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