in 100% of CTRL colonies, 66.67% of ACE, GLY, and MET colonies, and 0% of OEO colonies. Figure 3 . Number of eggs in Bombus terrestris colonies after four weeks of pesticide exposure. Honey syrup treatments were: (CTRL) uncontaminated (control; 0 µg/ml a.i.); (ACE) contaminated with acetamiprid (0.01 µg/ml a.i.); (OEO) contaminated with sweet orange essential oil (476 µg/ml a.i.); (GLY) contaminated with glyphosate (30 µg/ml a.i); and (MET) contaminated with metalaxyl-M (0.05 µg/ml a.i.). Each colony was considered a treatment replicate, with three replicates per treatment, totaling 15 observed colonies. The asterisk indicates a significant difference between pesticide-exposed colonies in comparison to control ( p < 0.05). 3.2 Individual-level pesticide exposure The survival of bumble bee workers individually exposed did not differ among treatments (χ² = 5.9, df = 4, p = 0.2) (Fig. S1 ). The LT₅₀ could not be calculated for CTRL and GLY due to high survival at the end of the experiment. For ACE, OEO, and MET, the LT₅₀ was 96 hours. Food ingestion over pesticide exposure was significantly affected by treatment (χ² = 53.757, df = 4, p < 0.001) and time (χ² = 13.398, df = 1, p < 0.001), with no interaction between treatment and time (χ² = 1.791, df = 4, p = 0.774) (Fig. 4 ). The amount of food ingested every 24 hours by workers exposed to ACE, GLY, and MET was similar to that of the CTRL on all days (Fig. 4 A). However, workers exposed to OEO ingested less food than the CTRL group on all days (Fig. 4 A). Individual food ingestion tended to increase over time for CTRL and ACE bees (CTRL: Z = 2.517, p = 0.012; ACE: Z = 2.002, p = 0.045) (Fig. 4 B). Time had no significant effect on food ingestion for OEO, GLY, and MET bees (OEO: Z = 0.545, p = 0.586; GLY: Z = 1.661, p = 0.097; MET: Z = 1.655, p = 0.098) (Fig. 4 B). Food ingestion after pesticide exposure was also significantly affected by treatment (χ² = 63.450, df = 4, p < 0.001), bumble bee mass (χ² = 26.449, df = 1, p < 0.001), and the interaction between treatment and bee mass (χ² = 16.131, df = 4, p = 0.003) (Fig. 4 C). The amount of honey syrup ingested tended to increase with bee mass for CTRL and GLY workers (CTRL: Z = 4.154, p < 0.001; GLY: Z = 5.130, p < 0.001). Food ingestion of workers exposed to ACE, OEO, and MET was not affected by bee mass (ACE: Z = 0.831, p = 0.406; OEO: Z = 0.299, p = 0.765; MET: Z = 0.74, p = 0.459) (Fig. 4 C). Figure 4 . Food ingestion by Bombus terrestris individuals during 96 hours of exposure to different pesticides. ( A) Daily amount of food ingested by individuals per treatment. (B) Generalized Linear Model (GLM) regression of the amount of food ingested by individuals per treatment as a function of hours of exposure. (C) Amount of food ingested by Bombus terrestris individuals as a function of bee mass. Honey syrup treatments were: (CTRL) uncontaminated (control); (ACE) contaminated with acetamiprid; (OEO) contaminated with sweet orange essential oil; (GLY) contaminated with glyphosate; and (MET) contaminated with metalaxyl-M. Each colony was considered a treatment replicate, with three replicates per treatment, totaling 15 colonies. Five workers were collected from each colony for the individual-level test, totaling 75 individuals observed. The asterisk indicates a significant effect of exposure duration on the amount of food ingested by individuals within the treatment ( p < 0.05). 3.3 Behavioral effects 3.3.1 Colonies and individuals after one week of exposure Exposure to pesticides at the colony level significantly affected multiple locomotor behaviors in bumble bee workers (Fig. 5 ; Table S1 ). Specifically, distance walked (χ² = 32.464, df = 4, p < 0.001), mean speed (χ² = 32.464, df = 4, p < 0.001), meandering (χ² = 39.562, df = 4, p < 0.001), resting time (χ² = 13.35, df = 4, p = 0.009), and mean fast time (χ² = 28.506, df = 4, p 0.05) (Fig. 5 ). Non-treated bees walked further and faster than all pesticide-exposed groups, while meandering behavior varied depending on the specific treatment (Fig. 5 ; Table S1 ). Notably, OEO-exposed bees exhibited significantly lower mean fast time compared to controls (Fig. 5 ; Table S1 ). At the individual level, pesticide exposure did not significantly affect most behavioral parameters, except for mean fast time (χ² = 11.082, df = 4, p = 0.026), which was increased in MET-exposed bees (Fig. 5 ; Table S1 ). The level of social exposure (colony vs. individual) played a critical role in our results, significantly influencing all behaviors except group density network (DW: χ² = 6.429, df = 1, p = 0.011; MS: χ² = 6.586, df = 1, p = 0.01; ME: χ² = 16.289, df = 1, p < 0.001; RT: χ² = 8.086, df = 1, p = 0.004; MT: χ² = 6.402, df = 1, p = 0.011; MF: χ² = 12.426, df = 1, p < 0.001; GN: χ² = 2.43, df = 1, p = 0.119) (Fig. 5 ). For CTRL and GLY treatments, bees exposed at the colony level showed greater distance walked and mean speed, but lower meandering than individually exposed bees (Fig. 5 ; Table S2). Additionally, GLY exposure at the colony level reduced resting time and increased mean movement time compared to individual exposure (Fig. 5 ; Table S2). Mean fast time was higher for colony-exposed bees in the CTRL and GLY treatments but reversed in MET-exposed bees (Fig. 5 ; Table S2). Figure 5 . Walking behavior of Bombus terrestris workers submitted to different pesticides at the colony and individual levels for one week. Honey syrup treatments were: (CTRL) uncontaminated (control); (ACE) contaminated with acetamiprid (0.01 µg/ml); (OEO) contaminated with sweet orange essential oil (476 µg/ml); (GLY) contaminated with glyphosate (30 µg/ml); and (MET) contaminated with metalaxyl-M (0.05 µg/ml). Each colony was considered a treatment replicate, with three replicates per treatment, totaling 15 colonies. Five workers were collected from each colony for the individual-level test, totaling 47 surviving individuals which were used for the behavioral bioassay. The “Colony” column shows the walking behavior of bees exposed to treatments at the colony level, while the “Individual” column shows the walking behavior of bees exposed at the individual level. The “Colony × Individual” column compares the walking behavior of bees exposed at both levels. In the “Colony” and “Individual” columns, asterisks indicate significant differences between pesticide-exposed treatments in relation to control ( p < 0.05). In the “Colony × Individual” column, asterisks indicate significant differences within treatments (CTRL, ACE, OEO, GLY, and MET) between bees exposed at the colony level and those exposed at the individual level ( p < 0.05). 3.3.2 Colonies after four weeks of exposure The number of weeks of exposure significantly influenced several locomotor behaviors in bees exposed at the colony level (Fig. 6 ; Table S3). Distance walked, mean speed, resting time, mean movement time, and mean fast time all showed significant temporal changes, while meandering and group density network remained unaffected (Fig. 6 ; Table S3). Notably, these effects were treatment-specific: increases in distance walked and mean speed were observed only in MET-exposed bees, whereas reductions in resting time occurred in OEO-, GLY-, and MET-exposed bees over time (Fig. 6 ; Table S3). Mean movement time increased exclusively in OEO-exposed bees, while mean fast time decreased in ACE-exposed bees and increased in OEO- and MET-exposed bees (Fig. 6 ; Table S3). No behavioral changes were detected in CTRL bees over the exposure period (Fig. 6 ). Figure 6 . Walking behavior of Bombus terrestris workers from colonies submitted to different pesticides over four weeks. (A) Distance walked. (B) Mean speed. (C) Resting time. (D) Mean movement time. (E) Mean fast time. Honey syrup treatments were: (CTRL) uncontaminated (control); (ACE) contaminated with acetamiprid; (OEO) contaminated with sweet orange essential oil; (GLY) contaminated with glyphosate; and (MET) contaminated with metalaxyl-M. Each colony was considered a treatment replicate, with three replicates per treatment, totaling 15 colonies. Five workers were collected per week per colony, totaling 300 individuals for the behavioral bioassay. Asterisks indicate a significant effect of the number of weeks on the behavior for the respective treatment ( p < 0.05). 4 DISCUSSION Our results demonstrate that not only synthetic insecticides but also biopesticides, herbicides, and fungicides can pose a threat to pollinator populations, at both field (biopesticides) and residual concentrations (other compounds). The tested pesticides caused different lethal and sublethal effects on bumble bee colonies and individuals, depending on the exposure period and the social level (colony or individual). Therefore, standard toxicological assessments focused on acute mortality of individuals are insufficient to perform risk assessments of pesticides on social bees. We observed lethal effects just in colonies and individuals exposed to the field concentration of the botanical biopesticide, probably because the concentration used for this product was higher than that of other treatments. As a consequence, mortality was higher in colonies treated with sweet orange essential oil, which is concerning, as increased forager death rates can lead to colony failure once a critical threshold is exceeded (Khoury et al., 2013 ; Russell et al., 2013 ). Despite using a higher concentration with the biopesticides, tests were based on the field-recommended dose, providing evidence that precautions are necessary before their field application. This information is particularly important because some countries have considered reducing certain requirements in biopesticide registration, such as toxicological testing (Soetopo and Alouw, 2023 ). The sublethal effects of pesticide treatments at the colony level depended on the specific pesticide treatment and the duration of exposure. Over time, non-treated colonies tended to gain mass and reduce food ingestion, as previously demonstrated (Incorvaia et al., 2022 ). In contrast, exposure to sweet orange essential oil led to a decrease in both colony mass and food ingestion, indicating colony weakening (Crone and Williams, 2016 ; Rotheray et al., 2017 ; Klatt et al., 2020 ; Capela et al., 2023 ). We also observed that non-treated colonies ingested more food as the temperature increased similar to previous research (Kenna et al., 2021 ). Since the behavior and physiology of bumblebees are affected by temperature (Uthoff and Ruxton, 2022 ; Karbassioon et al., 2023 ; Kuo et al., 2023 ), the lack of behavioral responses to temperature variation in colonies exposed to all pesticides tested here suggests a disruption of B. terrestris physiology, possibly due to metabolic alterations caused by the pesticides (Gooley and Gooley, 2020 ; Cullen et al., 2023 ; Fischer et al., 2024 ). The reduction in egg laying observed in all pesticide-exposed colonies suggests that pesticide exposure decreased queen fertility, negatively impacting colony reproduction and the production of new workers, ultimately contributing to colony failure (Rangel et al., 2013 ; Banks et al., 2020 ). The lower egg laying could also be linked to queen loss, which occurred only in pesticide-treated colonies. The negative effects of pesticides on bumble bee colony reproduction and queen survival have been observed previously (Richardson et al., 2024 ; Rondeau and Raine, 2024 ) and can be caused by disruption of ovary development and pesticide-induced malnutrition (Moreira et al., 2022 ). Unlike what was observed at the colony level, bumble bee workers exposed individually to the treatments for one week exhibited only sublethal effects. This contrast between the lethal effects of the biopesticide on bumble bee workers at the colony and individual levels highlights the need for long-term ecotoxicological studies to assess pesticide impacts on bees of different social contexts (Stanley et al., 2015 ; Hendriksma et al., 2019 ; Main et al., 2020 ; Thompson et al., 2022 ; Weidenmüller et al., 2022 ; Zioga et al., 2023 ). For example, in our data food ingestion was affected only in individual bumble bees treated with the biopesticide. As observed at the colony level, these individuals consumed less food than the control workers such as previous observations (Ferreira et al., under review). In contrast, colonies treated with the other pesticides showed a reduction in food consumption after one week of exposure, whereas individually exposed workers did not exhibit this effect. This suggests that the reduction in food intake at the colony level in these treatments is due to decreased foraging effort by the colony rather than changes in individual foraging behavior (Stanley et al., 2015 ). Another change observed at the individual level was that non-treated bees tended to increase their daily food intake over time. This pattern was also seen in individuals exposed to acetamiprid but not in those exposed to the other pesticides. Reduced feeding has been previously reported in bumble bees orally exposed to pesticides and has been linked to a dose-dependent intensification of pesticide toxicity over time (Cresswell et al., 2012 , 2014 ; Thompson et al., 2014 ; Muth et al., 2020 ; Catania et al., 2024 ). Previous studies have shown that bumble bees do not avoid food contaminated with glyphosate or metalaxyl-M (Thompson et al., 2022 ; Motta and Moran, 2023 ; (Ferreira et al., under review). Therefore, the lack of an increase in daily food intake observed in individuals exposed to these pesticides may be a consequence of sublethal effects, such as impaired learning ability (Thompson et al., 2023 ; Kaakinen et al., 2024 ). In contrast, the absence of an increasing intake of food contaminated with sweet orange essential oil could be attributed to bumble bee avoidance of honey syrup mixed with this biopesticide (Ferreira et al., under review). The increasing daily food intake observed in acetamiprid-exposed bees further supports the idea that the known antifeeding properties of some neonicotinoids depends on the compound (Thompson et al., 2014 ) and concentration (Catania et al., 2024 ) tested. The positive correlation between food ingestion and bees body mass aligns with field observations (Goulson et al., 2002 ; Ings et al., 2005 ). However, larger bumble bees exposed to the other pesticides did not ingest a proportionally greater amount of honey syrup relative to their body mass and they might experience malnutrition. Interestingly, in control and glyphosate-treated bees, workers exposed at the colony level performed better in behavioral tests than those exposed individually. For other treatments, worker performance was similar regardless of the social level of exposure, suggesting that social isolation affects bees but that this effect is masked by pesticide exposure in most cases. This finding reinforces the importance of conducting ecotoxicological studies on eusocial insects using also colonies (Demirozer et al., 2022 ; Weidenmüller et al., 2022 ). If our bioassays had been conducted only at the individual level, the sublethal effects of pesticides on walking behavior would not have been detected. As social isolation can cause physiological and morphological disruptions in bumble bees, our results demonstrate that these disruptions may be linked to the impaired walking ability observed in individual workers from the control and glyphosate treatments (Richter et al., 2012 ; Wang et al., 2022 ; Hill et al., 2023 ). The effect of social exposure level was less pronounced in workers treated with acetamiprid, sweet orange essential oil, and metalaxyl-M. While workers exposed to these pesticides at the colony level walked more slowly and covered shorter distances than control workers, this difference was not observed in those exposed individually. Additionally, workers treated with sweet orange essential oil and metalaxyl-M at the colony level exhibited a higher degree of meandering compared to the control, suggesting that their navigation ability was impaired. However, this difference between pesticide-exposed and control workers was not observed at the individual level. These findings suggest an antagonistic interaction between pesticide exposure and social isolation in the acetamiprid, sweet orange essential oil, and metalaxyl-M treatments. In contrast, for the glyphosate treatment, the interaction between pesticides and social isolation appears to be synergistic. When bees were exposed to pesticides at the colony level, their walking activity was lower than that of control workers. Moreover, exposure to most of the tested pesticides led to an increase in walking activity over time, unlike control bees. These results suggest that, over the weeks, the detrimental effects of certain pesticides on walking ability may diminish. Workers exposed to acetamiprid at the colony level differed from all other treatments, displaying slower movements over time. However, it is important to note that we did not evaluate the same individuals each week. Therefore, the observed decrease in walking impairment was not an individual-level effect but rather a colony-level response, potentially due to social buffering (Crall et al., 2019 ). Overall, our data demonstrate that field-realistic concentrations of various pesticides induce a wide range of sublethal effects on both individual bumble bees and entire colonies, an important concern given their role as key pollinators. Although most of the pesticides we tested did not significantly increase bumble bee mortality, they may still threaten the long-term maintenance of bumble bee populations, as sustained sublethal effects of pesticides can lead to colony failure (Bryden et al., 2013 ; Rondeau et al., 2014 ; Mulvey and Cresswell, 2020 ). Furthermore, our findings on chronic exposure at both individual and colony levels provide additional evidence supporting the need for extended laboratory risk assessments of pesticide impacts on wild and managed pollinators (Tosi et al., 2021 ; Barascou et al., 2022 ). Additionally, these results emphasize the importance of considering social context in ecotoxicological bioassays of social insects (Hendriksma et al., 2019 ; Demirozer et al., 2022 ; Weidenmüller et al., 2022 ). 5 CONCLUSIONS Even minimal exposure to pesticides considered safe for bees (acetamiprid, glyphosate, and metalaxyl-M) induced multiple sublethal effects in a bee species characterized by a larger body mass. Additionally, social isolation impaired the behavior of bumble bee workers, highlighting the importance of considering the group context when conducting laboratory bioassays with social insects. Our results demonstrate that commonly held assumptions - such as the necessity of using only individual bees for pesticide risk assessments and the belief that non-insecticides or biopesticides are safe for bees - need to be reconsidered. Declarations Funding “This work was funded by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil (CAPES) through the Print program (Grant 88887.803570/2023-00 to LMNF; Grant 88887.571161/2020-00 to MAPL), the Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG) (Grant 5.12/2022 to LMNF; Grant BPQ-06544-24 to MAPL), and the University of Catania (University Research Funds PIACERI — Research Plan 2020/2022 to GM).” Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Data Availability “The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.” Compliance with Ethical Standards The ARRIVE guidelines were followed in this study, and insects are not protected by the U.K. Animals (Scientific Procedures) Act, 1986. In addition, our methods are consistent with commonly accepted norms of animal welfare. CRediT Author Statement Lívia Maria Negrini Ferreira: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Writing - Original Draft, Visualization, Project administration, Funding acquisition. Gaetana Mazzeo: Conceptualization, Methodology, Validation, Resources, Writing - Review & Editing, Supervision, Project administration, Funding acquisition. Maria Augusta Pereira Lima: Writing - Original Draft, Writing - Review & Editing, Supervision. References Arena, M.; Sgolastra, F. (2014). A meta-analysis comparing the sensitivity of bees to pesticides. 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09:38:45","extension":"png","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":113660,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8311248/v1/0af5b62190f3faa512aae37c.png"},{"id":100969270,"identity":"593b11de-bb8c-48a6-8cff-55527fb020e1","added_by":"auto","created_at":"2026-01-23 09:51:37","extension":"xml","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":224598,"visible":true,"origin":"","legend":"","description":"","filename":"5e9def9fb2fc4042ad4cd340ec2e5e451structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8311248/v1/062e1466bb43a9841042c8e8.xml"},{"id":101942709,"identity":"dc13a937-0be5-4982-b74c-c706f685e9e9","added_by":"auto","created_at":"2026-02-05 09:34:44","extension":"html","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":239891,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8311248/v1/9cd6319d6d3f7537d839dba0.html"},{"id":100969251,"identity":"b1e95226-d5bf-4af4-b94a-049dec76b022","added_by":"auto","created_at":"2026-01-23 09:51:37","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":739320,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of pesticides on bumble bees’ mortality and colony mass variation over four weeks of colony exposure.\u003c/strong\u003e (A) Mean number of dead bees per treatment after four weeks of exposure. (B) Generalized Linear Model regression of the number of dead bees per treatment as a function of weeks of exposure. (C) Total colony mass gain or loss per treatment after four weeks of exposure. (D) Generalized Linear Model regression of total mass gain or loss per treatment as a function of weeks of exposure. Honey syrup treatments were: (CTRL) uncontaminated (control); (ACE) contaminated with acetamiprid; (OEO) contaminated with sweet orange essential oil; (GLY) contaminated with glyphosate; and (MET) contaminated with metalaxyl-M. Each colony was considered a treatment replicate, with three replicates per treatment, totaling 15 observed colonies. The asterisk indicates a significant difference between pesticide-exposed colonies in comparison to control (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8311248/v1/75e3a701f64e0f3f3be495fe.png"},{"id":100969252,"identity":"a417e7b4-870f-41f6-a391-72ee680d05a4","added_by":"auto","created_at":"2026-01-23 09:51:37","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":846051,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFood ingestion by \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eBombus terrestris\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003ecolonies over four weeks of exposure to different pesticides.\u003c/strong\u003e (A) Weekly amount of food ingested by each colony per treatment. (B) Generalized Linear Model (GLM) regression of the weekly amount of food ingested by each colony per treatment as a function of exposure duration. (C) Amount of food ingested by colonies submitted to different pesticides as a function of temperature. Honey syrup treatments were: (CTRL) uncontaminated (control); (ACE) contaminated with acetamiprid; (OEO) contaminated with sweet orange essential oil; (GLY) contaminated with glyphosate; and (MET) contaminated with metalaxyl-M. Each colony was considered a treatment replicate, with three replicates per treatment, totaling 15 observed colonies. The asterisk indicates a significant difference between pesticide-exposed colonies in comparison to control (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8311248/v1/a53b7a4531b0a4b9d1ba9574.png"},{"id":101203276,"identity":"2955dcb2-6d12-4ca6-86d1-5b8b63ce1ff8","added_by":"auto","created_at":"2026-01-27 09:39:15","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":209987,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNumber of eggs in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eBombus terrestris\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e colonies after four weeks of pesticide exposure.\u003c/strong\u003e Honey syrup treatments were: (CTRL) uncontaminated (control; 0 µg/ml a.i.); (ACE) contaminated with acetamiprid (0.01 µg/ml a.i.); (OEO) contaminated with sweet orange essential oil (476 µg/ml a.i.); (GLY) contaminated with glyphosate (30 µg/ml a.i); and (MET) contaminated with metalaxyl-M (0.05 µg/ml a.i.). Each colony was considered a treatment replicate, with three replicates per treatment, totaling 15 observed colonies. The asterisk indicates a significant difference between pesticide-exposed colonies in comparison to control (\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8311248/v1/c613b74cadf4a525d9ce048a.png"},{"id":100969264,"identity":"3879df2f-ee4d-475d-8b5e-d0f8287dfd5c","added_by":"auto","created_at":"2026-01-23 09:51:37","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":594856,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFood ingestion by \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eBombus terrestris\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eindividuals during 96 hours of exposure to different pesticides. (\u003c/strong\u003eA) Daily amount of food ingested by individuals per treatment. (B) Generalized Linear Model (GLM) regression of the amount of food ingested by individuals per treatment as a function of hours of exposure. (C) Amount of food ingested by \u003cem\u003eBombus terrestris\u003c/em\u003e individuals as a function of bee mass. Honey syrup treatments were: (CTRL) uncontaminated (control); (ACE) contaminated with acetamiprid; (OEO) contaminated with sweet orange essential oil; (GLY) contaminated with glyphosate; and (MET) contaminated with metalaxyl-M. Each colony was considered a treatment replicate, with three replicates per treatment, totaling 15 colonies. Five workers were collected from each colony for the individual-level test, totaling 75 individuals observed. The asterisk indicates a significant effect of exposure duration on the amount of food ingested by individuals within the treatment (\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8311248/v1/03a2c3244f7ec121cd5cd697.jpeg"},{"id":100969273,"identity":"3c335dec-8113-4348-a324-dd2443a192f7","added_by":"auto","created_at":"2026-01-23 09:51:37","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1300079,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eWalking behavior of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eBombus terrestris\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e workers submitted to different pesticides at the colony and individual levels for one week.\u003c/strong\u003e Honey syrup treatments were: (CTRL) uncontaminated (control); (ACE) contaminated with acetamiprid (0.01 μg/ml); (OEO) contaminated with sweet orange essential oil (476 μg/ml); (GLY) contaminated with glyphosate (30 μg/ml); and (MET) contaminated with metalaxyl-M (0.05 μg/ml). Each colony was considered a treatment replicate, with three replicates per treatment, totaling 15 colonies. Five workers were collected from each colony for the individual-level test, totaling 47 surviving individuals which were used for the behavioral bioassay. The “Colony” column shows the walking behavior of bees exposed to treatments at the colony level, while the “Individual” column shows the walking behavior of bees exposed at the individual level. The “Colony × Individual” column compares the walking behavior of bees exposed at both levels. In the “Colony” and “Individual” columns, asterisks indicate significant differences between pesticide-exposed treatments in relation to control (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05). In the “Colony × Individual” column, asterisks indicate significant differences within treatments (CTRL, ACE, OEO, GLY, and MET) between bees exposed at the colony level and those exposed at the individual level (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8311248/v1/123807c10785f3774f3e0f54.png"},{"id":100969257,"identity":"1129e059-c742-44f0-911c-542da791f360","added_by":"auto","created_at":"2026-01-23 09:51:37","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":614538,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eWalking behavior of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eBombus terrestris\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eworkers from colonies submitted to different pesticides over four weeks.\u003c/strong\u003e(A) Distance walked. (B) Mean speed. (C) Resting time. (D) Mean movement time. (E) Mean fast time. Honey syrup treatments were: (CTRL) uncontaminated (control); (ACE) contaminated with acetamiprid; (OEO) contaminated with sweet orange essential oil; (GLY) contaminated with glyphosate; and (MET) contaminated with metalaxyl-M. Each colony was considered a treatment replicate, with three replicates per treatment, totaling 15 colonies. Five workers were collected per week per colony, totaling 300 individuals for the behavioral bioassay. Asterisks indicate a significant effect of the number of weeks on the behavior for the respective treatment (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8311248/v1/3cd4ffe90ec4d6e22dfa5825.jpeg"},{"id":101943949,"identity":"e2e27ca0-3267-4541-84ea-dba3674492bd","added_by":"auto","created_at":"2026-02-05 09:46:21","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5642473,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8311248/v1/e2ff7507-2ea0-4c67-8f32-d64978b78a79.pdf"},{"id":100969253,"identity":"005c808a-cf1b-4611-93b0-f515a288ca37","added_by":"auto","created_at":"2026-01-23 09:51:37","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":72070,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-8311248/v1/c525fe1b350d60bc9bfed137.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Beyond the obvious: a realistic and holistic approach reveals multiple disorders caused by reduced-risk pesticides on individuals and colonies of bumble bees","fulltext":[{"header":"1 INTRODUCTION","content":"\u003cp\u003ePollination is one of the most significant ecosystem services provided by insects, with bees being the primary pollinators among animals (Khalifa et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In the Mediterranean basin, bee diversity is particularly high (Michener, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Mazzeo et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), and bees are associated with increased crop productivity, even for self-fertile cultivars (Bartual et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Marqu\u0026eacute;s et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Kleftodimos et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Despite the importance of bees for Mediterranean crop production, some agricultural practices in the region, such as the use of pesticides, negatively impact bee composition and local populations of various species (Turrisi et al., \u003cspan citationid=\"CR93\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAlthough pesticides have long been recognized as a major threat to bees, their use remains widespread and requires increased regulation (Epstein, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Nicholson et al., \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The susceptibility of wild bees to pesticides differs from that of honey bees (\u003cem\u003eApis mellifera\u003c/em\u003e), which is the model species used for testing pesticide toxicity in bees (Arena and Sgolastra, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Cham et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Schmolke et al., \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Varga-Szilay and T\u0026oacute;th, \u003cspan citationid=\"CR95\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Bumble bees (\u003cem\u003eBombus\u003c/em\u003e), in particular, are important wild pollinators in temperate regions (Nicholson and Ricketts, \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; McGrady et al., \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), and studies have shown they can be more susceptible to pesticides than honey bees (Arena and Sgolastra, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Gradish et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Schmolke et al., \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Oral exposure, through the ingestion of contaminated pollen and nectar, is particularly concerning for bumble bees due to their life history. Bumble bee queens and larvae consume only unprocessed pollen and nectar, which may lead to higher oral pesticide doses relative to their body mass (Arena and Sgolastra, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Gradish et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Additionally, bumble bee larvae may consume up to 130 times more pollen per day than honey bee larvae, increasing the amount of contaminated food they ingest (Gradish et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eResidues of insecticides, fungicides, and herbicides have been found in the nectar and pollen of plants visited by bumble bees (Zioga et al., \u003cspan citationid=\"CR99\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), and residues detected in the colonies and bodies of these insects further confirm the collection, storage, and ingestion of pesticides (Bot\u0026iacute;as et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Main et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Zioga et al., \u003cspan citationid=\"CR100\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Nicholson et al., \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Oral exposure can occur even days after plants have been sprayed with pesticides, including wild plants that are not the target crops, and can persist for many days or even weeks (Zioga et al., \u003cspan citationid=\"CR100\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Kuivila et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Exposure to contaminated food can lead to the collapse of bee colonies, and even low-risk compounds or sublethal doses can cause colony morbidity due to bioaccumulation or interactions with chemical mixtures, nutritional stress, or diseases (Holder et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Calatayud-Vernich et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Magal et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2019\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Traynor et al., \u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn addition to the death of individuals caused by lethal doses, exposure to sublethal doses of pesticides can also harm the survival of colonies and populations of bees (Stanley et al., \u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Crall et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Weidenm\u0026uuml;ller et al., \u003cspan citationid=\"CR97\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). These sublethal doses can impair the proper functioning of bee colonies due to multiple effects on individual workers (Marques et al., \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Costa et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Miotelo et al., \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Although synthetic insecticides, particularly neonicotinoids, are commonly identified as the main pesticides harmful to bees, growing evidence suggests that herbicides, fungicides, and biopesticides can also pose risks to bees (Seide et al., \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Iwasaki and Hogendoorn, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Battisti et al., 2022; Catania et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Lima et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). In some cases, these compounds can be just as toxic to bees as synthetic insecticides (Barbosa et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Tom\u0026eacute; et al., \u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Bernardes et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Marques et al., \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Padilha et al., \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Piovesan et al., \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn this study, we investigated the lethal and sublethal effects of different types of pesticides on individuals and colonies of \u003cem\u003eBombus terrestris\u003c/em\u003e Linnaeus, 1758 (Apidae: Bombini) under laboratory conditions. We chronically exposed individuals and colonies to food contaminated with field-realistic doses of acetamiprid (a neonicotinoid insecticide), sweet orange essential oil (a biopesticide), glyphosate (an herbicide), or metalaxyl-M (a fungicide). Individuals were orally exposed for one week, and colonies for four weeks, with weekly assessments. Our study aimed to test the following hypotheses: (1) Pesticides from different groups decrease the survival of bumble bees; (2) The performance of bumble bee colonies is negatively affected by pesticides from different groups; (3) Pesticides from different groups alter the behavior of bumble bees; (4) The social level of exposure (individual or colony) affects the impact of pesticides on bumble bee behavior.\u003c/p\u003e"},{"header":"2 MATERIAL AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Bees and pesticides\u003c/h2\u003e \u003cp\u003eWe acquired large colonies of \u003cem\u003eB. terrestris\u003c/em\u003e containing a queen, workers, brood, and sugar water (Natupol Excel line from Koppert Biological Systems, Netherlands). The sugar water was removed before the experiments. The colonies were housed in a BugDorm-4E4590DH specimen handling cage (W93.0 \u0026times; D47.5 \u0026times; H47.5 cm). The bees had free access to exit the nest and collect honey syrup (1:1 v/v honey and distilled water) from feeders placed inside the cage. Honey bee pollen (Koppert Biological Systems, Netherlands) was supplied once a week and placed inside the colony.\u003c/p\u003e \u003cp\u003eIn all experiments, we used commercial formulations of acetamiprid (Epik\u0026reg; SL; Sipcam, a.i.: 4.67%, 50 g/l), metalaxyl-M (Ridomil\u0026reg; Gold SL; Syngenta, a.i.: 43.88%, 465 g/l), glyphosate (Taifun\u0026reg; MK CL; Adama, a.i.: 30.8%, 360 g/l) and sweet orange essential oil (Prev-Am\u0026reg; Plus; Oroagri International Ltd, a.i.: 5.88%, 60 g/l). For synthetic pesticides, we used field-realistic concentrations found between the range of previously reported concentrations found in nectar residuals, as follows: 0.01 \u0026micro;g/ml acetamiprid (Pohorecka et al., \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), 0.05 \u0026micro;g/ml of metalaxyl-M (Gong et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) and 30 \u0026micro;g/ml of glyphosate (Thompson et al., \u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Acetamiprid, glyphosate, and metalaxyl-M are systemic pesticides previously found in plants visited by bees and in the bees themselves (Kiljanek et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Thompson et al., \u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; El Agrebi et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Zioga et al., \u003cspan citationid=\"CR99\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, \u003cspan citationid=\"CR100\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Rondeau and Raine, \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). We used realistic concentrations based on studies that reported field doses of pesticides applied to the crop analyzed. Among the reported concentrations, we selected one that closely matched residue levels found in other studies for each active ingredient. Since no data are available on sweet orange essential oil residues in nectar, we used the recommended field concentration for tomato crops (476 \u0026micro;g/ml). This concentration simulates the deposition of the biopesticide on flowers when applied during flowering. Note that this commercial formulation is not recommended for use during flowering; therefore, we tested a worst-case scenario, assuming misuse of the product.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Pesticide oral exposure\u003c/h2\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003e2.2.1 Colony-level exposure\u003c/h2\u003e \u003cp\u003eEach colony was maintained in a translucent cage and provided with a single feeder containing uncontaminated honey syrup (control; CTRL) or honey syrup contaminated with acetamiprid (ACE), sweet orange essential oil (OEO), glyphosate (GLY), or metalaxyl-M (MET). Each colony represented one replicate, with three replicates per treatment, totaling 15 bumble bee colonies in the experiment.\u003c/p\u003e \u003cp\u003eEach colony had access to its designated feeder for four weeks. Feeders were cleaned, and food was replaced every 24 hours to prevent syrup fermentation and pesticide degradation. Feeders were weighed daily before and after exposure to estimate honey syrup consumption. Colonies were weighed once a week (N\u0026thinsp;=\u0026thinsp;4), when the dead bees outside the nest were counted and removed. At the end of the fourth week, colonies were opened to assess queen presence/absence and to count the number of eggs laid, estimating colony reproductive performance. Laboratory temperature and relative humidity (RH) were monitored using a data logger (EasyLog\u0026reg; EL-USB-2).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003e2.2.2 Individual-level exposure\u003c/h2\u003e \u003cp\u003eThe individual-level bioassays were conducted using the same colonies as the colony-level bioassays, and both were performed simultaneously. Each colony assigned to one of the five treatments (CTRL, ACE, OEO, GLY, MET) served as a replicate. Five adult bumble bee workers were collected from each colony and assigned to the same pesticide treatment as their colony of origin. Individuals were cold-anesthetized and weighed, and only those within the size range of 0.13\u0026ndash;0.36 g were included to standardize this variable, as bee size can influence pesticide sensitivity (Sgolastra et al., \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Linguadoca et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe selected workers were placed in individual 650 mL plastic cages (15.0 \u0026times; 15.0 \u0026times; 7.0 cm) with a perforated, mesh-covered lid for ventilation. A 3 mL syringe, containing honey syrup (same treatments and concentrations previously described) was inserted horizontally into each cage. Individual bumble bees had access to the honey syrup for one week. After one week, the procedure was repeated with five new workers from each colony, continuing until the four-week colony-level exposure ended. Syringes were replaced daily to prevent syrup fermentation and pesticide degradation. Each syringe was weighed before and after daily exposure to measure individual food consumption. Bees\u0026acute; survival was recorded every 24 hours. Bumble bees were kept in a dark room at 22\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C and 65% RH.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Assessment of behavioral effects on workers exposed at individual and colony-levels\u003c/h2\u003e \u003cp\u003eSurviving bees from the individual-level bioassay were filmed for behavioral analysis after one week of exposure. Bees were grouped according to pesticide treatment and filmed together, resulting in 15 videos (five pesticide treatments \u0026times; three replicates). For the colony-level exposure, behavioral assessments were conducted weekly for four weeks. In each week, we recorded the behavior of five workers from each pesticide-treatment colony, totaling 60 videos (five treatments \u0026times; three replicates \u0026times; four weeks).\u003c/p\u003e \u003cp\u003eVideos were recorded using a Logitech C922 Pro Stream Webcam and Logitech Capture software (version 2.08.11) at 30 fps and full HD resolution. For filming, each group was placed in a Petri dish (140 mm diameter \u0026times; 20 mm height) in a dark room illuminated with a fluorescent light source and recorded for 10 minutes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Data analysis\u003c/h2\u003e \u003cp\u003eTo analyze the effects of different treatments on foraging behavior, we used generalized linear models (GLMs) with the \u0026ldquo;GAMLSS\u0026rdquo; package (Rigby \u0026amp; Stasinopoulos, \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Pesticide treatments were compared to the control using Dunnett\u0026rsquo;s tests with the \u0026ldquo;emmeans\u0026rdquo; package (Lenth, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAt the colony level, we performed three GLMs to assess how pesticide treatment (categorical variable: CTRL, ACE, OEO, GLY, MET) and weeks of exposure (continuous variable: 1\u0026ndash;4) influenced (1) the number of dead bees outside the nest (\u0026ldquo;number of dead bees\u0026rdquo;), (2) colony mass variation (\u0026ldquo;colony mass variation\u0026rdquo;), and (3) honey syrup consumption (\u0026ldquo;food ingested\u0026rdquo;). Additionally, we performed two GLMs to evaluate the effect of pesticide treatment (categorical variable: CTRL, ACE, OEO, GLY, MET) on egg production (\u0026ldquo;number of eggs\u0026rdquo;) after four weeks of exposure. We calculated the correlation between \u0026ldquo;mass gain\u0026rdquo; and \u0026ldquo;food ingested\u0026rdquo; to ensure that pesticide effects were not confounded by a strong correlation between these variables. Finally, we performed a GLM to assess whether weekly honey syrup consumption (\u0026ldquo;food ingested per week\u0026rdquo;) was influenced by temperature (continuous variable) and pesticide treatment (categorical variable: CTRL, ACE, OEO, GLY, MET).\u003c/p\u003e \u003cp\u003eAt the individual level, we used Kaplan-Meier analysis to assess the effect of each pesticide treatment on bumble bee survival, estimating survival curves and median survival times (LT\u003csub\u003e50\u003c/sub\u003e). For this analysis, we considered only survival status (dead or alive) during the first 96 hours of exposure. Curve similarity was tested using the log-rank test, with comparisons adjusted using Bonferroni correction (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Pesticide-contaminated treatments were not compared to each other; instead, ACE, OEO, GLY, and MET survival curves were compared only to the CTRL curve. Analyses were performed using the R packages \u0026ldquo;survival\u0026rdquo; (Therneau \u0026amp; Grambsch, \u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Therneau, \u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), \u0026ldquo;survminer\u0026rdquo; (Kassambara et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), and \u0026ldquo;dplyr\u0026rdquo; (Wickham et al., \u003cspan citationid=\"CR98\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Additionally, we performed two GLMs to assess how honey syrup consumption (\u0026ldquo;food ingested\u0026rdquo;) was influenced by: (1) pesticide treatment (categorical variable: CTRL, ACE, OEO, GLY, MET) and exposure duration (continuous variable: 24\u0026ndash;96 h), and (2) pesticide treatment and bee mass (continuous variable: \u0026ldquo;bee mass\u0026rdquo;).\u003c/p\u003e \u003cp\u003eThe behaviors of bees across pesticide treatments and social levels (colony or individual) were analyzed using Ethoflow\u0026reg;, a software that employs computer vision and artificial intelligence (AI) to monitor behavioral parameters (Bernardes et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The AI k-means algorithm and combinatorial optimization were used to track individual behavior while preserving identity within the group. The measured behavioral variables included: distance walked (cm), mean walking speed (cm/s), meandering (average turning angle in degrees), resting time (s), mean movement time (proportion of time spent in intermediate activity: distance walked\u0026thinsp;\u0026gt;\u0026thinsp;0.07 and \u0026le;\u0026thinsp;0.4 cm/frame), mean fast time (proportion of time spent in high activity: distance walked\u0026thinsp;\u0026gt;\u0026thinsp;0.4 cm/frame), and group density network (interactions between an individual and others in the group).\u003c/p\u003e \u003cp\u003eBehavioral measurements obtained via Ethoflow\u0026reg; were analyzed using generalized linear models (GLMs) to assess the effects of pesticide treatment (categorical variable: CTRL, ACE, OEO, GLY, MET) on each behavioral parameter previously described, at both social levels (colony or individual). For colony-level exposure, we also examined how the number of weeks of exposure (continuous variable: 1 to 4) influenced behavioral parameters. Pesticide-contaminated treatments were not compared to each other; instead, ACE, OEO, GLY, and MET results were compared only to CTRL. The MET treatment was excluded from individual-level \u0026ldquo;group density network\u0026rdquo; analyses due to an insufficient number of surviving bees per group (minimum required\u0026thinsp;=\u0026thinsp;two).\u003c/p\u003e \u003cp\u003eAll analyses were conducted in R (version 4.3.2; R Core Team, \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), and graphs were generated using the \u0026ldquo;ggplot2\u0026rdquo; package.\u003c/p\u003e \u003c/div\u003e"},{"header":"3 RESULTS","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Colony-level pesticide exposure\u003c/h2\u003e \u003cp\u003eThe number of dead bees outside the nest was influenced by pesticide treatment (χ\u0026sup2; = 48.513, df\u0026thinsp;=\u0026thinsp;4, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and week (χ\u0026sup2; = 31.618, df\u0026thinsp;=\u0026thinsp;1, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), but there was no interaction between treatment and week (χ\u0026sup2; = 2.957, df\u0026thinsp;=\u0026thinsp;4, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.565) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Compared to the CTRL, the number of dead bees was higher in OEO-treated colonies (Z\u0026thinsp;=\u0026thinsp;5.358, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). In colonies treated with ACE, GLY, and MET, the number of dead bees did not differ from the CTRL (ACE: Z\u0026thinsp;=\u0026thinsp;0.520, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.925; GLY: Z\u0026thinsp;=\u0026thinsp;0.965, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.705; MET: Z = -2.164, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.102) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Over the weeks, the number of dead bees increased in all treatments except OEO (CTRL: Z\u0026thinsp;=\u0026thinsp;2.686, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.007; ACE: Z\u0026thinsp;=\u0026thinsp;2.438, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.015; OEO: Z\u0026thinsp;=\u0026thinsp;1.369, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.171; GLY: Z\u0026thinsp;=\u0026thinsp;2.308, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.021; MET: Z\u0026thinsp;=\u0026thinsp;2.504, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.012) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eThe variation in colony mass relative to the beginning of exposure was influenced by the interaction between treatment and the number of weeks (χ\u0026sup2; = 24.471, df\u0026thinsp;=\u0026thinsp;4, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). By the end of the four weeks of exposure, the total mass gain of MET colonies did not differ from CTRL colonies (t = -0.279, df\u0026thinsp;=\u0026thinsp;49, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.983) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). ACE and GLY colonies gained less mass than CTRL colonies, while OEO colonies lost mass (ACE: t = -2.793, df\u0026thinsp;=\u0026thinsp;49, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.026; OEO: t = -11.502, df\u0026thinsp;=\u0026thinsp;49, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; GLY: t = -3.541, df\u0026thinsp;=\u0026thinsp;49, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.003) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Over the weeks, CTRL and MET colonies tended to gain mass (CTRL: t\u0026thinsp;=\u0026thinsp;2.354, df\u0026thinsp;=\u0026thinsp;49, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.023; MET: t\u0026thinsp;=\u0026thinsp;2.668, df\u0026thinsp;=\u0026thinsp;49, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.01), while OEO colonies tended to lose mass (t = -3.984, df\u0026thinsp;=\u0026thinsp;49, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). The number of weeks had no significant effect on mass gain or loss in ACE and GLY colonies (ACE: t\u0026thinsp;=\u0026thinsp;1.913, df\u0026thinsp;=\u0026thinsp;49, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.062; GLY: t\u0026thinsp;=\u0026thinsp;0.241, df\u0026thinsp;=\u0026thinsp;49, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.81) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. \u003cb\u003eEffects of pesticides on bumble bees\u0026rsquo; mortality and colony mass variation over four weeks of colony exposure.\u003c/b\u003e (A) Mean number of dead bees per treatment after four weeks of exposure. (B) Generalized Linear Model regression of the number of dead bees per treatment as a function of weeks of exposure. (C) Total colony mass gain or loss per treatment after four weeks of exposure. (D) Generalized Linear Model regression of total mass gain or loss per treatment as a function of weeks of exposure. Honey syrup treatments were: (CTRL) uncontaminated (control); (ACE) contaminated with acetamiprid; (OEO) contaminated with sweet orange essential oil; (GLY) contaminated with glyphosate; and (MET) contaminated with metalaxyl-M. Each colony was considered a treatment replicate, with three replicates per treatment, totaling 15 observed colonies. The asterisk indicates a significant difference between pesticide-exposed colonies in comparison to control (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003eThe amount of food ingested per week was affected by the interaction between treatment and the number of weeks (χ\u0026sup2; = 13.854, df\u0026thinsp;=\u0026thinsp;4, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.008) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The amount of food ingested each week was higher in CTRL colonies compared to all pesticide-exposed treatments during the first, second, and third weeks (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). In the fourth week, food ingestion in CTRL colonies did not differ from that of ACE, GLY, and MET colonies but remained higher than in OEO colonies (ACE: t = -1.939, df\u0026thinsp;=\u0026thinsp;48, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.182; OEO: t = -7.306, df\u0026thinsp;=\u0026thinsp;48, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; GLY: t = -0.405, df\u0026thinsp;=\u0026thinsp;48, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.958; MET: t = -1.062, df\u0026thinsp;=\u0026thinsp;48, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.649) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Over the weeks, the weekly amount of food ingested tended to decrease in CTRL, ACE, and OEO colonies (CTRL: t = -4.103, df\u0026thinsp;=\u0026thinsp;48, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; ACE: t = -2.070, df\u0026thinsp;=\u0026thinsp;48, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.044; OEO: t = -7.734, df\u0026thinsp;=\u0026thinsp;48, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). The number of weeks did not affect food ingestion in GLY and MET colonies (GLY: t = -1.585, df\u0026thinsp;=\u0026thinsp;48, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.12; MET: t = -0.598, df\u0026thinsp;=\u0026thinsp;48, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.553) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Weekly colony mass loss or gain was moderately correlated with weekly food ingestion (r\u0026thinsp;=\u0026thinsp;0.547; t\u0026thinsp;=\u0026thinsp;4.977, df\u0026thinsp;=\u0026thinsp;58, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Ambient temperature ranged from 18.62\u0026deg;C to 25.17\u0026deg;C, and its interaction with treatment significantly affected food ingestion by the colonies (χ\u0026sup2; = 12.066, df\u0026thinsp;=\u0026thinsp;4, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.017) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). The amount of food ingested by CTRL colonies tended to increase with temperature (t\u0026thinsp;=\u0026thinsp;4.095, df\u0026thinsp;=\u0026thinsp;49, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), whereas temperature had no significant effect on pesticide-exposed treatments (ACE: t = -1.148, df\u0026thinsp;=\u0026thinsp;49, p\u0026thinsp;=\u0026thinsp;0.257; OEO: t\u0026thinsp;=\u0026thinsp;0.505, df\u0026thinsp;=\u0026thinsp;49, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.616; GLY: t\u0026thinsp;=\u0026thinsp;1.165, df\u0026thinsp;=\u0026thinsp;49, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.25; MET: t = -1.097, df\u0026thinsp;=\u0026thinsp;49, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.278) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. \u003cb\u003eFood ingestion by\u003c/b\u003e \u003cb\u003eBombus terrestris\u003c/b\u003e \u003cb\u003ecolonies over four weeks of exposure to different pesticides.\u003c/b\u003e (A) Weekly amount of food ingested by each colony per treatment. (B) Generalized Linear Model (GLM) regression of the weekly amount of food ingested by each colony per treatment as a function of exposure duration. (C) Amount of food ingested by colonies submitted to different pesticides as a function of temperature. Honey syrup treatments were: (CTRL) uncontaminated (control); (ACE) contaminated with acetamiprid; (OEO) contaminated with sweet orange essential oil; (GLY) contaminated with glyphosate; and (MET) contaminated with metalaxyl-M. Each colony was considered a treatment replicate, with three replicates per treatment, totaling 15 observed colonies. The asterisk indicates a significant difference between pesticide-exposed colonies in comparison to control (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003eThe number of eggs in the colony after four weeks of exposure was significantly affected by treatment (χ\u0026sup2; = 23.696, df\u0026thinsp;=\u0026thinsp;4, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Egg numbers were significantly higher in the CTRL colonies compared to all pesticide-exposed colonies (ACE: Z = -3.9, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; OEO: Z = -6.524, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; GLY: Z = -5.885, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; MET: Z = -2.824, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.017) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). At the end of the four-week exposure period, the number of queens present in the pesticide-treated colonies (ACE, OEO, GLY, and MET) was insufficient to permit a statistical comparison of queen mass with the CTRL group. Queen presence was recorded in 100% of CTRL colonies, 66.67% of ACE, GLY, and MET colonies, and 0% of OEO colonies.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. \u003cb\u003eNumber of eggs in\u003c/b\u003e \u003cb\u003eBombus terrestris\u003c/b\u003e \u003cb\u003ecolonies after four weeks of pesticide exposure.\u003c/b\u003e Honey syrup treatments were: (CTRL) uncontaminated (control; 0 \u0026micro;g/ml a.i.); (ACE) contaminated with acetamiprid (0.01 \u0026micro;g/ml a.i.); (OEO) contaminated with sweet orange essential oil (476 \u0026micro;g/ml a.i.); (GLY) contaminated with glyphosate (30 \u0026micro;g/ml a.i); and (MET) contaminated with metalaxyl-M (0.05 \u0026micro;g/ml a.i.). Each colony was considered a treatment replicate, with three replicates per treatment, totaling 15 observed colonies. The asterisk indicates a significant difference between pesticide-exposed colonies in comparison to control (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Individual-level pesticide exposure\u003c/h2\u003e \u003cp\u003eThe survival of bumble bee workers individually exposed did not differ among treatments (χ\u0026sup2; = 5.9, df\u0026thinsp;=\u0026thinsp;4, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.2) (Fig.\u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The LT₅₀ could not be calculated for CTRL and GLY due to high survival at the end of the experiment. For ACE, OEO, and MET, the LT₅₀ was 96 hours.\u003c/p\u003e \u003cp\u003eFood ingestion over pesticide exposure was significantly affected by treatment (χ\u0026sup2; = 53.757, df\u0026thinsp;=\u0026thinsp;4, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and time (χ\u0026sup2; = 13.398, df\u0026thinsp;=\u0026thinsp;1, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), with no interaction between treatment and time (χ\u0026sup2; = 1.791, df\u0026thinsp;=\u0026thinsp;4, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.774) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The amount of food ingested every 24 hours by workers exposed to ACE, GLY, and MET was similar to that of the CTRL on all days (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). However, workers exposed to OEO ingested less food than the CTRL group on all days (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Individual food ingestion tended to increase over time for CTRL and ACE bees (CTRL: Z\u0026thinsp;=\u0026thinsp;2.517, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.012; ACE: Z\u0026thinsp;=\u0026thinsp;2.002, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.045) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Time had no significant effect on food ingestion for OEO, GLY, and MET bees (OEO: Z\u0026thinsp;=\u0026thinsp;0.545, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.586; GLY: Z\u0026thinsp;=\u0026thinsp;1.661, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.097; MET: Z\u0026thinsp;=\u0026thinsp;1.655, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.098) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Food ingestion after pesticide exposure was also significantly affected by treatment (χ\u0026sup2; = 63.450, df\u0026thinsp;=\u0026thinsp;4, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), bumble bee mass (χ\u0026sup2; = 26.449, df\u0026thinsp;=\u0026thinsp;1, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), and the interaction between treatment and bee mass (χ\u0026sup2; = 16.131, df\u0026thinsp;=\u0026thinsp;4, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.003) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). The amount of honey syrup ingested tended to increase with bee mass for CTRL and GLY workers (CTRL: Z\u0026thinsp;=\u0026thinsp;4.154, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; GLY: Z\u0026thinsp;=\u0026thinsp;5.130, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Food ingestion of workers exposed to ACE, OEO, and MET was not affected by bee mass (ACE: Z\u0026thinsp;=\u0026thinsp;0.831, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.406; OEO: Z\u0026thinsp;=\u0026thinsp;0.299, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.765; MET: Z\u0026thinsp;=\u0026thinsp;0.74, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.459) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. \u003cb\u003eFood ingestion by\u003c/b\u003e \u003cb\u003eBombus terrestris\u003c/b\u003e \u003cb\u003eindividuals during 96 hours of exposure to different pesticides. (\u003c/b\u003eA) Daily amount of food ingested by individuals per treatment. (B) Generalized Linear Model (GLM) regression of the amount of food ingested by individuals per treatment as a function of hours of exposure. (C) Amount of food ingested by \u003cem\u003eBombus terrestris\u003c/em\u003e individuals as a function of bee mass. Honey syrup treatments were: (CTRL) uncontaminated (control); (ACE) contaminated with acetamiprid; (OEO) contaminated with sweet orange essential oil; (GLY) contaminated with glyphosate; and (MET) contaminated with metalaxyl-M. Each colony was considered a treatment replicate, with three replicates per treatment, totaling 15 colonies. Five workers were collected from each colony for the individual-level test, totaling 75 individuals observed. The asterisk indicates a significant effect of exposure duration on the amount of food ingested by individuals within the treatment (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Behavioral effects\u003c/h2\u003e \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003ch2\u003e3.3.1 Colonies and individuals after one week of exposure\u003c/h2\u003e \u003cp\u003eExposure to pesticides at the colony level significantly affected multiple locomotor behaviors in bumble bee workers (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e; Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Specifically, distance walked (χ\u0026sup2; = 32.464, df\u0026thinsp;=\u0026thinsp;4, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), mean speed (χ\u0026sup2; = 32.464, df\u0026thinsp;=\u0026thinsp;4, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), meandering (χ\u0026sup2; = 39.562, df\u0026thinsp;=\u0026thinsp;4, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), resting time (χ\u0026sup2; = 13.35, df\u0026thinsp;=\u0026thinsp;4, p\u0026thinsp;=\u0026thinsp;0.009), and mean fast time (χ\u0026sup2; = 28.506, df\u0026thinsp;=\u0026thinsp;4, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) were all significantly altered, whereas mean movement time and group density network showed no significant effects (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Non-treated bees walked further and faster than all pesticide-exposed groups, while meandering behavior varied depending on the specific treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e; Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Notably, OEO-exposed bees exhibited significantly lower mean fast time compared to controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e; Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). At the individual level, pesticide exposure did not significantly affect most behavioral parameters, except for mean fast time (χ\u0026sup2; = 11.082, df\u0026thinsp;=\u0026thinsp;4, p\u0026thinsp;=\u0026thinsp;0.026), which was increased in MET-exposed bees (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e; Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe level of social exposure (colony vs. individual) played a critical role in our results, significantly influencing all behaviors except group density network (DW: χ\u0026sup2; = 6.429, df\u0026thinsp;=\u0026thinsp;1, p\u0026thinsp;=\u0026thinsp;0.011; MS: χ\u0026sup2; = 6.586, df\u0026thinsp;=\u0026thinsp;1, p\u0026thinsp;=\u0026thinsp;0.01; ME: χ\u0026sup2; = 16.289, df\u0026thinsp;=\u0026thinsp;1, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; RT: χ\u0026sup2; = 8.086, df\u0026thinsp;=\u0026thinsp;1, p\u0026thinsp;=\u0026thinsp;0.004; MT: χ\u0026sup2; = 6.402, df\u0026thinsp;=\u0026thinsp;1, p\u0026thinsp;=\u0026thinsp;0.011; MF: χ\u0026sup2; = 12.426, df\u0026thinsp;=\u0026thinsp;1, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; GN: χ\u0026sup2; = 2.43, df\u0026thinsp;=\u0026thinsp;1, p\u0026thinsp;=\u0026thinsp;0.119) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). For CTRL and GLY treatments, bees exposed at the colony level showed greater distance walked and mean speed, but lower meandering than individually exposed bees (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e; Table S2). Additionally, GLY exposure at the colony level reduced resting time and increased mean movement time compared to individual exposure (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e; Table S2). Mean fast time was higher for colony-exposed bees in the CTRL and GLY treatments but reversed in MET-exposed bees (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e; Table S2).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. \u003cb\u003eWalking behavior of\u003c/b\u003e \u003cb\u003eBombus terrestris\u003c/b\u003e \u003cb\u003eworkers submitted to different pesticides at the colony and individual levels for one week.\u003c/b\u003e Honey syrup treatments were: (CTRL) uncontaminated (control); (ACE) contaminated with acetamiprid (0.01 \u0026micro;g/ml); (OEO) contaminated with sweet orange essential oil (476 \u0026micro;g/ml); (GLY) contaminated with glyphosate (30 \u0026micro;g/ml); and (MET) contaminated with metalaxyl-M (0.05 \u0026micro;g/ml). Each colony was considered a treatment replicate, with three replicates per treatment, totaling 15 colonies. Five workers were collected from each colony for the individual-level test, totaling 47 surviving individuals which were used for the behavioral bioassay. The \u0026ldquo;Colony\u0026rdquo; column shows the walking behavior of bees exposed to treatments at the colony level, while the \u0026ldquo;Individual\u0026rdquo; column shows the walking behavior of bees exposed at the individual level. The \u0026ldquo;Colony \u0026times; Individual\u0026rdquo; column compares the walking behavior of bees exposed at both levels. In the \u0026ldquo;Colony\u0026rdquo; and \u0026ldquo;Individual\u0026rdquo; columns, asterisks indicate significant differences between pesticide-exposed treatments in relation to control (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In the \u0026ldquo;Colony \u0026times; Individual\u0026rdquo; column, asterisks indicate significant differences within treatments (CTRL, ACE, OEO, GLY, and MET) between bees exposed at the colony level and those exposed at the individual level (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e \u003ch2\u003e3.3.2 Colonies after four weeks of exposure\u003c/h2\u003e \u003cp\u003eThe number of weeks of exposure significantly influenced several locomotor behaviors in bees exposed at the colony level (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e; Table S3). Distance walked, mean speed, resting time, mean movement time, and mean fast time all showed significant temporal changes, while meandering and group density network remained unaffected (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e; Table S3). Notably, these effects were treatment-specific: increases in distance walked and mean speed were observed only in MET-exposed bees, whereas reductions in resting time occurred in OEO-, GLY-, and MET-exposed bees over time (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e; Table S3). Mean movement time increased exclusively in OEO-exposed bees, while mean fast time decreased in ACE-exposed bees and increased in OEO- and MET-exposed bees (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e; Table S3). No behavioral changes were detected in CTRL bees over the exposure period (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e. \u003cb\u003eWalking behavior of\u003c/b\u003e \u003cb\u003eBombus terrestris\u003c/b\u003e \u003cb\u003eworkers from colonies submitted to different pesticides over four weeks.\u003c/b\u003e (A) Distance walked. (B) Mean speed. (C) Resting time. (D) Mean movement time. (E) Mean fast time. Honey syrup treatments were: (CTRL) uncontaminated (control); (ACE) contaminated with acetamiprid; (OEO) contaminated with sweet orange essential oil; (GLY) contaminated with glyphosate; and (MET) contaminated with metalaxyl-M. Each colony was considered a treatment replicate, with three replicates per treatment, totaling 15 colonies. Five workers were collected per week per colony, totaling 300 individuals for the behavioral bioassay. Asterisks indicate a significant effect of the number of weeks on the behavior for the respective treatment (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"4 DISCUSSION","content":"\u003cp\u003eOur results demonstrate that not only synthetic insecticides but also biopesticides, herbicides, and fungicides can pose a threat to pollinator populations, at both field (biopesticides) and residual concentrations (other compounds). The tested pesticides caused different lethal and sublethal effects on bumble bee colonies and individuals, depending on the exposure period and the social level (colony or individual). Therefore, standard toxicological assessments focused on acute mortality of individuals are insufficient to perform risk assessments of pesticides on social bees.\u003c/p\u003e \u003cp\u003eWe observed lethal effects just in colonies and individuals exposed to the field concentration of the botanical biopesticide, probably because the concentration used for this product was higher than that of other treatments. As a consequence, mortality was higher in colonies treated with sweet orange essential oil, which is concerning, as increased forager death rates can lead to colony failure once a critical threshold is exceeded (Khoury et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Russell et al., \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Despite using a higher concentration with the biopesticides, tests were based on the field-recommended dose, providing evidence that precautions are necessary before their field application. This information is particularly important because some countries have considered reducing certain requirements in biopesticide registration, such as toxicological testing (Soetopo and Alouw, \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe sublethal effects of pesticide treatments at the colony level depended on the specific pesticide treatment and the duration of exposure. Over time, non-treated colonies tended to gain mass and reduce food ingestion, as previously demonstrated (Incorvaia et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In contrast, exposure to sweet orange essential oil led to a decrease in both colony mass and food ingestion, indicating colony weakening (Crone and Williams, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Rotheray et al., \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Klatt et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Capela et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). We also observed that non-treated colonies ingested more food as the temperature increased similar to previous research (Kenna et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Since the behavior and physiology of bumblebees are affected by temperature (Uthoff and Ruxton, \u003cspan citationid=\"CR94\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Karbassioon et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Kuo et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), the lack of behavioral responses to temperature variation in colonies exposed to all pesticides tested here suggests a disruption of \u003cem\u003eB. terrestris\u003c/em\u003e physiology, possibly due to metabolic alterations caused by the pesticides (Gooley and Gooley, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Cullen et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Fischer et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe reduction in egg laying observed in all pesticide-exposed colonies suggests that pesticide exposure decreased queen fertility, negatively impacting colony reproduction and the production of new workers, ultimately contributing to colony failure (Rangel et al., \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Banks et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The lower egg laying could also be linked to queen loss, which occurred only in pesticide-treated colonies. The negative effects of pesticides on bumble bee colony reproduction and queen survival have been observed previously (Richardson et al., \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Rondeau and Raine, \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) and can be caused by disruption of ovary development and pesticide-induced malnutrition (Moreira et al., \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eUnlike what was observed at the colony level, bumble bee workers exposed individually to the treatments for one week exhibited only sublethal effects. This contrast between the lethal effects of the biopesticide on bumble bee workers at the colony and individual levels highlights the need for long-term ecotoxicological studies to assess pesticide impacts on bees of different social contexts (Stanley et al., \u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Hendriksma et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Main et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Thompson et al., \u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Weidenm\u0026uuml;ller et al., \u003cspan citationid=\"CR97\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Zioga et al., \u003cspan citationid=\"CR100\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). For example, in our data food ingestion was affected only in individual bumble bees treated with the biopesticide. As observed at the colony level, these individuals consumed less food than the control workers such as previous observations (Ferreira et al., under review). In contrast, colonies treated with the other pesticides showed a reduction in food consumption after one week of exposure, whereas individually exposed workers did not exhibit this effect. This suggests that the reduction in food intake at the colony level in these treatments is due to decreased foraging effort by the colony rather than changes in individual foraging behavior (Stanley et al., \u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAnother change observed at the individual level was that non-treated bees tended to increase their daily food intake over time. This pattern was also seen in individuals exposed to acetamiprid but not in those exposed to the other pesticides. Reduced feeding has been previously reported in bumble bees orally exposed to pesticides and has been linked to a dose-dependent intensification of pesticide toxicity over time (Cresswell et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2012\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Thompson et al., \u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Muth et al., \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Catania et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Previous studies have shown that bumble bees do not avoid food contaminated with glyphosate or metalaxyl-M (Thompson et al., \u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Motta and Moran, \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; (Ferreira et al., under review). Therefore, the lack of an increase in daily food intake observed in individuals exposed to these pesticides may be a consequence of sublethal effects, such as impaired learning ability (Thompson et al., \u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Kaakinen et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). In contrast, the absence of an increasing intake of food contaminated with sweet orange essential oil could be attributed to bumble bee avoidance of honey syrup mixed with this biopesticide (Ferreira et al., under review). The increasing daily food intake observed in acetamiprid-exposed bees further supports the idea that the known antifeeding properties of some neonicotinoids depends on the compound (Thompson et al., \u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) and concentration (Catania et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) tested. The positive correlation between food ingestion and bees body mass aligns with field observations (Goulson et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Ings et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). However, larger bumble bees exposed to the other pesticides did not ingest a proportionally greater amount of honey syrup relative to their body mass and they might experience malnutrition.\u003c/p\u003e \u003cp\u003eInterestingly, in control and glyphosate-treated bees, workers exposed at the colony level performed better in behavioral tests than those exposed individually. For other treatments, worker performance was similar regardless of the social level of exposure, suggesting that social isolation affects bees but that this effect is masked by pesticide exposure in most cases. This finding reinforces the importance of conducting ecotoxicological studies on eusocial insects using also colonies (Demirozer et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Weidenm\u0026uuml;ller et al., \u003cspan citationid=\"CR97\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). If our bioassays had been conducted only at the individual level, the sublethal effects of pesticides on walking behavior would not have been detected. As social isolation can cause physiological and morphological disruptions in bumble bees, our results demonstrate that these disruptions may be linked to the impaired walking ability observed in individual workers from the control and glyphosate treatments (Richter et al., \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR96\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Hill et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe effect of social exposure level was less pronounced in workers treated with acetamiprid, sweet orange essential oil, and metalaxyl-M. While workers exposed to these pesticides at the colony level walked more slowly and covered shorter distances than control workers, this difference was not observed in those exposed individually. Additionally, workers treated with sweet orange essential oil and metalaxyl-M at the colony level exhibited a higher degree of meandering compared to the control, suggesting that their navigation ability was impaired. However, this difference between pesticide-exposed and control workers was not observed at the individual level. These findings suggest an antagonistic interaction between pesticide exposure and social isolation in the acetamiprid, sweet orange essential oil, and metalaxyl-M treatments. In contrast, for the glyphosate treatment, the interaction between pesticides and social isolation appears to be synergistic.\u003c/p\u003e \u003cp\u003eWhen bees were exposed to pesticides at the colony level, their walking activity was lower than that of control workers. Moreover, exposure to most of the tested pesticides led to an increase in walking activity over time, unlike control bees. These results suggest that, over the weeks, the detrimental effects of certain pesticides on walking ability may diminish. Workers exposed to acetamiprid at the colony level differed from all other treatments, displaying slower movements over time. However, it is important to note that we did not evaluate the same individuals each week. Therefore, the observed decrease in walking impairment was not an individual-level effect but rather a colony-level response, potentially due to social buffering (Crall et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOverall, our data demonstrate that field-realistic concentrations of various pesticides induce a wide range of sublethal effects on both individual bumble bees and entire colonies, an important concern given their role as key pollinators. Although most of the pesticides we tested did not significantly increase bumble bee mortality, they may still threaten the long-term maintenance of bumble bee populations, as sustained sublethal effects of pesticides can lead to colony failure (Bryden et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Rondeau et al., \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Mulvey and Cresswell, \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Furthermore, our findings on chronic exposure at both individual and colony levels provide additional evidence supporting the need for extended laboratory risk assessments of pesticide impacts on wild and managed pollinators (Tosi et al., \u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Barascou et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Additionally, these results emphasize the importance of considering social context in ecotoxicological bioassays of social insects (Hendriksma et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Demirozer et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Weidenm\u0026uuml;ller et al., \u003cspan citationid=\"CR97\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e"},{"header":"5 CONCLUSIONS","content":"\u003cp\u003eEven minimal exposure to pesticides considered safe for bees (acetamiprid, glyphosate, and metalaxyl-M) induced multiple sublethal effects in a bee species characterized by a larger body mass. Additionally, social isolation impaired the behavior of bumble bee workers, highlighting the importance of considering the group context when conducting laboratory bioassays with social insects. Our results demonstrate that commonly held assumptions \u003cb\u003e-\u003c/b\u003e such as the necessity of using only individual bees for pesticide risk assessments and the belief that non-insecticides or biopesticides are safe for bees \u003cb\u003e-\u003c/b\u003e need to be reconsidered.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026ldquo;This work was funded by the Coordena\u0026ccedil;\u0026atilde;o de Aperfei\u0026ccedil;oamento de Pessoal de N\u0026iacute;vel Superior \u0026ndash; Brasil (CAPES) through the Print program (Grant 88887.803570/2023-00 to LMNF; Grant 88887.571161/2020-00 to MAPL), the Funda\u0026ccedil;\u0026atilde;o de Amparo \u0026agrave; Pesquisa do Estado de Minas Gerais (FAPEMIG) (Grant 5.12/2022 to LMNF;\u0026nbsp;Grant\u0026nbsp;BPQ-06544-24 to MAPL), and the University of Catania (University Research Funds PIACERI \u0026mdash; Research Plan 2020/2022 to GM).\u0026rdquo;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026ldquo;The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.\u0026rdquo;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompliance with Ethical Standards\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe ARRIVE guidelines were followed in this study, and insects are not protected by the U.K. Animals (Scientific Procedures) Act, 1986. In addition, our methods are consistent with commonly accepted norms of animal welfare.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCRediT Author Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eL\u0026iacute;via Maria Negrini Ferreira: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Writing - Original Draft, Visualization, Project administration, Funding acquisition. Gaetana Mazzeo: Conceptualization, Methodology, Validation, Resources, Writing - Review \u0026amp; Editing, Supervision, Project administration, Funding acquisition. Maria Augusta Pereira Lima: Writing - Original Draft, Writing - Review \u0026amp; Editing, Supervision.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eArena, M.; Sgolastra, F. (2014). A meta-analysis comparing the sensitivity of bees to pesticides. Ecotoxicology, 23: 324\u0026ndash;334. 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Doi: 10.1016/j.scitotenv.2023.166214.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-pest-science","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pest","sideBox":"Learn more about [Journal of Pest Science](https://www.springer.com/journal/10340)","snPcode":"10340","submissionUrl":"https://submission.nature.com/new-submission/10340/3","title":"Journal of Pest Science","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Chronic exposure, Biopesticide, Herbicide, Fungicide, Neonicotinoid, Bee Colony Health","lastPublishedDoi":"10.21203/rs.3.rs-8311248/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8311248/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThere is growing evidence of the negative impacts of prolonged exposure to sublethal concentrations of pesticides that are considered safe for bees, such as biopesticides, herbicides, and fungicides. In this study, we investigated the effects of four different types of pesticides on individuals and colonies of \u003cem\u003eBombus terrestris\u003c/em\u003e, using a holistic approach. Bees were orally and chronically exposed to either pure honey syrup (control, CTRL) or honey syrup contaminated with the neonicotinoid acetamiprid (ACE), the herbicide glyphosate (GLY), the fungicide metalaxyl-M (MET), or the biopesticide sweet orange essential oil (OEO). Sublethal effects were observed for all pesticide treatments at both individual and colony levels; however, lethal effects were only observed in colonies treated with OEO, likely due to the higher concentration tested for this product. Pesticide-exposed colonies experienced negative impacts on mass gain (ACE, OEO, GLY), food ingestion (all pesticide treatments), and number of eggs (all pesticide treatments). Pesticide-exposed individuals showed negative impacts on food ingestion (OEO). The walking behavior of workers was affected by pesticide exposure at the colony level, and by social isolation when treated individually. Overall, our results demonstrate that chronic exposure to pesticides considered safe for bees can cause detrimental sublethal effects on bumble bees, potentially contributing to the decline of pollinators. We also highlight the importance of considering the social context when assessing pesticide toxicity in social insects.\u003c/p\u003e","manuscriptTitle":"Beyond the obvious: a realistic and holistic approach reveals multiple disorders caused by reduced-risk pesticides on individuals and colonies of bumble bees","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-23 09:51:31","doi":"10.21203/rs.3.rs-8311248/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-02-17T20:36:28+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"210499109393046608574697108971937142609","date":"2026-01-21T19:19:19+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-01-21T17:35:01+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-09T05:02:34+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-12-09T05:01:42+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Pest Science","date":"2025-12-08T21:56:19+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-pest-science","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pest","sideBox":"Learn more about [Journal of Pest Science](https://www.springer.com/journal/10340)","snPcode":"10340","submissionUrl":"https://submission.nature.com/new-submission/10340/3","title":"Journal of Pest Science","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"8069634e-5d53-455e-8e1a-ed9ec041bfdc","owner":[],"postedDate":"January 23rd, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-01-23T09:51:32+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-23 09:51:31","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8311248","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8311248","identity":"rs-8311248","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}