Expe imen al p o ocol o acu e o al oxici y es ing in he ho e ly
E is alinus aeneus (Scopoli, 1763) (Dip e a, Sy phidae)
Milica Radenko ić1, Jelena Pu ać1, Nikola K i okuća1, Danijela Kojić1, Dimi y Win e man el2,
Julia Os e man3,4, An e Vujić1
1 Depa men o BiologyandEcology,Facul yo Sciences,Uni e si yo No iSad,T gD.Ob ado ića2,No iSad,Se bia
2 Uni e si y o F eibu g, Na u e Conse a ion and Landscape Ecology, S e an-Meie -S . 76, F eibu g, Ge many
3 Uni e si y o Go henbu g, Depa men o Biological and En i onmen al Sciences, Medicina ega an 7B, Go henbu g, Sweden
4 Cen e o Fu u e Chemical Risk Assessmen and Managemen (FRAM), Medicina ega an 7B, Uni e si y o Go henbu g, Go henbu g, Sweden
Co esponding au ho : Danijela Kojić ([email p o ec ed])
Academic edi o : Gio anni Bu gio ♦
Recei ed
8 Augus 2025 ♦
Accep ed
28 Oc obe 2025 ♦
Published
21 No embe 2025
Abs ac
Ho e lies (Sy phidae) play a key ole in pollina ion se ices ac oss ecosys ems bu a e unde ep esen ed in pes icide oxici y esea ch.
Labo a o y oxici y es ing is a undamen al s ep owa d e alua ing pes icide isks, ye es ablished p o ocols o ho e lies a e lacking.
This s udy aims o ill his gap by de eloping an acu e o al oxici y es o E is alinus aeneus, a cosmopoli an ho e ly. A no el o al ox-
ici y es was in oduced using a cus om-designed es cage wi h a i icial lowe s, allowing p ecise measu emen o ood consump ion
and dose assessmen . Bo h indi idual and g oup eeding se ups we e es ed, wi h g oup eeding p o ing mo e easible, o e coming key
limi a ions o indi idual eeding, including di icul y in loca ing ood and e apo a ion o liquid die s. As an addi ional alida ion s ep,
an o al oxici y es was conduc ed wi h dime hoa e — a e e ence insec icide commonly used in oxicological s udies — using h ee
concen a ions (0.007, 0.0014, and 0.0007 g/L). The es ima ed LC50 alues o E. aeneus we e ~0.00166 g/L (24 h) and ~0.00137 g/L
(48 h), wi h co esponding LD50 alues o ~0.56 and ~0.46 μg/g ly. Ou esul s indica e ha E. aeneus is highly sensi i e o dime hoa e
and sligh ly mo e sensi i e han Apis melli e a. These indings unde sco e he need o include E. aeneus and o he sy phids in u u e
oxici y es ing and highligh he alue o he de eloped me hodology o eco oxicological s udies and conse a ion e o s.
Key Wo ds
A i icial lowe s, dime hoa e, labo a o y es , pollina o s, isk assessmen , sy phids
In oduc ion
Ho e lies (Sy phidae) a e a di e se amily o Dip e a
comp ising o e 6,000 species wo ldwide (Sommaggio
1999), and among he mos impo an non-bee pollina-
o s, con ibu ing subs an ially o bo h wild plan and c op
pollina ion (Klecka e al. 2018; Dunn e al. 2020). In ad-
di ion o hei ole as pollina o s, some species also con-
ibu e o na u al pes con ol by eeding on aphids (Dunn
e al. 2020). Like many o he pollina ing insec s, ho e -
lies a e inc easingly exposed o a ious en i onmen al
s esso s. Al hough he global decline o pollina o s ais-
es conce ns o biodi e si y and ag icul u e (Senapa hi e
al. 2015; B eeze e al. 2016; Po s e al. 2016; Neo e al.
2021), ho e lies emain unde ep esen ed in pollina o
esea ch, which has adi ionally ocused on bees.
Pes icide exposu e is conside ed a key d i e o pol-
lina o declines (Bo ías e al. 2015; Dicks e al. 2021;
Bi kenbach e al. 2024). While egula o y es ing p o o-
cols ha e been well es ablished o honeybees (Apis mel-
li e a L. 1758) (OECD 1998a, 1998b), and mo e ecen ly
o bumblebees and soli a y bees (OECD 2017a, 2017b;
OECD 2024), ho e lies emain la gely excluded om
such amewo ks. This gap pe sis s despi e g owing e -
idence o conside able in e - and in aspeci ic a ia ion
in insec sensi i i y o pes icides (Ha ds one and Sco
2010; Sgolas a e al. 2019; Hells öm e al. 2023; Na-
gloo e al. 2024).
Copy igh Alma Ma e S udio um – Uni e si à di Bologna. This is an open access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion License (CC BY 4.0), which
pe mi s un es ic ed use, dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal au ho and sou ce a e c edi ed.
Bulle in o Insec ology 78 2025, 121–130
DOI 10.3897/bull.insec ology.168212
Resea ch A icle
Bulle in o
Insec ology
bulle ino insec ology.o g
Milica Radenko ić e al.: Acu e o al oxici y es ing in E is alinus aeneus122
Toxicological esea ch on ho e lies emains limi ed
in bo h scope and axonomic co e age. S udies on la -
al exposu e ha e shown ha some insec icides inc ease
mo ali y and nega i ely a ec ep oduc ion in aphidoph-
agous species (Jansen e al. 2011; Moens e al. 2011),
whe eas o he s, such as hiaclop id, hiame hoxam, and
ace amip id, showed no e ec s on adul s a ield- ealis ic
doses (Jansen e al. 2011; Basley e al. 2018; Bi kenbach
e al. 2024). Howe e , s anda dized me hodologies o
adul o al oxici y es ing a e s ill lacking.
Some ecen p og ess has been made owa d add ess-
ing his gap. Nagloo e al. (2023) de eloped an o al ox-
ici y bioassay o E is alis enax L. 1758 using 3D-p in -
ed a i icial lowe s, demons a ing a much highe
ole ance o imidaclop id compa ed o A. melli e a.
Howe e , he equi emen o specialized equipmen
limi s he b oade applica ion o his me hod. Simila ly,
Hen iques Ma ins e al. (2024) assessed con ac oxici y
o an imidaclop id o mula ion (Con ido ®) in Sphae o-
pho ia ueppellii (Wiedemann 1820) and E is alinus ae-
neus (Scopoli 1763), inding ha hese species we e less
sensi i e han bees.
Gi en he lack o s anda dized and easily applicable
me hods o o al oxici y es ing in ho e lies, we aimed
o de elop a simple and ep oducible acu e o al oxici y
bioassay o E is alinus aeneus. This species was selec -
ed due o i s wide geog aphic dis ibu ion, high pollina-
ion e iciency, and po en ial o labo a o y ea ing and
comme cializa ion (Campoy e al. 2020). Speci ically,
ou objec i es we e o: (a) es di e en eeding designs
(wi h o wi hou a i icial lowe ) o de e mine which
design yields he highes eeding a e unde indi idual
eeding condi ions, and (b) assess whe he g oup eeding
enhances eeding a es compa ed o indi idual se ups. To
alida e he me hod, we es ed he oxici y o dime hoa e,
a s anda d posi i e con ol commonly used o alida e
expe imen al design and con i m es sensi i i y in ox-
ici y s udies (Gough e al. 1994; OECD 1998a, b; OECD
2017a, b; OECD 2024), and es ima ed he app oxima e
24- and 48-hou o al LC50 and LD50 o E. aeneus based
on a h ee-dose concen a ion ange. These p elimina y
alues p o ide a e e ence o selec ing app op ia e con-
cen a ions o dime hoa e as a posi i e con ol in u u e
s udies assessing he oxici y o o he pes icides and al-
low o e en ual compa isons wi h o he species, p ima -
ily A. melli e a and bumblebees.
Ma e ial and me hods
P e- ea men insec ea ing
Pupae o E is alinus aeneus we e ob ained om he
Uni e si y o Alican e (Spain) and andomly placed in
pe o a ed comme cial plas ic boxes (2L, ~30 pupae
pe box) on a plas ic ay (see Suppl. ma e ial 1). Each
box was supplied wi h a 50% w/ suc ose solu ion and
wa e in sepa a e con aine s. Eme ging adul s (wi hou
sepa a ing emales and males) we e collec ed daily in o
new boxes (~10 lies pe box) p o ided wi h suc ose
solu ion and wa e . Du ing acclima iza ion, he lies
we e kep a oom empe a u e (app oxima ely 22°C)
unde na u al ligh condi ions. Dead o mo ibund indi-
iduals we e emo ed daily, and ood was eplenished
as needed. Expe imen s began once a su icien numbe
o adul s had eme ged (~5 days). E. aeneus is also com-
me cially a ailable, making b oade applica ion o he
me hod easible.
Tes eeding designs
Comme cial plas ic boxes (375 mL) wi h needle-punched
pe o a ions we e modi ied o easy manipula ion o he
ood supply. To c ea e cages, he boxes we e in e ed so
ha he lid became he bo om. A 10 mm hole was d illed
in he cen e, sealed wi h an Eppendo cap se ing as a
eede uni , allowing easy emo al o eplacemen wi h-
ou opening he box (Fig. 1A, Suppl. ma e ial 9). Cages
con ained a i icial adially symme ical lowe s, ei he
2D (whi e o g een base) o 3D (na ow o wide ele a -
ed pe als on he g een base) (Fig. 1B–E). G een bases
we e in oduced o imi a e a g assy a ea (Fig. 1B, D, E).
Since ho e lies exhibi colou p e e ences (Klecka e al.
2018), pe als we e yellow o whi e, and he lowe s we e
unscen ed o p e en po en ial odo -induced sex bias
(Mish a e al. 2025). Thei adial symme y aligned wi h
he lo al complexi y p e e ence o E. aeneus and many
o he pollina o species (Ladu ne e al. 2005b).
Feeding ials
All eeding ials used a 50% w/ suc ose solu ion as
eeding solu ion. Flies we e indi idually es ed on i e
di e en eeding designs, ou lowe designs (Fig. 1B–E)
and one eede wi hou pe als (20 lies pe design, 100 in
o al). G oup eeding (5 lies pe cage) was es ed on he
3D lowe wi h wide pe als in 20 eplica es (100 lies o-
al, Fig. 1E and Fig. 2B, C). Two days be o e es ing, lies
we e alloca ed o es cages. G oup- ed lies we e b ie ly
chilled o immobilize hem and ma ked in ou colou s
(yellow, blue, ed, g een), wi h one ly le unma ked pe
cage o acking (Fig. 2, Suppl. ma e ial 10). Flies we e
p e- ed on suc ose-soaked co on wool o amilia ize
hem wi h he eeding p ocess, ollowed by an o e nigh
s a a ion pe iod o s anda dize hunge le els.
On he es day, caps wi h 20 µL (indi idual) o
100 µL (g oup) suc ose solu ion we e weighed and
placed in cages. Flies we e moni o ed un il hei i s
eeding (maximum h ee hou s, as p oposed by OECD
guidelines 1998b, 2017b) and classi ied as eede s o
non- eede s (based on isual con i ma ion o consump-
ion). A e h ee hou s, caps we e weighed and eplaced
wi h suc ose-soaked co on wool. E apo a ion was con-
olled using ly- ee cages, each wi h suc ose- illed
Bulle in o Insec ology 78 2025, 121–130
bulle ino insec ology.o g
123
caps con aining he app op ia e olume ( wen y caps o
20 µL and wen y caps o 100 µL). Since E. aeneus does
no exhibi sexual dimo phism in body size o o aging
ac i i y (Mish a e al. 2025), bo h sexes we e used in he
expe imen , ensu ing an equal dis ibu ion o emales and
males in he es g oups.
Assessmen o sensi i i y o dime hoa e – i s
se up
Dime hoa e (Pens anal®, Sigma-Ald ich) was dissol ed
in ace one o p epa e he s ock solu ion (100 g/L), which
was hen se ially dilu ed (de ails in Suppl. ma e ial 3).
Figu e 1. Cages wi h eeding uni s and lowe se ups. Tes cages: small, anspa en , pe o a ed plas ic boxes (375 mL olume)
wi h a 10 mm diame e hole in he cen e o he bo om. The hole was sealed wi h a mic o ube cap, which se es as a ood supply
uni and allows easy manipula ion wi hou opening he box (A). A i icial lowe models wi h la pape pe als a anged a ound he
cap (2D lowe s) on a g een su ace (B) and a whi e su ace (C), o ele a ed pe als a ound he cap (3D lowe s) wi h na ow (D) o
wide (E) pe als on a g een su ace.
Figu e 2. Ma ked ho e lies in es cage. Fi e ma ked indi iduals o E is alinus aeneus o g oup eeding: ou ma ked wi h di -
e en colou s and one unma ked (A), in a es cage wi h a 3D lowe ea u ing wide pe als (B and C).
bulle ino insec ology.o g
Milica Radenko ić e al.: Acu e o al oxici y es ing in E is alinus aeneus124
The ea men solu ions we e p epa ed in 50% w/ su-
c ose, ensu ing ha he ace one concen a ion did no
exceed 4% / (OECD 2017b). Al hough dime hoa e is
cu en ly banned in he Eu opean Union, i is commonly
used as a posi i e con ol in oxicological s udies o al-
ida e expe imen al design and con i m sensi i i y o he
es species (Gough e al. 1994; OECD 1998a, b; OECD
2017a, b; OECD 2024).
Based on pilo ials (da a no shown), h ee dime h-
oa e concen a ions we e es ed: D1 (0.007 g/L), D2
(0.0014 g/L), and D3 (0.0007 g/L). Each concen a ion
(100 µL pe cage) was es ed in a cage con aining i e
ma ked lies and a 3D lowe wi h wide pe als, wi h wo
eplica es pe concen a ion (10 lies pe concen a ion).
The se up was used as a p elimina y ange- inding es
a he han a de ini i e LD50 de e mina ion. Addi ionally,
wo con ol cages (C) wi h i e indi iduals we e included,
in which lies we e ed wi h 50% w/ suc ose solu ion wi h
4% ace one a inal concen a ion. In o al, ea ed and con-
ol g oups included 40 indi iduals. To accoun o e apo-
a ion, wo ly- ee con ol cages we e also included. A e
h ee hou s, he caps con aining he ea men solu ions
we e emo ed, weighed, and eplaced wi h suc ose-soaked
co on wool. The solu ion consumed pe cage was calcu-
la ed by sub ac ing he pos -exposu e cap weigh om he
p e-exposu e weigh , co ec ed o e apo a ion. Consump-
ion pe ly in g oup eeding was calcula ed by di iding
o al consump ion by he numbe o eede s. Mo ali y was
eco ded a 4, 24, and 48 hou s pos -exposu e.
Assessmen o sensi i i y o dime hoa e –
second se up
Based on esul s om he i s se up, he second ocused
on he D2 concen a ion, which had caused 50% mo al-
i y a e 24 hou s, using a la ge sample size o e ine he
es ima es o he median le hal concen a ion/dose. Fi e
eplica e cages, each con aining i e lies, we e exposed
o he D2 solu ion. A con ol g oup (C) was also included,
consis ing o h ee eplica e cages and wo ly- ee cages
as e apo a ion con ols.
S a is ical analysis
To de e mine how eeding success was a ec ed by eeding
design, only da a om indi idually caged lies we e anal-
ysed o a oid con ounding wi h po en ial e ec s o g oup
eeding. Feeding success ( ed s. no ed) was modelled us-
ing wo gene alized linea models (GLMs) wi h binomial
e o dis ibu ion and a logi link, implemen ed ia he glm
unc ion in R’s “s a s” package. The i s GLM es ed he
e ec o using pe als in gene al and con ained pe al p es-
ence (yes s no) as he only p edic o . The second GLM
es ed whe he he exac design a ec ed eeding success
and con ained eeding design as he sole p edic o . Pai wise
di e ences among he i e eeding designs we e es ed us-
ing es ima ed ma ginal means de i ed wi h he “emmeans”
unc ion and compa ed using he “pai s” unc ion o he
“emmeans” package. P- alues we e adjus ed o mul iple
es ing using he alse disco e y a e (FDR) me hod.
To assess whe he eeding success di e ed be ween
g oup and indi idual eeding, a gene alized linea
mixed-e ec s model (GLMM) was applied o he wo
ea men s sha ing he same eeding design (3D low-
e wi h wide pe als). Feeding success ( ed s. no ed)
was modeled using a gene alized linea mixed-e ec s
model wi h binomial e o dis ibu ion and a logi link,
implemen ed ia he “glmmTMB” unc ion o he “glm-
mTMB” package. This model con ained eeding design
as ixed ac o and cage ID as andom e ec .
Fo isualiza ion, eeding success in each ea men
(design o indi idual/g oup eeding) was exp essed as he
pe cen age o indi iduals ha ed, and he co esponding
95% con idence in e als we e calcula ed using he “em-
means” unc ion om he “emmeans” package.
To es ima e he median le hal dose (LD50) and concen-
a ion (LC50) a 24 hou s and 48 hou s, wo-pa ame e
log-logis ic dose– esponse models we e i ed using he
unc ion “d m” om he “d c” package (Ri z e al. 2015)
and da a om bo h se ups. The esponse a iable was he
p opo ion o dead indi iduals ou o hose ha had ed,
wi h he numbe o eede s included as weigh s o accoun
o a ia ion in sample size among ea men s. A bino-
mial e o dis ibu ion was assumed. The dose- esponse
ela ionships and concen a ion- esponse ela ionships
we e modelled using he log-logis ic unc ion speci ied by
LL2.2, wi h he lowe limi ixed a ze o and bo h he uppe
limi and slope es ima ed om he da a. LD50 and LC50 al-
ues and hei 95% con idence in e als we e de i ed using
he unc ion ED om he “d c” package wi h he a gumen
“in e al” = “ ls”, which calcula es con idence limi s on
he log- ans o med dose scale and back- ans o ms hem
o he o iginal scale. The con ol g oup (0 ng/ ly; 0 µg/
µL) was speci ied as he e e ence dose/concen a ion. Fo
compa ison o o he species, LD50 alues we e no malized
o body mass by di iding by he mean adul body mass o
E is alinus aeneus in his expe imen (0.043 g).
All analyses we e conduc ed in R ( e sion 4.4.2)
(Team RDC 2024), and he R sc ip is p o ided in Suppl.
ma e ial 2.
Resul s and discussion
Toxicological s udies on sy phids a e sca ce, despi e hei
impo an ole as pollina o s. Resea ch on acu e oxici y
has mos ly ocused on cosmopoli an species, wi h s udies
assessing o al oxici y (Nagloo e al. 2023) and con ac ox-
ici y (Hen iques Ma ins e al. 2024). As he me hodology
o o al oxici y es ing in sy phids is s ill in de elopmen
and gi en ha o al exposu e is a c i ical ou e o pes icide
exposu e, we p opose a simple and eliable acu e o al ox-
ici y es o E is alinus aeneus. Ou es was designed o be
easily implemen ed unde s anda d labo a o y condi ions.
Bulle in o Insec ology 78 2025, 121–130
bulle ino insec ology.o g
125
Feeding ials
Indi idual eeding was es ed i s , as i allows o p e-
cise measu emen o ea men solu ion in ake, which is
essen ial o calcula ing he exac dose o ac i e ing edi-
en consumed. The main me hodological challenge in o al
oxici y es ing is ha some species do no eadily con-
sume a i icial die s in labo a o y se ings. One app oach
o o e coming hese challenges in ol es using a i icial o
na u al lowe s as eeding pla o ms (Ladu ne e al. 2003,
2005a, 2005b; Mille e al. 2010; Nagloo e al. 2023). Na -
u al lowe -based me hods ha e demons a ed highe eed-
ing success a es (Ladu ne e al. 2003, 2005a), bu hei
widesp ead applica ion is hinde ed by he a ailabili y o
lo al esou ces. A simpli ied al e na i e using a single pe -
al has shown p omise o imp o ing eeding consis ency
in Osmia spp. (Azpiazu e al. 2023; Hells öm e al. 2023).
In ou s udy, we e alua ed a i icial lowe s wi h pape
pe als as a simple and p ac ical app oach. The eeding i-
als included indi idual eeding in cages wi h 2D and 3D
lowe designs (Fig. 1) and cages wi hou pe als. Feeding
success was measu ed in e ms o he lies’ abili y o loca e
he ood sou ce and consume he ood. Gene ally, eeding
success was highe wi h a i icial lowe s han wi h he pe -
al- ee design (76% s 35%, P < 0.001). The e was howe -
e no signi ican di e ence among di e en a i icial low-
e designs (P > 0.5). The highes eeding a e (85%) was
obse ed wi h he 3D lowe wi h wide downwa d-sloping
pe als, ollowed by he 3D lowe wi h na owe pe als and
he 2D lowe wi h a g een base (bo h 75%). These h ee
designs yielded a signi ican ly highe eeding a e o in-
di idual eeding han he pe al- ee design (FDR-adjus ed
Wald es on binomial GLMM, 3D wide pe als: P = 0.026,
o he wo designs: P = 0.046; Fig. 3, Suppl. ma e ials 4, 5).
The 2D lowe wi h a whi e base had also a wice as high
obse ed eeding a e (70%) han he pe al- ee design
(35%), bu he di e ence was no s a is ically signi ican
(P = 0.075) (see Suppl. ma e ial 5).
Du ing indi idual eeding ials, wo main challenges
eme ged. The i s issue s emmed om he lies’ beha iou :
in some cases, hey emained s a iona y o he majo i y, i
no he en i e y o he h ee-hou pe iod, se e ely limi ing
hei abili y o loca e ood. The second, majo challenge
was e apo a ion. In con ol expe imen s (Suppl. ma e i-
als 6, 7), he 100 µL suc ose solu ion los app oxima e-
ly 26% o i s mass due o e apo a ion o e h ee hou s,
whe eas he 20 µL solu ion los a ound 50% o i s mass as
he sol en e apo a ed and emaining suga c ys allized,
which p e en ed he lies om ha ing access o a liquid
ood sou ce. Fo o al oxici y es s, which ypically las
up o h ee hou s (OECD 1998b; OECD 2017b), he eed-
ing me hod mus ensu e e icien ood in ake h oughou
he exposu e pe iod. Due o hese issues, he indi idual
eeding ials we e ul ima ely deemed unsuccess ul. Gi -
en he challenges o wo king wi h wild pollina o s, whe e
specimen a ailabili y may be limi ed, op imizing eeding
me hods is essen ial o ob aining eliable da a.
These issues we e success ully esol ed by imple-
men ing g oup eeding and eeding success appea ed o
imp o e unde g oup eeding condi ions. When i e lies
we e p o ided wi h a 3D lowe wi h wide pe als (Fig. 1E)
and a 100 µL suc ose eede , eeding success inc eased
om 85% unde indi idual eeding o 97% unde g oup
eeding (P = 0.04; Fig. 3, Suppl. ma e ial 4).
Inc eased in e ac ion among lies led o g ea e mo e-
men and enhanced eeding success. Due o inc eased ly
ac i i y, ood was mos ly loca ed du ing he i s hou o ex-
posu e. Addi ionally, he la ge olume o eeding solu ions
Figu e 3. Feeding success o E is alinus aeneus ac oss di e en cage designs unde indi idual eeding condi ions, wi h one addi-
ional g oup eeding ea men included o compa ison. Poin s ep esen obse ed eeding success (pe cen o lies ha ed), wi h
e o ba s indica ing 95% con idence in e als. Colou s dis inguish be ween g oup and indi idual eeding.
bulle ino insec ology.o g
Milica Radenko ić e al.: Acu e o al oxici y es ing in E is alinus aeneus126
mi iga ed e apo a ion, ensu ing ha he lies had su icien
ime o consume he es solu ion wi hin he exposu e pe-
iod. To acili a e indi idual acking o consump ion, lies
we e ma ked (Fig. 2), allowing o he dis inc ion be ween
eede s and non- eede s. This app oach ensu ed ha only
lies ha consumed om he solu ion we e conside ed o
he ea men . As a esul , g oup eeding wi h i e ma ked
lies pe cage and 3D lowe s wi h wide pe als was used o
all subsequen es ing in his s udy.
While he g oup eeding me hod o e s se e al ad an-
ages, including po en ially highe a es o eeding suc-
cess, ease o implemen a ion and sui abili y o labo a o-
ies wi h a ying le els o esou ces, i is impo an o no e
i s limi a ions. One key limi a ion is ha consump ion pe
ly is calcula ed as an a e age: assuming ha all lies con-
sumed he same amoun o ood. This app oach may lead
o inaccu acies in es ima ing indi idual consump ion, as i
assumes uni o m eeding beha iou and does no accoun
o a iabili y among indi iduals. Howe e , his limi a-
ion is pa ially mi iga ed by he use o small g oups o
only i e indi iduals, which helps o educe he ex en o
in e -indi idual a ia ion. Addi ionally, he me hod elies
on isual obse a ion o h ee hou s o classi y lies as
eede s o non- eede s, which may be ime consuming.
Despi e hese limi a ions, he g oup eeding me hod e-
mains a p ac ical and ep oducible app oach o o al ox-
ici y es ing in E is alinus aeneus. This me hod may also
be applicable o o he Sy phidae species, o e ing a alu-
able ool o u u e eco oxicological s udies. I e ec i ely
add esses he challenges associa ed wi h indi idual eed-
ing, such as low eeding success and signi ican e apo a-
ion and/o c ys alliza ion o he es solu ion.
O al oxici y es ing o dime hoa e
To es he alidi y o he o al oxici y es ing me hod, we
measu ed he oxici y o h ee concen a ions o dime h-
oa e (0.007, 0.0014, and 0.0007 g/L, labelled as D1, D2,
and D3, espec i ely) on E is alinus aeneus using wo
expe imen al se ups (Fig. 4, Suppl. ma e ial 8). Th ee
concen a ions we e chosen as he es aimed o alida e
Figu e 4. Dose- esponse cu es o acu e o al oxici y in E is alinus aeneus-based on mo ali y a e 24 and 48 hou s. Poin s ep e-
sen obse ed mo ali y a each dose le el (µg/ ly)/concen a ion (g/L), wi h poin size indica ing he numbe o lies ha ed. Black
lines show i ed log-logis ic models, and ed poin s indica e es ima ed LD50/LC50 alues wi h ho izon al ed e o ba s ep esen ing
95% con idence in e als.
Bulle in o Insec ology 78 2025, 121–130
bulle ino insec ology.o g
127
he expe imen al design and con i m me hod sensi i i y
using a s anda d posi i e con ol, a he han o pe o m
a ull egula o y LC50 assessmen . In he i s se up, high
mo ali y was obse ed a he highes concen a ion (D1),
eaching 100% a e 48 hou s. Mode a e mo ali y was
eco ded o D2, wi h 60% o lies a ec ed a e 48 hou s,
while D3 caused no mo ali y. Simila mo ali y pa e ns
and inges ed doses we e obse ed in he second se up o
D2 concen a ion. The high success o he eeding ap-
p oach u he alida ed ou me hodology as a s aigh -
o wa d assessmen o o al oxici y in E is alinus aeneus.
Dime hoa e, an o ganophospha e insec icide, is a com-
monly used s anda d in oxici y s udies, and es ima es o
i s oxici y o di e en species a e essen ial o u u e eco-
oxicological esea ch. Despi e he ac ha s anda dized
p ocedu es we e no ollowed in his s udy (mo ali y was
assessed o only h ee concen a ions), and conside ing all
limi a ions o he g oup eeding app oach men ioned abo e,
app oxima e es ima es based on ou da a sugges ha he
acu e o al LC50 o E is alinus aeneus is 0.001659 g/L a
24 h and 0.001366 g/L a 48 h. Co esponding LD50 alues
a e 0.56 μg/g ly (0.024 µg/ ly) a e 24 h, and 0.46 μg/g ly
(0.020 µg/ ly) a e 48 h (Fig. 4). Fo compa ison, he LD50
o acu e o al oxici y o dime hoa e o Apis melli e a ang-
es om 1.1–3.3 μg/g (0.11 o 0.33 µg/bee) a e 24 h, wi h
simila alues epo ed o 48 h (Gough e al. 1994). These
indings indica e ha E. aeneus is sligh ly mo e sensi i e
compa ed o Apis melli e a, exhibi ing app oxima ely wo-
old g ea e sensi i i y. These esul s con as wi h p e ious
s udies on sy phid species (Nagloo e al. 2023; Hen iques
Ma ins e al. 2024), which indica ed ha sy phids a e mo e
obus han A. melli e a. As p e iously men ioned, Nagloo
e al. (2023) assessed o al oxici y o imidaclop id in E is a-
lis enax, while Hen iques Ma ins e al. (2024) ocused on
con ac oxici y o imidaclop id o mula ions in E. aeneus.
Since ou s udy assessed he o al oxici y o dime hoa e in
E. aeneus, di e ences ac oss pes icide classes, exposu e
ypes (o al s. con ac ), o ly species could be he eason
o he obse ed disc epancies in sensi i i y (Nagloo e al.
2024). Fu he esea ch explo ing sensi i i y dis ibu ions
o ho e lies using compa able me hodologies a e u gen ly
needed o be e unde s and he po en ial e ec s o pes i-
cides on his impo an pollina o g oup.
In conclusion, his s udy demons a es ha g oup eed-
ing in E is alinus aeneus is mo e e ec i e han indi idual
eeding, wi h he a i icial lowe design p o iding a eliable
me hod o o al oxici y es ing. The s udy design ocused
on es ablishing a ep oducible and con olled o al exposu e
se up o ho e lies unde labo a o y condi ions. Gi en he
ecological impo ance o ho e lies as pollina o s, u he
esea ch is needed o unde s and hei sensi i i y dis ibu-
ion and exposu e pa hways o pes icides. The p elimina y
oxici y da a o dime hoa e p o ide a e e ence poin o
selec ing i s app op ia e concen a ion as a posi i e con-
ol in u u e s udies. The obse ed highe sensi i i y o
E. aeneus compa ed o Apis melli e a addi ionally unde -
sco es he need o e alua e he e ec s o comme cially
used pes icides on ho e lies and o he wild pollina o s.
Acknowledgemen s
This wo k was suppo ed by he Eu opean Union’s Ho i-
zon Eu ope esea ch and inno a ion p og amme unde
g an ag eemen No. 101135238 (WildPosh), and Min-
is y o Science, Technological De elopmen and Inno-
a ion o he Republic o Se bia (G an s No. 451-03-
137/2025-03/ 200125 & 451-03-136/2025-03/ 200125).
The au ho s would also like o hank he membe s o Wo k
Package 2 o he WildPosh p ojec o hei aluable sug-
ges ions, pa icula ly P o . D . Robe Pax on, du ing he
expe imen al se up and he w i ing o his pape .
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Supplemen a y ma e ial 1
Holding cage
Au ho s: Milica Radenko ić, Jelena Pu ać, Nikola
K i okuća, Danijela Kojić, Dimi y Win e man el, Ju-
lia Os e man, An e Vujić
Da a ype: pd
Explana ion no e: Pupae o E is alinus aeneus in a la ge
pe o a ed (2L) plas ic box, p o ided wi h a 50% w/
suga solu ion (yellow ay) and wa e (blue ay). The
same ype o cage was used as a holding cage o new-
ly eme ging adul s.
Copy igh no ice: This da ase is made a ailable unde
he Open Da abase License (h p://openda acommons.
o g/licenses/odbl/1.0). The Open Da abase License
(ODbL) is a license ag eemen in ended o allow us-
e s o eely sha e, modi y, and use his Da ase while
main aining his same eedom o o he s, p o ided
ha he o iginal sou ce and au ho (s) a e c edi ed.
Link: h ps://doi.o g/10.3897/bull.insec ology.168212.suppl1
Bulle in o Insec ology 78 2025, 121–130
bulle ino insec ology.o g
129
Supplemen a y ma e ial 2
R sc ip
Au ho s: Milica Radenko ić, Jelena Pu ać, Nikola
K i okuća, Danijela Kojić, Dimi y Win e man el, Ju-
lia Os e man, An e Vujić
Da a ype: pd
Explana ion no e: S a is ical analysis.
Copy igh no ice: This da ase is made a ailable unde
he Open Da abase License (h p://openda acommons.
o g/licenses/odbl/1.0). The Open Da abase License
(ODbL) is a license ag eemen in ended o allow us-
e s o eely sha e, modi y, and use his Da ase while
main aining his same eedom o o he s, p o ided
ha he o iginal sou ce and au ho (s) a e c edi ed.
Link: h ps://doi.o g/10.3897/bull.insec ology.168212.suppl2
Supplemen a y ma e ial 3
Dime hoa e ea men solu ions and doses
Au ho s: Milica Radenko ić, Jelena Pu ać, Nikola
K i okuća, Danijela Kojić, Dimi y Win e man el, Ju-
lia Os e man, An e Vujić
Da a ype: pd
Explana ion no e: Concen a ions o ace one-based wo king
and ea men solu ions, and a ge ed inges ed doses o
dime hoa e o acu e o al sensi i i y es ing in E. aeneus.
Copy igh no ice: This da ase is made a ailable unde
he Open Da abase License (h p://openda acommons.
o g/licenses/odbl/1.0). The Open Da abase License
(ODbL) is a license ag eemen in ended o allow us-
e s o eely sha e, modi y, and use his Da ase while
main aining his same eedom o o he s, p o ided
ha he o iginal sou ce and au ho (s) a e c edi ed.
Link: h ps://doi.o g/10.3897/bull.insec ology.168212.suppl3
Supplemen a y ma e ial 4
Ra e o eeding success
Au ho s: Milica Radenko ić, Jelena Pu ać, Nikola
K i okuća, Danijela Kojić, Dimi y Win e man el, Ju-
lia Os e man, An e Vujić
Da a ype: pd
Explana ion no e: Ra e o eeding success in E is alinus
aeneus unde di e en eeding designs in indi idual
and g oup eeding.
Copy igh no ice: This da ase is made a ailable unde
he Open Da abase License (h p://openda acommons.
o g/licenses/odbl/1.0). The Open Da abase License
(ODbL) is a license ag eemen in ended o allow us-
e s o eely sha e, modi y, and use his Da ase while
main aining his same eedom o o he s, p o ided
ha he o iginal sou ce and au ho (s) a e c edi ed.
Link: h ps://doi.o g/10.3897/bull.insec ology.168212.suppl4
Supplemen a y ma e ial 5
Pai wise compa isons o eeding success
Au ho s: Milica Radenko ić, Jelena Pu ać, Nikola
K i okuća, Danijela Kojić, Dimi y Win e man el, Ju-
lia Os e man, An e Vujić
Da a ype: pd
Explana ion no e: Pai wise compa isons o eeding suc-
cess among cage designs unde indi idual eeding
condi ions.
Copy igh no ice: This da ase is made a ailable unde
he Open Da abase License (h p://openda acommons.
o g/licenses/odbl/1.0). The Open Da abase License
(ODbL) is a license ag eemen in ended o allow us-
e s o eely sha e, modi y, and use his Da ase while
main aining his same eedom o o he s, p o ided
ha he o iginal sou ce and au ho (s) a e c edi ed.
Link: h ps://doi.o g/10.3897/bull.insec ology.168212.suppl5
Supplemen a y ma e ial 6
Con ol o e apo a ion o 100 μL
Au ho s: Milica Radenko ić, Jelena Pu ać, Nikola
K i okuća, Danijela Kojić, Dimi y Win e man el, Ju-
lia Os e man, An e Vujić
Da a ype: pd
Explana ion no e: Con ol o e apo a ion o 100 μL o
50% (w/ ) suc ose solu ion o e a 3-hou pe iod.
Copy igh no ice: This da ase is made a ailable unde
he Open Da abase License (h p://openda acommons.
o g/licenses/odbl/1.0). The Open Da abase License
(ODbL) is a license ag eemen in ended o allow us-
e s o eely sha e, modi y, and use his Da ase while
main aining his same eedom o o he s, p o ided
ha he o iginal sou ce and au ho (s) a e c edi ed.
Link: h ps://doi.o g/10.3897/bull.insec ology.168212.suppl6
Supplemen a y ma e ial 7
Con ol o e apo a ion o 20 μL
Au ho s: Milica Radenko ić, Jelena Pu ać, Nikola
K i okuća, Danijela Kojić, Dimi y Win e man el, Ju-
lia Os e man, An e Vujić
Da a ype: pd
Explana ion no e: Con ol o e apo a ion o 20 μL o
50% (w/ ) suc ose solu ion o e a 3-hou pe iod.
Copy igh no ice: This da ase is made a ailable unde
he Open Da abase License (h p://openda acommons.
o g/licenses/odbl/1.0). The Open Da abase License
(ODbL) is a license ag eemen in ended o allow us-
e s o eely sha e, modi y, and use his Da ase while
main aining his same eedom o o he s, p o ided
ha he o iginal sou ce and au ho (s) a e c edi ed.
Link: h ps://doi.o g/10.3897/bull.insec ology.168212.suppl7
