Bees exposed to climate change are more sensitive to pesticides

6248 | Glob Change Biol. 2023;29:6248–6260.wileyonlinelib a y.com/jou nal/gcb Recei ed: 4 May 2023 | Accep ed: 13 Augus 2023 DOI: 10.1111/gcb.16928 RESEARCH ARTICLE Bees exposed o clima e change a e mo e sensi i e o pes icides Se gio Albace e1,2  | Gonzalo Sancho1,2  | Celes e Azpiazu1,3  | Anselm Rod igo1,2  | Robe o Molowny- Ho as1,2  | Fabio Sgolas a4 | Jo di Bosch1,2 This is an open access a icle unde he e ms o he C ea i e Commons A ibu ion License, which pe mi s use, dis ibu ion and ep oduc ion in any medium, p o ided he o iginal wo k is p ope ly ci ed. © 2023 The Au ho s. Global Change Biology published by John Wiley & Sons L d. 1Uni e si a Au ònoma de Ba celona, Bella e a, Spain 2Cen e o Ecological Resea ch and Fo es y Applica ions (CREAF), Bella e a, Spain 3Ins i u e o E olu iona y Biology (CSIC— Uni e si a Pompeu Fab a), Ba celona, Spain 4Dipa imen o di Scienze e Tecnologie Ag o- Alimen a i, Alma Ma e S udio um Uni e si à di Bologna, Bologna, I aly Co espondence Se gio Albace e, Uni e si a Au ònoma de Ba celona and CREAF (Cen e o Ecological Resea ch and Fo es y Applica ions), 08193 Bella e a, Spain. Email: s.albace e@c ea .uab.ca Funding in o ma ion Spanish Minis y o Science and Inno a ion, G an /Awa d Numbe : PRE2019- 090375, PRE2019- 088817, PID2021- 128938OB- I00 and RTI2018- 098399- B- I00; Spanish Minis y o Uni e si ies, G an /Awa d Numbe : Ma ga i a- Salas- schola ship Abs ac Bee popula ions a e exposed o mul iple s esso s, including land- use change, bio- logical in asions, clima e change, and pes icide exposu e, ha may in e ac syne gis i- cally. We analyze he combined e ec s o clima e wa ming and suble hal insec icide exposu e in he soli a y bee Osmia co nu a. P e ious Osmia s udies show ha wa m win e ing empe a u es cause body weigh loss, lipid consump ion, and a body de- ple ion. Because he a body plays a key ole in xenobio ic de oxi ica ion, we ex- pec ed ha bees exposed o clima e wa ming scena ios would be mo e sensi i e o pes icides. We exposed O. co nu a emales o h ee win e ing ea men s: cu en sce- na io (2007– 2012 empe a u es), nea - u u e (2021– 2050 p ojec ed empe a u es), and dis an - u u e (2051– 2080). Upon eme gence in sp ing, bees we e o ally exposed o h ee suble hal doses o an insec icide (Close , a.i. sul oxa lo ; 0, 4.55 and 11.64 ng a.i./bee). We measu ed he combined e ec s o win e ing and insec icide exposu e on pho o ac ic esponse, sy up consump ion, and longe i y. Win e ing ea men by i sel did no a ec win e mo ali y, bu body weigh loss inc eased wi h inc eas- ing win e ing empe a u es. Simila ly, win e ing ea men by i sel ha dly in luenced pho o ac ic esponse o sy up consump ion. Howe e , bees win e ed a he wa mes empe a u es had sho e longe i y, a s ong ecundi y p edic o in Osmia. Insec icide exposu e, especially a he high dose, impai ed he abili y o bees o espond o ligh , and esul ed in educed sy up consump ion and longe i y. The combina ion o he wa mes win e and he high insec icide dose esul ed in a 70% longe i y dec ease. Smalle bees, esul ing om smalle pollen– nec a p o isions, had sho e longe i y sugges ing nu i ional s ess may u he comp omise ecundi y in O. co nu a. Ou esul s show a syne gis ic in e ac ion be ween wo majo d i e s o bee declines, and indica e ha bees will become mo e sensi i e o pes icides unde he cu en global wa ming scena io. Ou indings ha e impo an implica ions o pes icide egula ion and unde sco e he need o conside mul iple s esso s o unde s and bee declines. KEYWORDS body size, clima e change, global wa ming, longe i y, nu i ional s ess, Osmia co nu a, pes icide exposu e, sul oxa lo , syne gis ic e ec s, oxici y | 6249 ALBACETE e al. 1 | INTRODUCTION Bees play a key ole in he unc ioning o e es ial ecosys ems and p o ide essen ial pollina ion se ices o ag icul u al c ops (Klein e al., 2007; Olle on e al., 2011). Howe e , bees ha e ex- pe ienced signi ican declines in abundance and di e si y o e he las cen u y (Goulson e al., 2015; Po s e al., 2010; Sánchez- Bayo & Wyckhuys, 2019). The causes o hese declines a e a leas pa - ially known, and include land- use change, biological in asions, cli- ma e change, and he inc easing use o pes icides associa ed wi h ag icul u al in ensi ica ion (Goulson e al., 2015; IPBES, 2021). Impo an ly, hese s esso s do no ac in isola ion and some imes in e ac syne gis ically (Cas elli e al., 2020; Goulson e al., 2015; Si i e e al., 2021). Va ious s udies show ha he combined expo- su e o mul iple s esso s a suble hal le els may cause le hal e ec s (Double e al., 2015; Vanbe gen, 2021). Fo example, disease- induced s ess may esul in inc eased sensi i i y o pes icides, and, a he same ime, suble hal exposu e o oxican s may cause immu- nosupp ession and inc eased ulne abili y o pa hogenic in ec ions (Au au e e al., 2012; Double e al., 2015; G assl e al., 2018; Pe is e al., 2012; Vidau e al., 2011). Insec icide exposu e and nu i ional s ess may also in e ac syne gis ically, esul ing in educed he mo- egula ion capabili y, ood consump ion, ligh abili y, and su i al (Cas le e al., 2022; Linguadoca e al., 2021; Tong e al., 2019; Tosi e al., 2017). These s udies unde sco e he need o add ess he com- bined e ec s o a ious s esso s o unde s and he d i e s o bee declines. Like o he ec o he ms, insec s a e highly dependen on en i- onmen al empe a u es o adequa e g ow h, de elopmen , and ep oduc ion, which makes hem pa icula ly ulne able o global wa ming (Deu sch e al., 2008; Po ne , 2002). In gene al, expo- su e o wa m empe a u es inc eases me abolic ac i i y in insec s, a ec ing de elopmen a es and o e all physiological unc ion- ing (González- Tokman e al., 2020; Ne en, 2000). These e ec s a e accompanied by an inc ease in ene gy expendi u e (B own e al., 2004), esul ing in he mobiliza ion and consump ion o s o ed me abolic esou ces (S o ey & S o ey, 2004), ul ima ely a ec ing body size, longe i y, and ecundi y (Blanckenho n & Hensele , 2005; Fische e al., 2014; Kie a e al., 2017; Ves e - lund e al., 2014; Zhang e al., 2015). Se e al s udies show ha he mal s ess enhances he deple ion o a body ese es in di - e en insec g oups (Jean e al., 1990; Klepsa el e al., 2016; Wil- liams e al., 2012), including bees (Bosch e al., 2010; Fliszkiewicz e al., 2012; Sgolas a e al., 2011). Fa bodies play a c ucial ole no only in he s o age and elease o ene gy in esponse o me abolic demands (A ese & Soulages, 2010), bu also in he unc ioning o key physiological p ocesses such as diapause de elopmen (Hahn & Denlinge , 2007), ho mone egula ion, and immune esponse (Keeley, 1985; Skow onek e al., 2021). Impo an ly, a bodies a e also he main issue in which de oxi ica ion enzymes, such as cy och ome P450 monooxygenases, a e p oduced and exp essed (Da id e al., 2006; Pe e sen e al., 2001). The e o e, he al e a ion o a bodies caused by hea s ess may also impai de oxi ica ion p ocesses in insec s (Cos a e al., 2020; Linguadoca e al., 2021; Lyce e al., 2006). In his s udy we explo ed he combined e ec s o inc easingly wa m win e ing empe a u es associa ed wi h he cu en scena io o global wa ming and exposu e o suble hal le els o insec icide in he soli a y bee Osmia co nu a. We expec ed exposu e o wa m em- pe a u es o esul in inc eased sensi i i y o he insec icide. To he bes o ou knowledge, he in e ac ion be ween pes icide exposu e and clima e change has no been p e iously add essed in soli a y bees. Osmia spp. a e app op ia e o ganisms o a s udy o his so because bo h hei win e ing ecophysiology and hei sensi i i y o insec icides ha e been s udied in some de ail. Osmia spp. o e win- e on a ixed ene ge ic budge (Bosch e al., 2010). Adul eclosion akes place in au umn, bu adul s emain inside hei cocoons wi hin hei na al nes , wi hou access o ood un il nex sp ing. The e o e, win e ing adul s ely o ally on he ene gy ese es de i ed om he ood p o isions inges ed by he la al s age in ea ly summe . This is in con as o bumblebees and honey bees, which o e win e as eme ged adul s and he e o e a e able o eed be o e, and some imes du ing, he win e . P e ious Osmia s udies ha e shown ha wa m p e- win e ing and win e ing empe a u es enhance a body deple- ion, lipid consump ion, and body weigh loss (Bosch e al., 2000, 2010; Bosch & Kemp, 2003, 2004; Fliszkiewicz e al., 2012; Sgolas- a e al., 2011). Osmia spp. nes abo e g ound, and he e o e a e mo e di ec ly exposed o ambien empe a u es han g ound nes - ing bees (Do ian e al., 2022). O. co nu a has a s ong a ini y o ui ee pollen (Jaumejoan e al., 2023) and, along wi h o he Osmia species, is comme cially managed o o cha d pollina ion, esul ing in equen exposu e o pes icides (Bosch & Kemp, 2002). In 2013, he Eu opean Food Sa e y Au ho i y ecommended he use o O. co nu a and O. bico nis as model species in pes icide isk assessmen (EFSA, 2013), and in o ma ion on he e ec s o pes icides on hese species has no ably inc eased since hen (Sgolas a e al., 2019). The aim o his s udy is o unde s and whe he clima e wa ming may exace ba e he sensi i i y o soli a y bees o pes icides. We ex- posed O. co nu a emales o h ee simula ed win e ing ea men s and hen assessed hei sensi i i y o acu e o al exposu e o he in- sec icide Close TM® (a o mula ion o sul oxa lo ) a wo suble hal concen a ions. We hen measu ed he combined e ec s o hese wo s ess ac o s on he p opensi y o he bees o espond o a ligh s imulus (pho o ac ic esponse), eeding beha io , and longe i y. 2 | MATERIALS AND METHODS 2.1  |  Li e his o y o O. co nu a O. co nu a, known as he Eu opean o cha d bee o he ho ned mason bee, occu s in mos o cen al and sou he n Eu ope. I s popula ions ly in la e win e and ea ly sp ing. Females a e ac i e o app oxi- ma ely 20 days (Bosch & Vicens, 2006), du ing which ime hey build one o mo e nes s in p e- exis ing ca i ies. Each nes consis s o a linea se ies o cells, delimi ed by mud pa i ions, each con aining a 13652486, 2023, 22, Downloaded om h ps://onlinelib a y.wiley.com/doi/10.1111/gcb.16928 by Csic O ganización Cen al Om (O icialia Mayo ) (U ici), Wiley Online Lib a y on [20/02/2024]. See he Te ms and Condi ions (h ps://onlinelib a y.wiley.com/ e ms-and-condi ions) on Wiley Online Lib a y o ules o use; OA a icles a e go e ned by he applicable C ea i e Commons License 6250 | ALBACETE e al. pollen– nec a p o ision and an egg. A e consuming he p o ision, he la a spins a cocoon and unde goes a summe diapause in he p epupal s age (Sgolas a e al., 2012). Pupa ion occu s in mid o la e summe , and adul eclosion in la e summe o ea ly au umn. Eclosed adul s emain inside hei cocoons and lowe hei me abolic a es (Bosch e al., 2010; Sgolas a e al., 2010). Adul s equi e exposu e o cold empe a u es (win e ing) o comple e diapause and eme ge he ollowing sp ing as empe a u es inc ease (Bosch & Kemp, 2004; Sgolas a e al., 2010). 2.2  |  Win e ing and eme gence We used he p ogeny o an O. co nu a popula ion ha nes ed in Feb ua y– Ma ch 2021 in an almond o cha d nea Lleida (Ca alo- nia, Spain). In mid- Sep embe , when bees eached he adul s age, we dissec ed a subse o nes s and collec ed 825 emale cocoons. Cocoon sex can be eliably es ablished based on size and posi ion wi hin he nes (Bosch, 1994). The collec ed cocoons we e indi- idually weighed and andomly assigned o one o h ee simula ed win e ing ea men s (n = 275 emales pe ea men ): (a) Cu en scena io (a e age hou ly empe a u es eco ded in he a ea o o i- gin o he popula ion du ing 2007– 2012); (b) nea - u u e scena io (p ojec ed empe a u es o 2021– 2050); and (c) dis an - u u e sce- na io (p ojec ed empe a u es o 2051– 2080). Fu u e win e ing ea men s we e based on he empe a u e p ojec ions o he 8.5 Rep esen a i e Concen a ion Pa hways (RCPs) used in he IPCC AR5 assessmen epo on clima e change (Moss e al., 2010). Ou p ojec ions co e a 30- yea pe iod and e lec a mean empe a u e inc ease o 0.6 and 2.9°C o he nea - u u e and dis an - u u e sce- na ios, espec i ely, compa ed o he win e empe a u es o 2007– 2012 (Table 1). Da a o he a ea o o igin o he s udy popula ion we e ex ac ed om he Escena ios- PNACC 2017 p ojec (h p:// escen a ios.adap ecca.es). We simula ed daily empe a u e cu es o each ea men and mon h (Figu e S1; Suppo ing In o ma ion Me hods). Cocoons we e indi idually placed in mul i- well pla es and ans e ed o empe a u e- con olled chambe s on 1 Oc obe un il eme gence he ollowing yea . Ac ual empe a u es wi hin he h ee chambe s we e moni o ed wi h da a logge s h oughou he win e - ing pe iod. Bees o he clima e change ea men s we e expec ed o eme ge be o e bees o he cu en ea men (Bosch & Blas, 1994a; Bosch & Kemp, 2003, 2004). Cocoons we e checked daily, and when eme - gence o a gi en ea men eached 5%, cocoons o ha ea men we e ans e ed o an incuba ion chambe in which hey we e ex- posed o 15°C o 24 h and subsequen ly o 20°C un il 100% eme - gence. Be o e being ans e ed o he incuba ion chambe cocoons we e again indi idually weighed o ob ain a measu e o weigh loss o e he win e . 2.3  |  Pes icide exposu e Upon eme gence, bees we e indi idually kep o 24 h in anspa - en plas ic con aine s (diame e : 11 cm; heigh : 7 cm) capped wi h a pin- pe o a ed lid o allow hem o deposi he meconium. Bees we e hen o ally and acu ely exposed o one o h ee insec icide ea men s using he “pe al me hod” (Azpiazu e al., 2023). Each bee was o e ed 20 μL o es solu ion. Only bees ha consumed 100% o he es solu ion wi hin 3 h (≥90% in all ea men s) we e included in he analyses. Following his exposu e phase, bees we e le in he plas ic cages and ed ad libi um wi h a 1- mL calib a ed sy inge illed wi h a eeding solu ion (hence o h sy up; 33% w/w suc ose– wa e ). A pe al o Eu yops spp. (As e aceae) was a ached o he ip o he sy inge o enhance p omp loca ion by he bees (Azpiazu e al., 2023). Du ing he expe imen , es con aine s we e main ained a 20 ± 2°C and 50%– 70% ela i e humidi y, and e- cei ed indi ec na u al ligh . We wo ked wi h he comme cial o mula ion o sul oxa lo Clos- e TM® (Sul oxa lo 11.43% SC, Co e a Ag iscience, Spain S.L.U.). We es ed wo doses o sul oxa lo (SUL), 4.55 ng/bee (hence o h low dose) and 11.64 ng/bee (high dose), along wi h a con ol (0 ng/ bee; 0 dose). The wo SUL doses co espond o he LD10 and LD50 a 72 h a e exposu e o emale O. bico nis (Azpiazu e al., 2021), adjus ed o he g ea e body weigh o O. co nu a (0.149 g ± 0.0142; Bosch & Vicens, 2002). To ob ain he es solu ions (20 μL/bee), we p epa ed a p ima y s ock solu ion o 1.16 g/L SUL by dissol ing Close in dis illed wa e . This solu ion was subsequen ly dilu ed and inally mixed wi h sy up a a a io o 50 μL/mL o achie e he desi ed doses. TABLE 1 Mean weigh loss and % win e mo ali y in Osmia co nu a emales exposed o h ee win e ing ea men s ep esen ing inc easingly wa m clima e change scena ios. Win e ing ea men N Tempe a u es (°C) ( X ± SE) ( ange)1,2 Win e ing du a ion (days)1 P e- win e ing weigh (mg) ( X ± SE)3 % Win e mo ali y ( X ± SE)3 % Weigh loss ( X ± SE)3 Cu en 275 9.4 ± 0.3 (2.6– 20.1) 141 224.2 ± 1.6a 2.2 ± 0.8a 3.9 ± 0.04a Nea - u u e 275 10.0 ± 0.4 (3.2– 22.5) 134 224.0 ± 1.6a 1.8 ± 0.6a 5.8 ± 0.05b Dis an - u u e 275 12.3 ± 0.4 (4.4– 25.3) 128 227.0 ± 1.6a 4.0 ± 1.2a 8.4 ± 0.09c 1F om 1 Oc obe un il 5% emale eme gence. 2Daily empe a u e cu es p o ided in Figu e S1. 3Values ollowed by di e en le e s a e signi ican ly di e en (Tukey HSD es , p > .05). 13652486, 2023, 22, Downloaded om h ps://onlinelib a y.wiley.com/doi/10.1111/gcb.16928 by Csic O ganización Cen al Om (O icialia Mayo ) (U ici), Wiley Online Lib a y on [20/02/2024]. See he Te ms and Condi ions (h ps://onlinelib a y.wiley.com/ e ms-and-condi ions) on Wiley Online Lib a y o ules o use; OA a icles a e go e ned by he applicable C ea i e Commons License | 6251 ALBACETE e al. 2.4  |  Pho o axis es Two hou s a e he exposu e phase, app oxima ely 25 indi iduals o each win e ing- pes icide ea men we e subjec ed o a pho o ac ic es in which bees in a da k chambe we e exposed o a ligh s imu- lus. The esponse o each bee was sco ed as ei he posi i e ( he bee walked o he ligh sou ce wi hin 60 s) o nega i e ( he bee walked bu did no each he ligh sou ce; see Suppo ing In o ma ion Me h- ods o de ails). 2.5  |  Sy up consump ion and su i al Tes cages we e inspec ed daily o moni o sy up consump ion (assessed by checking he le el o sy up in he calib a ed sy inge; accu acy: 0.01 mL) and bee mo ali y. Th ee addi ional con ain- e s wi hou bees we e used as con ols o measu e and accoun o changes in sy up le els due o e apo a ion. The sy up solu ion was enewed e e y 3– 4 days un il all he bees died. Sample sizes o sy up consump ion and su i al we e app oxima ely 60 indi iduals pe win e ing ea men and insec icide dose. 2.6  |  Da a analysis To es he e ec o win e ing ea men on win e mo ali y we used a binomial gene alized linea model (GLM). To de e mine he e ec o win e ing ea men on pe cen weigh loss, we used a be a eg ession model, wi h indi idual p e- win e ing weigh as a co a ia e (be a eg package; C iba i- Ne o & Zeileis, 2010). We used GLMs o analyze he e ec s o win e ing ea men , insec icide exposu e and hei in e ac ion on pho o ac ic esponse (binomial e o dis ibu ion and iden i y link unc ion), daily sy up consump ion (ze o- in la ed Gamma e o dis ibu ion and log link unc ion), and longe i y (Poisson e o dis ibu ion and log link unc ion). Because body size may a ec ood consump ion (Azpiazu e al., 2019; Sgolas a e al., 2018) and pes icide sensi i i y (Thomp- son, 2016), p e- win e ing weigh was added as a co a ia e in he sy up consump ion and longe i y analyses. Pai wise compa isons we e done wi h Tukey's p- alue adjus men me hod (emmeans- package; Len h e al., 2019). We used Kaplan– Meie (K- M) su i al cu es o illus a e he combined e ec s o win e ing and insec icide ea men s on pos - exposu e su i al. Then, we an a log- ank omnibus es o ex- plo e o e all di e ences among ea men s (su di unc ion o he su i al R package wi h ρ = 0; The neau e al., 2020). Pai wise compa isons be ween su i al cu es we e done wi h Holm mul i- compa ison co ec ions and ρ = 0 (pai wise_su di unc ion o he su mine package; Kassamba a e al., 2020). To explo e po en ial syne gis ic e ec s be ween win e ing and insec icide exposu e on su i al we used he Bliss d ugs' indepen- dence c i e ion (Demidenko & Mille , 2019) o compa e he obse ed su i al cu es o he nea - and dis an - u u e popula ions (a he di e en insec icide doses) wi h he co esponding expec ed su - i al p obabili y cu es, buil assuming no in e ac ion be ween he wo ac o s. The analyses (de ails p o ided in Suppo ing In o ma- ion Me hods) we e conduc ed wi h he Fh es cc unc ion o he Fh es package (Olle & Langoh , 2017). All analyses we e conduc ed in R (R Co e Team, 2020). P ima y da a and associa ed R sc ip s a e accessible on he eposi o y D yad (Albace e e al., 2023). 3 | RESULTS 3.1  |  Win e mo ali y and weigh loss As expec ed, bees exposed o clima e change win e ing ea men s eme ged ea lie han bees o he cu en clima e scena io (Table 1). Win e mo ali y was e y low and was no a ec ed by win e ing ea men (GLM, χ2 = 2.8; d  = 2; p = .2516). On he o he hand, body weigh loss inc eased wi h win e ing empe a u e (GLM, χ2 = 2311.8; d  = 2; p < .0001). Bees o he dis an - u u e scena io los wice as much body weigh as bees o he cu en scena io (Table 1). Re- ga dless o he win e ing ea men , bees wi h lowe p e- win e ing weigh los a g ea e p opo ion o body weigh (GLM, χ2 = 16.7; d  = 1; p < .0001). 3.2  |  Pho o ac ic esponse Pho o ac ic esponse was a ec ed by win e ing ea men (GLM, χ2 = 14.7; d  = 2; p = .001), insec icide dose (GLM, χ2 = 109.2; d  = 2; p < .0001), and hei in e ac ion (GLM, χ2 = 16.1; d  = 4; p = .003). The as majo i y o bees no exposed o he insec icide (0 dose) esponded posi i ely o ligh , i espec i e o he win e ing ea men (Figu e 1). The pe cen age o bees ha esponded posi i ely o ligh dec eased wi h inc easing insec icide dose, and his dec ease was especially p onounced in bees o he dis an - u u e scena io (Fig- u e 1). In he cu en and nea - u u e scena ios, he p opo ion o posi i ely esponding bees was ha dly a ec ed by he low insec i- cide dose, bu d opped o ca. 50% when bees we e exposed o he high dose. In bees o he dis an - u u e scena io, he p opo ion o posi i ely esponding bees wen om 95.8% (0 dose) o 73.1% (low dose) and 11.1% (high dose). 3.3  |  Sy up consump ion Daily consump ion o he eeding solu ion was no a ec ed by win e ing ea men (GLM, χ2 = 2.5; d  = 2; p = .2827) bu a ied wi h insec icide exposu e (GLM, χ2 = 65.6; d  = 2; p < .0001). The in e ac ion be ween win e ing and pes icide exposu e was signi i- can (GLM, χ2 = 17.7; d  = 4; p = .0014) because di e ences in sy up consump ion ac oss insec icide doses we e especially p onounced in bees o he dis an - u u e ea men (Figu e 2A). The e ec o 13652486, 2023, 22, Downloaded om h ps://onlinelib a y.wiley.com/doi/10.1111/gcb.16928 by Csic O ganización Cen al Om (O icialia Mayo ) (U ici), Wiley Online Lib a y on [20/02/2024]. See he Te ms and Condi ions (h ps://onlinelib a y.wiley.com/ e ms-and-condi ions) on Wiley Online Lib a y o ules o use; OA a icles a e go e ned by he applicable C ea i e Commons License 6252 | ALBACETE e al. he high insec icide dose on sy up consump ion was e iden om day 1 (Figu e S2). We expec ed la ge bees o consume mo e sy up han small bees, bu he ela ionship be ween p e- win e ing body weigh and daily sy up was no signi ican (GLM, χ2 = 1.6; d  = 1; p = .1987). 3.4  |  Su i al p obabili y and longe i y Su i al cu es di e ed signi ican ly among ea men s (log- ank es : χ2 = 244.0; d  = 8; p < .0001; Figu e 3). Fo a gi en insec icide dose, he cu en and nea - u u e su i al cu es we e simila (p > .05). Su i al p obabili y d opped as e in bees o he dis an - u u e scena io han in bees o he cu en and nea - u u e scena ios a all he insec icide doses es ed, including 0 ng/bee. Consequen ly, longe i y di e ed signi ican ly ac oss di e en ea men s (Fig- u e 2B). Longe i y was a ec ed by win e ing ea men (GLM, χ2 = 262.1; d  = 2; p < .0001), insec icide exposu e (GLM, χ2 = 613.3; d  = 2; p < .0001), and hei in e ac ion (GLM, χ2 = 39.6; d  = 4; p < .0001). P e- win e ing body weigh had a signi ican e ec on lon- ge i y (GLM, χ2 = 27.7; d  = 1; p < .0001), wi h la ge bees li ing longe han small ones. To assess po en ial syne gism be ween win e ing ea men and insec icide exposu e on su i al p obabili y, we compa ed he ob- se ed su i al cu es o he nea and dis an - u u e popula ions a he wo insec icide doses wi h he expec ed su i al cu es based on he Bliss c i e ion o d ugs independence. We ound ha he combina ion o he dis an - u u e scena io and exposu e o he high insec icide dose esul ed in a syne gis ic dec ease in su i al (Figu e 4a). We also ound a signi ican an agonis ic e ec in bees o he nea - u u e scena io exposed o he low insec icide dose (Figu e 4b). 4 | DISCUSSION The aim o his s udy was o es ablish whe he clima e wa ming could a ec he abili y o adul bees o cope wi h pes icide expo- su e. We used clima e change p ojec ions om IPCC RCP8.5 and ealis ic concen a ions o a widely used insec icide o assess he combined e ec s o inc easingly wa m win e ing empe a u es and suble hal insec icide exposu e on he beha io and longe i y o a soli a y bee. Ou esul s demons a e syne gis ic e ec s be ween ealis ic scena ios o wo o he main d i e s o bee declines. Impo - an ly, hese e ec s we e ob ained ollowing acu e o al exposu e o a single insec icide. In ield condi ions bees may expe ience epea ed and/o ch onic exposu e o a a ie y o chemicals ia di e en ex- posu e ou es (Knapp e al., 2023; Sanchez- Bayo & Goka, 2014). A he same ime, he empe a u es o ou mos pessimis ic clima e scena io a e al eady occu ing in pa icula ly wa m win e s (win e empe a u es in Eu ope in 2019– 2020 we e 3.4°C highe han he mean o 1981– 2010; AEMET, 2021; Cope nicus, 2020). The e o e, bo h ou pes icide and clima e scena ios may be conside ed con- se a i e. Ou esul s unde sco e he need o s udy combina ions o s esso s o ully unde s and he e ec s o global change on bee popula ions (Goulson e al., 2015). Ou indings also ha e impo an implica ions o pes icide egula ion. Pes icide isk assessmen is based on median le hal and e ec doses (LD50 and ED50). Gi en FIGURE 1 Model- es ima ed means and 95% con idence in e als o pe cen posi i e esponse o a pho o axis es in Osmia co nu a emales exposed o h ee win e ing ea men s ep esen ing inc easingly wa m clima e change scena ios, and h ee o ally adminis e ed doses o he insec icide sul oxa lo (0, 4.55 and 11.64 ng/bee). Di e en le e s deno e signi ican di e ences (Tukey HSD es , p < .05). N = 24– 29 indi iduals pe win e ing scena io and dose. 13652486, 2023, 22, Downloaded om h ps://onlinelib a y.wiley.com/doi/10.1111/gcb.16928 by Csic O ganización Cen al Om (O icialia Mayo ) (U ici), Wiley Online Lib a y on [20/02/2024]. See he Te ms and Condi ions (h ps://onlinelib a y.wiley.com/ e ms-and-condi ions) on Wiley Online Lib a y o ules o use; OA a icles a e go e ned by he applicable C ea i e Commons License | 6253 ALBACETE e al. ou esul s, cu en LD50 and ED50 alues may no ep esen su i- cien ly p o ec i e h esholds as bee popula ions a e con on ed wi h inc easingly wa m empe a u es. Win e ing ea men did no ha e a signi ican e ec on win- e mo ali y, which was e y low and simila o mo ali y alues ound in s udies in which Osmia popula ions we e win e ed unde na u al condi ions (Bosch & Blas, 1994b; Bosch & Kemp, 2000; Ca aDonna e al., 2018; Sgolas a e al., 2012). Simila ly, win e ing ea men by i sel did no impai he abili y o emales o espond o ligh . On he o he hand, win e ing ea men had an impo an impac on weigh loss, which, in ag eemen wi h p e ious s udies, inc eased wi h win e ing empe a u es. Because body weigh loss du ing win e ing is accompanied by he deple ion o ene gy e- se es in Osmia (Fliszkiewicz e al., 2012; Sgolas a e al., 2011), we expec ed ha bees exposed o clima e wa ming would consume mo e sy up o ebuild hei me abolic ese es (Nes el e al., 2016). FIGURE 2 Model- es ima ed means and 95% con idence in e als o daily sy up consump ion (a) and longe i y (b) in Osmia co nu a emales exposed o h ee win e ing ea men s ep esen ing inc easingly wa m clima e change scena ios, and h ee o ally adminis e ed doses o he insec icide sul oxa lo (0, 4.55, and 11.64 ng/bee). Di e en le e s deno e signi ican di e ences (Tukey HSD es , p < .05). N = 53– 65 indi iduals pe win e ing scena io and dose. 13652486, 2023, 22, Downloaded om h ps://onlinelib a y.wiley.com/doi/10.1111/gcb.16928 by Csic O ganización Cen al Om (O icialia Mayo ) (U ici), Wiley Online Lib a y on [20/02/2024]. See he Te ms and Condi ions (h ps://onlinelib a y.wiley.com/ e ms-and-condi ions) on Wiley Online Lib a y o ules o use; OA a icles a e go e ned by he applicable C ea i e Commons License 6254 | ALBACETE e al. Con a y o his expec a ion, win e ing ea men did no a ec daily sy up consump ion. So a , hese indings sugges ha he clima e change scena ios es ed would no comp omise win e su i al, eeding success and pos - eme gence pe o mance in O. co nu a. Howe e , e en wi h simila eeding a es, bees win e ed a he wa mes empe a u e egime had signi ican ly educed mean longe i y (10 days compa ed o 14– 15 days in bees o he o he wo win e ing scena ios), a s ong p edic o o ealized ecundi y in Osmia (Bosch & Vicens, 2006; Sgolas a e al., 2016). Longe i y was also signi ican ly a ec ed by body size, wi h small indi iduals ha ing sho e li e spans. Adul body size in Osmia is di ec ly de- penden on he amoun o ood p o ision alloca ed o he la a by he nes ing emale (Bosch & Vicens, 2002). The e o e, his o ical declines in bee body size a ibu ed o sca ci y o lo al esou ces in simpli ied landscapes may be u he comp omising bee longe - i y and ealized ecundi y (Chole e al., 2019; G ab e al., 2019; He e a e al., 2023; Oli ei a e al., 2016). Like mos soli a y bees, Osmia spp. o e win e on a ixed bud- ge , and expe ience a conside able deple ion o lipid ese es du ing win e ing (Fliszkiewicz e al., 2012; Sgolas a e al., 2011). Upon eme gence, adul s ly o nea by lowe s and ake a i s , long, nec- a meal ha p esumably allows hem o ebuild hei ene ge ic e- sou ces (Bosch & Kemp, 2001; Nes el e al., 2016). In ag icul u al en i onmen s, howe e , he nec a inges ed in his i s meal may con ain suble hal le els o pes icides (Da id e al., 2016; Di ely & Kamel, 2012; Helle e al., 2020; Hladik e al., 2016). To mimic his scena io, we exposed newly eme ged bees o wo suble hal doses o Close , a widely used insec icide. The doses used co espond o concen a ions o 0.58 and 0.23 ppm o ac i e ing edien , which all wi hin he ange o sul oxa lo esidues ound in he nec a o a ious c op lowe s 2– 7 days a e applica ion (USEPA, 2019). Ou esul s show ha hese ealis ic le els o exposu e, especially he high dose, impai ed he abili y o bees o espond o ligh esul ing in a d as ic educ ion o pho o ac ic esponse in bees o he dis an - u u e clima e scena io (Figu e 1). The inabili y o espond o ligh is indica i e o an al e a ion o he sensi i i y o he unc ioning o he isual sys em (Klein e al., 2017). Exposu e o suble hal doses o im- idaclop id, a neonico inoid insec icide wi h a mode o ac ion simila o sul oxa lo (Spa ks e al., 2013), inc eases he exp ession o genes ela ed o biogenic amine ecep o s, such as se o onin, y amine, and oc opamine, associa ed wi h a lack o pho o ac ic esponse in D osophila (Ma elli e al., 2020). High le els o oc opamine ha e also been linked o educed esponse o ligh in honey bees (Bloch & Meshi, 2007; Scheine e al., 2014). The in e ac ion be ween win e ing and pes icide exposu e was also e iden in he a e o daily sy up consump ion. Exposu e o he insec icide dep essed eeding a es in bees o he h ee win- e ing ea men s, bu his dec ease was especially p onounced in FIGURE 3 Cumula i e su i al p obabili y o Osmia co nu a emales exposed o h ee win e ing ea men s ep esen ing inc easingly wa m clima e change scena ios, and h ee o ally adminis e ed doses o he insec icide sul oxa lo (0, 4.55, and 11.64 ng/bee). Cu es wi h di e en le e s a e signi ican ly di e en (pai wise compa isons, log- ank es , p < .05). 13652486, 2023, 22, Downloaded om h ps://onlinelib a y.wiley.com/doi/10.1111/gcb.16928 by Csic O ganización Cen al Om (O icialia Mayo ) (U ici), Wiley Online Lib a y on [20/02/2024]. See he Te ms and Condi ions (h ps://onlinelib a y.wiley.com/ e ms-and-condi ions) on Wiley Online Lib a y o ules o use; OA a icles a e go e ned by he applicable C ea i e Commons License | 6255 ALBACETE e al. bees exposed o he wa mes win e (Figu e 2A). The inhibi o y e - ec o sul oxa lo on sy up consump ion has been documen ed in bo h soli a y and social bees (Azpiazu e al., 2022; Li e al., 2021; Si i e e al., 2020). Longe i y was educed by ca. 50% in bees o he cu en and nea - u u e scena ios exposed o he high insec i- cide dose. In bees o he dis an - u u e scena io, exposu e o he low dose led o an es ima ed 11% educ ion in longe i y and exposu e o he high dose esul ed in a syne gis ic 70% educ ion (Figu e 3). As men ioned, he cogni i e abili ies o he bees o he la e ea - men we e signi ican ly impai ed, u he educing he e ec i e ime ame a ailable o ep oduc ion (Bosch & Vicens, 2006; Sgolas a e al., 2016). We also ound ha bees exposed o he nea - u u e scena io and he low insec icide dose li ed longe han expec ed. Howe e , his an agonis ic e ec did no esul in an inc ease in lon- ge i y compa ed o bees o he nea - u u e scena io exposed o he 0 insec icide dose. An agonis ic in e ac ions a e no uncommon in s udies add essing in e ac ions be ween s esso s (Bi d e al., 2021; B uckne e al., 2023; Si i e e al., 2021; Za agoza- T ello e al., 2021). The inc eased sensi i i y o Close in bees exposed o clima e wa ming scena ios may be media ed by he e ec s o wa m win e empe a u es on he consump ion o ene gy ese es and a body deple ion in Osmia (Bosch e al., 2000, 2010; Bosch & Kemp, 2003, 2004; Fliszkiewicz e al., 2012; Sgolas a e al., 2011). The a body is a dynamic, “li e - like”, issue ha ac s as an o gan o ene gy s o age and o he me abolic unc ions in insec s (A ese & Soul- ages, 2010). S udies on bu e lies and mosqui oes ha e ound ha exposu e o xenobio ics inc eases he exp ession o cy och ome P450 monooxygenases genes, in ol ed in de oxi ica ion, in he a body (Da id e al., 2006; Pe e sen e al., 2001). In addi ion, s udies on bumblebees ha e ound ha he exp ession o hese genes in he a body is enhanced by a suc ose- ich die (Cos a e al., 2020), and ha queens, which ha e signi ican ly highe a body ese es han males and wo ke s, a e mo e esilien o pes- icides (Linguadoca e al., 2022). In honey bees, a ack by he mi e Va oa des uc o , ha eeds on he a body o adul s (Ramsey e al., 2019) dis up s de oxi ica ion abili y and inc eases sensi i i y FIGURE 4 Obse ed ( ed/yellow lines) and expec ed (black dashed lines) su i al p obabili y cu es o Osmia co nu a emales exposed o dis an - (a) and nea - u u e (b) clima e change scena ios and o wo o ally adminis e ed doses o he insec icide sul oxa lo (4.55 and 11.64 ng/bee). In e ac ion signi icance is based on he Bliss c i e ion o d ugs independence (FH s a is ic dis ibu ion >0 indica es syne gism be ween he wo s esso s; FH s a is ic dis ibu ion <0 indica es an agonism). 13652486, 2023, 22, Downloaded om h ps://onlinelib a y.wiley.com/doi/10.1111/gcb.16928 by Csic O ganización Cen al Om (O icialia Mayo ) (U ici), Wiley Online Lib a y on [20/02/2024]. See he Te ms and Condi ions (h ps://onlinelib a y.wiley.com/ e ms-and-condi ions) on Wiley Online Lib a y o ules o use; OA a icles a e go e ned by he applicable C ea i e Commons License 6256 | ALBACETE e al. o pes icides (Blanken e al., 2015; Schwa z e al., 2021; Zhu e al., 2022). Based on his line o e idence, we sugges ha he abili y o p oduce de oxi ica ion enzymes o con on suble hal le els o insec icide is dep essed in soli a y bees eme ging wi h deple ed a bodies as a esul o wa m win e empe a u es. Ou indings indica e ha ongoing global wa ming may exace ba e he impac o pes icides on bee heal h and comp omise bee ep o- duc i e success, wi h po en ially impo an consequences on popu- la ion dynamics. The magni ude o hese impac s will depend on ou abili y o educe he dependence o ag icul u e on pes icides (Si i e e al., 2023; Sponsle e al., 2019) and on he ex en o which bee pop- ula ions a e able o adjus o he new clima e scena io h ough adap a- ion and/o pheno ypic plas ici y. The dis ibu ion o O. co nu a anges om no he n A ica o sou he n Sweden (Mülle , 2022), sugges ing ha he species has he capaci y o adap o local clima es. Tempe a- u e inc eases such as hose simula ed in ou s udy a e likely o ha e a s onge impac in popula ions om al eady wa m en i onmen s, li ing close o he he mal sa e y limi s o he species, han in no h- e n popula ions (Ca aDonna e al., 2018). Bees may also mi iga e he e ec s o clima e wa ming by ac i ely selec ing coole nes ing si es, as shown in O. bico nis (Os ap- Chec e al., 2021), o h ough la i udinal o al i udinal mig a ion o coole a eas (Ke e al., 2015). A any a e, he ac ha empe a u e inc eases simila o hose o ou mos pessimis- ic scena io ha e been eco ded in ecen yea s (AEMET, 2021; Cope - nicus, 2020) sugges s ha he combined e ec s o clima e wa ming and pes icide exposu e a e al eady a ec ing bee popula ion dynamics. ACKNOWLEDGMENTS The au ho s a e g a e ul o h ee anonymous e iewe s o hei cons uc i e commen s. The au ho s a e also g a e ul o M. Ba na- das, E. Se a osa and P. Sole o hei echnical assis ance h ough- ou he expe imen . The s udy was unded by he Spanish Minis y o Science and Inno a ion, h ough p ojec s RTI2018- 098399- B- I00 and PID2021- 128938OB- I00, and PhD schola ships o SA and GS (PRE2019- 088817 and PRE2019- 090375). CA was suppo ed by a Ma ga i a Salas pos doc o al ellowship om he Spanish Minis y o Uni e si ies h ough he EU Nex Gene a ion p og am. CONFLICT OF INTEREST STATEMENT The au ho s decla e hey ha e no con lic o in e es . DATA AVAILABILITY STATEMENT The da a ha suppo indings o his s udy a e a ailable on D yad a h ps://doi.o g/10.5061/d yad.dz08k ps3 . ORCID Se gio Albace e h ps://o cid.o g/0000-0001-7696-623X Gonzalo Sancho h ps://o cid.o g/0000-0001-7711-846X Celes e Azpiazu h ps://o cid.o g/0000-0001-8217-9347 Anselm Rod igo h ps://o cid.o g/0000-0001-6341-0363 Robe o Molowny- Ho as h ps://o cid.o g/0000-0003-2626-6379 Fabio Sgolas a h ps://o cid.o g/0000-0002-2845-8297 Jo di Bosch h ps://o cid.o g/0000-0002-8088-9457 REFERENCES Agencia Es a al de Me eo ología (AEMET). (2021). 2020, he wa mes yea in Spain, in Eu ope and in he wo ld. h ps://www.aeme .es/es/ no ic ias/2022/10/sep i emb e_2022_ esum en_clima Albace e, S., Sancho, G., Azpiazu, C., Rod igo, A., Molowny- Ho as, R., Sgolas a, F., & Bosch, J. (2023). Bees exposed o clima e change a e mo e sensi i e o pes icides. D yad Da ase . h ps://doi.o g/ 10.5061/d yad.dz08k ps3 A ese, E. L., & Soulages, J. L. (2010). Insec a body: Ene gy, me ab- olism, and egula ion. Annual Re iew o En omology, 55, 207– 225. h p s : / / d o i . o g / 1 0 . 1 1 4 6 / a n n u e - e n o - 1 1 2 4 0 8 - 0 8 5 3 5 6 Au au e, J., Bi on, D. G., Vidau, C., Fon bonne, R., Roudel, M., Diogon, M., Viguès, B., Belzunces, L. P., Delbac, F., & Blo , N. (2012). Pa asi e- insec icide in e ac ions: A case s udy o Nosema ce anae and ip onil syne gy on honeybee. Scien i ic Repo s, 2(1), 326. h ps://doi.o g/10.1038/s ep0 0326 Azpiazu, C., Bosch, J., Bo olo i, L., Med zycki, P., Tepe , D., Molowny- Ho as, R., & Sgolas a, F. (2021). Toxici y o he insec icide sul - oxa lo alone and in combina ion wi h he ungicide luxapy oxad in h ee bee species. Scien i ic Repo s, 11(1), 6821. h ps://doi. o g / 1 0 . 1 0 3 8 / s 4 1 5 9 8 - 0 2 1 - 8 6 0 3 6 - 1 Azpiazu, C., Bosch, J., Ma ins, C., & Sgolas a, F. (2022). E ec s o ch onic exposu e o he new insec icide sul oxa lo in combina ion wi h a SDHI ungicide in a soli a y bee. Science o he To al En i onmen , 850, 157822. h ps://doi.o g/10.1016/j.sci o en .2022.157822 Azpiazu, C., Bosch, J., Viñuela, E., Med zycki, P., Tepe , D., & Sgolas a, F. (2019). Ch onic o al exposu e o ield- ealis ic pes icide combina- ions ia pollen and nec a : E ec s on eeding and he mal pe o - mance in a soli a y bee. Scien i ic Repo s, 9(1), 13770. h ps://doi. o g / 1 0 . 1 0 3 8 / s 4 1 5 9 8 - 0 1 9 - 5 0 2 5 5 - 4 Azpiazu, C., Hina ejos, S., Sancho, G., Albace e, S., Sgolas a, F., Ma ins, C. A. H., Domene, X., Ben ezkallah, J., Rod igo, A., A nan, X., & Bosch, J. (2023). Desc ip ion and alida ion o an imp o ed me hod o eed soli a y bees (Osmia spp.) known amoun s o pes icides. Eco oxicology and En i onmen al Sa e y, 264, 115398. Bi d, G., Wilson, A. E., Williams, G. R., & Ha dy, N. B. (2021). Pa asi es and pes icides ac an agonis ically on honey bee heal h. Jou nal o Applied Ecology, 58(5), 997– 1005. h ps://doi. o g/10.1111/1365- 2664.13811 Blanckenho n, W. U., & Hensele , C. (2005). Tempe a u e- dependen o a iole and es is ma u a ion in he yellow dung ly. En omologia Expe imen alis e Applica a, 116(3), 159– 165. h ps://doi.o g/10.1111/ j.1570- 7458.2005.00316.x Blanken, L. J., an Lange elde, F., & an Doo emalen, C. (2015). In e ac ion be ween Va oa des uc o and imidaclop id educes ligh capaci y o honeybees. P oceedings o he Royal Socie y B: Biological Sciences, 282(1820), 20151738. h ps://doi.o g/10.1098/ spb.2015.1738 Bloch, G., & Meshi, A. (2007). In luences o oc opamine and ju enile ho mone on locomo o beha io and pe iod gene exp ession in he honeybee, Apis melli e a. Jou nal o Compa a i e Physiology A, 193, 181– 199. h p s : / / d o i . o g / 1 0 . 1 0 0 7 / s 0 0 3 5 9 - 0 0 6 - 0 1 7 9 - 5 / a b l e s / 6 Bosch, J. (1994). The nes ing beha iou o he mason bee Osmia co - nu a (La ) wi h special e e ence o i s pollina ing po en ial (hy- menop e a, Megachilidae). Apidologie, 25(1), 84– 93. h ps://doi. o g/10.1051/apido :19940109 Bosch, J., & Blas, M. (1994a). Fo aging beha iou and pollina ing e i- ciency o Osmia co nu a and Apis melli e a on almond (Hymenop e a, Megachilidae and Apidae). Applied En omology and Zoology, 29(1), 1 – 9 . h ps://doi.o g/10.1303/aez.29.1 Bosch, J., & Blas, M. (1994b). E ec o o e - win e ing and incuba- ion empe a u es on adul eme gence in Osmia co nu a La . (Hymenop e a, Megachilidae). Apidologie, 25(3), 265– 277. h ps:// doi.o g/10.1051/apido :19940301 13652486, 2023, 22, Downloaded om h ps://onlinelib a y.wiley.com/doi/10.1111/gcb.16928 by Csic O ganización Cen al Om (O icialia Mayo ) (U ici), Wiley Online Lib a y on [20/02/2024]. See he Te ms and Condi ions (h ps://onlinelib a y.wiley.com/ e ms-and-condi ions) on Wiley Online Lib a y o ules o use; OA a icles a e go e ned by he applicable C ea i e Commons License