Effects ofWolbachiaon Transposable Element Expression Vary BetweenDrosophila melanogasterHost Genotypes

GBE E ec s o Wolbachia on T ansposable Elemen Exp ession Va y Be ween D osophila melanogas e Hos Geno ypes Ana T. Eugénio 1 , Ma a S.P. Ma ial a 1 , and Pa ícia Beldade 1,2, * 1 Ins i u o Gulbenkian de Ciência, Oei as, Po ugal 2 cE3c (Cen e o Ecology, E olu ion and En i onmen al Changes) and CHANGE (Global Change and Sus ainabili y Ins i u e), Facul y o Sciences, Uni e si y o Lisbon, Lisbon, Po ugal *Co esponding au ho : E-mail: [email p o ec ed]. Accep ed: 19 Feb ua y 2023 Abs ac T ansposable elemen s (TEs) a e epe i i e DNA sequences capable o changing posi ion in hos genomes, he eby causing mu a ions. TE inse ions ypically ha e dele e ious e ec s bu hey can also be bene icial. Inc easing e idence o he con i- bu ion o TEs o adap i e e olu ion u he aises in e es in unde s anding wha ac o s impac TE ac i i y. Based on p e ious s udies associa ing he bac e ial endosymbion Wolbachia wi h changes in he abundance o piRNAs, a mechanism o TE ep ession, and o ansposi ion o speci ic TEs, we hypo hesized ha Wolbachia in ec ion would in e e e wi h TE ac i i y. We es ed his hypo hesis by s udying he exp ession o 14 TEs in a panel o 25 D osophila melanogas e hos geno ypes, na u ally in ec ed wi h Wolbachia and anno a ed o TE inse ions. The hos geno ypes di e ed signi ican ly in Wolbachia i e s inside indi idual lies, wi h b oad-sense he i abili y a ound 20%, and in he numbe o TE inse ions, which depended g ea ly on TE iden i y. By emo ing Wolbachia om he a ge hos geno ypes, we gene a ed a panel o 25 pai s o Wolbachia-posi i e and Wolbachia-nega i e lines in which we quan i ied ansc ip ion le els o ou a ge TEs. We ound a ia ion in TE exp ession ha was dependen on Wolbachia s a us, TE iden i y, and hos geno ype. Compa ing be ween pai s o Wolbachia-posi i e and Wolbachia-nega i e lies, we ound ha Wolbachia emo al a ec ed TE exp ession in 21.1% o he TE-geno ype combina ions es ed, wi h up o 2.3 imes di e ences in he median le el o ansc ip . Ou da a show ha Wolbachia can impac TE ac i i y in hos genomes, unde sco ing he impo ance his endosymbion can ha e in he gene a ion o gene ic no el y in hos s. Key wo ds: ansposable elemen ac i i y, Wolbachia in ec ion, D osophila melanogas e , DGRP, gene ic a ia ion. Signi icance Mobiliza ion o ansposable elemen s (TEs) gene a es mu a ions ha can con ibu e o adap i e e olu ion, making i all he mo e ele an o unde s and wha ac o s a ec TE ac i i y. We show ha in ec ion wi h a common endosymbio ic bac e ium, Wolbachia, a ec s TE ac i i y in D osophila melanogas e hos s, in a manne ha a ies depending on TE iden i y and on hos geno ype. © The Au ho (s) 2023. Published by Ox o d Uni e si y P ess on behal o Socie y o Molecula Biology and E olu ion. This is an Open Access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion-NonComme cial License (h ps://c ea i ecommons.o g/licenses/by-nc/4.0/), which pe mi s non-comme cial e-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. Fo comme cial e-use, please con ac [email p o ec ed] Genome Biol. E ol. 15(3) h ps://doi.o g/10.1093/gbe/e ad036 Ad ance Access publica ion 28 Feb ua y 2023 1 Downloaded om h ps://academic.oup.com/gbe/a icle/15/3/e ad036/7059559 by Uni e sidade de Lisboa use on 03 July 2023 Eugénio e al. GBE In oduc ion T ansposable elemen s (TEs) a e epe i i e DNA sequences capable o changing posi ion independen ly in he hos genome (Bou que e al. 2018; Mé el e al. 2020), and make up a signi ican ac ion o many euka yo ic genomes (Guio and González 2019). They a e di ided in o wo majo classes, depending on whe he he mechanism o anspos- i ion does ( o e o ansposons) o does no ( o DNA ans- posons) in ol e an RNA in e media e ha is e e se ansc ibed be o e in eg a ing back in o he hos genome (Bou que e al. 2018). TE inse ions can cause mu a ions, which ypically ha e dele e ious e ec s because hey dis- up p ope gene unc ion in a a ie y o manne s (McFaddena and Knowlesb 1997; Hedges and Deininge 2007; Belancio e al. 2008; Aya padikannan and Kim 2014). Consequen ly, hos o ganisms ha e e ol ed me- chanisms o con ol and ep ess TE ac i i y, including he piRNA pa hway in animals (Tó h e al. 2016). On he o he hand, an inc easing numbe o s udies ha e been p o iding compelling examples o TE inse ions wi h posi i e e ec s on hos i ness, con ibu ing o adap a ion (González and Pe o 2009; González e al. 2010; an’ Ho e al. 2016), s ess esis ance (Guio e al. 2014; Pe ei a and Ryan 2019), and he o igin o no el ai s (Eme a and Wagne 2012; Benne zen and Wang 2014; San os e al. 2014; T izzino e al. 2017). Mo eo e , TEs migh also con ibu e o ep oduc i e isola ion, as in he case o TE-media ed hy- b id incompa ibili y (Pe o e al. 1995; Se a o-Capuchina and Ma u e 2018). TE con ibu ion o adap i e e olu ion and di e si ica ion aises in e es in unde s anding wha ac o s impac TE ac i i y. TE ac i i y di e s be ween TEs (Venne e al. 2009; Mé el e al. 2020) and be ween hos geno ypes (Ande son e al. 2019; Signo 2020; Wang e al. 2022). Fu he mo e, s ud- ies on di e en o ganisms ha e shown ha TE ac i i y can be a ec ed by ex e nal en i onmen al ac o s, including empe a u e (Chen e al. 2018), adia ion (Newman e al. 2014), hea y me als (Habibi e al. 2014), s a a ion (Rep e al. 2005), and a ious o he s esso s (Miousse e al. 2015). No much is known abou how hese ac o s can a - ec he molecula mechanisms esponsible o TE egula- ion, including he piRNA pa hway. On he o he hand, Wolbachia, a common endosymbio ic bac e ium, has been shown o a ec he abundance o some piRNAs (in Aedes aegyp i mosqui oes; Mayo al e al. 2014) and he a e o ansposi ion o he e o ansposon gypsy (in D osophila melanogas e ; Tou e e al. 2014). Mo eo e , he in asion o he DNA ansposon P-elemen in popula- ions o D osophila epo edly co-occu ed wi h a eplace- men o Wolbachia s ain in ec ing hose lies (Riegle e al. 2005). Howe e , he e has been no sys ema ic ana- lysis o he e ec s o Wolbachia on he ac i i y o di e en TEs in di e en hos geno ypes. Wolbachia is a ma e nally inhe i ed endosymbion ha is p e alen in in e eb a es, including insec s, a achnids, and nema odes (We en e al. 2008; Kau e al. 2021). Mul iple s udies ha e documen ed Wolbachia p e alence (Cla k e al. 2005; Riegle e al. 2005; Weeks e al. 2007) and load (López-Mad igal and Dua e 2019; Liu and Li 2021) in na u al and labo a o y popula ions o D osophila hos s. Associa ed wi h i s mode o ansmission, Wolbachia can ha e impo an e ec s on hos ep oduc ion, being espon- sible o phenomena such as cy oplasmic incompa ibili y, eminiza ion, and male killing (We en e al. 2008; Kau e al. 2021). Wolbachia can also a ec o he aspec s o hos biology, including esis ance o i al in ec ion (Teixei a e al. 2008), gu mic obiome composi ion (Simhad i e al. 2017), he mal p e e ence (T ui e al. 2019), sleep beha io (Bi e al. 2018), and ecundi y and li espan (Se ga e al. 2021). A he molecula le el, Wolbachia is known o a ec hos gene exp ession (Baião e al. 2019; Biwo e al. 2020), and meio ic ecombina ion a e (Singh 2019), as well as he a o emen ioned TE- ela ed p ope ies (Riegle e al. 2005; Mayo al e al. 2014; Tou e e al. 2014). He e, we es he impac o Wolbachia on TE exp ession by using hos lines whe e Wolbachia is p esen e sus whe e i was emo ed. Speci ically, we use lies om he D osophila melanogas e Gene ic Re e ence Panel (DGRP), a panel o isogenic lines de i ed om a na u al popula ion, whose genomes ha e been ully sequenced and anno a ed o TE inse ions (Mackay e al. 2012; Rahman e al. 2015). We selec ed 25 DGRP lines ha we e na u ally in ec ed wi h Wolbachia o which we es i- ma ed Wolbachia loads in indi idual lies and eco ded he numbe o TE inse ions o 14 TEs, ep esen ing di e - en amilies. We ound di e ences in Wolbachia loads and in numbe o TE inse ions be ween geno ypes, as well as an associa ion be ween he wo. We hen gene a ed a Wolbachia- ee coun e pa o each o he 25 a ge gen- o ypes and used ou panel o 25 pai ed Wolbachia-posi i e and Wolbachia-nega i e lines o quan i y ansc ip ion le- els o he 14 a ge TEs. We ound a ia ion in TE exp es- sion depending on hos geno ype, TE iden i y, and Wolbachia s a us. Whe he Wolbachia emo al led o in- c eased o dec eased TE exp ession appea ed o be mo e o a p ope y o hos geno ype han o TE iden i y. Resul s and Discussion To in es iga e he e ec o Wolbachia in ec ion on TE ex- p ession, we ocused on 25 D. melanogas e geno ypes, o which we documen ed di e ences in Wolbachia loads and in numbe o inse ions o 14 a ge TEs ( ig. 1). We o- cused speci ically on wha we e called “no el inse ions” (Mackay e al. 2012; Rahman e al 2015), which co espond 2Genome Biol. E ol. 15(3) h ps://doi.o g/10.1093/gbe/e ad036 Ad ance Access publica ion 28 Feb ua y 2023 Downloaded om h ps://academic.oup.com/gbe/a icle/15/3/e ad036/7059559 by Uni e sidade de Lisboa use on 03 July 2023 E ec s o Wolbachia on T ansposable Elemen Exp ession GBE o TE inse ions ound in he DGRP genomes bu no in Release 6 o he D. melanogas e ’s e e ence genome (coun s c . Rahman e al. 2015). We hen gene a ed a co - esponding panel o 25 lines om which Wolbachia was clea ed, and compa ed exp ession le el o ou a ge TEs in adul emales be ween he pai s o Wolbachia-posi i e (Wolb+) and Wolbachia-nega i e (Wolb−) lies ( ig. 2). Hos Geno ypes Di e in Wolbachia Loads and in Numbe o TE Inse ions We andomly chose 25 o he 85 DGRP lines known o be in ec ed wi h he wMel s ain o Wolbachia (Mackay e al. 2012; Richa dson e al. 2012). Fo each o hese lines, we measu ed Wolbachia loads in i e indi idual adul emales 10 days pos -eclosion, he same sex and age used o meas- u e TE exp ession. Fo his, we used quan i a i e eal- ime polyme ase chain eac ion (qPCR) wi h p ime s o one Wolbachia-speci ic gene (wsp), o es ima e numbe o bac- e ial cells, and o one hos -speci ic gene (ac in), o assess numbe o hos cells. Ac oss he ∼125 lies assayed indi idually, Wolbachia loads a ied be ween a minimum o 3.5 and a maximum o 51 Wolbachia cells pe hos cell. Only six indi iduals, o di e en geno ypes, had >20 Wolbachia pe hos cell. These es ima es o Wolbachia densi y all along he same o de o magni ude as hose ound h ough sequencing o he DGRP lines (0.9–17.1 copies pe hos cell; Richa dson e al. 2012), o h ough qPCR o whole bodies (Béna d e al. 2021; Ch os ek e al. 2021) and gonadal is- sues (Co ea and Balla d 2012) o o he D. melanogas e geno ypes, as well as o o he Wolbachia s ains (Ch os ek and Teixei a 2015). We ound di e ences in Wolbachia loads be ween hos geno ypes, wi h median alues anging om 5 o 15 copies o Wolbachia pe hos cell ( ig. 1A), and es ima ed FIG. 1.—Cha ac e iza ion o ou 25 a ge hos lines in ela ion o Wolbachia load in indi idual lies (A) and o he numbe o no el TE inse ions in hei genomes (B). The 25 geno ypes a e o ganized along he y-axis in o de o he median alue o Wolbachia load. (A) Wolbachia load ela i e o numbe o hos cells (x-axis). Each blue do is a biological eplica e and ep esen s one single emale. Wolbachia load is signi ican ly di e en ac oss geno ypes (ANOVA; F 24,5508 = 4.5e + 27, P < 2e−16). (B) Hea map ep esen ing he p edic ed numbe o no el inse ions o ou 14 a ge TEs. TEs a e o ganized in he x-axis by median numbe o no el inse ions ac oss geno ypes. The scale o g ay, om whi e o da k g ay, ep esen s, espec i ely, om he lowes o highes numbe o no el inse ions. The TE pogo in geno ype RAL-21 is ou o he scale, wi h 216 no el inse ions anno a ed. The e was no in o ma ion in Rahman e al. (2015) o RAL-855. The numbe o no el inse ions di e ed signi ican ly be ween geno ypes (ANCOVA; geno ype: F 22,4974 = 1.3e + 28, P < 2e−16, TEs: F 13,4974 = 1.7e + 29, P < 2e−16, and Wolbachia i e s: F 1,4974 = 2.2e + 27, P < 2e−16). Genome Biol. E ol. 15(3) h ps://doi.o g/10.1093/gbe/e ad036 Ad ance Access publica ion 28 Feb ua y 2023 3 Downloaded om h ps://academic.oup.com/gbe/a icle/15/3/e ad036/7059559 by Uni e sidade de Lisboa use on 03 July 2023 Eugénio e al. GBE FIG. 2.—Exp ession le els o 14 TEs in adul emale lies o 25 geno ypes wi h e sus wi hou Wolbachia. TEs a e o de ed om le o igh by median (and a e age, o ied medians) numbe o no el inse ions. S a is ical signi icance o exp ession di e ences be ween Wolb+ and Wolb− is shown as * o P < 0.05, ** o P < 0.01, and *** o P < 0.001 (ANOVA, see Ma e ial and Me hods). (A) Exp ession o he 14 TEs in geno ypes RAL-21, RAL-181, RAL-712, and RAL-737. The same plo s o all o he geno ypes can be ound in supplemen a y igu e S2, Supplemen a y Ma e ial online. (B) Exp ession o di e en TEs ac oss a ious geno ypes, illus a ing cases whe e exp ession le els a e s a is ically di e en be ween Wolbachia s a us. Each do in plo s (A) and (B) ep esen s a biological eplica e, co esponding o a pool o en emale lies. (C) Hea map ep esen ing di e ences in exp ession le el o he 14 a ge TEs be ween Wolb + and Wolb− lies o all 25 di e en geno ypes. Geno ypes in he y-axis a e o de ed by Wolbachia load (as in ig. 1B). Cells a e displayed in a g adien o colo , ep esen ing e ec size (colo in ensi y) and whe he exp ession is highe in Wolb+ ela i e o Wolb− (blue shades; op hal o he g adien legend igh o he hea map) o he o he way a ound (pink shades; bo om hal o he g adien legend igh o he hea map). Unde lined as e isks ep esen signi ican di e - ences a e Benjamini–Hochbe g co ec ion o mul iple compa isons. (D) Volcano plo ep esen ing he e ec size (x-axis) and P- alue ( es ing o log2 old- change o TE exp ession di e ences be ween Wolb+ and Wolb−; y-axis). Do s ela i e o la ges e ec sizes (blas opia in RAL-321 and blood in RAL-595), and o nonsigni ican e ec size below −1, co esponding o 100% di e ence in TE exp ession a e Wolbachia emo al (blas opia in RAL-440 and Quasimodo in RAL-440), a e labelled. The dashed g ey line ep esen s he h eshold o s a is ical signi icance o di e ences in TE exp ession le els (P = 0.05). E ec sizes in (C) and (D) co espond o he di e ence o he log2 old-change in TE exp ession be ween Wolb+ and Wolb− pai ed geno ypes. 4Genome Biol. E ol. 15(3) h ps://doi.o g/10.1093/gbe/e ad036 Ad ance Access publica ion 28 Feb ua y 2023 Downloaded om h ps://academic.oup.com/gbe/a icle/15/3/e ad036/7059559 by Uni e sidade de Lisboa use on 03 July 2023 E ec s o Wolbachia on T ansposable Elemen Exp ession GBE b oad-sense he i abili y (H 2 ) o 0.22 (among-line a i- ance = 10.3, wi hin-line a iance = 36.0). Al hough li le is known abou wha hos loci ha bo na u al allelic a i- a ion con ibu ing o a ia ion in Wolbachia loads, we do know ha loads a y wi h en i onmen al ac o s, including empe a u e (Wiwa ana a anabu and Ki ayapong 2009), hos die (Pon on e al. 2015; Se bus e al. 2015), and i al in ec ion (Kau e al. 2020). Fi s , we alida ed in silico p edic ions o inse ions (Mackay e al. 2012) by PCR wi h p ime s o he ly genom- ic sequence lanking 132 p edic ed no el inse ions in 11 geno ypes (supplemen a y able S1, Supplemen a y Ma e ial online). The amplicons om each o he inse ions we e sized (aga ose gel) and sequenced o con i m he p esence, leng h, and iden i y o he inse ed DNA (supplemen a y able S1, Supplemen a y Ma e ial online). Fo 100% o he p edic ed inse ion loca ions we es ed, we con i med he p esence o a TE inse ion, and, in mos cases, we also con i med ha he size and he sequence o he inse ed DNA co esponded o he p edic ed TE iden- i y (supplemen a y ig. S1A, Supplemen a y Ma e ial on- line). Fo 113 (85.6%) o he inse ions es ed, he inse ed TE co esponded o he mos likely expec ed iden- i y (c . he p edic ions made om he whole-genome se- quence da a), and o 16, i co esponded o he second mos likely TE (Mackay e al. 2012). We obse ed ha 67 in- se ions (50.8%) had he size co esponding o he ex- pec ed ull leng h o ha TE, 44 (33.3%) we e smalle , and 21 (15.9%) we e la ge (supplemen a y ig. S1A and able S1, Supplemen a y Ma e ial online). Wi h p edic ions o no el inse ions alida ed, we used da a om he TIDAL-FLY 1.0 ool o Rahman e al. (2015) o ga he in o ma ion abou he numbe o no el in- se ions o each o ou 14 a ge TEs in 24 o ou 25 s udy geno ypes ( he e we e no da a o geno ype RAL-855). We ound signi ican di e ences in he numbe o no el inse - ions be ween geno ypes and Wolbachia i e s. The e o- ansposons C 1a, gypsy5, and Ide ix had he lowes numbe o p edic ed no el inse ions (wi h ze o o he ma- jo i y o he lines), whe eas he DNA ansposons 1360, pogo, and P-elemen gene ally had he highes numbe o no el inse ions, in acco dance wi h o he s udies de- sc ibing DNA ansposons as mos ac i e (Bou que e al. 2018). Fo mos indi idual TEs, he es ima ed numbe o no el inse ions a ied be ween 0 and 44, wi h he excep- ion o he TE pogo, p edic ed o ha e 216 no el inse ions in he line RAL-21 ( ig. 1B; Rahman e al. 2015). No e ha he in silico p edic ions o he numbe o TE inse ions a e likely o be unde es ima es o he ac ual numbe o inse - ions. The DGRP lines we e o iginally sequenced using a combina ion o Illumina and 454 sequencing echnologies (Mackay e al. 2012), which gene a e sho - eads and, as such, a e no ideal o de ec ing TE inse ions (Fis on-La ie e al. 2015; Goe ne -Po in and Bou que 2018; Panda and Slo kin 2020; Rech e al. 2022). Mo eo e , new inse ions may also ha e occu ed a e se- quencing. We con i med expe imen ally mul iple “ alse ne- ga i es” in TE inse ion p edic ions o he DGRPs. By unning PCRs wi h TE-speci ic p ime s and DNA om eigh DGRP geno ypes p edic ed in Mackay e al. (2012) o ha e no inse ions (no el o sha ed) o pa icula TEs. In all 17 cases es ed, we e i ied he p esence o hose TEs (supplemen a y ig. S1B, Supplemen a y Ma e ial online). Howe e , e en i p edic ions a e unde es ima es o ac ual numbe o inse ions, he e ec s should be simila / andom ac oss TEs and geno ypes wi h equi alen sequence co e - age dep h. TE T ansc ip ion Le el Va ies Wi h Wolbachia S a us in a Hos Geno ype-dependen Manne Using qPCR wi h TE-speci ic p ime s and a e e ence hos gene, we quan i ied he exp ession o ou 14 a ge TEs in eigh eplica e pools o en 10-day-old emales each, o each o he 25 Wolb+ and Wolb− pai s o geno ypes (Cq da a in supplemen a y able S2, Supplemen a y Ma e ial online). Exp ession le els di e ed signi ican ly (analysis o a iance, ANOVA), be ween TEs (F 13,4796 = 4.75, P = 2.97e−08), geno ypes (F 24,4796 = 26.63, P < 2.2e−16), and wi h Wolbachia s a us (F 1,4796 = 28.79, P = 8.5e−08), wi h all in e ac ions being signi ican (P < 0.0001 in all cases; ig. 2). Gi en he signi ican e ec o Wolbachia s a us on TE ex- p ession, we hen speci ically compa ed exp ession o each o he 14 TEs in each o he 25 hos geno ypes wi h e sus wi hou Wolbachia. We ound s a is ically signi ican di e - ences in TE exp ession be ween Wolb+ and Wolb− lines o a o al o 74 o he 350 (21.1%) geno ype-TE combina ions es ed ( ig. 2; supplemen a y ig. S2 and able S3, Supplemen a y Ma e ial online). We obse ed dis inc scen- a ios depending on TE and geno ype: highe exp ession in Wolb− lies o 47 in 350 cases (13.4%) and highe exp es- sion in Wolb+ lies o 26 in 350 cases (7.4%). Fo any gi en TE, he e ec o Wolbachia on exp ession was no he same ac oss geno ypes, and, o any gi en geno ype, he e ec o Wolbachia on TE exp ession was no he same ac oss TEs. Howe e , o some geno ypes, we obse ed some consis ency in he e ec s o Wolbachia on TE exp ession. Fo geno ypes RAL-142 and RAL-181, when s a is ically signi ican ly di e en be ween Wolb+ and Wolb− lies, TE exp ession was always highe in Wolb+ (blue shades in ig. 2C) ela i e o Wolb− lies. Con e sely, o geno ypes RAL-712, RAL-21, and RAL-321, when signi ican ly di e en , TE exp ession was al- ways highe in Wolb− (pink shades in ig. 2C) ela i e o Wolb+ lies. Genome Biol. E ol. 15(3) h ps://doi.o g/10.1093/gbe/e ad036 Ad ance Access publica ion 28 Feb ua y 2023 5 Downloaded om h ps://academic.oup.com/gbe/a icle/15/3/e ad036/7059559 by Uni e sidade de Lisboa use on 03 July 2023 Eugénio e al. GBE E ec Size o Wolbachia Remo al on TE Exp ession Le els Fo each TE in each Wolb+/Wolb− geno ype pai , he aw e ec size o Wolbachia emo al was calcula ed by sub ac ing he median log2 TE exp ession (no malized o e e ence gene) in he Wolb− lies om he median log2 TE exp ession in he Wolb+ lies ( ig. 2C and D; supplemen a y able S5, Supplemen a y Ma e ial online). The e we e mo e cases in which Wolbachia emo al e- sul ed in an inc ease in TE exp ession han he e e se (i.e., mo e cases wi h signi ican ly highe TE exp ession in Wolb− ela i e o Wolb+ lies; pink shades in ig. 2Cand pink do s in 2D). Also, he size o he e ec o Wolbachia emo al on TE exp ession ended o be la ge when his e- mo al esul ed in inc eased exp ession ela i e o when i esul ed in dec eased exp ession (i.e., u he om ze o in he x-axis o ig. 2D). The median e ec size o s a is ically signi ican ly highe exp ession in Wolb− ela i e o Wolb+ (i.e., pink do s in ig. 2D) was app oxima ely −1.0, co e- sponding o almos 100% inc ease in exp ession upon Wolbachia emo al. On he o he hand, he median e ec size o s a is ically signi ican lowe exp ession in Wolb− ela i e o Wolb+ (i.e., blue do s in ig. 2D) was a ound +0.5, co esponding o a ound 71% educ ion in exp es- sion upon Wolbachia emo al. In 24 o he 350 TE-geno ype combina ions es ed, Wolbachia emo al e- sul ed in an inc ease in TE exp ession >100% inc ease (e - ec size <−1.0 in ig. 2D). O hese 24 cases, only wo we e no s a is ically signi ican (blas opia and Quasimodo, bo h in RAL-440; highligh ed in ig. 2D). In e es ingly, wo gen- o ypes, RAL-21 and RAL-712, s ood ou o ha ing among he highes Wolbachia loads ( ig. 1A) and a gene al inc ease in exp ession o mos TEs a e Wolbachia emo al ( ig. 2A and C), wi h median e ec sizes ac oss TEs o a ound −1.1 (107% inc ease) and a ound −0.9 (93% inc ease), espec i ely. In h ee o he 350 TE-geno ype combina ions es ed, Wolbachia emo al esul ed in a >100% educ ion (e ec size >+1.0) in TE exp ession. O hese, blood in RAL-595 s ood ou wi h an e ec size a ound 2.3, co esponding o a 246% educ ion in exp ession a e Wolbachia emo al ( ig. 2D). Conclusion In his s udy, we in es iga ed he hypo hesis o a ela ion- ship be ween Wolbachia in ec ion and TE ac i i y. Wolbachia is a p e alen endosymbio ic bac e ium whose impac on TE mobiliza ion was sugges ed by dis inc lines o e idence, including: (1) e ec on piRNA exp ession (Mayo al e al. 2014), (2) e ec on a e o ansposi ion o e o ansposon gypsy (Tou e e al. 2014), and (3) Wolbachia s ain- eplacemen co-occu ing wi h in asion o DNA ansposon P-elemen (Riegle e al. 2005). We es ed whe he he exp ession o 14 di e se TEs was di e - en be ween Wolbachia-in ec ed and Wolbachia- ee D. melanogas e lies o 25 dis inc geno ypes di e ing in Wolbachia loads and in numbe o TE inse ions. We o- cused on TE exp ession, which is o en used as a p oxy o TE ac i i y (e.g., Becking e al. 2020; To es e al. 2021), ea- soning ha highe exp ession c ea es mo e oppo uni ies o inse ions. TE ansc ip le els we e quan i ied using qPCR, wi h e o pu in o ca ying ou and explaining in de- ail da a s uc u e, quali y con ol, and analyses (see Ma e ials and Me hods). Howe e , ansc ip ion is only one, albei necessa y, s ep in TE mobiliza ion, and Wolbachia, o o he ac o s, may impac TE in eg a ion pos - ansc ip ionally. We ound s a is ically signi ican di e ences in le els o TE ansc ip be ween Wolb+ and Wolb− lies in 21.1% o he 350 geno ype-TE combina ions analyzed, and a max- imum e ec size o 2.3 lowe exp ession upon Wolbachia emo al (supplemen a y able S5, Supplemen a y Ma e ial online). The obse ed e ec s o Wolbachia emo al we e no uni o m o any gi en TE (i.e., one same TE could in- c ease, dec ease, o no change exp ession depending on geno ype) no o mos geno ypes (i.e., one same geno ype could ha e TEs ha inc eased, TEs ha dec eased, and TEs ha did no change exp ession). Howe e , some geno ypes did s and ou in ha ing mul iple TEs o which he di ec ion Wolbachia e ec on exp ession was he same. In pa icula , geno ypes RAL-21 and RAL-712 showed bo h some o he highes Wolbachia loads and signi ican inc ease in exp es- sion o mos TEs when Wolbachia was emo ed. Va ious ac o s can po en ially lead o Wolbachia emo al a ec ing TE exp ession. Wolbachia may a ec piRNAs, as has been shown o a na ow se o piRNAs in A. aegyp i mosqui oes (Mayo al e al. 2014), which a e na u ally de oid o Wolbachia. Wolbachia emo al migh also ac as a s ess ac o o hos geno ypes, which migh ha e adjus ed o ha ing Wolbachia. This s udy was pe o med using a subse o he DGRP lines, a panel o isogenic and ully sequenced geno ypes ha p o ide he possibili y o looking a geno ypic a i- a ion. E en hough he geno ypes a e no na u ally occu - ing, in ha hey we e highly isogenized pos -collec ion o a na u al popula ion, hey ep esen na u ally seg ega - ing allelic a ian s. Many s udies showed di e ences be ween DGRP geno- ypes o a ious di e en ypes o ai s (e.g., Magwi e e al. 2012; Webe e al. 2012; Du lam e al. 2014; I ano e al. 2015; Howick and Lazza o 2017; La uen e e al. 2018; Mackay and Huang 2018). Ou esul s highligh di - e ences be ween geno ypes in Wolbachia loads and num- be o TE inse ions, as well as in he e ec o Wolbachia emo al on TE ac i i y. The in e -geno ype di e ences u - he emphasize he impo ance o analyzing mul iple 6Genome Biol. E ol. 15(3) h ps://doi.o g/10.1093/gbe/e ad036 Ad ance Access publica ion 28 Feb ua y 2023 Downloaded om h ps://academic.oup.com/gbe/a icle/15/3/e ad036/7059559 by Uni e sidade de Lisboa use on 03 July 2023 E ec s o Wolbachia on T ansposable Elemen Exp ession GBE geno ypes o ha e a mo e comple e unde s anding o any biological phenomena. S udies ha only ocus on a single o a ew geno ypes may miss o mis ep esen gene al p ope ies. No el gene ic a ian s c ea ed by TE mobiliza ion can be, and a e o en, dele e ious (McFaddena and Knowlesb 1997; Hedges and Deininge 2007; Belancio e al. 2008; Aya padikannan and Kim 2014). As such, high TE ac i i y can pu na u al popula ions unde s able condi ions a a disad an age. On he o he hand, TE inse ions can also be bene icial and, pa icula ly in condi ions o en i onmen- al pe u ba ion, TE ac i i y could con ibu e o no el gene - ic a ian s be e adjus ed o he changed condi ions (e.g., Rey e al. 2016). The ques ion o which and how in insic and ex insic ac o s a ec TE ac i i y is a undamen ally in- e es ing and la gely un esol ed ques ion, especially o ani- mal when compa ing wi h plan TEs (Thieme e al. 2017). Ou s udy shows ha he ma e nally inhe i ed Wolbachia endosymbion , which is p e alen in insec s and nema- odes, a ec ed TE exp ession in D. melanogas e . We ex- pec u u e s udies o p o ide insigh abou which and how di e en ac o s a ec TE mobiliza ion; including Wolbachia and o he en i onmen al ac o s in mul iple hos s. Ma e ials and Me hods Con i ming in silico P edic ions o TE Inse ions in DGRP Lines We looked o alida e in silico p edic ions in e ms o bo h po en ial alse posi i es ( ocusing on speci ic inse ions) and po en ial alse nega i es ( ocusing on pa icula TEs deemed as ha ing no inse ions in some geno ypes). TE in- se ions in he DGRPs ha e been classi ied as “sha ed” e - sus “no el” depending on whe he hey we e e sus we e no p esen in he e e ence genome, elease 6 (Mackay e al. 2012; Rahman e al. 2015). Al hough non e e ence inse ions migh no necessa ily be no el, we kep he e - minology om he o iginal a icles ha documen ed TE in- se ions in he DGRPs, and which is used in a ious o he s udies e e ing o hose da a. Fi s , we selec ed 132 o he p edic ed no el inse ions in 12 o he DGRP lines and designed p ime s o he sequence lanking hose inse ions (supplemen a y able S1, Supplemen a y Ma e ial online). Fo each o he lines, we ex ac ed gDNA om pools o en males (homogenized using pes les), using DNeasy Blood and Tissue ki (Qiagen), ollowing manu ac u e ’s ins uc ions. We hen used 4 ng o his gDNA in 15 μl long PCRs wi h 0.5 μM p i- me s, 2% DMSO, 0.5 mM dNTPs mix, 0.21 μl o GoTaq en- zyme (P omega). The mocycle condi ions included 2 min a 92 °C; 10 cycles o 92 °C o 10 s, 60 °C o 15 s, 68 °C o 10 min; 30 cycles o 92 °C o 15 s, 60 °C o 30 s, 68 °C o 10 min + 20 s cycle elonga ion o each successi e cycle; 7 min a 68 °C. Amplicons we e sized (1% aga ose gel elec opho esis) and sequenced (The moFishe BigDye Te mina o 1.1, o SUPREME un™ om; same o wa d p ime s used o ampli ica ion) and hese we e NZYTech compa ed wi h he size and sequence o he canonical D osophila ansposons (Flybase e sion 9.42). Second, we es ed he absence o speci ic TEs in geno- ypes anno a ed as ha ing no inse ions o ha TE. We an PCR wi h p ime s speci ic o each o se en TEs (blood, copia, gypsy5, H-elemen , jockey, opus, pogo; supplemen a y able S4, Supplemen a y Ma e ial online) and gDNA ex ac ed om pools o en adul emales (ex ac ions as desc ibed abo e) o eigh geno ypes (RAL-109, RAL-161, RAL-237, RAL-350, RAL-362, RAL-555, RAL-776, and RAL-808) p e- dic ed o no ha e one o mo e o hose TEs (Mackay e al. 2012), con i ming he p esence o absence o inse - ion band in 1% aga ose gel (supplemen a y ig. S1B, Supplemen a y Ma e ial online). Wi h he gDNA om each o he a ge geno ypes, we an wo ypes o posi i e con ols: (1) wi h TE-speci ic p ime s wi h gDNA ex ac ed om a geno ype (RAL-321) p edic ed o ha e inse ions o all six TEs, and (2) wi h p ime s o he D osophila gene RPL32 (supplemen a y able S4, Supplemen a y Ma e ial online) p esen in e e y line. gDNA ex ac ed as desc ibed abo e was used in 10 µl PCRs con aining 0.4 ng gDNA, 0.25 U GoTaq (P omega), 1.5 mM MgCl 2 , and 0.5 μM o each p ime . The he mal cycling p o ocol was: 10 min a 95 °C; 35 cycles o 95 °C o 30 s, 60 °C o 1 min, 72 °C o 30 s; 5 min a 72 °C. Fly Lines and Husband y We andomly chose 25 DGRP lines desc ibed o be in ec ed wi h Wolbachia (Mackay e al. 2012) and none we e de- sc ibed o be in ec ed wi h he endosymbion Spi oplasma (Richa dson e al. 2012). See he comple e lis o a ge DGRP geno ypes in supplemen a y ables S2 and S3, Supplemen a y Ma e ial online. Fo each o he lines selec ed, we gene a ed a Wolbachia- ee e sion ol- lowing p ocedu es desc ibed in Teixei a e al. (2008) and Ch os ek e al. (2013). In sho , lies we e i s id o Wolbachia by eeding on ood supplemen ed wi h e acyc- line an ibio ic (0.05 mg/ml) o wo gene a ions. Thei gu lo a was hen es o ed by placing s e ilized eggs o Wolbachia-clea ed lies (10 min in 50% bleach ollowed by washing in s e ilized wa e ) on ood supplemen ed wi h a bac e ial inoculum (150 µl o a mix p epa ed by mixing 2 ml o s e ile wa e wi h 1 g o a mon h-old ood il e ed o emo e eggs and la ae) o each espec - i e un ea ed (Wolbachia-posi i e) line. Flies we e Wolbachia- ee and gu mic obio a-homogenized o a leas i e gene a ions be o e he expe imen s we e ini ia ed. Genome Biol. E ol. 15(3) h ps://doi.o g/10.1093/gbe/e ad036 Ad ance Access publica ion 28 Feb ua y 2023 7 Downloaded om h ps://academic.oup.com/gbe/a icle/15/3/e ad036/7059559 by Uni e sidade de Lisboa use on 03 July 2023 Eugénio e al. GBE Flies we e ea ed a 25 °C and 12 h:12 h ligh :da k cycle, in ials wi h co nmeal-aga ood (45 g/l molasses, 75 g/l whi e suga , 70 g/l co n lou , 20 g/l yeas ex ac , 10 g/l aga -aga , and 25 ml Nipagin a 10%) and simila densi y condi ions. Fo ou expe imen s, we ans e ed newly eclosed adul lies o ials in g oups o en emales and six males. Females we e sampled o ex ac ion o DNA ( o quan i ica ion o Wolbachia) o o RNA ( o quan i ica ion o TE exp ession) a 10 days o age. Wolbachia P esence and Loads We used Wolbachia-speci ic p ime s agains he Wolbachia su ace p o ein gene (wsp; sequence om Teixei a e al. 2008) o con i m ha he e acycline- ea ed Wolb− lines we e indeed Wolbachia ee and o quan i y Wolbachia loads in he un ea ed Wolb+ lines. We con i med he absence o Wolbachia in each o he e acycline- ea ed lines in 10 µl PCRs, con aining 0.4 ng gDNA empla e, 0.25 U GoTaq (P omega), 1.5 mM MgCl 2 , and 0.5 μM o each p ime (wsp). We used gDNA ex ac ed (Qiagen’s DNeasy Blood and issue ki , ollowing manu ac u e ’s indica ions) om 3 pools o 10 emales (mixed ages) om each o he 25 Wolb− lines, homoge- nized using Qiagen Tissue Lyse II (2 min a 23 s/ ). As posi- i e con ol, we ex ac ed gDNA om he 25 Wolb+ lines (same p o ocol) and used hose samples as empla e. The mal cycle was 4 min a 95 °C; 35 cycles o 95 °C o 30 s, 60 °C o 1 min and 72 °C o 30 s; 5 min a 72 °C. PCR amplicons we e checked by elec opho esis gel (1% aga ose) and we con i med he success ul emo al o Wolbachia (no amplicon) in all 25 a ge DGRP lines. We measu ed Wolbachia loads in 5 indi idual emales (10 days pos -eclosion) om each o ou 25 a ge Wolb+ lines. Indi idual emales we e homogenized in Qiagen ATL Bu e in 96 well-pla es wi h a s e ile glass bead pe well on a Tissue Lyse II (Qiagen) a 23 s/ o 2 min, be o e DNA was ex ac ed using he Quick-DNA™ 96 ki (Zymo Resea ch), ollowing manu ac u e ’s ins uc ions. DNA was elu ed in 200 µl bu e AE om he ki and s o ed a −20 °C un il qPCR, which was un using p ime s (supplemen a y able S4, Supplemen a y Ma e ial online) ei he o a Wolbachia gene (wsp; measu ing Wolbachia load) o o a hos gene (ac in, p oxy o numbe o hos cells) as desc ibed below. RNA Ex ac ion and cDNA Syn hesis o TE Exp ession Quan i ica ion by qPCR To quan i y TE exp ession, we ex ac ed RNA om eigh eplica e pools o 10 co-housed, 10-day-old emales, o each o he 25 pai s o Wolb+ and Wolb− geno ypes ( o al o 50 lines). Whole bodies we e homogenized in 400 µl TRIzol (In i ogen) using a s e ile glass bead in mic ocen i- uge ubes and a Tissue Lyse II (Qiagen) a 26 s/ o 1 min. Homogena es we e s o ed a −80 °C un il u he p ocess- ing. Once hawed, we added 80 µl o chlo o o m, cen i- uged (12,000 × g o 15 min a 4 °C) and collec ed he supe na an aqueous phase con aining he RNA ( o a oid ca ying ly issues and a o he RNA ex ac ion s ep), and hen we added 400 µl mo e TRIzol. To al RNA was hen ex ac ed using he Di ec -zol™ 96 RNA Ki (Zymo Resea ch), ollowing manu ac u e ins uc ions. We used 4 µg o RNA o syn hesize cDNA wi h NZY Fi s -S and cDNA Syn hesis Ki (NZYTech), ollowing manu ac u e ’s in- s uc ions. cDNA was hen dilu ed 1:10 in s e ile wa e (Sigma) o be used as empla e in qPCR wi h p ime s (supplemen a y able S4, Supplemen a y Ma e ial online) agains each o he 14 a ge TEs (412, 1360, blas opia, blood, copia, C 1a, gypsy5, Ide ix, Juan, mdg1, opus, Quasimodo, P-elemen , pogo) o agains one e e ence gene, EF1, chosen om a numbe o candida es (18S, Ac 5c, ac in, EF1, ELF2, Gapdh1, Mn , Rpl32, Rps20, TBP, ubulin) using No m inde (Ande sen e al. 2004) and se- lec ing a gene wi h Cq alues simila o ha o he TEs being es ed (qPCR eagen s and he mocycle as desc ibed below). qPCR Wi h S anda d Cu es We used qPCR o measu e bo h Wolbachia i e s (gDNA empla e and p ime s o one Wolbachia-speci ic gene, wsp, and one hos -speci ic e e ence gene, ac in) and TE ex- p ession (cDNA empla e and p ime s o each o he 14 a - ge TEs and one e e ence gene). Templa e p epa a ion and p ime s we e desc ibed abo e. Ou qPCR s udies ollowed MIQE guidelines (Taylo e al. 2010), including echnical and biological eplica ion, ensu ing empla e quali y, ca e- ul selec ion o e e ence genes, and co ec ion o di e - ences in p ime e iciency. Mo eo e , all samples being di ec ly compa ed we e an oge he and using he same ba ch o eagen s. Fo each biological eplica e sample, we an wo ech- nical eplica e eac ions in an Quan S udio™ 7 Flex Real-Time PCR Sys em (Applied Biosys ems™). We used 4 µl o genomic empla e, 0.5 µl o each p ime (0.2 µM) and 5 µl o SYBR G een I® (Bio Rad), and he ollowing he - mal cycling condi ions: 2 min a 50 °C; 10 min a 95 °C; 40 cycles o 95 °C o 30 s, 60 °C o 1 min and 72 °C o 30 s. We disca ded biological eplica es o which he s anda d de ia ion be ween Cq alues o he wo echnical eplica es was >0.5, and calcula ed he mean Cq alue be ween ech- nical eplica es o each o all o he biological eplica es. P ocessing o Cq da a o biological eplica es is de ailed below. Fo each gene and each TE, we also ob ained s anda d cu es ela ing amoun o empla e and Cq alues. These we e ob ained by using as empla e a 1:10 se ial dilu ion (8 dilu ions) o a cleaned (Mache ey-Nagel’s NucleoSpin 8Genome Biol. E ol. 15(3) h ps://doi.o g/10.1093/gbe/e ad036 Ad ance Access publica ion 28 Feb ua y 2023 Downloaded om h ps://academic.oup.com/gbe/a icle/15/3/e ad036/7059559 by Uni e sidade de Lisboa use on 03 July 2023 E ec s o Wolbachia on T ansposable Elemen Exp ession GBE Gel and PCR Clean-up) and quan i ied (In i ogen’s Qubi ™) PCR p oduc (ob ained by PCR on gDNA ex ac ed om lies om he s anda d line O egon R). We used he equa ions o he linea eg ession o log quan i y o s a - ing empla e (x-axis) and Cq alue (y-axis) o: (1) do abso- lu e quan i ica ion o wsp and ac in, as he e in no ob ious calib a o sample o analysis o Wolbachia loads, and (2) calcula e p ime e iciency equi ed o he ela i e quan i ica ion o TE exp ession using he P a l me hod (2001). P ocessing qPCR Cq Da a o Quan i y Wolbachia and TE Exp ession Fo he absolu e quan i ica ion o Wolbachia loads, we used he mean Cq alues o each biological sample and he s anda d cu es o wsp and ac in o de e mine he quan i y o each o he genes in he sample used as em- pla e: quan i y = 10 ((Cq−b)/m) , whe e b is he in e cep and m is he slope o he linea eg ession equa ion. We es i- ma ed he quan i y o bo h wsp and ac in in each sample and hen calcula ed he a io be ween he wo (quan i y o wsp/quan i y o ac in) as a measu emen o Wolbachia load in ela ion o hos cells. Fo he ela i e quan i ica ion o TE exp ession, we used he P a l me hod: exp ession a io = E (TE) ΔCq(TE) /E (EF1) ΔCq(EF1) . E is he ampli ica ion e iciency o each p ime pai and is cal- cula ed based on he equa ion o he linea eg ession o he espec i e s anda d cu e: E = 10 −1/slope (p ime e icien- cies in supplemen a y able S4, Supplemen a y Ma e ial on- line). Fo all TE × geno ype samples, ΔCq e e s o he di e ence in Cq alues be ween a calib a o sample (a e age o same-geno ype Wolb+ samples) and each sample o ha geno ype (Wolb− and Wolb+). S a is ical Analysis All s a is ical analyses we e pe o med in R ( e sion 4.3.1), using Rs udio ( e sion 2022.07.2). We es ima ed b oad sense he i abili y (H 2 ) o Wolbachia loads as H 2 = σ 2 A/(σ 2 A + σ 2 W), whe e σ 2 A is he among- lines a iance and σ 2 W is he wi hin-line a iance. Va iance componen s we e ex ac ed using he VCA R package (Schue zenmeis e and Du ey 2020). We es ed o di e ences be ween he 25 a ge DGRP geno ypes in: (1) Wolbachia loads, using ANOVA wi h geno ype as ixed ac o : ao (Wolbachia load ∼ geno ype) in R syn ax, and (2) he numbe o no el TE inse ions, using ANCOVA wi h Wolbachia load as co a ia e, and geno ype and TE as ixed ac o s: ao (no el TE inse ions ∼ mean Wolbachia load + geno ype * TE) in R syn ax. To accoun o a ia ion in TE exp ession wi h Wolbachia s a us (Wolb+/Wolb−), we used ANOVA wi h TE, geno- ype, and Wolbachia s a us as ixed ac o s: ao (log2 TE ex- p ession no malized o e e ence gene exp ession ∼ TE * geno ype * Wolbachia s a us) in R syn ax. Then, o each TE in each pai ed Wolb+/Wolb- geno ype, we compa ed TE exp ession be ween Wolb+ and Wolb− lies using ANOVA wi h Wolbachia s a us as ixed ac o : ao (log2 TE exp ession no malized o e e ence gene exp ession o TE exp ession ∼ Wolbachia s a us) in R syn ax. We plo ed he esiduals o he models and ound ha hei dis ibu- ions we e su icien ly close o no mal o jus i y pa ame ic es s. Howe e , we also applied a nonpa ame ic es (K uskal–Wallis es ), which ga e mos ly equi alen esul s (supplemen a y able S3, Supplemen a y Ma e ial online). Supplemen a y Ma e ial Supplemen a y da a a e a ailable a Genome Biology and E olu ion online (h p://www.gbe.ox o djou nals.o g/). Acknowledgmen s The au ho s hank Rena o Al es o helping wi h bioin o - ma ic ools, Daniel Sob al o aligning nex gene a ion se- quencing eads agains TE canonical sequences, Jo ge Ca nei o o c ucial help wi h he s a is ical analysis, he Fly Facili y a Ins i u o Gulbenkian de Ciência o ly main- enance, and Élio Sucena and Isabel Go do o se ing as hesis commi ee membe s on ATE’s PhD wo k. This wo k was suppo ed by he Po uguese science unding agency, Fundação pa a a Ciência e Tecnologia (FCT): PhD ellow- ships o AT Eugénio (SFRH/BD/115535/2016, COVID/BD/ 151707/2021), PhD ellowships o MSP Ma ial a (SFRH/ BD/51882/2012), and esea ch g an s o P Beldade (PTDC/BIA-EVF/0017/2014, PTDC/BIA-EVL/0321/2021). Da a A ailabili y Da a submi ed in supplemen a y able S2, Supplemen a y Ma e ial online. Li e a u e Ci ed Ande sen CL, Jensen JL, O n o TF. 2004. No maliza ion o eal- ime quan i a i e e e se ansc ip ion-PCR da a: a model-based a i- ance es ima ion app oach o iden i y genes sui ed o no maliza- ion, applied o bladde and colon cance da a se s. Cance Res. 64:5245–5250. Ande son SN, e al. 2019. Dynamic pa e ns o ansc ip abundance o ansposable elemen amilies in maize. G3 (Be hesda) 9: 3673–3682. Aya padikannan S, Kim HS. 2014. The impac o ansposable ele- men s in genome e olu ion and gene ic ins abili y and hei impli- ca ions in a ious diseases. Genomics In o m. 12:98–104. Baião GC, Schneide DI, Mille WJ, Klasson L. 2019. The e ec o Wolbachia on gene exp ession in D osophila paulis o um and i s implica ions o symbion -induced hos specia ion. BMC Genomics. 20:465. Becking T, Gilbe C, Co daux R. 2020. Impac o ansposable ele- men s on genome size a ia ion be ween wo closely ela ed c us - acean species. Annal Biochem. 600:113770. Genome Biol. E ol. 15(3) h ps://doi.o g/10.1093/gbe/e ad036 Ad ance Access publica ion 28 Feb ua y 2023 9 Downloaded om h ps://academic.oup.com/gbe/a icle/15/3/e ad036/7059559 by Uni e sidade de Lisboa use on 03 July 2023