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Glob Change Biol. 2023;29:6248–6260.wileyonlinelib a y.com/jou nal/gcb
Recei ed: 4 May 2023
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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
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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
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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
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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
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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.
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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.
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6254
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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).
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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).
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6256
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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
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