Agricultura Scientia 21: No 2 35-45(2024) https://doi.org/10.18690/agricsci. 21.2.4 *Correspondence to: E-mail: cecdomingues@gmail.com 35 The Adverse Impacts of a Single Exposure to the Fungicide Picoxystrobin during the Larval Stage on Africanized Apis mellifera Caio Eduardo da Costa DOMINGUES 1* , Leticia Salvioni ANSALONI 1 , Aleš GREGORC 1 , Elaine Cristina MATHIAS DA SILVA 2 1 University of Maribor, Faculty of Agriculture and Life Sciences, Pivola 10, 2311 Hoče, Slovenia 2 Federal University of São Carlos – UFSCar, Department of Biology (DBio), Laboratory of Ecotoxicology and Environmental Integrity Analysis (LEIA), Sorocaba 18052-780, São Paulo State, Brazil ABSTRACT Pesticide use remains a problem in agriculture, contaminating natural ecosystems and affecting bees. Fungicides have been widely used worldwide, and honey bees can bring contaminated pollen and nectar to the colony, exposing the larvae. Studies on larval exposure to fungicides are still rare. Therefore, this work aimed to evaluate the effects of larval exposure to the fungicide picoxystrobin on biological parameters and cellular stress in the fat body. The larvae were single exposure on the fourth day (D4) to picoxystrobin at concentrations of 5 ng a.i./ μL (PCX5), 45 ng a.i./ μL (PCX45), 135 ng a.i./ μL (PCX135), and 400 ng a.i./ μL (PCX400). The effects on larval and pupal mortality, pupation rate, and emergence were evaluated. Additionally, cellular stress in the fat body was assessed in newly emerged bees. Exposure to PCX400 increased larval mortality by 26% and reduced the emergence of adult bees. The other concentrations did not affect larval and pupal mortality, or pupation and emergence rates. A cytotoxicity effect was observed in newly emerged bees from PCX400, indicated by positive immunolabeling of HSP70. Thus, a single exposure to picoxystrobin can impair larval development, induce a cellular stress response, and may interfere with colony dynamics. Keywords: development, honey bee, non-target organism, strobilurin, toxicity INTRODUCTION Brazil is the largest country in South America and is recognized as an essential food supplier in global agriculture. It produces a substantial amount of the food consumed worldwide (Calil and Ribera, 2019). The country has vast arable lands, abundant resources, and a favorable climate, making the cultivating of many crops feasible (Martinelli et al., 2010). The most cultivated crops include soybeans, sugarcane, maize, coffee, oranges, rice, cotton, beans, and tobacco (Bordonal et al., 2018; Toloi et al., 2021; Valdes, 2022), contributing to Brazil achieving US$ 125 billion in agricultural export value in 2021 (Valdes, 2022). Even with great importance in agriculture, Brazil still faces internal challenges such as inefficient agricultural sub-sectors, land distribution inequality, environmental concerns, and the need for sustainable practices (Martinelli et al., 2010). Among these challenges, the use of pesticides has raised concerns among researchers regarding the damage to human health and the risk to the environment, as the country is one of the top consumers of pesticides worldwide (Tang et al., 2022). Many studies have warned about the harmful effects of pesticides on human health and the potential risks of related diseases (Paumgartten, 2020; Islam et al., 2021; Lopes- Ferreira et al., 2022). Insecticides, herbicides, and fungicides are the most frequently used pesticides in Brazil (Lopes- Ferreira et al., 2022), and their usage has also been associated with terrestrial and aquatic contamination (Daam et al., 2019; Fernandes et al., 2020; Guarda et al., 2020; Brovini et al., 2021). Additionally, the impact of pesticide use extends to pollinators, e.g., bees, posing significant threats to ecosystems and biodiversity (Goulson et al., 2015; Sgolastra et The Adverse Impacts of a Single Exposure to the Fungicide Picoxystrobin during the Larval Stage on Africanized Apis mellifera 36 al., 2020), and efforts must be made to mitigate this. The global bee population demonstrates high diversity, with over 20,000 described species (Orr et al., 2021), and Brazil significantly contributes to this richness with more than 3,000 bee species (Silveira et al., 2002). However, Brazil's most well-known bee species is the poly-hybrid Africanized Apis mellifera (non-native), resulting from crossbreeding European and African subspecies (Sheppard et al., 1991). These managed bees have a high defense capability, remain active in foraging for extended periods, and are more efficient in resource collection compared to European subspecies (Winston and Katz, 1982; Malaspina and Stort, 1987). Furthermore, A. mellifera serves as a model for pesticide regulation in Brazil (Cham et al., 2017). Pesticide use in the country, however, is closely linked to the weakness and collapse of Africanized A. mellifera colonies (Pires et al., 2016). Among pesticides, fungicides are widely used worldwide (Gikas et al., 2022). Nevertheless, studies on the effects of fungicides on non-target organisms receive less attention compared to insecticides and herbicides (Wood and Goulson, 2017; Zubrod et al., 2019). This is concerning, as field concentrations of fungicide residues may exceed levels considered safe by regulatory agencies (Rondeau and Raine, 2022). Cullen et al. (2019) suggest that further research is needed, employing diverse approaches, various species, and a wide range of compounds to reduce the current knowledge gap. Picoxystrobin (C18H16F3NO4) is a fungicide from the strobilurin group; it acts by inhibiting the mitochondrial respiration (halting the production of ATP) of fungi (Bartlett et al., 2002). Nevertheless, previous studies have revealed that picoxystrobin can also be harmful to amphibians (Li et al., 2016), fish (Jia et al., 2018), soil animals (Schnug et al., 2015), and bees (Domingues et al., 2017; Batista et al., 2020). Adult workers of Africanized A. mellifera exposed continuously to the fungicide picoxystrobin had their lifespan reduced by 51.76%, along with an overload of the hepato-nephrocitic system (Domingues et al., 2017). Cytotoxic effects of picoxystrobin exposure after 24, 48, 72, and 96 hours were also observed in the midgut of Africanized A. mellifera, which can affect the individual performance of bees and may impact the colony as a whole (Batista et al., 2020). In the environment, bees can be exposed to picoxystrobin and other strobilurins through direct spray application or by residues found in pollen, nectar, and water that they collect (Pettis et al., 2013; Simon-Delso et al., 2014; Samarghandi et al., 2017; Rondeau and Raine, 2022). This exposure may pose a potential risk to honey bee larvae as well. Additionally, picoxystrobin has been detected in crops visited by A. mellifera (Rondeau and Raine, 2022). Benuszak et al. (2017) highlighted the need to use larvae in studies on honey bees' exposure to pesticides. From this perspective, it is essential to study honey bee larvae, as the ingestion of fungicide residues can cause stress, disturb their post-embryonic development, and potentially weaken the colony. Furthermore, this stress can activate cellular defense mechanisms and induce the expression of heat shock proteins (HSPs) (Tkáčová and Angelovičová, 2012). According to Silva et al. (2006), HSPs are valuable cellular biomarkers for pesticide exposure. Based on the information mentioned above and considering that research assessing the effects of fungicides on A. mellifera larvae is still scarce compared to studies on insecticides (Aupinel et al., 2007; Silva et al., 2015; Tavares et al., 2015; Dai et al., 2017; Friol et al., 2017; Tavares et al., 2019; Tesovnik et al., 2020; Begna et al., 2023; Carneiro et al., 2023; Ke et al., 2023), although adverse effects have been reported (Simon-Delso et al., 2017; Tadei et al., 2019; Tadei et al., 2020; Zhang et al., 2020; Domingues et al., 2021). The present study aimed to evaluate the effects of larval exposure to the active ingredient of fungicide picoxystrobin through biological parameters. The response to cellular stress in the fat body was evaluated by detection of HSP70. It is crucial to determine whether exposure to picoxystrobin adversely affects larval development and induces stress responses, as this can help predict possible negative effects on honey bee colonies and their ecological and economic roles. In addition, it can guide regulatory decisions on fungicide use in agriculture and support strategies to protect bees and other pollinators. MATERIALS AND METHODS Colonies of Africanized A. mellifera The honey bee larvae used in the present study were sampled from three different healthy colonies at an apiary located in the rural area of Piedade, São Paulo State (23°37 ′5.506"S, 47°29 ′7.926"W). The physiological status of the colonies were known, and no chemical treatments was applied to manage the colonies before or during the study period. In Brazil, research on invertebrates does not require animal ethics approval. Chemicals: fungicide picoxystrobin and insecticide dimethoate The picoxystrobin Pestanal® analytical standard (CAS number 117428-22-5, ≥ 98.0%) and dimethoate Pestanal® analytical standard (CAS number 1219794-81-6, ≥ 95.0%) were used for the larval toxicity tests. These standards were purchased from the Pestanal® product line, a registered trademark of Merck KGaA, Darmstadt, Germany. The Adverse Impacts of a Single Exposure to the Fungicide Picoxystrobin during the Larval Stage on Africanized Apis mellifera 37 Honey bee larval toxicity test, single exposure to picoxystrobin The methodology followed the Organisation for Economic Co-operation and Development No. 237 protocol (OECD, 2013). Initially, a brood comb from each of the three colonies was collected and taken to the “Laboratory of Ecotoxicology and Environmental Integrity Analysis (LEIA)” at the “Federal University of São Carlos (UFScar)” in Sorocaba, São Paulo State, where the larval bioassay was performed. The first instar larvae were individually transferred to sterilized polystyrene grafting cells (1 x 1 x 1 cm) with a wetted paintbrush (number 0), with each cell holding 20 µl of the standardized artificial diet A. The diet was composed of 50% by weight of fresh royal jelly and 50% by weight of an aqueous solution containing D-(+)-glucose (≥99.5%), D-(−)- fructose (≥99%), and yeast extract, as described by Aupinel et al. (2005). The polystyrene grafting cells were placed in cell culture plates (48 wells), each containing a piece of cotton soaked in 500 μl of sterilization solution (0.2% w/v methylbenzethonium chloride) enhanced with 15% w/v glycerol at the bottom of the wells. The plates containing the larvae were then placed into an acrylic desiccator cabinet (Thermo Scientific™ Nalgene™, 178 x 305 x 305 mm), where beakers containing a saturated solution of potassium sulphate (K2SO4) were also added to maintain humidity. The acrylic desiccator cabinet was kept in an incubator set at 34±2 ºC, with a relative humidity of 90±5 %, under dark conditions. The larvae were fed once a day until the sixth day (D6), and the diets and volumes were adapted at different stages of development, as described by Aupinel et al. (2005). On the fourth day of the experiment (D4), the larvae were single exposed to picoxystrobin concentrations (Fig. 1). First, a stock solution of picoxystrobin (1000 ng a.i./ μL) was prepared in autoclaved distilled water (60%) and acetone (40%) and diluted serially to obtain the working concentrations of 5, 45, 135, and 400 ng a.i./ μL. Since the fungicide picoxystrobin is not completely soluble in water (3.1 mg/L at 20 °C), acetone was used as an organic solvent, and a solvent control (CAC) was also added following the protocol described by the OECD No. 237 (2013), not exceeding 5% of the final diet volume (1.5 μL of acetone for a diet volume of 30 μL on D4). The control group (CTL) received only the larval diet without adding additional chemicals. Dimethoate (DMT) was used as a toxic reference chemical (8.8±0.5 μg a.i./larva) to ensure the reliability of the experiment (OECD, 2013). D – day; PCX – picoxystrobin Figure 1: Schematic representation of the larval stage feeding period adapted from OECD No. 237 protocol for larval toxicity test, single exposure (OECD, 2013). The diets A, B, and C were based on Aupinel et al. (2005). On the day of the single exposure (D4), the honey bee larvae were divided into the following experimental groups: picoxystrobin at 5 ng a.i./ μL (PCX5), picoxystrobin at 45 ng a.i./ μL (PCX45), picoxystrobin at 135 ng a.i./ μL (PCX135), picoxystrobin at 400 ng a.i./ μL (PCX400), control (CTL), solvent control (CAC), and dimethoate positive control (DMT). Fourteen honey bee larvae were used from each of the three selected healthy colonies per experimental group. This resulted in 42 larvae per experimental group, meeting the OECD No. 237 (OECD, 2013) requirement of a minimum of 36 honey bee larvae per group. The specific concentrations used in this study were based on preliminary studies conducted in the LEIA at UFSCar. Evaluation of the biological effects of single exposure After pesticide exposure on the fourth day (D4), the larval mortality rate of all experimental groups was monitored for up to 72 hours (D5-D7). The pupation mortality and pupation rates were monitored from the eighth to the fifteenth day (D8-D15), and the cumulative emergence rate was recorded on the twenty-second day (D22). Immunofluorescence "in totum" for HSP70 detection Three newly emerged bees (up to 48 hours old) that had been exposed to picoxystrobin during the larval stage were sampled from CTL, CAC, and PCX400 groups. They were then anesthetized by exposure to a low temperature (4 °C) for one minute and dissected in a sodium chloride (0.9%) using a stereomicroscope (Leica EZ4 HD) to remove the dorsal vessel along with the parietal fat body. The Adverse Impacts of a Single Exposure to the Fungicide Picoxystrobin during the Larval Stage on Africanized Apis mellifera 38 The dissected organs from all selected groups were placed individually on positively charged silanized slides (ImmunoSlide, EasyPath), where drops of the fixative solution (paraformaldehyde 4% in phosphate-buffered saline (PBS), 0.1 mol L 1 , pH 7.4) were added for 24 hours at 4 °C and covered with a plastic coverslip to spread the solution. The entire procedure was carried out in a black incubation tray for immunohistochemistry (EasyPath). After the fixation period, the slides containing the organs were washed in PBS and then incubated for 10 minutes in PBS with 0.05% Tween ® 20 (pH 7.4). The organs were subsequently permeabilized using a solution of 0.5% Triton X-100 in PBS for 30 minutes, followed by three washes in PBS with 0.05% Tween ® 20, with a five-minute incubation during the final wash. Nonspecific antigenic sites were blocked using PBS with 0.05% Tween ® 20 and 3% bovine serum albumin (BSA) solution for one hour at room temperature. The slides with organs were then washed three times in PBS with 0.05% Tween ® 20 and incubated with a primary antibody solution (monoclonal anti-heat shock protein 70, antibody produced in mouse, Clone BRM-22, H5147 - Sigma-Aldrich™), diluted 1:100, for five days in a black incubation tray in the fridge at 4 ºC. After incubation with the primary antibody, the slides containing the organs were washed in PBS with 0.05% Tween ® 20 for 30 minutes. Incubation was then carried out with the secondary antibody (rabbit anti-mouse IgG (H+L) cross-adsorbed, conjugated with Alexa Fluor™ 488, Invitrogen - Thermo Fisher Scientific, A-11059), diluted 1:100, for one hour at room temperature. Following this incubation, the slides were washed three times in PBS buffer and mounted with an aqueous fluorescence mounting medium (Dako) using glass coverslips. Two negative reaction controls were also performed (without primary and secondary antibodies). Immunofluorescence analyses were conducted to localize HSP70 using a laser scanning confocal microscope (LEICA TCS-SP8) with Leica Application Suite X software (LAS X, version 3.5.5), following the configurations described by Domingues et al. (2017). Three slides, each prepared from a single bee, were analyzed per group. Statistical analysis Data analysis was performed using R software, version 4.2.2. Survival data from larval and pupal stages were analyzed using the Log-rank test from the “survival” package (Therneau, 2021). The occurrence of bee pupation and emergence for each individual was computed up to the fifteenth day (D15) and twenty-second day (D22), respectively. Then, the pupation and emergence events were analyzed using generalized linear models with quasibinomial and binomial distributions, with the experimental groups as independent variables. The goodness of fit of the statistical models to the data was checked by half-normal plots (Moral et al., 2017). The pupation and emergence proportions of each experimental group were contrasted with the control group using estimation of effect size analysis with 5,000 resamples from the “dabestr” package (version 2023.9.12, Ho et al., 2019) generating Cohen’s h and p-value from a two-sided permutation t-test. RESULTS Biological effects of a single exposure to picoxystrobin The larval exposure to pesticides, considering DMT, increased the mortality of Africanized honey bees during the larval stage ( χ²=109, df=6, p<0.001), but did not influence the survival probability during the pupal stage ( χ²=1.3, df=5, p=0.9), as shown in Figure 2. During the larval stage, larvae from the CAC, PCX5, PCX45, and PCX135 groups showed similar survival probabilities to the CTL group (p>0.91). Exposure to PCX400 increased larval mortality by 26% compared to the CTL group (p=0.013).. The highest larval mortality was observed in the DMT group, which reduced survival probability by 69% compared to the CTL group (p<0.001), validating the larval toxicity test according to the OECD No. 237 protocol (OECD, 2013). CTL – Control; CAC – solvent control; PCX5 – picoxystrobin at 5 ng a.i./ μL; PCX45 – picoxystrobin at 45 ng a.i./ μL; PCX135 – picoxystrobin at 135 ng a.i./ μL; PCX400 – picoxystrobin at 400 ng a.i./ μL; DMT – dimethoate as a positive control. n = 42 honey bee larvae per experimental group. Figure 2: Survival probability of Africanized honey bees during the larval and pupal stages after single pesticide exposure. The pupation rate was not impaired by picoxystrobin exposure (Quasibinomial GLM, χ²=9.98, df=5, p=0.087). Compared to pupae from the CTL group, pupae from all groups exhibited a weak Cohen’s h with values ranging from -0.5 to 0.2 (Fig. 3). However, a negative influence of picoxystrobin exposure was observed on the emergence rate (Binomial GLM, χ²=21.311, df=5, p=0.0007), with a reduction in The Adverse Impacts of a Single Exposure to the Fungicide Picoxystrobin during the Larval Stage on Africanized Apis mellifera 39 the number of newly emerged adults when exposed to PCX400 (p=0.0001), as depicted in Figure 4. CTL – Control; CAC – solvent control; PCX5 – picoxystrobin at 5 ng a.i./ μL; PCX45 – picoxystrobin at 45 ng a.i./ μL; PCX135 – picoxystrobin at 135 ng a.i./ μL; PCX400 – picoxystrobin at 400 ng a.i./ μL. Figure 3: Proportion of Africanized honey bees that reached the pupal stage after larval exposure to picoxystrobin. The inferior axis displays 95% effect size bootstraps of Cohen’s h values obtained by comparing the experimental groups with the control group (indicated by the horizontal black line). CTL – Control; CAC – solvent control; PCX5 – picoxystrobin at 5 ng a.i./ μL; PCX45 – picoxystrobin at 45 ng a.i./ μL; PCX135 – picoxystrobin at 135 ng a.i./ μL; PCX400 – picoxystrobin at 400 ng a.i./ μL. Figure 4: Proportion of Africanized honey bees that reached the adult stage after larval exposure to picoxystrobin. The inferior axis displays 95% effect size bootstraps of Cohen’s h values obtained by comparing the experimental groups with the control group (indicated by the horizontal black line). Detection of HSP70 in the fat body Figure 5 shows the cellular stress response following exposure to picoxystrobin, as evidenced by the detection of HSP70 in the fat body of newly emerged Africanized A. mellifera. The oenocytes and trophocytes of bees from the CTL and CAC groups exhibited similar response patterns, characterized by either basal levels or the absence of immunolabeling of HSP70 (Fig. 5A and Fig. 5B). Furthermore, HSP70 labeling was not observed in the cell nuclei. Regarding the fat body of bees from the PCX400 group, positively immunolabeled regions were observed (Fig. 5C). These regions were not identified in the CTL and CAC groups. The response pattern of oenocytes was also altered in bees from the PCX400 group, with evidence of labeled HSP70 in the cytoplasm, specifically in the perinuclear region (Fig. 5D), a feature not observed in the CTL and CAC groups. A – Control (CTL); B – solvent control (CAC); C – picoxystrobin at 400 ng a.i./ μL (PCX400); fb – fat body; n – nuclei; oe – oenocyte; tr – trophocyte; white arrow – positive labeling of HSP70; n – three newly emerged honey bees per experimental group. Figure 5: Detection of HSP70 in the fat body of newly emerged Africanized honey bees exposed to the fungicide picoxystrobin during the larval stage. DISCUSSION The results presented in this study highlight that larval exposure to the fungicide picoxystrobin can increase larval mortality and reduce bee emergence, even if only at the highest concentration (400 ng a.i./ μL). This finding is concerning, as bees may be exposed to high concentrations of fungicide through pollen, nectar, and water (Pettis et al., 2013; Zubrod et al., 2019; Zioga et al., 2020). According to Thompson et al. (2014), the toxicity of fungicides may increase in a dose-dependent manner due to ingestion by honey bees. In that regard, studies focusing on the prolonged contact of larvae and adult bees with fungicides are needed to better understand disruptions in developmental processes and physiological responses linked to cellular stress. Regarding the other picoxystrobin concentrations used in this study, neither larval mortality rates nor post- embryonic development were significantly affected.. The absence of adverse effects on these parameters was similarly The Adverse Impacts of a Single Exposure to the Fungicide Picoxystrobin during the Larval Stage on Africanized Apis mellifera 40 observed in studies performed with the active ingredient pyraclostrobin (Tadei et al., 2019; Domingues et al., 2021) and its commercial formulation (Tadei et al., 2020). Fungicide pyraclostrobin belongs to the strobilurin chemical class, similar to picoxystrobin (Bartlett et al., 2002). On the other hand, when fungicides were combined with insecticides, larvae were less likely to survive to adulthood (Wade et al., 2019). In addition to the observed effects on the development parameters in the PCX400 group, oenocytes from the parietal fat body of newly emerged bees exhibited positive immunolabeling for HSP70, indicating a cellular stress response. Similar findings were described in the intestine after larval exposure to the fungicide pyraclostrobin, where positive labeling for HSP70 was observed (Tadei et al., 2020). According to Malaspina and Silva-Zacarin (2006), proteins from the HSP family are essential biomarkers and can be used to assess cellular responses to pesticide exposure in bees. Due to its sensitivity, this cellular marker has been widely used in ecotoxicology studies to evaluate stress response, particularly in the fat body of various bee species (Balsamo et al., 2023; Farder-Gomes et al., 2024a; Farder- Gomes et al., 2024b). The fat body is a multifunctional organ found around the organs (perivisceral) and adjacent to the tegument (parietal) in insects, composed of trophocytes and oenocytes (Roma et al., 2010). Among the several functions of the fat body are the storage of organic molecules, synthesis of vitellogenin, hemolymph regulation, immune response, and detoxification (Roma et al., 2010; Arrese and Soulages, 2010; Abdalla and Domingues, 2015). According to the literature, oenocytes are linked to cellular stress response after pesticide exposure (Domingues et al., 2017; Assis et al., 2022; Inoue et al., 2022), supporting the findings observed in this study. During the larval stage of bees, the fat body exhibits distinct characteristics and is more abundant than in adults due to developmental adaptations specific to this stage (Cruz-Landim, 2009). Despite its abundance, we observed that bees exposed to the highest concentration of picoxystrobin exhibited effects on HSP70 in newly emerged bees. This may suggest that the fungicide remained bioavailable throughout development, leading to a late cellular stress response in this parameter. Similar late effects have also been reported for other fungicides (Tadei et al., 2019; Domingues et al., 2021). Based on the findings discussed, this research may support future risk assessment programs for bees concerning fungicides, which have received less attention compared to insecticides and herbicides. However, it is important to highlight that this study was conducted under laboratory conditions, which might not take field conditions into account. Future research should look at long-term effects and test these findings in field settings to ensure their applicability in natural environments. CONCLUSIONS Considering that the biological parameters of Africanized honey bee larvae were impacted by a single exposure to the highest concentration of fungicide picoxystrobin and based on the knowledge gap in the research field, studies like this reinforce the relevance of intensifying efforts to develop protective actions against larval exposure to fungicides. ACKNOWLEDGEMENTS We would like to thank the beekeeper Edson Sampaio for maintaining hives and providing support for bee sampling, Thamiris Porto Sipriano Nascimento and Rafaela Tadei for their technical help, as well as the PPGBMA (“Programa de Pós-Graduação em Biotecnologia e Monitoramento Ambiental”, UFSCar Sorocaba) for the use of Laser Scanning Confocal Microscopy (Pró-Equipamentos: 3420/2013-17, 2610/2014-90), and DBio (Department of Biology, UFSCar Sorocaba) by infrastructure for the confocal microscopy. This research was supported by “São Paulo Research Foundation” (FAPESP) [grant numbers 2013/09419-4, 2014/04697-9, 2016/15743-7], and the “Brazilian National Council for Scientific and Technological Development” (CNPq) [grant number 490379/2011-7]. This research was co- funded by the Slovenian Research and Innovation Agency (ARIS), Research core funding No. P4-164; Research for improvement of safe food and health, by the project CRISPR- B number N4-0192. REFERENCES 1. Abdalla, F. C., & Domingues, C. E. C. (2015). Hepato- nephrocitic system: A novel model of biomarkers for analysis of the ecology of stress in environmental biomonitoring. Plos ONE, 10(7), e0132349. Retrieved from: https://doi.org/10.1371/journal.pone.0132349 2. Arrese, E. L., & Soulages, J. L. (2010). Insect fat body: energy, metabolism, and regulation. Annual Review of Entomology, 55, 207-225. Retrieved from: https://doi.org/10.1146/annurev-ento-112408-085356 3. Assis, J. C., Domingues, C. E. C., Tadei, R., da Silva, C. I., Lima, H. M. S., Decio, P., & Silva-Zacarin, E. C. (2022). Sublethal doses of imidacloprid and pyraclostrobin impair fat body of solitary bee Tetrapedia diversipes (Klug, 1810). Environmental Pollution, 304, 119140. Retrieved from: https://doi.org/10.1016/j.envpol.2022.119140 4. Aupinel, P., Fortini, D., Dufour, H., Tasei, J., Michaud, B., Odoux, J., & Pham-Delegue, M. (2005). Improvement of The Adverse Impacts of a Single Exposure to the Fungicide Picoxystrobin during the Larval Stage on Africanized Apis mellifera 41 artificial feeding in a standard in vitro method for rearing Apis mellifera larvae. Bulletin of Insectology, 58(2), 107-111. 5. Aupinel, P., Fortini, D., Michaud, B., Marolleau, F., Tasei, J. N., & Odoux, J. F. (2007). Toxicity of dimethoate and fenoxycarb to honey bee brood (Apis mellifera), using a new in vitro standardized feeding method. Pest Management Science, 63(11), 1090-1094. Retrieved from: https://doi.org/10.1002/ps.1446 6. Balsamo, P. J., de Andrade Nogueira, F. L., Ceschi-Bertoli, L., Salla, R. F., Maia, F. M. C., Irazusta, S. P., Boeing, G. A. N. S., & Abdalla, F. C. (2023). Effect of a 110 ppb mercury exposition on neotropical bumble bee workers, Bombus atratus: In situ localization of Hsp70 and Hsp90 and general morphological changes of hepato-nephrocitic cells. Journal of Apicultural Research, 62(4), 953-961. Retrieved from: https://doi.org/10.1080/00218839.2021.2024696 7. Bartlett, D. W., Clough, J. M., Godwin, J. R., Hall, A. A., Hamer, M., & Parr ‐Dobrzanski, B. (2002). The strobilurin fungicides. Pest Management Science, 58(7), 649-662. Retrieved from: https://doi.org/10.1002/ps.520 8. Batista, A. C., Domingues, C. E. D. C., Costa, M. J., & Silva- Zacarin, E. C. M. (2020). Is a strobilurin fungicide capable of inducing histopathological effects on the midgut and Malpighian tubules of honey bees? Journal of Apicultural Research, 59(5), 834-843. Retrieved from: https://doi.org/10.1080/00218839.2020.1724678 9. Begna, T., Ulziibayar, D., Bisrat, D., & Jung, C. (2023). Acute and sublethal effects of acetamiprid alone and in mixture with emamectin benzoate on honeybee, Apis mellifera. Journal of Asia-Pacific Entomology, 26(4), 102125. Retrieved from: https://doi.org/10.1016/j.aspen.2023.102125 10. Benuszak, J., Laurent, M., & Chauzat, M. P. (2017). The exposure of honey bees (Apis mellifera; Hymenoptera: Apidae) to pesticides: Room for improvement in research. Science of The Total Environment, 587, 423-438. Retrieved from: https://doi.org/10.1016/j.scitotenv.2017.02.062 11. Bordonal, R. D. O., Carvalho, J. L. N., Lal, R., De Figueiredo, E. B., De Oliveira, B. G., & La Scala, N. (2018). Sustainability of sugarcane production in Brazil. A review. Agronomy for sustainable development, 38, 1-23. Retrieved from: https://doi.org/10.1007/s13593-018-0490- x 12. Brovini, E. M., de Deus, B. C. T., Vilas-Boas, J. A., Quadra, G. R., Carvalho, L., Mendonça, R. F., Pereira, R. O., & Cardoso, S. J. (2021). Three-bestseller pesticides in Brazil: Freshwater concentrations and potential environmental risks. Science of the Total Environment, 771, 144754. Retrieved from: https://doi.org/10.1016/j.scitotenv.2020.144754 13. Calil, Y. C. D., & Ribera, L. (2019). Brazil’s agricultural production and its potential as global food supplier. Choices, 34(3), 1-12. Retrieved from: https://www.jstor.org/stable/26964933 14. Carneiro, L. S., Santos, C. G., de Resende, M. T. C. S., de Souza, D. L. L., dos Santos Souza, D., da Cruz Souza, A. M., Motta, J. V. O., Nere, P. H. A., Oliveira, A. H., & Serrão, J. E. (2023). Effects of the insecticide imidacloprid on the post-embryonic development of the honey bee Apis mellifera (Hymenoptera: Apidae). Science of the Total Environment, 905, 167278. Retrieved from: https://doi.org/10.1016/j.scitotenv.2023.167278 15. Cham, K. O., Rebelo, R. M., Oliveira, R. P., Ferro, A. A., Viana-Silva, F. E. C., Borges, L. O., Saretto, C. O. S. D., Tonelli, C. A. M., Macedo, T. C. (2017). Manual de avaliação de risco ambiental de agrotóxicos para abelha. Brasília, Brasília: Editora Ibama/Diqua. 16. Cruz-Landim, C. (2009). Abelhas: Morfologia e Funções de Sistemas. São Paulo, Brazil: Editora UNESP. 17. Cullen, M. G., Thompson, L. J., Carolan, J. C., Stout, J. C., & Stanley, D. A. (2019). Fungicides, herbicides and bees: A systematic review of existing research and methods. Plos ONE, 14(12), e0225743. Retrieved from: https://doi.org/10.1371/journal.pone.0225743 18. Daam, M. A., Chelinho, S., Niemeyer, J. C., Owojori, O. J., De Silva, P. M. C., Sousa, J. P., van Gestel, C. A. M., & Römbke, J. (2019). Environmental risk assessment of pesticides in tropical terrestrial ecosystems: test procedures, current status and future perspectives. Ecotoxicology and Environmental Safety, 181, 534-547. Retrieved from: https://doi.org/10.1016/j.ecoenv.2019.06.038 19. Dai, P., Jack, C. J., Mortensen, A. N., & Ellis, J. D. (2017). Acute toxicity of five pesticides to Apis mellifera larvae reared in vitro. Pest Management Science, 73(11), 2282- 2286. Retrieved from: https://doi.org/10.1002/ps.4608 20. Domingues, C. E., Abdalla, F. C., Balsamo, P. J., Pereira, B. V., de Alencar Hausen, M., Costa, M. J., & Silva-Zacarin, E. C. (2017). Thiamethoxam and picoxystrobin reduce the survival and overload the hepato-nephrocitic system of the Africanized honeybee. Chemosphere, 186, 994-1005. Retrieved from: https://doi.org/10.1016/j.chemosphere.2017.07.133 21. Domingues, C. E. C., Tadei, R., Inoue, L. V. B., Silva- Zacarin, E. C. M., & Malaspina, O. (2021). Effects of larval exposure to the fungicide pyraclostrobin on the post- embryonic development of Africanized Apis mellifera workers. Environmental Advances, 4, 100069. Retrieved from: https://doi.org/10.1016/j.envadv.2021.100069 22. Farder-Gomes, C. F., Grella, T. C., Malaspina, O., & Nocelli, R. F. C. (2024). Exposure to sublethal concentrations of imidacloprid, pyraclostrobin, and glyphosate harm the behavior and fat body cells of the stingless bee Scaptotrigona postica. Science of The Total Environment, 907, 168072. Retrieved from: https://doi.org/10.1016/j.scitotenv.2023.168072 23. Farder-Gomes, C. F., de Oliveira, M. A., Malaspina, O., & Nocelli, R. F. C. (2024). Exposure of the stingless bee Melipona scutellaris to imidacloprid, pyraclostrobin, and glyphosate, alone and in combination, impair its walking activity and The Adverse Impacts of a Single Exposure to the Fungicide Picoxystrobin during the Larval Stage on Africanized Apis mellifera 42 fat body morphology and physiology. Environmental Pollution, 348, 123783. Retrieved from: https://doi.org/10.1016/j.envpol.2024.123783 24. Fernandes, C. L. F., Volcão, L. M., Ramires, P. F., De Moura, R. R., & Júnior, F. M. R. D. S. (2020). Distribution of pesticides in agricultural and urban soils of Brazil: a critical review. Environmental Science: Processes & Impacts, 22(2), 256-270. Retrieved from: https://doi.org/10.1039/c9em00433e 25. Friol, P. S., Catae, A. F., Tavares, D. A., Malaspina, O., & Roat, T. C. (2017). Can the exposure of Apis mellifera (Hymenoptera, Apiadae) larvae to a field concentration of thiamethoxam affect newly emerged bees? Chemosphere, 185, 56-66. Retrieved from: https://doi.org/10.1016/j.chemosphere.2017.06.113 26. Gikas, G. D., Parlakidis, P., Mavropoulos, T., Vryzas, Z. (2022). Particularities of fungicides and factors affecting their fate and removal efficacy: A review. Sustainability, 14, 4056. Retrieved from: https://doi.org/10.3390/su14074056 27. Goulson, D., Nicholls, E., Botías, C., & Rotheray, E. L. (2015). Bee declines driven by combined stress from parasites, pesticides, and lack of flowers. Science, 347(6229), 1255957. Retrieved from: https://doi.org/10.1126/science.1255957 28. Guarda, P. M., Pontes, A. M., Domiciano, R. D. S., Gualberto, L. D. S., Mendes, D. B., Guarda, E. A., & da Silva, J. E. (2020). Assessment of ecological risk and environmental behavior of pesticides in environmental compartments of the Formoso River in Tocantins, Brazil. Archives of Environmental Contamination and Toxicology, 79(4), 524-536. Retrieved from: https://doi.org/10.1007/s00244-020-00770-7 29. Ho, J., Tumkaya, T., Aryal, S., Choi, H., & Claridge-Chang, A. (2019). Moving beyond P values: data analysis with estimation graphics. Nature methods, 16(7), 565-566. Retrieved from: https://doi.org/10.1038/s41592-019-0470- 3 30. Inoue, L. V., Domingues, C. E. C., Gregorc, A., Silva- Zacarin, E. C., & Malaspina, O. (2022). Harmful effects of pyraclostrobin on the fat body and pericardial cells of foragers of Africanized honey bee. Toxics, 10(9), 530. Retrieved from: 31. Islam, M. S., Azim, F., Saju, H., Zargaran, A., Shirzad, M., Kamal, M., Fatema, K., Rehman, S., Azad, M. A. M., & Ebrahimi-Barough, S. (2021). Pesticides and Parkinson’s disease: Current and future perspective. Journal of Chemical Neuroanatomy, 115, 101966. Retrieved from: https://doi.org/10.1016/j.jchemneu.2021.101966 32. Jia, W., Mao, L., Zhang, L., Zhang, Y., & Jiang, H. (2018). Effects of two strobilurins (azoxystrobin and picoxystrobin) on embryonic development and enzyme activities in juveniles and adult fish livers of zebrafish (Danio rerio). Chemosphere, 207, 573-580. Retrieved from: https://doi.org/10.1016/j.chemosphere.2018.05.138 33. Ke, L., Chen, X., Dai, P., & Liu, Y. J. (2023). Chronic larval exposure to thiacloprid impairs honeybee antennal selectivity, learning and memory performances. Frontiers in Physiology, 14, 1114488. Retrieved from: https://doi.org/10.3389/fphys.2023.1114488 34. Li, D., Liu, M., Yang, Y., Shi, H., Zhou, J., & He, D. (2016). Strong lethality and teratogenicity of strobilurins on Xenopus tropicalis embryos: Basing on ten agricultural fungicides. Environmental Pollution, 208, 868-874. Retrieved from: https://doi.org/10.1016/j.envpol.2015.11.010 35. Lopes-Ferreira, M., Maleski, A. L. A., Balan-Lima, L., Bernardo, J. T. G., Hipolito, L. M., Seni-Silva, A. C., Batista- Filho, J., Falcao, M. A. P., & Lima, C. (2022). Impact of pesticides on human health in the last six years in Brazil. International Journal of Environmental Research and Public Health, 19(6), 3198. Retrieved from: https://doi.org/10.3390/ijerph19063198 36. Malaspina, O., & Stort, A.C., (1987). Sucrose syrup- collecting behavior in Africanized and Caucasian bees and in the descendants of their crossings. Revista Brasileira de Genética, 10, 459-469. 37. Malaspina, O., & Silva-Zacarin, E. C. M. (2017). Cell markers for ecotoxicological studies in target organs of bees. Brazilian Journal of Morphological Sciences, 23(3- 4), 303-309. 38. Martinelli, L. A., Naylor, R., Vitousek, P. M., & Moutinho, P. (2010). Agriculture in Brazil: impacts, costs, and opportunities for a sustainable future. Current Opinion in Environmental Sustainability, 2(5-6), 431-438. Retrieved from: https://doi.org/10.1016/j.cosust.2010.09.008 39. Moral, R. A., Hinde, J., & Demétrio, C. G. B. (2017). Half- normal plots and overdispersed models in R: the hnp package. Journal of Statistical Software, 81(10), 1-23. Retrieved from: https://doi.org/10.18637/jss.v081.i10 40. OECD, (2013). Test No. 237: honey bee (Apis mellifera) larval toxicity test, single exposure. OECD Guidel Test Chem, 23, 1-10. 41. Orr, M. C., Hughes, A. C., Chesters, D., Pickering, J., Zhu, C. D., & Ascher, J. S. (2021). Global patterns and drivers of bee distribution. Current Biology, 31(3), 451-458. Retrieved from: https://doi.org/10.1016/j.cub.2020.10.053 42. Paumgartten, F. J. (2020). Pesticides and public health in Brazil. Current opinion in Toxicology, 22, 7-11. Retrieved from: https://doi.org/10.1016/j.cotox.2020.01.003 43. Pettis, J. S., Lichtenberg, E. M., Andree, M., Stitzinger, J., Rose, R., & vanEngelsdorp, D. (2013). Crop pollination exposes honey bees to pesticides which alters their susceptibility to the gut pathogen Nosema ceranae. Plos ONE, 8(7), e70182. Retrieved from: https://doi.org/10.1371/journal.pone.0070182 44. Pires, C. S. S., Pereira, F. D. M., Lopes, M. T. D. R., Nocelli, R. C. F., Malaspina, O., Pettis, J. S., & Teixeira, É. W. (2016). Enfraquecimento e perda de colônias de abelhas no The Adverse Impacts of a Single Exposure to the Fungicide Picoxystrobin during the Larval Stage on Africanized Apis mellifera 43 Brasil: há casos de CCD? Pesquisa Agropecuária Brasileira, 51, 422-442. Retrieved from: https://doi.org/10.1590/S0100-204X2016000500003 45. Roma, G. C., Bueno, O. C., & Camargo-Mathias, M. I. (2010). Morpho-physiological analysis of the insect fat body: a review. Micron, 41(5), 395-401. Retrieved from: https://doi.org/10.1016/j.micron.2009.12.007 46. Rondeau, S., & Raine, N. E. (2022). Fungicides and bees: a review of exposure and risk. Environment International, 165, 107311. Retrieved from: https://doi.org/10.1016/j.envint.2022.107311 47. Samarghandi, M. R., Mohammadi, M., Karami, A., Tabandeh, L., Dargahi, A., & Amirian, F. (2017). Residue analysis of pesticides, herbicides, and fungicides in various water sources using gas chromatography-mass detection. Polish Journal of Environmental Studies, 26(5), 2189-2195. Retrieved from: https://doi.org/10.15244/pjoes/70387 48. Schnug, L., Ergon, T., Jakob, L., Scott-Fordsmand, J. J., Joner, E. J., & Leinaas, H. P. (2015). Responses of earthworms to repeated exposure to three biocides applied singly and as a mixture in an agricultural field. Science of the Total Environment, 505, 223-235. Retrieved from: https://doi.org/10.1016/j.scitotenv.2014.09.089 49. Sgolastra, F., Medrzycki, P., Bortolotti, L., Maini, S., Porrini, C., Simon-Delso, N., & Bosch, J. (2020). Bees and pesticide regulation: lessons from the neonicotinoid experience. Biological Conservation, 241, 108356. Retrieved from: https://doi.org/10.1016/j.biocon.2019.108356 50. Sheppard, W. S., Soares, A. E. E., DeJong, D., & Shimanuki, H. (1991). Hybrid status of honey bee populations near the historic origin of Africanization in Brazil. Apidologie, 22(6), 643-652. Retrieved from: https://doi.org/10.1051/apido:19910607 51. Silva-Zacarin, E. C., Gregorc, A., & de Moraes, R. L. S. (2006). In situ localization of heat-shock proteins and cell death labelling in the salivary gland of acaricide- treated honeybee larvae. Apidologie, 37(5), 507-516. Retrieved from: https://doi.org/10.1051/apido:2006030 52. Silva, C. A., Silva-Zacarin, E., Domingues, C. E. C., Abdalla, F. C., Malaspina, O., Nocelli, R. C. (2015). Fipronil effect on the frequency of anomalous brood in honeybee reared in vitro. Julius Kuhn-Archiv. 450, 140. 53. Silveira, F. A., Melo, G. A. & Almeida, E. A. (2002). Abelhas Brasileiras: Sistemática e Identificação. Belo Horizonte, Belo Horizonte: Fundação Araucária. 54. Simon-Delso, N., San Martin, G., Bruneau, E., Minsart, L. A., Mouret, C., & Hautier, L. (2014). Honeybee colony disorder in crop areas: The role of pesticides and viruses. Plos ONE, 9(7), e103073. Retrieved from: https://doi.org/10.1371/journal.pone.0103073 55. Simon-Delso, N., San Martin, G., Bruneau, E., Hautier, L., & Medrzycki, P. (2017). Toxicity assessment on honey bee larvae of a repeated exposition of a systemic fungicide, boscalid. Bulletin of Insectology, 70(1), 83-89. 56. Tadei, R., Domingues, C. E., Malaquias, J. B., Camilo, E. V., Malaspina, O., & Silva-Zacarin, E. C. (2019). Late effect of larval co-exposure to the insecticide clothianidin and fungicide pyraclostrobin in Africanized Apis mellifera. Scientific Reports, 9(1), 3277. Retrieved from: https://doi.org/10.1038/s41598-019-39383-z 57. Tadei, R., Menezes-Oliveira, V. B., & Silva-Zacarin, E. C. (2020). Silent effect of the fungicide pyraclostrobin on the larval exposure of the non-target organism Africanized Apis mellifera and its interaction with the pathogen Nosema ceranae in adulthood. Environmental Pollution, 267, 115622. Retrieved from: https://doi.org/10.1016/j.envpol.2020.115622 58. Tang, F. H., Malik, A., Li, M., Lenzen, M., & Maggi, F. (2022). International demand for food and services drives environmental footprints of pesticide use. Communications Earth & Environment, 3(1), 272. Retrieved from: https://doi.org/10.1038/s43247-022- 00601-8 59. Tavares, D. A., Roat, T. C., Carvalho, S. M., Silva- Zacarin, E. C. M., & Malaspina, O. (2015). In vitro effects of thiamethoxam on larvae of Africanized honey bee Apis mellifera (Hymenoptera: Apidae). Chemosphere, 135, 370-378. Retrieved from: https://doi.org/10.1016/j.chemosphere.2015.04.090 60. Tavares, D. A., Roat, T. C., Silva-Zacarin, E. C. M., Nocelli, R. C. F., & Malaspina, O. (2019). Exposure to thiamethoxam during the larval phase affects synapsin levels in the brain of the honey bee. Ecotoxicology and Environmental Safety, 169, 523-528. Retrieved from: https://doi.org/10.1016/j.ecoenv.2018.11.048 61. Tesovnik, T., Zorc, M., Ristanić , M., Glavinić, U., Stevanović, J., Narat, M., & Stanimirović, Z. (2020). Exposure of honey bee larvae to thiamethoxam and its interaction with Nosema ceranae infection in adult honey bees. Environmental Pollution, 256, 113443. Retrieved from: https://doi.org/10.1016/j.envpol.2019.113443 62. Therneau, T. M. (2021). Survival Analysis [R package survival version 3.2-13]. 63. Thompson, H. M., Fryday, S. L., Harkin, S., & Milner, S. (2014). Potential impacts of synergism in honeybees (Apis mellifera) of exposure to neonicotinoids and sprayed fungicides in crops. Apidologie, 45, 545-553. Retrieved from: https://doi.org/10.1007/s13592-014-0273- 6 64. Tkáčová, J., & Angelovičová, M. (2012). Heat shock proteins (HSPs): a review. Scientific Papers Animal Science and Biotechnologies, 45(1), 349-349. 65. Toloi, M. N. V., Bonilla, S. H., Toloi, R. C., Silva, H. R. O., & Nääs, I. D. A. (2021). Development indicators and soybean production in Brazil. Agriculture, 11(11), 1164. Retrieved from: https://doi.org/10.3390/agriculture11111164 66. Valdes, C. (2022). Brazil's Momentum as a Global Agricultural Supplier Faces Headwinds. Amber Waves: The Adverse Impacts of a Single Exposure to the Fungicide Picoxystrobin during the Larval Stage on Africanized Apis mellifera 44 The Economics of Food, Farming, Natural Resources, and Rural America, 2022. Retrieved from: https://doi.org/10.22004/ag.econ.338866 67. Wade, A., Lin, C. H., Kurkul, C., Regan, E. R., & Johnson, R. M. (2019). Combined toxicity of insecticides and fungicides applied to California almond orchards to honey bee larvae and adults. Insects, 10(1), 20. Retrieved from: https://doi.org/10.3390/insects10010020 68. Winston, M. L., & Katz, S. J. (1982). Foraging differences between cross-fostered honeybee workers (Apis mellifera) of European and Africanized races. Behavioral Ecology and Sociobiology, 10, 125-129. 69. Wood, T. J., Goulson, D., (2017). The environmental risks of neonicotinoid pesticides: a review of the evidence post 2013. Environmental Science and Pollution Research, 24, 17285-17325. Retrieved from: https://doi.org/10.1007/s11356-017-9240-x. 70. Zhang, C., Zhou, T., Xu, Y., Du, Z., Li, B., Wang, J., Wang, J., & Zhu, L. (2020). Ecotoxicology of strobilurin fungicides. Science of the Total Environment, 742, 140611. Retrieved from: https://doi.org/10.1016/j.scitotenv.2020.140611 71. Zioga, E., Kelly, R., White, B., & Stout, J. C. (2020). Plant protection product residues in plant pollen and nectar: A review of current knowledge. Environmental research, 189, 109873. Retrieved from: https://doi.org/10.1016/j.envres.2020.109873 72. Zubrod, J. P., Bundschuh, M., Arts, G., Bru ̈hl, C. A., Imfeld, G., Kna ̈bel, A., Payraudeau, S., Rasmussen, J. J., Rohr, J., Scharmüller, A., Smalling, K., Stehle, S., Schulz, R., & Scha ̈fer, R. B. (2019). Fungicides: an overlooked pesticide class? Environmental Science & Technology, 53(7), 3347-3365. Retrieved from: https://doi.org/10.1021/acs.est.8b04392 The Adverse Impacts of a Single Exposure to the Fungicide Picoxystrobin during the Larval Stage on Africanized Apis mellifera 45 Škodljivi učinki enkratne izpostavljenosti ličink afrikanizirane čebele (Apis mellifera) fungicidu pikoksistrobin IZVLEČEK Uporaba pesticidov ostaja problem v kmetijstvu, saj onesnažuje naravne ekosisteme in vpliva na čebele. Fungicidi se pogosto uporabljajo po vsem svetu, medonosne čebele pa lahko v svojo čebeljo družino prinesejo kontaminiran cvetni prah in nektar ki vpliva na razvoj ličink. Študije o izpostavljenosti ličink fungicidom so še redke. Zato je bilo to delo namenjeno oceni učinkov izpostavljenosti ličink fungicidu pikoksistrobin na biološke parametre in celični stres v maščobnem telesu. Ličinke so bile četrti dan (D4) enkrat izpostavljene pikoksistrobinu pri koncentracijah 5 ng a.i./ μL (PCX5), 45 ng a.i./ μL (PCX45), 135 ng a.i./ μL (PCX135) in 400 ng a.i./ μL (PCX400). Ocenjeni so bili učinki na umrljivost ličink in bub, ter učinki na stopnjo zabubljenja in izleganja. Poleg tega je bil pri na novo izleženih čebelah ocenjen celični stres v maščobnem telesu. Izpostavljenost PCX400 je povečala smrtnost ličink za 26 % in zmanjšala stopnjo izleganja čebel. Druge koncentracije niso vplivale na umrljivost ličink in bub ali na stopnje zabubljenja in izleganja čebel. Učinek citotoksičnosti je bil ugotovljen v novo izleženih čebelah, tretiranih s PCX400, na kar kaže pozitivni imunski test na HSP70. Enkratna izpostavljenost pikoksistrobinu vpliva na slabši razvoj ličink, povzroči celični stresni odziv in potencialno moti dinamiko razvoja čebelje družine. Ključne besede: razvoj, medonosna čebela, neciljni organizem, strobilurin, toksičnost