First insights into the Aurelia aurita transcriptome response upon manipulation of its microbiome

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. Weiland-B äue e al. 10.3389/ micb.2023.1183627 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.