Received: 4 May 2023 | Accepted: 13 August 2023 DOI: 10.1111/gcb.16928 RESEARCH ARTICLE Bees exposed to climate change are more sensitive to pesticides Sergio Albacete1,2 | Gonzalo Sancho1,2 | Celeste Azpiazu1,3 | Anselm Rodrigo1,2 | Roberto Molowny-­Horas1,2 | Fabio Sgolastra4 | Jordi Bosch1,2 1 Universitat Autònoma de Barcelona, Bellaterra, Spain Abstract 2 Centre for Ecological Research and Bee populations are exposed to multiple stressors, including land-­use change, bio- Forestry Applications (CREAF), Bellaterra, Spain logical invasions, climate change, and pesticide exposure, that may interact synergisti- 3 Institute of Evolutionary Biology (CSIC—­ cally. We analyze the combined effects of climate warming and sublethal insecticide Universitat Pompeu Fabra), Barcelona, exposure in the solitary bee Osmia cornuta. Previous Osmia studies show that warm Spain 4 wintering temperatures cause body weight loss, lipid consumption, and fat body de- Dipartimento di Scienze e Tecnologie Agro-­Alimentari, Alma Mater Studiorum pletion. Because the fat body plays a key role in xenobiotic detoxification, we ex- Università di Bologna, Bologna, Italy pected that bees exposed to climate warming scenarios would be more sensitive to Correspondence pesticides. We exposed O. cornuta females to three wintering treatments: current sce- Sergio Albacete, Universitat Autònoma nario (2007–­2012 temperatures), near-­future (2021–­2050 projected temperatures), de Barcelona and CREAF (Centre for Ecological Research and Forestry and distant-­future (2051–­2080). Upon emergence in spring, bees were orally exposed Applications), 08193 Bellaterra, Spain. to three sublethal doses of an insecticide (Closer, a.i. sulfoxaflor; 0, 4.55 and 11.64 ng Email: s.albacete@creaf.uab.cat a.i./bee). We measured the combined effects of wintering and insecticide exposure Funding information on phototactic response, syrup consumption, and longevity. Wintering treatment by Spanish Ministry of Science and Innovation, Grant/Award Number: itself did not affect winter mortality, but body weight loss increased with increas- PRE2019-­090375, PRE2019-­0 88817, ing wintering temperatures. Similarly, wintering treatment by itself hardly influenced PID2021-­128938OB-­I 00 and RTI2018-­ 098399-­B-­I 00; Spanish Ministry of phototactic response or syrup consumption. However, bees wintered at the warmest Universities, Grant/Award Number: temperatures had shorter longevity, a strong fecundity predictor in Osmia. Insecticide Margarita-­Salas-­scholarship exposure, especially at the high dose, impaired the ability of bees to respond to light, and resulted in reduced syrup consumption and longevity. The combination of the warmest winter and the high insecticide dose resulted in a 70% longevity decrease. Smaller bees, resulting from smaller pollen–­nectar provisions, had shorter longevity suggesting nutritional stress may further compromise fecundity in O. cornuta. Our results show a synergistic interaction between two major drivers of bee declines, and indicate that bees will become more sensitive to pesticides under the current global warming scenario. Our findings have important implications for pesticide regulation and underscore the need to consider multiple stressors to understand bee declines. KEYWORDS body size, climate change, global warming, longevity, nutritional stress, Osmia cornuta, pesticide exposure, sulfoxaflor, synergistic effects, toxicity This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2023 The Authors. Global Change Biology published by John Wiley & Sons Ltd. 6248 |  wileyonlinelibrary.com/journal/gcb Glob Change Biol. 2023;29:6248–6260. | 13652486, 2023, 22, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/gcb.16928 by Readcube (Labtiva Inc.), Wiley Online Library on [24/10/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License ALBACETE et al. 6249 1 | I NTRO D U C TI O N processes in insects (Costa et al., 2020; Linguadoca et al., 2021; Lycett et al., 2006). Bees play a key role in the functioning of terrestrial ecosystems In this study we explored the combined effects of increasingly and provide essential pollination services for agricultural crops warm wintering temperatures associated with the current scenario (Klein et al., 2007; Ollerton et al., 2011). However, bees have ex- of global warming and exposure to sublethal levels of insecticide in perienced significant declines in abundance and diversity over the the solitary bee Osmia cornuta. We expected exposure to warm tem- last century (Goulson et al., 2015; Potts et al., 2010; Sánchez-­Bayo peratures to result in increased sensitivity to the insecticide. To the & Wyckhuys, 2019). The causes of these declines are at least par- best of our knowledge, the interaction between pesticide exposure tially known, and include land-­use change, biological invasions, cli- and climate change has not been previously addressed in solitary mate change, and the increasing use of pesticides associated with bees. Osmia spp. are appropriate organisms for a study of this sort agricultural intensification (Goulson et al., 2015; IPBES, 2021). because both their wintering ecophysiology and their sensitivity to Importantly, these stressors do not act in isolation and sometimes insecticides have been studied in some detail. Osmia spp. overwin- interact synergistically (Castelli et al., 2020; Goulson et al., 2015; ter on a fixed energetic budget (Bosch et al., 2010). Adult eclosion Siviter et al., 2021). Various studies show that the combined expo- takes place in autumn, but adults remain inside their cocoons within sure of multiple stressors at sublethal levels may cause lethal effects their natal nest, without access to food until next spring. Therefore, (Doublet et al., 2015; Vanbergen, 2021). For example, disease-­ wintering adults rely totally on the energy reserves derived from the induced stress may result in increased sensitivity to pesticides, and, food provisions ingested by the larval stage in early summer. This at the same time, sublethal exposure to toxicants may cause immu- is in contrast to bumblebees and honey bees, which overwinter as nosuppression and increased vulnerability to pathogenic infections emerged adults and therefore are able to feed before, and sometimes (Aufauvre et al., 2012; Doublet et al., 2015; Grassl et al., 2018; Pettis during, the winter. Previous Osmia studies have shown that warm et al., 2012; Vidau et al., 2011). Insecticide exposure and nutritional pre-­wintering and wintering temperatures enhance fat body deple- stress may also interact synergistically, resulting in reduced thermo- tion, lipid consumption, and body weight loss (Bosch et al., 2000, regulation capability, food consumption, flight ability, and survival 2010; Bosch & Kemp, 2003, 2004; Fliszkiewicz et al., 2012; Sgolas- (Castle et al., 2022; Linguadoca et al., 2021; Tong et al., 2019; Tosi tra et al., 2011). Osmia spp. nest above ground, and therefore are et al., 2017). These studies underscore the need to address the com- more directly exposed to ambient temperatures than ground nest- bined effects of various stressors to understand the drivers of bee ing bees (Dorian et al., 2022). O. cornuta has a strong affinity for declines. fruit tree pollen (Jaumejoan et al., 2023) and, along with other Osmia Like other ectotherms, insects are highly dependent on envi- species, is commercially managed for orchard pollination, resulting ronmental temperatures for adequate growth, development, and in frequent exposure to pesticides (Bosch & Kemp, 2002). In 2013, reproduction, which makes them particularly vulnerable to global the European Food Safety Authority recommended the use of O. warming (Deutsch et al., 2008; Portner, 2002). In general, expo- cornuta and O. bicornis as model species in pesticide risk assessment sure to warm temperatures increases metabolic activity in insects, (EFSA, 2013), and information on the effects of pesticides on these affecting development rates and overall physiological function- species has notably increased since then (Sgolastra et al., 2019). ing (González-­Tokman et al., 2020; Neven, 2000). These effects The aim of this study is to understand whether climate warming are accompanied by an increase in energy expenditure (Brown may exacerbate the sensitivity of solitary bees to pesticides. We ex- et al., 2004), resulting in the mobilization and consumption of posed O. cornuta females to three simulated wintering treatments stored metabolic resources (Storey & Storey, 2004), ultimately and then assessed their sensitivity to acute oral exposure to the in- affecting body size, longevity, and fecundity (Blanckenhorn & secticide CloserTM® (a formulation of sulfoxaflor) at two sublethal Henseler, 2005; Fischer et al., 2014; Kierat et al., 2017; Vester- concentrations. We then measured the combined effects of these lund et al., 2014; Zhang et al., 2015). Several studies show that two stress factors on the propensity of the bees to respond to a light thermal stress enhances the depletion of fat body reserves in dif- stimulus (phototactic response), feeding behavior, and longevity. ferent insect groups (Jean et al., 1990; Klepsatel et al., 2016; Wil- liams et al., 2012), including bees (Bosch et al., 2010; Fliszkiewicz et al., 2012; Sgolastra et al., 2011). Fat bodies play a crucial role not 2 | M ATE R I A L S A N D M E TH O DS only in the storage and release of energy in response to metabolic demands (Arrese & Soulages, 2010), but also in the functioning of 2.1 | Life history of O. cornuta key physiological processes such as diapause development (Hahn & Denlinger, 2007), hormone regulation, and immune response O. cornuta, known as the European orchard bee or the horned mason (Keeley, 1985; Skowronek et al., 2021). Importantly, fat bodies bee, occurs in most of central and southern Europe. Its populations are also the main tissue in which detoxification enzymes, such as fly in late winter and early spring. Females are active for approxi- cytochrome P450 monooxygenases, are produced and expressed mately 20 days (Bosch & Vicens, 2006), during which time they build (David et al., 2006; Petersen et al., 2001). Therefore, the alteration one or more nests in pre-­existing cavities. Each nest consists of a of fat bodies caused by heat stress may also impair detoxification linear series of cells, delimited by mud partitions, each containing a | 13652486, 2023, 22, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/gcb.16928 by Readcube (Labtiva Inc.), Wiley Online Library on [24/10/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 6250 ALBACETE et al. pollen–­nectar provision and an egg. After consuming the provision, Bees of the climate change treatments were expected to emerge the larva spins a cocoon and undergoes a summer diapause in the before bees of the current treatment (Bosch & Blas, 1994a; Bosch & prepupal stage (Sgolastra et al., 2012). Pupation occurs in mid or late Kemp, 2003, 2004). Cocoons were checked daily, and when emer- summer, and adult eclosion in late summer or early autumn. Eclosed gence of a given treatment reached 5%, cocoons of that treatment adults remain inside their cocoons and lower their metabolic rates were transferred to an incubation chamber in which they were ex- (Bosch et al., 2010; Sgolastra et al., 2010). Adults require exposure posed to 15°C for 24 h and subsequently to 20°C until 100% emer- to cold temperatures (wintering) to complete diapause and emerge gence. Before being transferred to the incubation chamber cocoons the following spring as temperatures increase (Bosch & Kemp, 2004; were again individually weighed to obtain a measure of weight loss Sgolastra et al., 2010). over the winter. 2.2 | Wintering and emergence 2.3 | Pesticide exposure We used the progeny of an O. cornuta population that nested in Upon emergence, bees were individually kept for 24 h in transpar- February–­March 2021 in an almond orchard near Lleida (Catalo- ent plastic containers (diameter: 11 cm; height: 7 cm) capped with nia, Spain). In mid-­September, when bees reached the adult stage, a pin-­perforated lid to allow them to deposit the meconium. Bees we dissected a subset of nests and collected 825 female cocoons. were then orally and acutely exposed to one of three insecticide Cocoon sex can be reliably established based on size and position treatments using the “petal method” (Azpiazu et al., 2023). Each within the nest (Bosch, 1994). The collected cocoons were indi- bee was offered 20 μL of test solution. Only bees that consumed vidually weighed and randomly assigned to one of three simulated 100% of the test solution within 3 h (≥90% in all treatments) were wintering treatments (n = 275 females per treatment): (a) Current included in the analyses. Following this exposure phase, bees were scenario (average hourly temperatures recorded in the area of ori- left in the plastic cages and fed ad libitum with a 1-­mL calibrated gin of the population during 2007–­2012); (b) near-­future scenario syringe filled with a feeding solution (henceforth syrup; 33% w/w (projected temperatures for 2021–­2050); and (c) distant-­future sce- sucrose–­water). A petal of Euryops spp. (Asteraceae) was attached nario (projected temperatures for 2051–­2080). Future wintering to the tip of the syringe to enhance prompt location by the bees treatments were based on the temperature projections of the 8.5 (Azpiazu et al., 2023). During the experiment, test containers were Representative Concentration Pathways (RCPs) used in the IPCC maintained at 20 ± 2°C and 50%–­70% relative humidity, and re- AR5 assessment report on climate change (Moss et al., 2010). Our ceived indirect natural light. projections cover a 30-­year period and reflect a mean temperature We worked with the commercial formulation of sulfoxaflor Clos- increase of 0.6 and 2.9°C for the near-­future and distant-­future sce- erTM® (Sulfoxaflor 11.43% SC, Corteva Agriscience, Spain S.L.U.). narios, respectively, compared to the winter temperatures of 2007–­ We tested two doses of sulfoxaflor (SUL), 4.55 ng/bee (henceforth 2012 (Table 1). Data for the area of origin of the study population low dose) and 11.64 ng/bee (high dose), along with a control (0 ng/ were extracted from the Escenarios-­PNACC 2017 project (http:// bee; 0 dose). The two SUL doses correspond to the LD10 and LD50 escen​arios.adapt​ecca.es). We simulated daily temperature curves at 72 h after exposure of female O. bicornis (Azpiazu et al., 2021), for each treatment and month (Figure S1; Supporting Information adjusted to the greater body weight of O. cornuta (0.149 g ± 0.0142; Methods). Cocoons were individually placed in multi-­well plates and Bosch & Vicens, 2002). To obtain the test solutions (20 μL/bee), transferred to temperature-­controlled chambers on 1 October until we prepared a primary stock solution of 1.16 g/L SUL by dissolving emergence the following year. Actual temperatures within the three Closer in distilled water. This solution was subsequently diluted and chambers were monitored with data loggers throughout the winter- finally mixed with syrup at a ratio of 50 μL/mL to achieve the desired ing period. doses. TA B L E 1 Mean weight loss and % winter mortality in Osmia cornuta females exposed to three wintering treatments representing increasingly warm climate change scenarios. Wintering Temperatures (°C) duration Pre-­wintering weight % Winter mortality % Weight loss Wintering treatment N ( X ± SE) (range)1,2 (days)1 (mg) ( X ± SE)3 ( X ± SE)3 ( X ± SE)3 Current 275 9.4 ± 0.3 (2.6–­20.1) 141 224.2 ± 1.6a 2.2 ± 0.8a 3.9 ± 0.04a Near-­future 275 10.0 ± 0.4 (3.2–­22.5) 134 224.0 ± 1.6a 1.8 ± 0.6a 5.8 ± 0.05b Distant-­future 275 12.3 ± 0.4 (4.4–­25.3) 128 227.0 ± 1.6a 4.0 ± 1.2a 8.4 ± 0.09c 1 From 1 October until 5% female emergence. 2 Daily temperature curves provided in Figure S1. 3 Values followed by different letters are significantly different (Tukey HSD test, p > .05). | 13652486, 2023, 22, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/gcb.16928 by Readcube (Labtiva Inc.), Wiley Online Library on [24/10/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License ALBACETE et al. 6251 2.4 | Phototaxis test different insecticide doses) with the corresponding expected sur- vival probability curves, built assuming no interaction between the Two hours after the exposure phase, approximately 25 individuals of two factors. The analyses (details provided in Supporting Informa- each wintering-­pesticide treatment were subjected to a phototactic tion Methods) were conducted with the Fhtestrcc function of the test in which bees in a dark chamber were exposed to a light stimu- Fhtest package (Oller & Langohr, 2017). lus. The response of each bee was scored as either positive (the bee All analyses were conducted in R (R Core Team, 2020). Primary walked to the light source within 60 s) or negative (the bee walked data and associated R scripts are accessible on the repository Dryad but did not reach the light source; see Supporting Information Meth- (Albacete et al., 2023). ods for details). 3 | R E S U LT S 2.5 | Syrup consumption and survival 3.1 | Winter mortality and weight loss Test cages were inspected daily to monitor syrup consumption (assessed by checking the level of syrup in the calibrated syringe; As expected, bees exposed to climate change wintering treatments accuracy: 0.01 mL) and bee mortality. Three additional contain- emerged earlier than bees of the current climate scenario (Table 1). ers without bees were used as controls to measure and account Winter mortality was very low and was not affected by wintering for changes in syrup levels due to evaporation. The syrup solution treatment (GLM, χ2 = 2.8; df = 2; p = .2516). On the other hand, body was renewed every 3–­4 days until all the bees died. Sample sizes for weight loss increased with wintering temperature (GLM, χ2 = 2311.8; syrup consumption and survival were approximately 60 individuals df = 2; p < .0001). Bees of the distant-­future scenario lost twice as per wintering treatment and insecticide dose. much body weight as bees of the current scenario (Table 1). Re- gardless of the wintering treatment, bees with lower pre-­wintering weight lost a greater proportion of body weight (GLM, χ2 = 16.7; 2.6 | Data analysis df = 1; p < .0001). To test the effect of wintering treatment on winter mortality we used a binomial generalized linear model (GLM). To determine the 3.2 | Phototactic response effect of wintering treatment on percent weight loss, we used a beta regression model, with individual pre-­wintering weight as a covariate Phototactic response was affected by wintering treatment (GLM, (betareg package; Cribari-­Neto & Zeileis, 2010). χ2 = 14.7; df = 2; p = .001), insecticide dose (GLM, χ2 = 109.2; df = 2; We used GLMs to analyze the effects of wintering treatment, p < .0001), and their interaction (GLM, χ2 = 16.1; df = 4; p = .003). insecticide exposure and their interaction on phototactic response The vast majority of bees not exposed to the insecticide (0 dose) (binomial error distribution and identity link function), daily syrup responded positively to light, irrespective of the wintering treatment consumption (zero-­inflated Gamma error distribution and log link (Figure 1). The percentage of bees that responded positively to light function), and longevity (Poisson error distribution and log link decreased with increasing insecticide dose, and this decrease was function). Because body size may affect food consumption (Azpiazu especially pronounced in bees of the distant-­future scenario (Fig- et al., 2019; Sgolastra et al., 2018) and pesticide sensitivity (Thomp- ure 1). In the current and near-­future scenarios, the proportion of son, 2016), pre-­wintering weight was added as a covariate in the positively responding bees was hardly affected by the low insecti- syrup consumption and longevity analyses. Pairwise comparisons cide dose, but dropped to ca. 50% when bees were exposed to the were done with Tukey's p-­value adjustment method (emmeans-­ high dose. In bees of the distant-­future scenario, the proportion of package; Lenth et al., 2019). positively responding bees went from 95.8% (0 dose) to 73.1% (low We used Kaplan–­Meier (K-­M) survival curves to illustrate the dose) and 11.1% (high dose). combined effects of wintering and insecticide treatments on post-­ exposure survival. Then, we ran a log-­rank omnibus test to ex- plore overall differences among treatments (survdiff function of 3.3 | Syrup consumption the survival R package with ρ = 0; Therneau et al., 2020). Pairwise comparisons between survival curves were done with Holm multi-­ Daily consumption of the feeding solution was not affected by comparison corrections and ρ = 0 (pairwise_survdiff function of the wintering treatment (GLM, χ2 = 2.5; df = 2; p = .2827) but varied survminer package; Kassambara et al., 2020). with insecticide exposure (GLM, χ2 = 65.6; df = 2; p < .0001). The To explore potential synergistic effects between wintering and interaction between wintering and pesticide exposure was signifi- insecticide exposure on survival we used the Bliss drugs' indepen- cant (GLM, χ2 = 17.7; df = 4; p = .0014) because differences in syrup dence criterion (Demidenko & Miller, 2019) to compare the observed consumption across insecticide doses were especially pronounced survival curves of the near-­ and distant-­future populations (at the in bees of the distant-­f uture treatment (Figure 2A). The effect of | 13652486, 2023, 22, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/gcb.16928 by Readcube (Labtiva Inc.), Wiley Online Library on [24/10/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 6252 ALBACETE et al. F I G U R E 1 Model-­estimated means and 95% confidence intervals of percent positive response to a phototaxis test in Osmia cornuta females exposed to three wintering treatments representing increasingly warm climate change scenarios, and three orally administered doses of the insecticide sulfoxaflor (0, 4.55 and 11.64 ng/bee). Different letters denote significant differences (Tukey HSD test, p < .05). N = 24–­29 individuals per wintering scenario and dose. the high insecticide dose on syrup consumption was evident from (Figure 4a). We also found a significant antagonistic effect in bees day 1 (Figure S2). We expected large bees to consume more syrup of the near-­future scenario exposed to the low insecticide dose than small bees, but the relationship between pre-­wintering body (Figure 4b). weight and daily syrup was not significant (GLM, χ2 = 1.6; df = 1; p = .1987). 4 | DISCUSSION 3.4 | Survival probability and longevity The aim of this study was to establish whether climate warming could affect the ability of adult bees to cope with pesticide expo- Survival curves differed significantly among treatments (log-­rank sure. We used climate change projections from IPCC RCP8.5 and test: χ2 = 244.0; df = 8; p < .0001; Figure 3). For a given insecticide realistic concentrations of a widely used insecticide to assess the dose, the current and near-­future survival curves were similar combined effects of increasingly warm wintering temperatures and (p > .05). Survival probability dropped faster in bees of the distant-­ sublethal insecticide exposure on the behavior and longevity of a future scenario than in bees of the current and near-­future scenarios solitary bee. Our results demonstrate synergistic effects between at all the insecticide doses tested, including 0 ng/bee. Consequently, realistic scenarios of two of the main drivers of bee declines. Impor- longevity differed significantly across different treatments (Fig- tantly, these effects were obtained following acute oral exposure to ure 2B). Longevity was affected by wintering treatment (GLM, a single insecticide. In field conditions bees may experience repeated 2 2 χ = 262.1; df = 2; p < .0001), insecticide exposure (GLM, χ = 613.3; and/or chronic exposure to a variety of chemicals via different ex- df = 2; p < .0001), and their interaction (GLM, χ2 = 39.6; df = 4; posure routes (Knapp et al., 2023; Sanchez-­Bayo & Goka, 2014). At p < .0001). Pre-­wintering body weight had a significant effect on lon- the same time, the temperatures of our most pessimistic climate gevity (GLM, χ2 = 27.7; df = 1; p < .0001), with large bees living longer scenario are already occurring in particularly warm winters (winter than small ones. temperatures in Europe in 2019–­2020 were 3.4°C higher than the To assess potential synergism between wintering treatment and mean of 1981–­2010; AEMET, 2021; Copernicus, 2020). Therefore, insecticide exposure on survival probability, we compared the ob- both our pesticide and climate scenarios may be considered con- served survival curves of the near and distant-­future populations at servative. Our results underscore the need to study combinations the two insecticide doses with the expected survival curves based of stressors to fully understand the effects of global change on bee on the Bliss criterion for drugs independence. We found that the populations (Goulson et al., 2015). Our findings also have important combination of the distant-­future scenario and exposure to the implications for pesticide regulation. Pesticide risk assessment is high insecticide dose resulted in a synergistic decrease in survival based on median lethal and effect doses (LD50 and ED50). Given | 13652486, 2023, 22, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/gcb.16928 by Readcube (Labtiva Inc.), Wiley Online Library on [24/10/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License ALBACETE et al. 6253 F I G U R E 2 Model-­estimated means and 95% confidence intervals of daily syrup consumption (a) and longevity (b) in Osmia cornuta females exposed to three wintering treatments representing increasingly warm climate change scenarios, and three orally administered doses of the insecticide sulfoxaflor (0, 4.55, and 11.64 ng/bee). Different letters denote significant differences (Tukey HSD test, p < .05). N = 53–­65 individuals per wintering scenario and dose. our results, current LD50 and ED50 values may not represent suffi- treatment by itself did not impair the ability of females to respond ciently protective thresholds as bee populations are confronted with to light. On the other hand, wintering treatment had an important increasingly warm temperatures. impact on weight loss, which, in agreement with previous studies, Wintering treatment did not have a significant effect on win- increased with wintering temperatures. Because body weight loss ter mortality, which was very low and similar to mortality values during wintering is accompanied by the depletion of energy re- found in studies in which Osmia populations were wintered under serves in Osmia (Fliszkiewicz et al., 2012; Sgolastra et al., 2011), we natural conditions (Bosch & Blas, 1994b; Bosch & Kemp, 2000; expected that bees exposed to climate warming would consume CaraDonna et al., 2018; Sgolastra et al., 2012). Similarly, wintering more syrup to rebuild their metabolic reserves (Nestel et al., 2016). | 13652486, 2023, 22, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/gcb.16928 by Readcube (Labtiva Inc.), Wiley Online Library on [24/10/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 6254 ALBACETE et al. F I G U R E 3 Cumulative survival probability of Osmia cornuta females exposed to three wintering treatments representing increasingly warm climate change scenarios, and three orally administered doses of the insecticide sulfoxaflor (0, 4.55, and 11.64 ng/bee). Curves with different letters are significantly different (pairwise comparisons, log-­rank test, p < .05). Contrary to this expectation, wintering treatment did not affect contain sublethal levels of pesticides (David et al., 2016; Dively & daily syrup consumption. So far, these findings suggest that the Kamel, 2012; Heller et al., 2020; Hladik et al., 2016). To mimic this climate change scenarios tested would not compromise winter scenario, we exposed newly emerged bees to two sublethal doses survival, feeding success and post-­emergence performance in O. of Closer, a widely used insecticide. The doses used correspond to cornuta. However, even with similar feeding rates, bees wintered at concentrations of 0.58 and 0.23 ppm of active ingredient, which the warmest temperature regime had significantly reduced mean fall within the range of sulfoxaflor residues found in the nectar of longevity (10 days compared to 14–­15 days in bees of the other various crop flowers 2–­7 days after application (USEPA, 2019). Our two wintering scenarios), a strong predictor of realized fecundity results show that these realistic levels of exposure, especially the in Osmia (Bosch & Vicens, 2006; Sgolastra et al., 2016). Longevity high dose, impaired the ability of bees to respond to light resulting was also significantly affected by body size, with small individuals in a drastic reduction of phototactic response in bees of the distant-­ having shorter life spans. Adult body size in Osmia is directly de- future climate scenario (Figure 1). The inability to respond to light is pendent on the amount of food provision allocated to the larva by indicative of an alteration of the sensitivity or the functioning of the the nesting female (Bosch & Vicens, 2002). Therefore, historical visual system (Klein et al., 2017). Exposure to sublethal doses of im- declines in bee body size attributed to scarcity of floral resources idacloprid, a neonicotinoid insecticide with a mode of action similar in simplified landscapes may be further compromising bee longev- to sulfoxaflor (Sparks et al., 2013), increases the expression of genes ity and realized fecundity (Chole et al., 2019; Grab et al., 2019; related to biogenic amine receptors, such as serotonin, tyramine, Herrera et al., 2023; Oliveira et al., 2016). and octopamine, associated with a lack of phototactic response in Like most solitary bees, Osmia spp. overwinter on a fixed bud- Drosophila (Martelli et al., 2020). High levels of octopamine have get, and experience a considerable depletion of lipid reserves during also been linked to reduced response to light in honey bees (Bloch & wintering (Fliszkiewicz et al., 2012; Sgolastra et al., 2011). Upon Meshi, 2007; Scheiner et al., 2014). emergence, adults fly to nearby flowers and take a first, long, nec- The interaction between wintering and pesticide exposure was tar meal that presumably allows them to rebuild their energetic re- also evident in the rate of daily syrup consumption. Exposure to sources (Bosch & Kemp, 2001; Nestel et al., 2016). In agricultural the insecticide depressed feeding rates in bees of the three win- environments, however, the nectar ingested in this first meal may tering treatments, but this decrease was especially pronounced in | 13652486, 2023, 22, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/gcb.16928 by Readcube (Labtiva Inc.), Wiley Online Library on [24/10/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License ALBACETE et al. 6255 F I G U R E 4 Observed (red/yellow lines) and expected (black dashed lines) survival probability curves of Osmia cornuta females exposed to distant-­(a) and near-­future (b) climate change scenarios and to two orally administered doses of the insecticide sulfoxaflor (4.55 and 11.64 ng/bee). Interaction significance is based on the Bliss criterion of drugs independence (FH statistic distribution >0 indicates synergism between the two stressors; FH statistic distribution <0 indicates antagonism). bees exposed to the warmest winter (Figure 2A). The inhibitory ef- The increased sensitivity to Closer in bees exposed to climate fect of sulfoxaflor on syrup consumption has been documented in warming scenarios may be mediated by the effects of warm winter both solitary and social bees (Azpiazu et al., 2022; Li et al., 2021; temperatures on the consumption of energy reserves and fat body Siviter et al., 2020). Longevity was reduced by ca. 50% in bees of depletion in Osmia (Bosch et al., 2000, 2010; Bosch & Kemp, 2003, the current and near-­future scenarios exposed to the high insecti- 2004; Fliszkiewicz et al., 2012; Sgolastra et al., 2011). The fat body cide dose. In bees of the distant-­future scenario, exposure to the low is a dynamic, “liver-­like”, tissue that acts as an organ of energy dose led to an estimated 11% reduction in longevity and exposure storage and other metabolic functions in insects (Arrese & Soul- to the high dose resulted in a synergistic 70% reduction (Figure 3). ages, 2010). Studies on butterflies and mosquitoes have found that As mentioned, the cognitive abilities of the bees of the latter treat- exposure to xenobiotics increases the expression of cytochrome ment were significantly impaired, further reducing the effective time P450 monooxygenases genes, involved in detoxification, in the frame available for reproduction (Bosch & Vicens, 2006; Sgolastra fat body (David et al., 2006; Petersen et al., 2001). In addition, et al., 2016). We also found that bees exposed to the near-­future studies on bumblebees have found that the expression of these scenario and the low insecticide dose lived longer than expected. genes in the fat body is enhanced by a sucrose-­r ich diet (Costa However, this antagonistic effect did not result in an increase in lon- et al., 2020), and that queens, which have significantly higher fat gevity compared to bees of the near-­future scenario exposed to the body reserves than males and workers, are more resilient to pes- 0 insecticide dose. Antagonistic interactions are not uncommon in ticides (Linguadoca et al., 2022). In honey bees, attack by the mite studies addressing interactions between stressors (Bird et al., 2021; Varroa destructor, that feeds on the fat body of adults (Ramsey Bruckner et al., 2023; Siviter et al., 2021; Zaragoza-­Trello et al., 2021). et al., 2019) disrupts detoxification ability and increases sensitivity | 13652486, 2023, 22, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/gcb.16928 by Readcube (Labtiva Inc.), Wiley Online Library on [24/10/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 6256 ALBACETE et al. to pesticides (Blanken et al., 2015; Schwartz et al., 2021; Zhu REFERENCES et al., 2022). Based on this line of evidence, we suggest that the Agencia Estatal de Meteorología (AEMET). 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