F on ie s in Mic obiology 01 on ie sin.o g
Fi s insigh s in o he Au elia au i a
ansc ip ome esponse upon
manipula ion o i s mic obiome
NancyWeiland-B äue
1‡, VasilikiKou sou eli
2‡,
DanielaLang eld
1† and Ru hA.Schmi z
1
*
1 Ins i u e o Gene al Mic obiology, Kiel Uni e si y, Kiel, Ge many, 2 GEOMAR Helmhol z Cen e o
Ocean Resea ch Kiel, Düs e nb ooke Weg, Kiel, Ge many
In oduc ion: The associa ed di e se mic obiome con ibu es o he o e all
i ness o Au elia au i a, pa icula ly o asexual ep oduc ion. Howe e , how A.
au i a main ains his speci ic mic obiome o eac s o manipula ions is unknown.
Me hods: In his epo , he esponse o A. au i a o manipula ions o i s
na i e mic obiome was s udied by a ansc ip omics app oach. Mic obiome-
manipula ed polyps we e gene a ed by an ibio ic ea men and challenging
polyps wi h a non-na i e, na i e, and po en ially pa hogenic bac e ium. To al RNA
ex ac ion ollowed by RNAseq esul ed in o e 155 million eads used o a de
no o assembly.
Resul s: The ansc ip ome analysis showed ha he an ibio ic-induced change
and esul ing educ ion o he mic obiome signi ican ly a ec ed he hos
ansc ip ome, e.g., genes in ol ed in p ocesses ela ed o immune esponse
and de ense mechanisms we e highly up egula ed. Simila ly, manipula ing he
mic obiome by challenging he polyp wi h a high load o bac e ia (2 × 107 cells/
polyp) esul ed in induced ansc ip ion o apop osis-, de ense-, and immune
esponse genes. A second ocus was on hos -de i ed quo um sensing in e e ence
as a po en ial de ense s a egy. Quo um Quenching (QQ) ac i i ies and he
espec i e encoding QQ-ORFs o A. au i a we e iden i ied by unc ional sc eening
a cDNA-based exp ession lib a y gene a ed in Esche ichia coli. Co esponding
sequences we e iden i ied in he ansc ip ome assembly. Mo eo e , gene
exp ession analysis e ealed di e en ial exp ession o QQ genes depending on
he ea men , s ongly sugges ing QQ as an addi ional de ense s a egy.
Discussion: O e all, his s udy allows i s insigh s in o A. au i a’s esponse o
manipula ing i s mic obiome, hus pa ing he way o an in-dep h analysis o he
basal immune sys em and addi ional undamen al de ense s a egies.
KEYWORDS
Au elia au i a, mic obiome, ansc ip ome, immune sys em, de ense mechanisms,
quo um quenching
1. In oduc ion
Cnida ians, such as he moon jelly ish Au elia au i a, a e dis ibu ed wo ldwide and play
essen ial oles in shaping ma ine ecosys ems (B ekhman e al., 2015). Cnida ia a e da ed back
o abou 700 million yea s and a e conside ed a sis e g oup o he Bila e ia (Pu nam e al., 2007;
Pa k e al., 2012). Thus, hey a e among he simples animals a he issue le el o ganiza ion
possessing wo ge m laye s (ec ode m and endode m) sepa a ed by he mesoglea (Ball e al.,
2004). In addi ion o hei mo phological simplici y, many Cnida ia, pa icula ly Scyphozoa,
ha e a high le el o de elopmen al plas ici y, allowing o an eno mous ole ance, egene a ion
OPEN ACCESS
EDITED BY
Alejand a P ie o-Da ó,
Na ional Au onomous Uni e si y o Mexico,
Mexico
REVIEWED BY
Ming Guo,
Ningbo Uni e si y, China
Ojas Na a ajan,
Uni e si y o Sou h Flo ida, Uni edS a es
*CORRESPONDENCE
Ru h A. Schmi z
[email p o ec ed]
†PRESENT ADDRESS
Daniela Lang eld ,
Ins i u e o Clinical Molecula Biology (IKMB),
Kiel Uni e si y, Kiel, Ge many
‡These au ho s ha e con ibu ed equally o his
wo k and sha e i s au ho ship
RECEIVED 10 Ma ch 2023
ACCEPTED 18 July 2023
PUBLISHED 10 Augus 2023
CITATION
Weiland-B äue N, Kou sou eli V,
Lang eld D and Schmi z RA (2023) Fi s
insigh s in o he Au elia au i a ansc ip ome
esponse upon manipula ion o i s mic obiome.
F on . Mic obiol. 14:1183627.
doi: 10.3389/ micb.2023.1183627
COPYRIGHT
© 2023 Weiland-B äue , Kou sou eli, Lang eld
and Schmi z. This is an open-access a icle
dis ibu ed unde he e ms o he C ea i e
Commons A ibu ion License (CC BY). The
use, dis ibu ion o ep oduc ion in o he
o ums is pe mi ed, p o ided he o iginal
au ho (s) and he copy igh owne (s) a e
c edi ed and ha he o iginal publica ion in his
jou nal is ci ed, in acco dance wi h accep ed
academic p ac ice. No use, dis ibu ion o
ep oduc ion is pe mi ed which does no
comply wi h hese e ms.
TYPE O iginal Resea ch
PUBLISHED 10 Augus 2023
DOI 10.3389/ micb.2023.1183627
Weiland-B äue e al. 10.3389/ micb.2023.1183627
F on ie s in Mic obiology 02 on ie sin.o g
po en ial, and asexual p oli e a ion du ing hei li e cycle (Richa dson
e al., 2009). Cnida ia ha e e ol ed and a e cons an ly exposed o
di e se mic oo ganisms (Liu e al., 2019). This close associa ion wi h
mic oo ganisms has p o ound e ec s on a ious hos unc ions.
Recen s udies ha e demons a ed ha speci ic hos -associa ed
mic obio a can con ibu e o a ious hos unc ions. Examples a e
hos me abolism (Ochsenkühn e al., 2017), de elopmen (Rook e al.,
2017), o gan mo phogenesis (Somme and Bäckhed, 2013), pa hogen
p o ec ion and immuni y (Mo an and Yun, 2015), beha io (Ezenwa
e al., 2012), en i onmen al sensing and adap a ion (Bang e al., 2018;
Ziegle e al., 2019), de elopmen al ansi ions (Webs e and Reusch,
2017; Woznica e al., 2017), and ep oduc ion (Chil on e al., 2015;
Jacob e al., 2015).
E iden ly, Cnida ians a e cons an ly exposed o mic obes in he
en i onmen ; consequen ly, molecula analyses ha e e ealed a a ie y
o molecula pa hways o espond o mic obial exposu e (Die king
and Pi a, 2020). In he i s s ep, ex acellula su ace ecep o s
ecognize mic obe-associa ed molecula pa e ns (MAMPs) du ing
mic obial epi helium coloniza ion (Chu and Mazmanian, 2013).
MAMPs include lipopolysaccha ides (LPSs), pep idoglycan (PGN),
lagellin, and mic obial nucleic acids (Rosens iel e al., 2009). In he
i s line o de ense, an imic obial pep ides (AMPs) egula e
es ablishing and main aining a speci ic mic obio a (Bosch, 2013;
Bosch and Zaslo , 2021). Toll-like ecep o s (TLRs) a he hos cell
su ace u he pe cei e he MAMP signal, ini ia ing MAMP- igge ed
immuni y (Augus in e al., 2010; Bosch, 2013). Downs eam o hose
conse ed signaling cascades a e s ess- esponsi e ansc ip ion
ac o s, including euka yo ic ansc ip ion ac o s o he p o eins’
NF-kappaB (NF-kB) amily (Zheng e  al., 2005). Recen s udies
e ealed ha euka yo ic hos s also use quo um quenching (QQ) as a
s a egy o espond o bac e ial coloniza ion (G andclâmen e al.,
2016). The hos s in e e e wi h he small molecule-dependen bac e ial
communica ion h ough enzyma ic deg ada ion o he au oinduce ,
blocking au oinduce p oduc ion, o i s ecep ion o con ol
popula ion-dependen beha io s like coloniza ion, bio ilm o ma ion,
and pa hogenesis (Ki an e al., 2017; Mukhe jee and Bassle , 2019). In
he Cnida ian Hyd a, he au oinduce signaling molecule
3-oxo-homose ine lac one has been shown o becon e ed in o he
inac i e 3-hyd oxy coun e pa by a hos -de i ed oxido educ ase
allowing hos coloniza ion o he main colonize Cu ibac e sp.
(Pie schke e al., 2017).
The Cnida ian A. au i a ha bo s a highly di e se and dynamic
mic obio a speci ic o he animal, he di e en sub-popula ions, and
li e s ages (Weiland-B äue e al., 2015a). In he absence o he speci ic
mic obial communi y, he i ness o A. au i a was signi ican ly
comp omised, and no ably, asexual ep oduc ion was almos hal ed
(Weiland-B äue e al., 2020a). This mic obial impac is c ucial a he
polyp li e s age be o e en e ing he p ocess o asexual o sp ing
p oduc ion (s obila ion) o ensu e a no mal p ogeny ou pu (Jensen
e al., 2023). Mo eo e , in A. au i a, h ee p o eins in e e ing wi h
bac e ial QS we e iden i ied (Weiland-B äue e al., 2019). Incuba ion
o na i e animals wi h po en ially pa hogenic bac e ia induced he
exp ession o he iden i ied QQ-ORFs, s ongly sugges ing a hos
de ense s a egy.
Despi e he g owing knowledge abou he impac o mic obes on
he hos and he undamen al s a egies o he hos o espond,
esea ch o unde s and how mic obiomes in luence hos gene
exp ession is s ill in i s in ancy (Nichols and Da enpo , 2021). Many
s udies o model o ganisms and humans demons a ed an in e linkage
be ween he mic obiome and he hos ’s gene exp ession. Howe e , he
di ec ion o causali y mainly emained unanswe ed (Nichols and
Da enpo , 2021). Compa ing con en ional (mic obiome-con aining)
o ge m- ee sys ems is one way o assess whe he he mic obiome
plays a causa i e ole in egula ing gene exp ession (Bäckhed e al.,
2012; Al-Asmakh and Zadjali, 2015; Fu e al., 2017; Pie e, 2022).
Genome-wide ansc ip omic analyses a e now ou inely used o
quan i y he changing le els o each ansc ip unde di e en
condi ions (Conesa e al., 2016).
In he p esen s udy, weaimed o de e mine he in luence o he
associa ed mic obio a on A. au i a’s gene exp ession. A e mic obiome
manipula ion (by an ibio ic ea men o bac e ial challenge), RNA
was ex ac ed om polyps, ollowed by RNA-Seq and a de no o
ansc ip ome assembly. Gene on ology ca ego ies and gene exp ession
pa e ns we e analyzed o elucida e how A. au i a ecognizes and
esponds o he manipula ion o i s na i e mic obiome and he
p esence o po en ial pa hogens. A pa icula ocus was on hos -
de i ed QQ ac i i ies as an addi ional po en ial de ense s a egy.
2. Ma e ials and me hods
2.1. Au elia au i a polyp husband y
Husband y is desc ibed in de ail by Weiland-B äue e al. (2015a,
2020a). B ie ly, polyps o he sub-popula ion No h A lan ic (Rosco ,
F ance) we e kep in he lab in 2-li e plas ic anks in 3% a i icial
seawa e (ASW) ( opical sea sal s; T opic Ma in). Polyps we e ed
wice a week wi h eshly ha ched A emia salina (HOBBY, G a scha -
Gelsdo , Ge many).
2.2. Reduc ion o he na i e Au elia au i a
polyp mic obio a by an ibio ics
Single na i e polyps we e placed in 48 well mul iwell pla es in
1 mL ASW supplemen ed wi h an an ibio ic mix u e (P o asoli’s
an ibio ic mix u e wi h inal concen a ions o 360,000 U/L penicillin
G, 1.5 mg/L chlo amphenicol, 1.8 mg/L neomycin, and 9,000 U/L
polymyxin B; all componen s om Ca l Ro h, Ka ls uhe, Ge many).
No ood was p o ided du ing he an ibio ic ea men . The educ ion
and consequen change o he mic obio a we e es ed by pla ing a
single homogenized polyp (10 eplica es) on Ma ine Bouillon aga
pla es (Ca l Ro h, Ka ls uhe, Ge many). Pla es we e incuba ed o
5 days a 20°C. Colony o ming uni s (c u) we e calcula ed, and an
87 ± 9% educ ion pe polyp was de e mined.
2.3. Bac e ial g ow h condi ions and
mic obial challenge o polyps
Bac e ia (Pseudoal e omonas espejiana GenBank accession No.
MK967174, and Vib io anguilla um GenBank accession No.
MK967055) o he mic obial challenge we e isola ed om A. au i a
polyps (Weiland-B äue e al., 2020b). S ains we e g own in Ma ine
Bouillon (MB; Ca l Ro h, Ka ls uhe, Ge many) a 30°C and 120 pm
o op ical u bidi y a 600 nm o 0.8. Klebsiella oxy oca M5aI (DSM No.
Weiland-B äue e al. 10.3389/ micb.2023.1183627
F on ie s in Mic obiology 03 on ie sin.o g
7342) was simila ly g own in Lu ia-Be ani (LB) medium. Bac e ial
cell numbe s we e de e mined using a Neubaue coun chambe
(Assis an , Sondheim o de Röhn, Ge many). Pools o 20 na i e
A. au i a polyps we e sepa a ed in 6-well mul iwell pla es (G eine ,
K emsmüns e , Aus ia) in 4 mL 3% ASW a e washing hem wice
wi h s e ile ASW. A pool o 20 na i e A. au i a polyps was incuba ed
wi h 108 cells/mL (in 4 mL) o he espec i e s ain a 20°C o 30 min.
Nex , polyps we e washed wice wi h s e ile ASW o emo e he
bac e ia and used o isola e o al RNA.
2.4. Expe imen al design o ansc ip ome
analysis
The No h A lan ic sub-popula ion polyps we e gene a ed om a
single mo he polyp by clonal budding. Pools o 20 daugh e polyps
we e sepa a ed in o 6-well pla es in 4 mL 3% ASW. Polyps we e kep
in i e condi ions wi hou ood supply: 1. na i e polyps wi hou
ea men , 2. an ibio ic (AB)- ea ed polyps, 3. na i e polyps
challenged wi h 108 cells/mL Klebsiella oxy oca M5aI, 4. na i e polyps
challenged wi h 10
8
cells/mL Pseudoal e omonas espejiana, and 5.
na i e polyps challenged wi h 10
8
cells/mL Vib io anguilla um
(Figu e 1). Th ee eplica es (3 × 20 polyps) we e used o each
condi ion. Fo he bac e ial challenges, 20 polyps in 4 mL ASW we e
supplemen ed wi h 4×108 cells and incuba ed o 30 min (Figu e1).
The o al RNA was ex ac ed om each pool o polyps.
2.5. RNA isola ion
To al RNA o a pool o 20 A. au i a polyps was isola ed wi h an
adap ed p o ocol o Gold e al. (2019). In mo e de ail, polyps we e
washed h ee imes wi h s e ile ASW ( o emo e an ibio ic esidues)
and homogenized wi h a mo o ized pes le. RiboLock RNase Inhibi o
(40 U/μL, The mo Fishe Scien i ic, Wal ham/Massachuse s,
Uni ed S a es), 200 μL lysis solu ion (100 mM T is/HCl, pH 5.5,
10 mM disodium EDTA, 0.1 M NaCl, 1% SDS, 1%
ß-me cap oe hanol), and 2 μL P o einase K (25 mg/mL, The mo
Fishe Scien i ic, Wal ham/Massachuse s, Uni edS a es) we e added
o he homogena e and incuba ed o 10 min a 55°C. Chilled
solu ions we e added wi h 5 μL o 3 M sodium ace a e (pH 5.2) and
250 μL phenol-chlo o o m-isoamyl alcohol (25:24:1) and incuba ed
o 15 min on ice p io o 15 min cen i uga ion a 12,000 x g a
4°C. The uppe phase was mixed wi h 1 olume 2-p opanol, and
p ecipi a ion occu ed a -80°C o e nigh . The p ecipi a e was
cen i uged o 15 min a 12,000 x g a 4°C. The pelle was washed
wice wi h 70% e hanol be o e he ai -d ied pelle was dissol ed in
25 μL RNase- ee wa e . DNA con amina ions we e emo ed wi h
Tu bo DNA- ee DNase (The mo Fishe Scien i ic, Wal ham/
Massachuse s, Uni edS a es). The RNA quali y and quan i y we e
assessed by NanoD op1000 (The mo Fishe Scien i ic, Wal ham/
Massachuse s, Uni edS a es) and 1.5% aga ose gel elec opho esis,
and he cDNA lib a y was p epa ed wi h DNA- ee hos RNA
(500 ng) using he T uSeq S anded mRNA Lib a y P epa a ion ki .
The lib a y was sequenced wi h he Nex Seq500 Sys em (Illumina,
San Diego/ Cali o nia, Uni edS a es).
2.6. T ansc ip ome analysis
The aw eads we e immed wi h T immoma ic (Bolge e al.,
2014) o emo e bad-quali y eads and he adap e s. A de no o
assembly wi h he immed sequences was conduc ed wi h he T ini y
package 2.8.4 (G abhe e al., 2011a,b). The quali y o he de no o
assembly was assessed wi h se e al pa ame e s, such as he N50 alue
and he pe cen age o he assembly-mapped eads, which was
calcula ed wi h Bow ie2. Finally, Benchma king Uni e sal Single-
Copy O hologs (BUSCO V2/3) agains me azoan casse es (Simão
e al., 2015) was used o e alua e he comple eness o he assembly
ega ding he co e genes ound in me azoans.
FIGURE1
Expe imen al se up. RNA ex ac ion wi h subsequen RNAseq and analysis was conduc ed on a pool o 20 polyps (3 biological eplica es). Polyps we e
kep unde na i e and mic obiome-manipula ed condi ions. C ea ed wi h BioRende s.
Weiland-B äue e al. 10.3389/ micb.2023.1183627
F on ie s in Mic obiology 04 on ie sin.o g
A Blas x o he ansc ip ome assembly was execu ed agains
he Swiss-P o da abase o me azoans. Fo he gene exp ession
analysis, eads we e mapped o he e e ence assembly wi h
Bow ie2 (Langmead and Salzbe g, 2012). T ansc ip quan i ica ion
was pe o med wi h RSEM (Li and Dewey, 2011). The di e en ial
gene exp ession (DGE) analysis was done wi h edgeR (Robinson
e al., 2010; McCa hy e al., 2012). Pai wise compa isons be ween
all expe imen al condi ions we e pe o med using FDR ≤ 0.001
and 2- old changes as s a is ical pa ame e s. Fu he mo e, GO
en ichmen analysis was conduc ed using a Fishe ’s Exac Tes in
Blas 2GOPRO (Conesa e al., 2005) wi h a alue o p h eshold o
≤0.05. He e, he o al anno a ion ile o he e e ence
ansc ip ome was used as he “ e e ence da ase ,” whe eas he
up egula ed genes in each condi ion se ed as he “ es da ase .”
Based on sequence dep h and quali y, only wo ou o h ee
biological eplica es we e used o he gene exp ession analysis o
he condi ions o na i e polyps and na i e polyps unde he
bac e ial challenges. T ansc ip ome da a is deposi ed unde he
BioP ojec ID PRJNA938117. Gene exp ession pa e ns in he
ansc ip ome da a se we e e i ied wi h qRT-PCRs o he
housekeeping gene elonga ion ac o 1 (EF1) (Weiland-B äue
e al., 2019; Jensen e al., 2023). EF1 showed simila ends in gene
exp ession o all ea men s when compa ing ansc ip ome da a
and qRT-PCR (Table1). Mo eo e , he p e iously published QQ
genes aaqq1, aaqq2, and aaqq3 o A. au i a (Weiland-B äue
e al., 2019) we e iden i ied in he ansc ip ome da a se . Thei
exp ession p o iles ha e been s udied using qRT-PCR in na i e,
an ibio ic- ea ed, and K. oxy oca- ea ed polyps [see s udy
(Weiland-B äue e al., 2019)]. Gene exp ession p o iles we e
simila in ansc ip ome da a and qRT-PCR, which we a ed and
conside ed as in e nal con ol o ou newly ob ained da a se and
i s alidi y (Table1).
2.7. Quo um quenching assay
Quo um quenching (QQ) assays using he epo e s ains
AI1-QQ.1 and AI2-QQ.1 we e pe o med wi h cell- ee
supe na an s and cell ex ac s o EST clones om he A. au i a EST
lib a y as desc ibed (Weiland-B äue e al., 2015b). B ie ly, QQ
sc eening pla es we e p epa ed wi h LB aga con aining 0.8%
aga a 50°C supplemen ed wi h inal concen a ions o
100 μM N-(β-ke ocap oyl)-l-homose ine lac one (3-oxo-C6-HSL)
(Sigma-Ald ich, Munich, Ge many), 100 μg/mL ampicillin, 30 μg/
mL kanamycin, and 10% ( ol/ ol) exponen ially g owing cul u e o
he epo e s ain AI1-QQ.1. Simila ly, AI-2 QQ sc eening pla es
we e p epa ed wi h inal concen a ions o 50 mM 4-hyd oxy-5-
me hyl-3- u anone (Sigma-Ald ich, Munich, Ge many), 100 μg/mL
ampicillin, 30 μg/mL kanamycin, and 5% ( ol/ ol) exponen ially
g owing cul u e o he epo e s ain AI2-QQ.1. LB aga pla es we e
coa ed wi h he aga mix u e. A e 10 min, 5 μL o he es
subs ances we e applied, ollowed by o e nigh incuba ion a
37°C. QQ ac i i ies we e isualized by he g ow h o he espec i e
epo e s ain. P epa a ion o cell ex ac s and cell- ee cul u e
supe na an s was conduc ed wi h 5 mL o e nigh cul u es o EST
clones g own a 37°C and 120 pm. Cells we e ha es ed by
cen i uga ion a 7,000 × g, and he cul u e supe na an was
subsequen ly il e ed using 0.2-μm cen i ugal il e uni s (Ca l
Ro h, Ka ls uhe, Ge many). The cell ex ac was p epa ed om he
cell pelle using he Geno/G inde 2000 (BT&C/OPS Diagnos ics,
B idgewa e , NJ). Cell ex ac s we e il e ed h ough 0.2-μm
il e uni s.
Following he manu ac u e ’s p o ocol, he plasmids o
iden i ied QQ-ac i e single EST clones we e pu i ied using he P es o
Mini Plasmid ki (GeneAid, New Taipeh Ci y, Taiwan). The
espec i e inse s we e Sange sequenced a he Ins i u e o Clinical
Molecula Biology in Kiel wi h he p ime se T7_P omo e
(5′-TAATACGACTCACTATAGGG-3′) and T7_Re e se (5’-TAGTT
ATTGCTCAGCGGTGG-3′). Sequences a e deposi ed a NCBI
GenBank unde Accession Nos. OQ581004- OQ581041.
3. Resul s
A ansc ip omics app oach was applied o gain insigh s in o
he esponse o A. au i a polyps o manipula ion o i s na i e,
associa ed mic obio a, wi h a pa icula ocus on quo um sensing
in e e ence as a po en ial hos de ense s a egy. In gene al, pools
o 20 polyps we e ea ed and p ocessed oge he in he
expe imen s, each wi h h ee biological eplica es using he
ollowing ea men s: na i e, an ibio ic- ea ed polyps esul ing in
87 ± 9% educed mic obial cells pe polyp (c u/polyp), and na i e
polyps challenged wi h Klebsiella oxy oca M5aI, Pseudoal e omonas
espejiana, o Vib io anguilla um (2 × 10
7
cells/polyp, Figu e1). The
TABLE1 Compa ison o ansc ip ome da a sco es and qRT-PCR C alues o e i ica ion.
da a o igin ORF na i e AB- ea ed K. oxy oca V. anguilla um P. espejiana
ansc ip ome da a EF1 5.6 5.3 2.6 1.9 1.1
aaqq1 1.1 0.7 0.1 nd nd
aaqq2 4.1 1.6 2.9 nd nd
aaqq3 3.9 2.5 0 nd nd
qRT-PCR EF1 20.4 20.1 13.6 14.2 11.3
aaqq1 11.9 8.6 5.5 nd nd
aaqq2 14.0 6.9 8.2 nd nd
aaqq3 6.3 6.9 5.4 nd nd
Fo he housekeeping gene elonga ion ac o 1 (EF1), he mean aw C alue o h ee biological and h ee echnical eplica es is shown. Values o QQ-ORFs ep esen he mean ΔC alue
( h ee biological and h ee echnical eplica es), no malized wi h he housekeeping gene ac in (Weiland-B äue e al., 2019).
nd, no de ec ed.
Weiland-B äue e al. 10.3389/ micb.2023.1183627
F on ie s in Mic obiology 05 on ie sin.o g
espec i e pools we e used o o al RNA ex ac ion ollowed by
RNAseq and ansc ip ome analysis.
3.1. S a is ics o he de no o ansc ip ome
assembly
The RNAseq app oach o e all esul ed in 167,269,257 aw eads.
A e quali y imming, 145,746,530 eads (87.1% o he o al eads)
emained o u he analysis and we e used o he de no o
ansc ip ome assembly (Supplemen a y Table S1A). The de no o
assembly esul ed in 213,897 ansc ip s and 160,700 genes
(Supplemen a y Table S1A) wi h an N50 alue o 1,170
(Supplemen a y Table S1B). 94.25% o eads we e success ully aligned
back o he assembly, while he comple eness o genes was 96.42%
acco ding o he BUSCO sco e o me azoans
(Supplemen a y Table S1B). 30% (597,796 ansc ip s) o he assembly
e ealed an anno a ion agains he Swiss-P o da abase o me azoans
(Supplemen a y Table S2).
3.2. Manipula ion o i s mic obiome a ec s
A. au i a’s ansc ip ome
T ansc ip ome analysis iden i ied i e sepa a ed clus e s
co esponding o he di e en condi ions, while biological eplica es
o a condi ion clus e oge he (Figu e2). I should beno ed ha h ee
eplica es we e only analyzed o an ibio ic (AB)- ea ed polyps, as all
o he condi ions esul ed in an unaccep able low sequence dep h o
one eplica e. No ably, wo clus e s we e iden i ied wi hin he
hie a chical clus e ing o ansc ip s. He e, na i e and AB- ea ed
condi ions yielded a emendous di e ence. AB- ea ed polyps
showed a massi e educ ion o he mic obial load (by 87 ± 9%),
assuming a igo ous change in he abundance and di e si y o
mic obial colonize s. Bac e ia-challenged polyps showed a mix u e o
na i e and AB- ea ed exp ession pa e ns (Figu e2). In mo e de ail,
he compa ison o na i e and AB- ea ed polyps esul ed in he highes
numbe o di e en ially exp essed genes (22,073 genes), wi h 10,451
up egula ed and 11,622 down- egula ed genes in AB- ea ed
compa ed o na i e polyps (Supplemen a y Table S3A). Acco ding o
FIGURE2
Holis ic exp ession pa e ns o A. au i a polyps. Hea map including he Di e en ial Exp essed (DE) genes in all he pai wise compa isons o he na i e,
AB- ea ed, and bac e ia-challenged polyps, including eplica es.

Weiland-B äue e al. 10.3389/ micb.2023.1183627
F on ie s in Mic obiology 06 on ie sin.o g
FIGURE3
GO en ichmen analysis o di e en ially exp essed genes o na i e and AB- ea ed A. au i a polyps. (A,C) Ba plo s wi h GO-en iched gene ca ego ies
when compa ing na i e and Ab- ea ed polyps. Ba plo s indica e he p opo ion (%) o DE gene sequences in ol ed in (A) up egula ed and (C) down-
egula ed GO-en iched ca ego ies compa ed o he e e ence (de no o assembly). (B,D) Hea map o up egula ed and down- egula ed genes wi h he
compa ison o na i e and AB- ea ed polyps ela ed o (B) immune esponse and apop osis (D) and common physiological p ocesses. Rela i e
exp ession le el inc ease om blue o ed.
he GO en ichmen analysis (Conesa e  al., 2005), he 10,451
up egula ed genes we e o e ep esen ed in p ocesses ela ed o
in lamma ion and immune esponse (e.g., acu e in lamma o y
esponse o an igenic s imuli, MAP-kinase ac i i y, cy okine
p oduc ion, and neu ophil cell ac i a ion) (Figu e 3A;
Supplemen a y Table S4A). Genes in ol ed in immune esponse and
apop osis included, e.g., Caspases, Apop osis egula o s, In e e ones,
and Toll-like ecep o s (Figu e3B; Supplemen a y Tables S3A, S4A).
On he con a y, down- egula ed genes in AB- ea ed polyps we e
en iched o p ocesses ela ed o de elopmen , mo phogenesis,
ep oduc ion, s imuli esponse, and signaling (Figu es 3C,D;
Supplemen a y Tables S3A, S4A).
When compa ing exp ession pa e ns be ween na i e and
bac e ia-challenged polyps, di e ences based on he bac e ial species
used in each challenge we e e ealed (Figu e4). The compa ison o
na i e polyps compa ed o hose challenged wi h V. anguilla um ga e
he highes numbe o di e en ially exp essed (DE) genes (7,193
genes, Figu e 4A; Supplemen a y Table S3B), ollowed by polyps
challenged wi h K. oxy oca (6,974 DE genes, Figu e 4A;
Supplemen a y Table S3C), and P. espejiana (2,721 genes, Figu es4A;
Supplemen a y Table S3D). Weobse ed 2,210 genes we e commonly
up egula ed among he ea men s compa ed o na i e polyps
(Figu e4A; Supplemen a y Table S3E). A he same ime, 52 genes
we e join ly down egula ed (Figu e4B; Supplemen a y Table S3E).
Weiland-B äue e al. 10.3389/ micb.2023.1183627
F on ie s in Mic obiology 07 on ie sin.o g
Genes ela ed o biological p ocesses, such as de ense, immune and
in lamma o y esponses, and he egula ion o apop o ic p ocesses,
we e up egula ed in bac e ia-challenged polyps (Figu e 4B;
Supplemen a y Table S3E). In con as , genes ela ed o o ganism
de elopmen , cell cycle p ocesses, and cy oskele on o ganiza ion we e
down- egula ed in challenged polyps (Figu e 4B;
Supplemen a y Table S3E). Mo eo e , exclusi ely up egula ed and
down- egula ed genes we e iden i ied o each bac e ial challenge
(Figu e4). 1,319 genes we e exclusi ely up egula ed in V. anguilla um-
challenged polyps (Figu e4A le panel; Supplemen a y Table S3F),
1,020 genes we e exclusi ely up egula ed in polyps challenged wi h
K. oxy oca (Figu e4A le panel; Supplemen a y Table S3G), and 160
genes exclusi ely up egula ed in P. espejiana-challenged polyps
(Figu e4A le panel; Supplemen a y Table S3H). Simila ly, exclusi ely
down- egula ed genes we e iden i ied (Figu e 4A, igh panel).
Analyzing he GO-en iched ca ego ies o each species
(Supplemen a y Table S4B–D), we ound ca ego ies common among
all ea men s (Supplemen a y Table S4E) as well as ca ego ies p esen
in wo di e en ea men s o only in one (Figu e 4B;
Supplemen a y Table S4B–D). Focusing on genes exclusi ely
up egula ed a e each species-speci ic challenge, weindeed obse ed
ha he same GO ca ego ies, e.g., immune esponse, MAPK signal
ansduc ion, au ophagy, and DNA epai , we e en iched o di e en
deg ees, hough ep esen ed by a ious genes (depic ed in Figu e5).
3.3. Bac e ial challenge o polyps a ec s
hos quo um quenching
Recen s udies e ealed ha in e e ing wi h Qu om sensing,
so-called quo um quenching (QQ), migh be a undamen al,
addi ional in e -phylum in e ac ion o main ain me ao ganismal
homeos asis. Consequen ly, weaimed o iden i y hos -de i ed QQ
ac i i ies. An exp essed sequence ag (EST) lib a y om A. au i a
FIGURE4
GO en ichmen and di e en ial exp ession analysis o genes di e gen ly ansc ibed in na i e compa ed o bac e ia-challenged polyps. (A) Venn
diag am indica ing he genes ha a e commonly and exclusi ely up egula ed (le panel) o down- egula ed ( igh panel) in he di e en bac e ia-
challenged condi ions. (B) Hea map o up-and down egula ed genes in na i e compa ed o bac e ia-challenged polyps. Rela i e exp ession le el
inc eases om blue o ed.
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F on ie s in Mic obiology 08 on ie sin.o g
FIGURE5
Exclusi ely up egula ed genes in na i e s. bac e ia-challenged polyps. Bubble plo showing he deg ee o en ichmen pe ca ego y o exclusi ely
up egula ed genes in each bac e ia-challenged compa ed o na i e condi ions.
polyps-de i ed mRNA was cons uc ed in E. coli SoluBL21 (Ladewig
e al., 2023). The lib a y consis ed o 29,952 clones wi h an inse ion
e iciency o app ox. 98% and an a e age inse size o 1.46 kbp,
esul ing in 43 Mbps cloned hos ansc ip ome, co esponding o an
es ima ed 11.4% co e age [calcula ed A. au i a genome size 376 Mbps
(Gold e  al., 2019)]. The A. au i a EST lib a y was successi ely
sc eened o QQ ac i i ies owa d acyl-homose ine lac ones (AHL)
and au oinduce -2 (AI-2) in cell- ee cell ex ac s and cul u e
supe na an s o he EST clones using es ablished E. coli-based epo e
sys ems (Weiland-B äue e al., 2015b). O e all, 37 ou o 29,952 EST
clones we e iden i ied as QQ ac i e (Table2). P edominan ly, QQ
ac i i ies agains he G am-nega i e signaling molecule AHL we e
de ec ed. Al hough hose hos -de i ed QQ ac i i ies we e iden i ied
in a unc ional sc een, hei biological ac i i ies in i o and hei
unc ions in A. au i a ha e o beexplo ed. In a i s a emp , plasmid
inse ions o QQ-ac i e single EST clones we e sequenced, and
sequence da a subsequen ly checked o homologies in he A. au i a
ansc ip ome assembly. All QQ-con e ing sequences we e iden i ied
in he ansc ip ome assembly wi h homologies anging om 83 o
100% (Supplemen a y Table S5). Fu he mo e, public da abases
(NCBI, UniP o , PFAM) we e used o homology p edic ions and
anno a ion (Supplemen a y Table S5). Unexpec edly, se e al
QQ-ORFs showed homologies o highly conse ed ibosomal
p o eins, sugges ing a moonligh ing unc ion o hose p o eins (Je e y,
2003; Singh and Bhalla, 2020). Secondly, analyzing ansc ip ion le els
o hose iden i ied QQ-ORFs in he gene a ed RNAseq da a se (see
abo e) allowed i s insigh s in o hei ansc ip ional egula ion in
esponse o he espec i e ea men s, which migh suppo p edic ing
hei biological ole. Rela i e exp ession o he 37 QQ-con e ing
genes was calcula ed o mic obiome-manipula ed e sus na i e
polyps. Hie a chical clus e ing obse ed ou clus e s o exp ession
p o iles. The i s clus e ep esen s QQ genes simila ly exp essed in
mic obiome-manipula ed polyps compa ed o na i e ones (Figu e6,
igh column highligh ed in g ey). The second clus e o genes showed
dec eased exp ession in all ea men s, excep when challenged wi h
P. espejiana (Figu e6, igh column ligh o ange clus e ). The hi d
Weiland-B äue e al. 10.3389/ micb.2023.1183627
F on ie s in Mic obiology 09 on ie sin.o g
clus e summa izes hose genes wi h inc eased exp ession ega dless
o he ype o manipula ion (Figu e6, igh column da k o ange
clus e ). The ou h clus e includes hose genes wi h an inc eased
exp ession a e adding po en ial pa hogens bu educed exp ession
when polyps we e AB- ea ed (Figu e6, igh column g een clus e ).
No ably, P. espejiana p ima ily showed inc eased exp ession o
QQ-ORFs; hus, hie a chal clus e ing showed he highes dissimila i y
compa ed o he o he ea men s.
4. Discussion
In he p esen s udy, we ob ained he i s insigh s in o he
ansc ip omic esponse o A. au i a o mic obiome manipula ion.
The manipula ions o he polyp mic obiome included a massi e
educ ion o iable bac e ial cells (87% educ ion) due o applying a
b oad-spec um an ibio ics mix u e. The mix u e con ained Penicillin,
Polymyxin B, Chlo amphenicol, and Neomycin (P o asoli and
Pin ne , 1980; Liu e al., 2017a; KleinJan e al., 2022), and has been
shown o only pa ially elimina e an en i e bac e ial communi y
(Azma e al., 2010; Liu e al., 2017a). Weassume ha by d as ically
educing he colony- o ming uni s by 87%, he di e si y and
abundance o communi y membe s on he polyp a e c ucially
changed. Consequen ly, he polyp was con on ed wi h he loss o
ce ain communi y membe s and a d as ic change in he ela i e
abundance o emaining ones. This ex eme change in mic obiome
composi ion esul ed in he up- egula ion o a ious immune
esponse mechanisms. Pa e n ecogni ion ecep o s (PRRs) like Toll-
like ecep o s and Ficolins we e up egula ed, inducing he i s line o
de ense (Figu e3). Fu he , Caspases we e en iched in AB- ea ed
polyps, po en ially main aining homeos asis h ough egula ing cell
dea h and in lamma ion (McIlwain e  al., 2013). Con a ily, all
p ocesses in ol ed in mo phogenesis and de elopmen , hus no
essen ial o su i al, we e down- egula ed in AB- ea ed polyps
(Figu e3).
Fu he mo e, he na i e mic obiome was a ec ed by challenging
he polyps wi h h ee di e en bac e ia in high cell numbe s: (i)
K. oxy oca has no been de ec ed in any li e s age o A. au i a and hus
ep esen s a non-na i e bac e ium (Weiland-B äue e  al., 2015a,
2019); (ii) V. anguilla um, an oppo unis ic pa hogen o a ious
in e eb a es and e eb a es, has been isola ed om an A. au i a
polyp (Aus in, 2010; F ans e al., 2011; Weiland-B äue e al., 2015a,b);
and (iii) P. espejiana was ini ially isola ed om seawa e bu was also
ound in high abundance associa ed wi h all li e s ages o A. au i a,
hus ep esen ing a na i e bac e ium (Isnanse yo and Kamei, 2009;
Weiland-B äue e  al., 2015a,b). Due o bac e ial challenges,
weobse ed he up- egula ion o de ense, immune, and in lamma o y
esponses, and apop osis ega dless o he bac e ial species. Based on
his inding, wehypo hesize ha he hos ecognized he non-na i e
and na i e bac e ia as a po en ial h ea , a leas when p esen in such
high cell numbe s. The immune sys em o A. au i a likely esponded
wi h he ou p ima y inna e immune sys em unc ions as
demons a ed o o he Cnida ians and al eady pa ly obse ed o
AB- ea ed polyps (Mille e  al., 2007; Pa isi e  al., 2020). Fi s ,
immune ecogni ion occu ed by PRRs, like Fucolec ins, Ficolins, and
NACHT-con aining domain p o eins, binding o bac e ial MAMPs/
PAMPs (Figu es4, 5). Subsequen ly, a ious ansc ip ion ac o s om
he NF-κB amily we e ac i a ed (Figu es4, 5). In acellula signaling
cascades (MAPK, in e leukin, cy okines) led o a ge gene
ansc ip ion o elimina e he h ea and mi iga e sel -ha m (Figu es4,
5). Las ly, au ophagy, DNA epai , and p og ammed cell dea h we e
up egula ed a e bac e ial challenge (Figu es4, 5). Ou obse a ions
TABLE2 Au elia au i a-de i ed quo um quenching ac i i ies de i ed om
an exp essed sequence ag (EST) lib a y.
QQ-ORF O iginal clone
designa ion
QQ ac i i y
AHL AI-2
QQ_Aa_1 115/H7 x x
QQ_Aa_2 118/E4 x –
QQ_Aa_3 118/G4 x x
QQ_Aa_4 127/C7 x x
QQ_Aa_5 164/A6 x –
QQ_Aa_6 164/E2 x x
QQ_Aa_7 184/A11 x –
QQ_Aa_8 184/B9 x –
QQ_Aa_9 202/G6 x –
QQ_Aa_10 208/E9 x –
QQ_Aa_11 208/A3 x –
QQ_Aa_12 213/E2 x –
QQ_Aa_13 213/F2 x –
QQ_Aa_14 217/H4 x –
QQ_Aa_15 221/C11 x x
QQ_Aa_16 284/H11 x –
QQ_Aa_17 115/A1 x –
QQ_Aa_18 115/A11 x x
QQ_Aa_19 115/G1 x –
QQ_Aa_20 118/F5 x x
QQ_Aa_21 164/B2 x –
QQ_Aa_22 164/C8 x –
QQ_Aa_23 164/D1 x –
QQ_Aa_24 164/D6 x –
QQ_Aa_25 164/F2 x –
QQ_Aa_26 164/G1 x –
QQ_Aa_27 127/A8 x x
QQ_Aa_28 127/A10 x –
QQ_Aa_30 127/C12 x –
QQ_Aa_31 127/F8 x –
QQ_Aa_32 184/A6 x –
QQ_Aa_33 213/B7 x –
QQ_Aa_34 223/E7 x –
QQ_Aa_35 270/C5 x x
QQ_Aa_36 273/A1 x –
QQ_Aa_37 273/F12 x x
QQ_Aa_38 284/A10 x x
Func ionally iden i ied QQ-ORFs a e lis ed wi h hei QQ ac i i y in he cell- ee cell ex ac
(CE) and cul u e supe na an (SN) agains acyl-homose ine lac ones (AHL) and
au oinduce -2 (AI-2) wi h hei po en ial anno a ions; x, ac i i y; −, no ac i i y.