Urban Lasius niger ants more readily accept low concentration sucrose solution than rural ants

Urbani sa tion c au se s bro ad ch ange s in b i otic a nd abio tic fac t or s, whi ch ar e often detr i m ental to a nimals . Whil e exten sive a tte ntion h a s been f ocus se d on f ood re s ource s for polli n ators , c omparativ ely l it t le r e s ea r c h ha s ex amined r e s ou rc e s fo r a phidop hag ou s in s e ct s . Many a nts, such a s Lasius niger , a re highly aphido ph agous , r ely ing on h oneyde w s ecre tion s f or th e majority of their c arbohydra te int ake. An ts wi ll als o of ten reje ct sugar solu tion w hich i s o f lower c o ncentra tion tha n tha t the y we r e expec ting. He re, we a sk w hether Lasius niger an t s from u r ba n e n vi r onme nt s show di ffe re nt i al ac c epta n ce to v ario us s uc r o se solu tion mola riti e s. W e of fe red ou tgoi n g ants o n ac tive forag ing t rail s drop s o f s u cro s e s ol u t i on s ov er a rang e o f mola r i tie s (0.1 25, 0.25 , 0 . 5, 1 , and 2 M ), a nd q ua n t if i e d ac c ept an ce . A c c ept an c e w as s co r e d us i n g a s im p le b eh a v i o ur a l m e as ure o f w het he r t h e a nt r e mai ned in con ta ct with t h e s uc ro s e d r op fo r o ver 3 s ec ond s (ful l ac cep tanc e ), broke awa y from the dr o p within 3 se cond s but r emain ed i n the ar ea and eve ntua lly dr ank to sati ati on (par tial a cc eptanc e ) , o r did not dr ink to s a t i a tion and walk ed aw ay (reje cti on) . Urban ant s showed a sign ific antly high er acc e ptanc e of all s uc r ose molariti e s tha n rural a n ts, w it h th i s di ffe r e nce e s pec i all y pronou nced a t lower mo l ariti e s . Thi s ma y indic ate tha t th ey a r e und er nut rition al str e ss fo r c arbohydra te s. Ant r e spon s e s t o suc ro se solu tion s may allow a s tr a igh tf or w ard m ea sure o f th e a vail ability of ca rbohy dra te r e s ou r c es in the en viro nment .

urba ni s a t i on – c arb ohydra te – nu tr it ion a l lan ds c ap e – an t s .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 22, 2025. ; https://doi.org/10.1101/2025.08.18.670873doi: bioRxiv preprint

Urbani sa tion and la nd -u s e al ter ation s are one o f t he main ca u s e s o f t he cu rren t g lobal bi odiver sity cri sis (Almo nd e t a l. 2022) . Although th e thr eat s to bio diver si t y are diverse (e.g ., oc ea n a lter atio n, t e r re s tr ia l l and u se, in s e c tici d es, inv asive sp ecie s ; (Ha ld -Morten sen 2023 ), the s e thre a ts l argely r e s ult from e nviro nment al cha ng es cau se d by huma n ac t i vitie s. Amo ng t he se, land u s e c hang e rep re sen t s one of t he mo st im portan t drive rs of bi odive rsity lo ss (Oli ve r and M o rec ro ft 2 014), primarily due to th e c ombined e f f ec t s of mul t i p le abio tic and bi o t i c pre s s u re s, s uc h a s habit at l oss, fragmen ta tio n (Zhang e t al. 2024 ) , a nd the in troduc t i on o f inv as i ve sp eci e s (M o llo t et a l. 2017) . Urbaniza tion r epr es ent s on e o f the mo s t ex t reme for m s o f la nd -u s e t r a n sforma tio n, a lthough i ts full i mpact on native bio dive rsity rem a in s insuffic i ently unde r s to od. As a re s ult, u rba n ecol ogic al re sea rch ha s gain ed inc re a s i ng rel ev ance in rece nt yea r s, driven by th e u r g ent n ee d to u nder stand the f unctioning o f th e se ar tifici a l ec o s y st ems and to inf or m con serva tion s t rat egies ai m ed at mi t i gating t he ir wide spr ead e colog ica l co nse que nce s (McIn tyre 2 000) . Urbani s ati on brings a bo ut profo und physica l and e nvironm ental ch an ges — f or ex ample, t h e con ve rs i o n of n atural me adow s i nt o imperme abl e surfac es . Le s s visible , yet equ a lly import a n t, ar e al tera tion s in soil chemi stry, i ncludi ng ele vate d l evel s of h ea vy metal s and othe r tox ic c ompound s (Sw ain 20 24 ). Ur b a nizati on a ls o ind uce s s hif t s in env ir o nmen tal c on dition s, such a s in cre as ed ex po s u re t o a rtifi cial light a t n ight (Hopk in s e t a l. 20 18) and the urba n hea t i s la nd ef fec t (Mac La chla n et al . 2 017). T o gethe r, th e se c hang es h ave trigg er ed a w ide rang e of r esp on s e s w ithin urb an b iotic c ommunit i es , sp anning fr om shi ft s in co mmunity c ompo sitio n and int er a cti on s in bi ologic al ne t w orks ( e .g. (Mil e s et a l . 2019 ; P at t e rs on et a l . 2023; Ma r c ac ci e t al . 202 3; Stuka lyuk and Maá k 202 3; Trigo s -P eral a nd Reye s -L óp ez 2 025) to beh a vioural and phy s iol ogic al a da pta tion s at the indiv idual l eve l (e .g. (Di tchk of f et al . 2006; Mei llèr e et a l. 2015 ; Bra n s e t al . 20 18; Mi nia s 20 23; Toug e ron and S a nder s 2023) . F or ins t a nc e, urba niz a t i on ha s b een linked t o decl ine s in epi phyte div er s i ty (Becke r et al. 20 17), reduc ed th e hea l th a nd per formanc e of bumbl e bee c ol onie s (The odoro u et al. 2022), i n t e n s i fie d the of fspring de fenc e i n urban popu la tion s o f th e Blac k Ki t e Milvus migrans (Kuma r e t al. 20 18 ), red uce d attra ction to l ight i n moths and s hyne ss in spid er s (Al terma tt an d Ebe rt 2016; Cz a czke s e t al. 2018) a nd alter atio ns in a nimals ph en ology like diurn al spi der s sw itching to hunt duri ng t he night fac ilit at ed by the a rtific i al i llumina t io n in c iti e s (Fr ank 20 09) or cha nges in a n t fo r a ging activ it y and c olony d eve lopmen t (Trigo s - P era l et al . 2024a ). While some o rga ni sm s ha ve th e capa city to a djus t the i r beh avi our and phy s i o logy i n re s pon se to u r ba n envi r onm en tal c on dition s (Lunia k 200 4; Cz e chow s k i et al . 2 012; Mc D on n ell a nd Hahs 20 15 ), enab l ing them t o p er s i st in t hes e nov el ec o s y s t ems , oth er s – lik e th o s e with l ow e colog ical pla stici ty – ha ve fac ed mo re s ev ere con seq ue nce s, inc ludi ng t h e loc al ex t i nction o f t h e ir po pulati on s (Ancil lo tto et al . 20 25, Dri et al. 2021, D unc an e t al . 201 1). .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 22, 2025. ; https://doi.org/10.1101/2025.08.18.670873doi: bioRxiv preprint As pr evi ou s ly mention ed , orga nism s re s p ond t o environm e nta l cha nge s in di ver s e way s , bot h i n form and t ime sc ale— ranging f r om r a p id beha vioural a djus tment s that h e lp ove r c ome tempo rary c ondition s to slow er ev oluti onary ada pta tion s tha t per si st a cro ss ge ne rat ion s . B eyond t h e cruc ial role tha t b o t h t y pe s o f r espon se s pla y in orga nism s s urviva l, rapid b ehav ioural re spon se s c an a l so of fe r prac tica l appli c ation s wi t h po ten tial b e n efi ts for hum a n s , namely bioindi ca tion . The us e of bi valv e mo llusc s r e s p on se to d e cre as e s in wa t e r q uality a s an e ff ec t i ve bio moni tor i ng me thod in wa t e r qu ality mo nito r i ng sy stem s g lob ally is a w idely cited e xampl e of a b ioindic a tor ( Ve r e yck en and Ald r id ge 202 3) . Howeve r , aquatic o rgani sm s are n ot th e on ly group capa bl e of s e rving t h is func tion ; soil o r g ani sms c an a l so pr ov ide v aluab l e insight s in to hab i t a t statu s an d qua lity ( Vi a na et al . 2010; Morell i e t al. 2021 ). S hi ft s i n th e func tion a l trai t s o f the ir po pulati on s of ten r efl ec t cha nges in e nvironmen tal condi t i o n s , o wing t o t h e strong a ssoci a tion be twe en speci e s ’ e colo g ical r e quirem ent s a nd habi ta t char ac t e r i stic s. T hi s i s p a r tic ularly s ig ni fica nt for o r g ani sms w ith limi t ed disp ersal c apac ity, w hich tend t o be h ighly s p e cial i z ed t o loca l cond i t i on s and , c on s eq u entl y, more sen sitive t o e nvironmen tal cha nge s— ma king t h e m e ff ectiv e biol ogica l indi c ator s (Mc Ge och 1 99 8). Althou gh all o rgani sms re spond t o chang es in e nv ir o nmen tal c onditi on s, th ose co nsid ered suit abl e a s bioi ndi cato r s must mee t a s p ecific s et o f crit eria . They sh ould be wi de ly dis trib uted , h ighly a bundan t, a nd r elative ly e a s y t o sa mple. In additi on, t h e y shoul d pla y a key functi onal role in e cosy ste ms, e xhibi t h igh sen si tivity to e n vironmental cha ng es , and d i s pla y c on s i st ent re sp on s e s th at c an be reliabl y in terp re ted ( McGe oc h 1998; Ander sen 1999). Th roug h the c a tegor ization in func tion al g r ou ps (And er sen 199 5; Su sil o e t al . 200 9) or direc tly us i ng t he spec i fic e colog y of the sp ec ie s, bi oindic ato rs provi de a v aluab l e inf ormat ion a bout the c har acte ri stic s and c on se r v ation st atu s o f t he ha bita t s . For ex ampl e, t h e occ urr e nc e of d esert a nt s indic a te s a x ero t he r mi c ha bi tat (de Pl etinc x e t a l. 202 4), t he pr ese nce o f odon atan s indi cate s pre se nce o f wate r bodie s n e arby ( Carc hini e t a l. 2007 ), the exi ste nce of fe rn s i n an a rea i s an in d ication o f high moisture a nd mild t e mp era tures (D ella 2022) , a nd dif fer ent sp ecie s of l ea f litt er fungi a re a s s oc iat ed t o sp ec ific s uc ce s s i on s ta g e of w oodland s (Pr ak a sh et al . 2015) . Inde ed, bioi n dica t o rs hav e b een u s e d widel y in e colog ica l st udi e s to e valu a t e c hang es in the env ir onment al c harac t e r i stic s o f th e e co sys t e m s or ev e n the p re se nc e of poll u t a n ts (Via na e t a l. 2010; Gha nnem e t a l. 2024 ) . Amon g all bioindic at or organi sm s, an t s a re wi dely reg ard ed a s one o f th e mo st va lua ble an d rel iabl e. Their se nsi t i vity to e n vironmen t al disturbanc e s, combi ned wi t h t he ir ea s e of samplin g and tax onomic re s ol u t i on, mak e s the m par tic ula r l y ef fec t i ve in dicato r s of h abita t qu ali ty a nd ec o s y stem c hang e (U n d er w ood an d Fi sh er 2006 ). In broad te rm s, ant sp eci e s a re tr a dition all y cla s si fie d i nto two ma in bioi ndica t o r g r ou p s ba s ed on thei r nic he pre fer enc e and tole r a nce to v aryin g environme nt a l .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 22, 2025. ; https://doi.org/10.1101/2025.08.18.670873doi: bioRxiv preprint c ondition s: ge n erali s ts, w hich exhibi t br o a d ec ologic al req uireme nts an d pla s ticity , and sp ec iali st s , w hich have narrow ec ologi cal niche s (An der sen 1999 ; Roig a n d Espa dal e r 2010; V a s c onc elo s et a l . 2 018; Trig os-P eral e t al. 2 020 ). Whil e t hi s c las s i f i c ation i s e ffe ctive f o r a ss es s ing ab iotic cha r a ct eri stic s of habi tat s, it prove s le s s e f ficien t i n det e ctin g biotic cha nge s, such a s shif t s i n the av ailab ility a nd qu ality of nu t ri tiona l r e s o ur c es. T he av ailab ility o f a bundan t, hi gh -quali ty food i s cruc ial f or orga ni s m s w it h limit ed ne st mo bility, suc h a s an t s . A s c a rcity of e ss en tial nut r i en ts w it hin thei r ne sti ng n iche can s ig ni fica nt l y i mpact a nt popu latio ns , a s a nut r ie n t - ric h diet i s vi t a l f or col ony dev elopm en t an d the ov era ll ph ysiologic al st atu s o f ant s an d co lonie s (Cs a t a and Du s s utou r 20 19). F or a phidic o lous an t s p ecie s li k e Lasius niger , ap hi d honey dew con sti tute s a key compone n t of t h eir di et . Thi s suga r y s e cr etion of fer s a c omplex mixture o f micronu trien ts impo rtan t to the an t s (Shik e t al. 2014 ) . As a re sul t, ant s a ctive ly sel ec t th e mo st nu t riti ou s hone y dew ava ilable t o t h em. H owev er, food so urce s elec t ion i s no t de ter m in ed s olely by nu t ri tiona l con t ent; p rev iou s f eeding expe r ie nce al so pla ys a r ol e (We ndt a nd Czaczke s 201 7; Wend t et al . 2019). :A nts acc ustome d t o r i che r foo d so urce s m a y ex hibit reduc ed a cc eptanc e of al ter nativ e s w ith co mpa ra tively low er nutr i tion a l qual ity, a s r e sult of a ne ga tive i ncentiv e co ntra st s (F lahe rty 19 96). A s s u c h, the w illing ne ss of a p hidico lou s to acc ept low molari ty suc r o s e c oncen trati on s s hould be a di rec t functi on of the s ug ar conc entra tio n or gene r a l qu ali t y of the hon eyde w they have be en c ollec ting, a nd thi s in turn may r e flec t th e produc t i vity – and thu s st re ss lev el s – o f th e pl an t the ap hi d s a r e feeding on . T his s tudy aim s to inve s tig a t e p oten tial s hift s in aphi d h oney dew qua li ty be t w e en urban and rural habi ta ts by u sing t he aphidi colou s a nt Lasius niger a s a mo d el s peci e s. Spec if ical ly, w e seek t o a sse s s w he the r L. niger c an se r v e a s a bi oindic ato r of the nu triti onal q uali ty o f av ai lable f o od s ou r c e s by ex amining differ ence s i n die tary p ref e renc e s by c omparing t h e ac cept anc e o f s ucros e s oluti on s of v arying conc entra tion s be tween an t pop ula t i on s orig in ating from hi ghly ur b ani sa tion or low urba ni s a t i on envi r on m ent s.

Species and Localities T o as s e s s whe ther a nts c an serve a s r elia ble bio indica t o rs o f fo od qua lity i n huma n-modi f i ed ha bita t s , we u sed th e gener al i s t Hola rc t i c ant sp ec ie s Lasius niger a s m odel speci es. This a phidic olou s s pe cie s r elie s on ho neyde w produc ed by aphid s a s it s main sou rce o f carbohy dr a te s (D ev igne a nd De tra in 2005 ; N o vgorod ova 2015). L. niger employ s m ass r ecruitm e nt to food s ourc e s , so w or k e r s form st able for ag ing trail s f ro m the ne st e ntr ance to the ex ploi ted sou rce (D ev igne and .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 22, 2025. ; https://doi.org/10.1101/2025.08.18.670873doi: bioRxiv preprint Det rain 2005; N ovg or od ova 20 15) and fo rag ing trail s c an r eac h up to 2. 9 m long ( Stukal yuk & Ak hmedov 2022), ma king t h em par ticul a rly ea s y to find . More ov er , th e ec ologic a l pla stic ity of Lasius niger en ab le s t h e m to col onize a w ide ra nge of habi ta t ty pe s ( S eif ert 2007; Cz ec h owski et al . 20 12; Ra dchen ko 201 6) , inc ludi ng h ighly modified habit at s s uc h a s urban a r ea s (S t u kaly uk and Maák 2023). I n s uc h ur ban h abi tat s, t he s p e cie s ex hibi ts a ma rke d domina nc e (T r ig o s-P eral e t a l. 2020), repr es enti ng nearly 5 0% of th e ob s e r v e d ant w or k er s (S t uk alyu k et al . 2020). T he stud y wa s p e r fo rmed in two ha b ita t type s dif feri ng i n the deg re e o f urb aniza t ion: urba n a nd rural a rea s . Urban a r e a s wer e c hara cteriz ed by green spac e s emb edde d wit h in t he city sca pe, i ncludi ng co ur tya r d s w ith pav ed road s, ti led pa vemen ts , and lawn s w ith pre se nce of a f ew t re e s; w herea s rur al are a s w ere d efin ed a s ar e as o f pa stu r e s , h e r b s, o r sp ot s surro unde d by orchard s. Bo th bi omes we re sampl ed ac r o s s five d i ffe re n t c itie s withi n t h e Kyiv distr ic t of Uk rain e: Bu cha, B oyarka , I r pin , G o s tom el, and Vi shnev e . The GPS coodi nat e s of al l sampl ed loc a tion s can be found i n th e raw da ta ( S1) Procedure An activ e Lasius niger fo r a ging trail wa s l oc ated by s e a r c hing t re e t ru nk s for a n ts shu tt li ng bac k a nd for t h . Sp ec ie s iden tity wa s confi r me d by ex amini ng sample s of a nts f rom eac h foragi ng t r a il un der a ste r e o microsc ope , u sing ide n t i f i cation keys in Ra dche nko (2016 ) and S eif ert (2007 ). T o pe r f orm th e behavi ou r a l a s s a y, a s ma ll a c etat e shee t (c . 10x10m m) was pla ced d i r e ctly o nto t he tr a il, a nd a drop o f suc r o se solu tion wa s plac e d on t he s he et . We t e s t ed t he followi ng s ucros e molari tie s: 0 .125, 0. 25, 0.5, 1, a nd 2M, with a ra ndo mised pr e sen tati on ord er. Only one dro p of one solu tion w as pre se nte d at a time . Af t e r 2 0 an t s e ncou nt e r ed th e s o luti on, th e a ce tat e sh e et w a s r em oved a nd a new s uc ro se s oluti on dr op o f a dif f eren t mol arity wa s pre s ent ed. T he ex pe rimen ter wa s bl ind to solu tion t y pe , al t h ou gh hig h molarity ( 1 M , 2M) s uc ro se is v i scou s and thu s c an b e d if f e re nt i ate d fr om the oth er mola riti e s eve n when unlab e ll ed. Th e r espo n s e o f outgoi ng ant s (h e adi ng tow ard s t he t re e ) w hen en count ering t h e sol ut i o n was no t ed. We r e cord ed on e of t hre e re spo nse s, foll owing W e nd t et a l. (2019 ): • F ull a cce ptanc e (sc ored a s 1): The a nt ma intai n s i ts mand ibl e s in c ontac t w it h the sucro se drop f o r 3 sec ond s upon fir st con tac t wit h the drop . • P artia l ac cep tan ce (sc ored a s 0 .5): The a nt break s co n t a ct wi t h t he dro p within 3 secon ds o f i nitial c ont act, b u t r em ain s in the v icini t y (c. 2 cm radi u s ) o f th e dr op, re t u rn s to i t freq uen tly , or e ventu all y drinks to sati ation . .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 22, 2025. ; https://doi.org/10.1101/2025.08.18.670873doi: bioRxiv preprint • Re jection (s c or e d a s 0 ): The an t bre aks c ont a ct w it h t h e dr op withi n 3 s econd s, a nd continu e s on it s ou t w ard s journ ey. S olution s ( all c onc en tr a tio n s ) w e r e o ff er ed in pa ttern ed manne r, so tha t al l s ol u t i ons wer e te s t e d a t eac h t e s ting bou t. All ex perimen t s wer e c onduc t e d in the shad e , u s i ng a para so l to provide s ha d e i f nece ssa ry, an d all da ta we r e coll e cte d be t w ee n 9:0 0 and 1 2:00, b e t w ee n the 1 4 th and 27 th Augu st 2 024. In additi on, th e ambien t a ir te mpe rature , ambi e nt humidi ty, t re e t ru nk dia meter , a nd an t trail tra ffic wa s mea s ur ed f or ea ch c olo ny. An t t ra f fic was me a s u red by c ounting al l a n ts p as s ing by a po int on th e tr ail fo r 2 minute s . We studi ed 9 urban c oloni e s and 9 rur al c olonie s fr om eac h munic ipal i t y , te stin g 20 a nts from e ach col ony on ea ch mol ari t y , re sul t i ng in a tot al of 90 00 an t s te sted . Statistical analysis All code u s e d in th e a naly si s, and t he cod e outpu t, i s provided in onli ne s up plem e nt S 1. We analy s ed t he da ta u sing Linea r Mixed -Ef fec t Model s, wit h the glmmTMB p ack a ge (Mag nu sson et a l. 202 0) in R (R Cor e Team 2 023) vi a R studi o (RS tudio T eam 20 15) . Acc ept anc e r es pons es w er e pred ict ed by an i nt e r a ctio n of food conc entra t i on a nd bi ome, w ith the addi t io n o f temp era ture and hu midity a s i nd epend ent v ari able s . Col o ny ID w as ad ded a s a ra ndom e ff ec t. O ur m ain an alysi s mo del fo rmula was : Acc eptanc e sco re (1 , 0 .5, 0) ~ fo od con ce ntra tion * biom e + r a nd om ef fec t ( c olon y ID) . Dif fe renc e s in sucro se ac cept anc e be twe en habi ta t t y pe s we re t e sted u sing a bin omia l model. I n a ddi tion, we ex plored t h e e f fec t o f e nv ironmental f acto rs (h umi dity and t e mperatu re ) a n d t re e di amete r, on suc r o s e ac ce pta nce, exami ning the t hr ee -w ay i nt e r a ctio ns wit h hu midity a nd us ing the mod el f or m ula: Ac cepta nc e_ scor e ~ s uc ro s e _m olari ty * Bi ot o p e * Humidity + Temp e r a ture + t re e_ diam ete r + rand om e ff ect (c ol onyI D) And exam ining the t h re e - w ay int er a c tion s with tem pera ture and u sing the mod el f o r mul a : Ac cepta nc e_ scor e ~ s uc ro s e _m olari ty * Bi ot o p e * Tem pe ratu r e + Humidi ty + t re e_ diam ete r + rand om e ff ect (c ol onyI D) .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 22, 2025. ; https://doi.org/10.1101/2025.08.18.670873doi: bioRxiv preprint F inall y, we expl or e d the e f fect o f tr ee c h arac te r i stic s on tr af fic. For t his, we ex clu ded data from thr ee tre e sp eci e s w hich w ere only sampl e d o nce, l eavin g three t re e spec ie s. We u s e d the follow ing GLM w ith a g aus s ia n di s t ribu tion: a nt_ t ra f fic ~ tr ee _di ame ter * tr ee_ sp ec i es P valu e s w ere lat er co rrec ted f or mul tip l e t e s ting u s in g a B enja mini- Hochbe r g co rrecti on (Benj amini an d H o ch be r g 1 9 95) . I nt e r a c t i o ns wer e a n a l ys e d b y s u bse tt i n g : f or ins ta n ce , i f th e i nt e r a ct i o n f oo d c once nt r ation * biome w a s s ig nifica n t , tw o s ub se t s were mad e , e ac h contai ni ng just on e biom e, and the a n alysi s we re r er u n o nly inc luding f ood c oncen t ra tion mea s u re . Fo r gr aph ic al v is u aliz ation , raw da ta figure s w e r e c rea ted u s ing ggpl o t 2 (W i ckha m et al. 2020 ), w hile mod el predi c tion s we r e v isualize d wi t h ggef fec t (Lüdec ke 2018) .

T he co mplete r aw data on whic h th e ana lysis wa s ba s ed is p rovid ed in on lin e s up pleme nt S1 . Th e c omplete an aly s i s doc ume nt, wi t h al l c ode a nd ana ly si s re sul t s , i s pr o vided in onl ine s up p lemen t S2 . We f o u nd a signifi can t int erac ti on be twe en sucr o se conc en tra tion and h abita t typ e on ac cept an ce sco re s (Χ 2 = 2 2.9 P < 0 .0001 ) . O v erall, a cce ptanc e s c o r e s i ncre a sed w it h s uc ro se c onc entra t io n (Χ 2 = 1 649, P < 0.000 1) a nd a nts from urb an h a bita ts e xhibited high er l evel s o f s ucro se ac ceptan ce c ompared to the rural coun terp ar ts (Χ 2 = 16.27 , P < 0.0001 ). Mo reov e r, th e inc r e a s e in ac ce p t a nc e w ith ris i ng sucro s e con cent rat ion w as m or e pron ounce d in urb an popula tion s t h an the r u ral on es. At the highe s t conc ent rati on te s t e d (2M) , a c ceptanc e le ve l c onverge d with n o s ig ni fi ca nt dif fe rence be t w ee n u r ba n an d rural a nt popul ation s (s ee figu r e 1) . .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 22, 2025. ; https://doi.org/10.1101/2025.08.18.670873doi: bioRxiv preprint Figure 1 ) Mean acceptance scores of ants from rural or urban areas in response to a range of sucrose solution molarities Symbols are means, whiskers are bootstrapped 95% confidence intervals. H u m i di t y h a d n o ef fe c t o n s u cr os e ac ce pt a n c e, e i t he r a l one o r i n a t hr ee- w a y i n ter ac t i o n bet we en hu midity, suc ro se c oncen tra tion, a nd bio tope (H umi dity: Χ 2 = 0.007, P = 0.93 , th r e e-way int eracti on: Χ 2 = 0.068, P = 0 .79) . Howev er, whi l e te mperatu re ha d n o predic t i ve pow er on s ucros e ac cept anc e a lone (Χ 2 = 1.51, P = 0.22 ), it did ha ve in fl u ence on how ant s i n di f fer ent biom es re spond di ffe re nt i ally to dif fe r e nt s uc ro s e m olari t ies (t h ree - w ay inte r a cti on, Χ 2 = 8.38, P < 0. 0038). E xploring the thr ee -way inte rac t io n by e x amini ng the predi cti on s fo r t h e minimu m, mean, and ma ximum t e mperatu re s ( fig ure 2), we s e e tha t in urban envir onme nts a t th e l owe st a nd hi ghe st temp era t u r e s, sucro s e ac cept an ce i s red uc ed c ompared to at me an temp era tur e s, e sp ecia lly f o r hi gh suc r o s e le vel s. .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 22, 2025. ; https://doi.org/10.1101/2025.08.18.670873doi: bioRxiv preprint Figure 2 ) P re dic te d acc ep tan ce sc ore s of an ts fr om ru ra l o r urb an ar eas i n res pon se to a ra ng e of su cro s e so lut io n m ol ari ti es , a t the m ini mum , max imu m, an d mea n te mpe ra tur e s . L ine s are g lmm m od el pr edi ct ion s, ri bbo ns are th e 95% c onf id en ce in ter v a l o f e ac h p re dic ti on . We fo und a po sitive c o rr e l ation b etw e en tree di a met er s ize a n d ant t ra ffic (Χ 2 = 8.6, P = 0.00 33), signific a nt di ff erenc e s i n tr a ffic betwe e n dif fer ent tre e speci e s (Χ 2 = 7.45, P = 0.0 24 ), and a non-signi fic ant in ter ac tion b etwe en sp ec ie s a n d diame ter (Χ 2 = 5.1, P = 0.078 ), in whi ch inc reasi ng dia mete r stro ngly increa s e s t raf fic on Acer platanoides but only s li ghtly i ncre a se s t ra ffic on Populus nigra an d Tilia cordata ( s e e figu re 3). F igure 3 s h ow s the int erac tion – a plo t sho wi ng the l inear re spon s e with out in ter ac t i on ca n b e fou nd in the c ode an d analy s i s sup pleme n t . .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 22, 2025. ; https://doi.org/10.1101/2025.08.18.670873doi: bioRxiv preprint Figure 3 ) R el ati on sh ip be twe e n tr ee di ame te r a nd a nt tr aff ic . Traf fi c inc rea se s o n lar ge r tre es , e s p eci all y in No rwa y Ma ple ( Acer palatanoides ). Lin es ar e l in ear r eg res si ons , dot s a re in di vid ua l val ue s. .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 22, 2025. ; https://doi.org/10.1101/2025.08.18.670873doi: bioRxiv preprint

T he swee te r a suc r o se solu tion i s , t h e more lik ely a nt s a re t o acc ept i t, wi t h ant s g enerall y reje cting low-mol a r ity s uc ro se s olution s ( Wendt et al . 2019). H ow eve r, w e de tec t e d a ma r k ed di ffe re nce i n th e re s p on se t o low -mola r i t y s uc ro s e solu tion s be tw een ur ban and r ural popu la tion s: Urban a n ts ex hibited a grea ter ac cep t a nc e for the se l ow er -quali ty re sourc es . S ugary s ub st ance s a re par t i cula r ly at tra ctive to many ant sp ecie s, e sp ec iall y aphi dicolo us spec i e s s uc h a s Lasius niger , a s t hey c on stitu te th ei r pr im a r y ca r bo hydra te— and t hus en ergy —sourc e (Wil son a nd Ei sn er 1957; Markin 19 70 ). Under na tural c on dition s , ant s prima rily obtain c arbohydra te s f rom aphi d hon ey dew, th e composi tion o f w hic h varie s signi fic an tl y dependin g on fa cto r s such a s s oil ch em istry, pl an t physi olog ical stat e, and ap h id sp ecie s ( Völk l e t al. 1999). One e s p ec iall y att ractiv e com pon ent o f h oney dew f o r a nts i s mel icito s e ( Corn eliu s et al . 1 996; Det rain a nd Pri eur 2014 ). The c onc en t r ation o f melic it o s e in hon ey dew i s se nsit ive t o e nvironmen tal f actor s a f fecting t h e ho s t plant's phy siolog ical condi t i on, of ten inc re a s i ng unde r c ondition s o f the rmal or hydric st re ss (S e e bur g er et al . 2022). Give n the se f actor s, it is plau sibl e t h a t ho neydew produc e d by sap - fee di ng ins e c t s in ur b a n envi r on m ent s c on tain s elev a ted le vel s o f me lici t o se c ompar ed to tha t fr om rural a r e a s . Thi s i s like ly due to the fac t th a t urb a n veg eta tion i s oft en s ubje ct t o grea ter thermal stre ss, d riv en by eleva t e d ambie nt t e mpe rat ur e s (Zh ou et al . , 2017), a nd reduc e d wa t e r a vail ability, a s impe rviou s sur fac es re stric t s oil inf iltr ation a nd promot e s u r f ac e runof f. T h en, an ts - kno wn fo r thei r s en s i t i vity to s ubtl e c hange s in the nu tri tional c omposi tion o f food (Cs a ta e t al. 2 020) – s h ould be lik el y t o detec t an d re s p ond to cha nge s in th e c once nt r atio n of c arbohydra te s in t h e honeyd ew . Ind e ed, Wendt an d Cza czke s (2017) d e mon s tr at e d that w orke r ant s w ere wi lling t o t r av el grea t e r d i stanc e s—thu s exp ending mor e energy and in cr ea s ing thei r ri sk of mo r tal i t y — w hen th e suga r conc en tr a t io n of a f ood s ource wa s hig he r. Fur the rmore, an t s ar e c apable of lea rning and f orming expec ta tion s abo ut foo d qu ality ba sed on prio r exp erie nce . For ex ampl e, Wend t e t al . (201 9) found that ant s ac cu sto med to hig h -suga r di et s w er e le s s incl ined to a cce p t fo od sou rce s w ith lowe r suga r conc entra t i on s. Ba sed on all ab ove info rmatio n, i t c ould be exp ected tha t urba n an t s will s how r e duced a cc eptanc e of low - suga r c once n t ra tion s. Cont rary to thi s ex pecta tio n , but in li ne wi t h pr e viou s pu blish ed work s, ou r st udy foun d t h at u rb an popul a tion s of L. niger we re mor e wi ll ing to a cce pt low c once ntratio n s o f suga r th a n th eir rura l cou nt e r pa rt s, s u gge sting a lowe r qu ality o f h oneyde w in urba n envi r onm e nts . A s an e xampl e o f t he nutri tional con s tr a in t s of u rba n a rea s, Garbe r et a l. (2024 ) repo rt tha t urb an L. niger colo n ie s de fen ded aphid c lu ste rs more fierc ely tha n t he ir counte rpa rt s in rural are a s, s ug ge st i ng a po t e n t i ally grea ter r elian ce on t hi s re so urce due to inc r e ase d fo od sca rc it y i n citie s . In lin e w ith th es e fin ding s, P enic k et al. (2015 ) al s o f oun d th at an t s from u r ba n ar ea s f ed on .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 22, 2025. ; https://doi.org/10.1101/2025.08.18.670873doi: bioRxiv preprint hu man wa ste, whic h is an ab undan t, e ne rgy -r i ch, a nd ac ce s s i bl e die t a r y alt erna tive in urban e nvironmen ts . . I n additi on t o gen eral di ff ere nce s in re so urce acc ept anc e, ru r a l popul a tion s o f Lasius niger e xhibited a mor e prono unc ed i ncr ea se in i nt e re st in suga r y sol ution s a s conc entra tio n leve l s r o s e. Pr e vi o us s t u di e s h av e s h o w n t h at u r ba n p l an t s a re m o r e s us c ept ib l e t o a p hi d i nf es ta t i o ns t h an t he i r rural coun terp art s (L ubiarz e t al. 2011; M ac kos -Iwa sz k o e t al . 2015 ), pote nti ally pro viding ants with a g r e at er abund an ce o f h oneyde w . Howev er, th e nut rition al quality o f t h i s h on eyde w may be c ompromis ed by t h e phy siolog ica l s t re s s e xperienc e d by urban pla n t s . Ou r finding s suppor t thi s i nt e r p re tati on: ur b a n ant s s ho wed a con si ste nt in tere s t in al tern ative suga r s ou rc e s r eg ardl e s s of c once ntratio n , indic ating a lowe r b a s e lin e qua lity o f na tu r a lly a vail able c a rbohydr ate s in u r b a n e nvironmen ts . Co nsi sten t w ith th e ob ser vation s of W end t et a l . (2019 ), t h i s pa t t e rn sugg e sts tha t the mo r e sel ec tive re s pon s e of rural an t s a n d their sha rp inc r e a se in sug ar c on s umpti on only at c once ntratio n s a b ove 0. 5 M might b e lin k ed to th eir ac ce s s to high e r-qua li ty honey dew. Simil arly, T r i gos-P e r a l e t al. (2024a) foun d tha t urb an ant p opul ation s o f Lasius niger in War s a w, P ol and, c onsumed mo re c arb ohydrat e -ric h ( s ug a r- w at er ) sol ut i o ns a nd le s s pr o t e in t han t heir rur al c ounter par ts. T he s e ha bita t -rela ted di et ary shi ft s w er e inte rpr ete d a s ev idence o f low er die tary qu ality i n urban e nv ironmen t s and w ere ac compan ied by physiol ogi cal dif fer ence s, w ith ur ban gy ne s e xhibited lowe r bo dy fa t c onte nt. T aken t o get her , the r e sult s r epor ted h e re and el sew he re suppo rt th e po tenti al of a nts a s bi oindic ato rs of di eta r y shi fts and enviro nme nt a l qual it y . A s pre viou sly n ot e d , ant s ar e wi dely rec ognized for their a ccu rac y a nd relia bi l ity as ind ica tor s o f envi r onm e ntal c on diti ons, owing to thei r hi gh sen si tivity to h abi tat al te rati on s and ecolo gica l dis tur b a nce s (A nder s en 1997 ). The findi ng s o f this s tudy ex tend the k nown s en sitivity o f ant s a s a bioi nd icato r group, d emon st r a ting their p ote ntial to d ete ct no t only struc tural o r abiotic ch an ges in the e nvir onment (Trigo s-Pe ral e t al. 20 24b) bu t a ls o s hi ft s in t he n utritio na l land sca p e. T hi s bro ad en s the ir a pplic abili t y ac ros s a wid er r a nge o f e colog ical re s e a r c h. F u r th ermor e, th e method ology employ ed in t hi s study i s low -co st, ea sy to i mplement, r api d, an d yields relia bl e re s ults w it hin a shor t time fram e, making it a prac tica l t ool f or mo nitor i ng env iro nment al qua lity, e ith er by trained s pec iali st s or citiz en scie nt i s t s. .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 22, 2025. ; https://doi.org/10.1101/2025.08.18.670873doi: bioRxiv preprint

T J C was sup por t e d by a Sta rte r G r ant fro m the E ur o pe an Re se arc h Counc il ( C ogni t iv eC on trol : 9 48181) a nd a Hei sen berg gran t fro m th e Deu t s c he Fo rschun g s g em ein scha ft ( DF G, G erman Re sea r c h F oundati on) — P roje ktnumme r 4621 01190.

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