{"paper_id":"3c2f3858-0cc0-4bad-a286-11a56c112fc2","body_text":"1 \n \nMicrobial education plays a crucial role in harnessing the 1 \nbeneficial properties of microbiota for infectious disease 2 \nprotection in Crassostrea gigas 3 \nLuc Dantan 1*, Prunelle Carcassonne 1, Lionel D égremont2, Benjamin Morga 2, Marie-4 \nAgnès Travers3, Bruno Petton 4, Mickael Mege 2, Elise Maurouard 2, Jean -François 5 \nAllienne1, Gaëlle Courtay3, Océane Romatif3, Juliette Pouzadoux3, Raphaël Lami5, 6 \nLaurent Intertaglia6, Yannick Gueguen7, Jeremie Vidal-Dupiol3, Eve Toulza 1, Céline 7 \nCosseau1* 8 \n1 IHPE, Univ. Montpellier, CNRS, IFREMER, Univ. Perpignan Via Domitia, Perpignan , 9 \nFrance 10 \n2 Ifremer, ASIM, F- 17390 La Tremblade, France 11 \n3 IHPE, Univ. Montpellier , CNRS, IFREMER, Univ. Perpignan Via Domitia, Montpellier, 12 \nFrance 13 \n4 Univ Brest, Ifremer, CNRS, IRD, LEMAR, F-29280 Plouzané, France 14 \n5 Sorbonne Université, CNRS, Laboratoire de Biodiversité et Biotechnologies Microbiennes, 15 \nObservatoire Océanologique de Banyuls -sur-Mer, Avenue Pierre Fabre, 66650, Banyuls -sur-16 \nMer, France  17 \n6 Sorbonne Université, CNRS, Fédération de Recherche, Observatoire Océanologique, 66650 18 \nBanyuls-sur-mer, France 19 \n7 MARBEC, Univ Montpellier, CNRS, Ifremer, IRD, Sète, France 20 \n*Corresponding authors 21 \nluc.dantan@gmail.com 22 \nceline.cosseau@univ-perp.fr  23 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted May 17, 2024. ; https://doi.org/10.1101/2024.05.17.594303doi: bioRxiv preprint \n\n2 \n \nAbstract 24 \nBackground: Recently, the frequency and severity of marine diseases have increased  in 25 \nassociation with global changes, and molluscs of economic interest are particularly concerned. 26 \nAmong them, the Pacific oyster (Crassostrea gigas) production faces challenges from several 27 \ndiseases such as the Pacific Oyster Mortality Syndrome (POMS) or vibriosis. Various strategies 28 \nsuch as  genetic selection or immune priming  have been developed to fight some of these 29 \ninfectious diseases. The microbial education, which consist of  exposing the host immune 30 \nsystem to beneficial microorganisms during early life stages is a promising approach against 31 \ndiseases. This study explores the concept of microbial education using controlled and pathogen-32 \nfree bacterial communities and  assesses its protective effects against POMS and Vibrio 33 \naestuarianus infections, highlighting potential applications in oyster production.  34 \nResults: We demonstrate that it is possible to educate the oyster immune system by adding 35 \nmicroorganisms during the larval stage. Adding culture based bacterial mixes to larvae protects 36 \nonly against the POMS disease while adding whole microbial communities from oyster donors 37 \nprotects against both POMS and vibriosis . The efficiency of the immune protection depends 38 \nboth on oyster origin and on the composition of the bacterial mixes  used for exposure . No 39 \npreferential protection was observed when the oysters were stimulated with their sympatric 40 \nstrains. We further show that the added bacteria were not maintained in the oyster microbiota 41 \nafter the exposure , but this bacterial addition induced long term changes in the microbiota 42 \ncomposition and oyster immune gene expression.  43 \nConclusion: Our study reveals successful immune system education of oysters by introducing 44 \nbeneficial micro-organisms during the larval stage. We improved the  long-term resistance of 45 \noysters against critical diseases (POMS  disease and Vibrio aestuarianus infections) 46 \nhighlighting the potential of microbial education in aquaculture. 47 \n 48 \nKey words: 49 \nCrassostrea gigas; Microbial education; Oyster holobiont; OsHV-1 µVar; Vibrio aestuarianus  50 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted May 17, 2024. ; https://doi.org/10.1101/2024.05.17.594303doi: bioRxiv preprint \n\n3 \n \nIntroduction: 51 \nThe Pacific oyster Crassostrea gigas (also known as Magallana gigas ) stands as the most 52 \nwidely cultivated oyster species in the world , underpinning a substantial proportion of the 53 \naquaculture industry (Food and Agriculture Organisation 2022). However, the production of C. 54 \ngigas faces significant challenges due to recurring infectious diseases, inflicting high 55 \nmortalities each year (Friedman et al. 2005; Cotter et al. 2010; Pernet et al. 2012; Azéma et al. 56 \n2015). Two prevalent infections - the Pacific Oyster Mortality Syndrome (POMS) caused by 57 \nthe Ostreid herpesvirus type 1 µVar iant (OsHV-1 µVar) and vibriosis initiated by Vibrio 58 \naestuarianus infection - are primarily responsible for these alarming mortalities . POMS is a 59 \ncomplex and polymicrobial disease  which preferentially affects younger oysters and can 60 \ndecimate up to 100% of the spat in French farms (Segarra et al. 2010; Petton et al. 2021) . The 61 \ninfection by OsHV -1 µVar marks a critical step in the progression of POMS, inducing an 62 \nimmunocompromised state in oysters by altering haemocytes physiology  (de Lorgeril et al. 63 \n2018; Petton et al. 2021) . This leads to a dysbiosis of oyster microbiota and results in  64 \ncolonisation by opportunistic bacteria and death of the oyster (de Lorgeril et al. 2018; King et 65 \nal. 2019a; Petton et al. 2021) . On the other hand, V. aestuarianus is another harmful primary 66 \npathogen with chronic mortality reaching a cumulative mortality rate up to 30%. This loss 67 \ninduces important economic consequences since it preferentially infects market size oysters 68 \nwhich have been raised for several years (Azéma et al. 2017; Lupo et al. 2019). 69 \nEfforts to combat these infectious diseases have spawned various approaches based on the 70 \nincreasing knowledge and resources available on oyster s. Genetic selection is a promising 71 \navenue which aims at selecting pathogen -resistant oysters (Dégremont et al. 2015, 2020) . 72 \nHowever, this approach exhibits limitations such as the potential selection of trade -offs which 73 \ncould counter select traits important for the commercial value of C. gigas . Moreover, t he 74 \ndemonstration of the existence of immune priming in C. gigas has opened up a whole new field 75 \nof applications based on the use of viral mimics  (Lafont et al. 2017, 2020; de Kantzow et al. 76 \n2023; Montagnani et al. 2024). However, this innovative approach only protects against POMS 77 \ninfections (Green and Montagnani 2013; Lafont et al. 2017) . A diversity of studies on oyster -78 \nmicrobiota interactions have also opened a new field of investigations consisting in identifying 79 \nbacteria beneficial for their associated host during adverse conditions  (King et al. 2019a; 80 \nClerissi et al. 2020; Delisle et al. 2022; Fallet et al. 2022) . Research on disease prevention in 81 \nmolluscs based on the use of probiotic s has been ongoing for decades but has yet to see 82 \nwidespread applications in farms (Yeh et al. 2020; Takyi et al. 2023, 2024; Muñoz-Cerro et al. 83 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted May 17, 2024. ; https://doi.org/10.1101/2024.05.17.594303doi: bioRxiv preprint \n\n4 \n \n2024). While several pre/probiotic -based methods to mitigate infectious diseases have 84 \ndemonstrated success in shrimp hatcheries  (Swain et al. 2009; Pham et al. 2014; Wen et al. 85 \n2014), their application in oyster farming, particularly in open-sea environments, faces distinct 86 \nchallenges and limitations. Oysters, as filter -feeding organisms, often face complex microbial 87 \ninteractions in their natural habitats  (Lokmer et al. 2016) . Consequently, achieving and 88 \nmaintaining a precise balance of beneficial microorganisms through probiotics addition can be 89 \nchallenging. Additionally, the ir culture in open-sea present limitations in the implementation 90 \nof probiotics.  91 \nThe concept of microbial education, consists in exposing the host immune system to beneficial 92 \nmicroorganisms during early development (Arrieta et al. 2014; Gensollen et al. 2016) . This is 93 \nbecause early life stages represent critical periods of growth and development where the host's 94 \nimmune system is still maturing (Renz et al. 2017). This strategy offers significant advantages 95 \non oysters, as it can confer a protective effect while allowing exposure in hatchery during the 96 \nlarval phase in controlled environments  (Dantan et al. 2024) . Numerous studies have shown 97 \nthat a proper host-microbiota interaction during the early development plays an important role 98 \nin the long term host immune responses in a wide range of  marine organisms (Chung et al. 99 \n2012; Galindo-Villegas et al. 2012; Abt and Artis 2013; Sommer and Bäckhed 2013) . In this 100 \ncontext, Fallet and colleagues  (Fallet et al. 2022)  explored the potential of using wild-101 \nmicrobiota to educate the immune system of C. gigas. Through a ten-day exposure of C. gigas 102 \nlarvae to a whole microbiota from donor oysters, they induced a long -term beneficial effect . 103 \nThe microbiota-exposed oysters exhibited enhanced resistance to OsHV -1 µVar, resulting in 104 \nimproved survival rates compared to non -exposed counterparts. This study underscored the 105 \ncrucial role of microbiota on oyster immune system education, suggesting potential applications 106 \nin commercial hatcheries. However, concerns regarding exposure to hazardous uncontrolled 107 \nmicrobial communities transferred from donor  oysters necessitate a cautious approach as it 108 \nmight contain primary or opportunistic pathogens. Indeed, prior to the recipient larvae exposure 109 \nperformed in Fallet et al. study, the donor oysters were placed in farming area during a non -110 \ninfectious period to allow oysters to capture the maximum diversity of field microorganisms. 111 \nThen, these donor oysters were placed in the rearing tanks during larval development where 112 \nthey transmitted their highly diverse microbial community to the recipient larvae. Although the 113 \ndonor oysters were considered healthy  (Le Roux et al. 2016; Fleury et al. 2020) , the presence 114 \nof undetectable pathogens cannot entirely be excluded 115 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted May 17, 2024. ; https://doi.org/10.1101/2024.05.17.594303doi: bioRxiv preprint \n\n5 \n \nHere, our study aimed to explore the feasibility of microbial education in oyster larvae while 116 \nconsidering and mitigating the risks associated with uncontrolled transfer of hazardous 117 \nmicroorganisms found in wild-microbiota. We investigated whether exposing oyster larvae to 118 \na controlled, pathogen -free bacterial community from donor oysters that had always been 119 \nmaintained in biosecured facilities could confer the protective effects against POMS and V. 120 \naestuarianus infection. Additionally, we examined the feasibility of microbial education using 121 \na reduced synthetic bacterial community composed of cultivable bacteria isolated from disease 122 \nresistant oysters. For this purpose, we developed and tested multi-strain bacterial mixes 123 \noriginating from the same geographical areas as the recipient oyster populations used in this 124 \nstudy. Our comprehensive assays encompassed three distinct oyster populations from the 125 \nAtlantic Ocean (Brest bay, La Tremblade in Marennes-Oleron bay, and Arcachon bay) and one 126 \nfrom the Mediterranean Sea (Thau lagoon), enabling an in -depth exploration of the potential 127 \ndifferential effects of bacterial exposure to either sympatric or allopatric oyster populations.  128 \n 129 \nMaterials and methods: 130 \nOyster sampling 131 \nOysters were collected along the French Atlantic coast s, during two different sampling 132 \ncampaigns (in February 2020 and November 2020) , while it was only in November 2020 for 133 \nthe Mediterranean site due to covid restrictions arisen earlier in the year. For the Atlantic coast, 134 \n3 sites were selected: the Brest bay (Brittany, France; lat 48.3349572; long -4.3189134), La 135 \nTremblade in  Marennes-Oleron bay (Nouvelle-Aquitaine, France ; lat 45.8029675; long -136 \n1.1534223) and the Arcachon bay (Nouvelle-Aquitaine, France ; lat 44.6813750; long -137 \n1.1402178). For the Mediterranean coast, the selected site was the Thau lagoon (Occitanie, 138 \nFrance; lat 43.39404; long 3.58092) . For each site, 5 oysters (average weight = 2.5 g) were 139 \nrandomly sampled. Hence, the sampled oysters  were located on sites with a high density of 140 \noysters (wild and farmed) and have therefore  survived an annual infectious episode of POMS 141 \nallowing us to assume they were resistant to the disease  but also in the window of 142 \npermissiveness for Vibrio aestuarianus infection (Azéma et al. 2016). Based on these facts, we 143 \nhypothesized that sampling bacteria from these disease -resistant oysters increases the 144 \nlikelihood of isolating beneficial bacteria.  145 \n 146 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted May 17, 2024. ; https://doi.org/10.1101/2024.05.17.594303doi: bioRxiv preprint \n\n6 \n \nIsolation of cultivable bacteria from Crassostrea gigas 147 \nThe five disease resistant oysters sampled on each site were carefully brushed and washed to 148 \nremove the sediments, epiphytes and epibionts present on the shell. The flesh of the animals 149 \nwas then individually crushed with an Ultra-Turrax T25 mixer (5 x 5 sec) in 15 ml falcon tubes. 150 \nThe homogenized tissues were then diluted at 1:10 , 1:100 and 1:1000  in sterile artificial 151 \nseawater. A hundred µL of each dilution were spread on two Marine Agar (MA) (Marine Agar 152 \nDifco 2216) plates and incubated at 15°C or 20°C.  153 \nAfter a minimum incubation period of 3 days, bacterial colonies were selected according to 154 \ntheir morphotypes . A maximum of different morphotypes were selected to maximise the 155 \nbiodiversity in our sampling and isolated by streaking a colony on a new MA plate and purified 156 \nby two successive subculturing. Then, the pure cultures of individual bacteria were transferred 157 \nonto Marine Broth (MB) tube (Marine Broth Difco 2216) at 15°C or 20°C and under a constant 158 \nagitation. After 48h of growth, 500 µL of these cultures was used for cryopreservation in 35% 159 \nglycerol (V/V) and put into a -80 °C freezer. About 1 ml of the liquid culture was pelleted for 160 \nfurther DNA extraction. 161 \n 162 \nDNA extraction and identification of the cultivable bacteria 163 \nDNA extraction of the bacterial strains isolated from oysters and cultivated on agar plates was 164 \ncarried with the Wizard® Genomic DNA Purification Kit (Promega) according to the 165 \nmanufacturer instructions. 16S rRNA gene sequencing was performed on these samples to 166 \nidentify each bacterium from the collection. The PCR and 16S rRNA gene sequencing was 167 \nperformed by the Genoscreen sequencing facilities (http://www.genoscreen.fr/fr/). Briefly, two 168 \npairs of primers P8/PC535 (P8 5' -AGAGTTTGATCCTGGCTCAG; PC535 5' - 169 \nGTATTACCGCGGCTGCTGGCAC) and 338F/1040R (338F 5' -CTCCTACGGGAGGCAG; 170 \n1040R 5'-GACACGAGCTGACGACA) were used for the PCR to amplify the V1-V3 and V3-171 \nV5 of the 16S r RNA gene . PCR products were then purified with Sephadex -G50 gel (GE 172 \nHealthcare) before analysis into ABI 3730XL capillary sequencer. The resulting sequences 173 \nwere then assembled by using the DNA baser sequence assembly software (v4) ( Heracle 174 \nBioSoft, www.DnaBaser.com) and then added in the E zbiocloud database (Yoon et al. 2017)  175 \nin order to identify the taxonomy of the isolated bacteria composing the collection. 176 \n 177 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted May 17, 2024. ; https://doi.org/10.1101/2024.05.17.594303doi: bioRxiv preprint \n\n7 \n \nLarval cytotoxic effect 178 \nTwo days old larvae (D stage) were distributed in wells of a 6-well plate filled with three ml of 179 \nsterile seawater at a density of 10 larvae per ml and maintained at a temperature of 20°C and a 180 \n12:12 day:night photoperiod. Treatment (bacterial challenge with a single bacterial strain) and 181 \ncontrol (only sterile seawater) was each conducted in duplicate. The bacteria were cultivated 182 \nfrom glycerol stock in 10 ml of Marine Broth (MB) for 24h at 20°C and then, 1 ml of each 183 \nbacterial culture was inoculated into 10 ml fresh MB media and incubated at 20°C under 184 \nconstant agitation. After 48 hours of incubation, the OD600 was measured, and the right amount 185 \nof bacteria was collected before being centrifuged at 4000 rpm for 2 minutes and the supernatant 186 \nwas discarded. The pellets were then resuspended in 10 ml sterile seawater. Larvae were 187 \nchallenged by addition of a target concentration of 10 7 CFU/ml of each bacterial strain 188 \n(Multiplicity of infection = 10 6 bacteria per larvae) . Larval mortality was recorded 48h post 189 \naddition of bacteria by evaluation of active swimming and/or gut and cilia movement under 190 \nbinocular microscope. 191 \n 192 \nMulti-strain bacterial mixes preparation for interaction with oysters 193 \nFive multi-strain bacterial mixes were tested (Table 1): four site-specific multi-strain bacterial 194 \nmixes composed of bacteria isolated from oysters sampled at each geographical site (Brest mix, 195 \nLa Tremblade mix, Arcachon mix and Thau mix ) and a multi -site bacterial composed of  196 \nbacteria isolated from oysters sampled on  all the different sites. The bacteria were cultivated 197 \nfrom glycerol stock in 10 ml of Marine Broth (MB) for 24h at 20°C and then, 1 ml of each 198 \nbacterial culture was inoculated into 50  ml fresh MB media and incubated at 20°C  under 199 \nconstant agitation. After 48 hours of incubation, the OD 600 was measured, and a quantity of 200 \n3.108 CFU was collected and pooled into a same mix for each cultivated bacterium. The mixes 201 \nwere then centrifuged at 4000 rpm for 2 minutes and the supernatant was discarded. The pellets 202 \nwere then resuspended in 10 ml sterile seawater and added immediately to 30  L larval rearing 203 \ntanks to a final concentration of 104 CFU/ml for each bacterium.  204 \n  205 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted May 17, 2024. ; https://doi.org/10.1101/2024.05.17.594303doi: bioRxiv preprint \n\n8 \n \nOyster reproduction 206 \n150 wild oysters were randomly sampled from each geographic site as described above (Brest 207 \nbay, La Tremblade in Marennes-Oleron bay, Arcachon bay, Thau lagoon) in order to generate 208 \n4 oyster populations (Brest, La Tremblade, Arcachon and Thau population s) accordingly to 209 \ncommercial oyster hatchery practices. Briefly, oyster genitors were transferred into the Ifremer 210 \nhatchery facility in La Tremblade. To avoid eventual horizontal transmission of pathogens 211 \namong populations, each was placed in separate tanks of 250 L in a flow through system with 212 \na water circulation of 500 L/h. Seawater temperature was gradually increased from 10 to 20°C 213 \nwithin one week and maintain to 20°C to favour the gametogenesis . Broodstock were fed ad 214 \nlibitum with a mixture of phytoplankton ( Isochrysis galbana, Tetraselmis suecica, and 215 \nSkeletonema costatum ). After 2 months, oysters were shucked and sexed by microscopic 216 \nobservation. Only fully mature oysters were used, representing between 20 to 23 genitors per 217 \npopulation (Supplementary File 1, Table S1). Spermatozoa and oocytes were collected by 218 \nstripping the gonad. For each population, sperm was collected individually for each male while 219 \noocytes of all females were pooled. Eggs were sieved on a 20 µm and 100 µm screens to remove 220 \nsmall and large debris, respectively, the eggs being retained on the 20 µm screen. Then, the 221 \npool of eggs was divided by the number of males, and each subgroup was fertilized by a male. 222 \nFifteen minutes after fertilization, all subgroups were mixed, and all fertilized and unfertilized 223 \neggs were placed in fourteen 30 L tanks at a density of 34 to 100 eggs per mL (Supplementary 224 \nFile 1, Table S2). Thus, depending on the population, between one to three million eggs were 225 \nadded into each 30 L conical tank. Tanks were in a batch system containing 26 °C filtered and 226 \nUV-treated seawater, supplemented with gentle air -bubbling. Larval farming density were 10 227 \nlarvae per ml at day 2, and 3 larvae per ml at day 7. Seawater was changed three times per week, 228 \nand larvae were fed daily with Isochrysis galbana, supplemented with Skeletonema costatum 229 \nfrom day 7. 230 \n 231 \nExposure of oyster larvae with microorganisms  232 \nFor each population, seven conditions were tested, each using two 30 L replicate tanks. Larvae 233 \nwere either unexposed or exposed to microbiota from donor oysters (ME seawater D0-D14) or 234 \nto the five different multi-strains bacterial mixes at two different larval developmental window 235 \n(Brest D0-D14, Brest D7-D14, La Tremblade D0-D14, La Tremblade D7-D14, Arcachon D0-236 \nD14, Thau D0-D14 and Multi -site D0-D14, Multi-site D7-D14 (Figure 1). For ME seawater 237 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted May 17, 2024. ; https://doi.org/10.1101/2024.05.17.594303doi: bioRxiv preprint \n\n9 \n \nD0-D14, larvae were exposed to the  whole natural microbiota coming from healthy donor 238 \noysters (Microorganism-Enriched seawater = ME seawater). This microorganism community 239 \nwas introduced thanks to donor oysters of microbiota which were placed into the rearing tanks. 240 \nOyster donors of microbiota were NSI ( Naissains Standardisés Ifremer , standardised Ifremer 241 \nspats) (Petton et al. 2013, 2015)  which were always kept in controlled facilities using UV -242 \ntreated seawater, strict biosecurity zoning and management procedures. The oysters were tested 243 \nnegative for  the three main pathogens (Vibrio coralliilyticus, OsHV-1 µVar and 244 \nHaplosporidium costale) of C. gigas from larvae to juveniles (Azéma et al. 2017; Dégremont 245 \net al. 2021). The microorganisms were added to the larvae either 3 hours post-fertilization (pf) 246 \nand at each water change until day 14 pf or from day 7 pf to day 14 pf (Figure 1). The water 247 \nchanges at day 14 was performed without addition of the bacterial mixes . In this sense, the 248 \nmicrobial exposure ended up at day 14. 249 \nLarval survival was determined by counting the larvae either at days 2, 7  and 18 for oysters 250 \nexposed from day 0 pf to day 14 pf or at day 18 for oysters exposed from day 7 pf to day 14 pf. 251 \nFixation rate was determined at day 25 pf for all conditions. Larvae (Pools of 10000 -20000 252 \nindividuals) were sampled either at days 7 pf or at day 14  pf , flash frozen in liquid nitrogen 253 \nand stored at -80°C for subsequent molecular analysis. After the rearing steps, only one replicate 254 \nwas kept to perform the experimental infections. 255 \nAll oyster populations were kept in controlled facilities  of the La Tremblade hatchery using 256 \nUV-treated seawater until experimental infections by OsHV-1 µVar or V. aestuarianus. 257 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted May 17, 2024. ; https://doi.org/10.1101/2024.05.17.594303doi: bioRxiv preprint \n\n10 \n \n 258 \nFigure 1: Overall experimental design for larval microbial exposure and experimental 259 \ninfections.  260 \nMulti-parental reproduction was performed for the four oyster populations and the larvae were 261 \nplaced in  30 L t anks in a batch system containing 26°C filtered and UV -treated seawater, 262 \nsupplemented with gentle air -bubbling. Three hours post-fertilisation (pf), larvae remained 263 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted May 17, 2024. ; https://doi.org/10.1101/2024.05.17.594303doi: bioRxiv preprint \n\n11 \n \nunexposed (2 tanks) or were exposed in duplicate to microbiota from donor oysters (ME  264 \nseawater D0-D14) or to the five different multi -strains bacterial mixes (Brest D0-D14, La 265 \nTremblade D0-D14, Arcachon D0-D14, Thau D0-D14 and Multi-site D0-D14). This 266 \nmicroorganism exposure was renewed three times per week and lasted for 14 days. In parallel, 267 \nexposure to three multi -strains bacterial mixes (Brest D7-D14, La Tremblade D7-D14 and 268 \nMulti-site D7-D14) was performed on older larvae between D7 and D14 pf. During the larval 269 \nstage, seawater and larvae were sampled at days 2, 7, 11, 14, 18 and 25 pf to perform growth 270 \nand mortality monitoring, or to perform molecular analysis. After the larval stage, spat grew in 271 \nour controlled facility. At day 213 pf (approximatively seven months old), a first set was used 272 \nto carry out an experimental infection to OsHV-1 µVar and at day 352 pf (approximatively one 273 \nyear), a second set was used to perform a V. Aestuarianus experimental infection. 274 \n 275 \nOsHV-1 µVar experimental infection by cohabitation 276 \nOsHV-1 µVar experimental infection was perform ed either on control or microorganisms 277 \nexposed oysters (seven-month-old, mean individual weight = 2.80 ± 0.69g). A randomized 278 \ncomplete block design composed of five 50 L tanks (replicates) filled with filtered and UV -279 \ntreated seawater and maintained at 20°C with adequate aeration and no food supply. Each tank 280 \ncontained 12 oysters of each population exposed to each condition (total: 420 oysters per tank) 281 \n(Supplementary File 2, Figure S1). A cohabitation protocol, adapted from (Schikorski et al. 282 \n2011) was used as described. This approach starts with the injection of 100 µL of OsHV-1 µVar 283 \nsuspension (10 5 OsHV-1 µVar genomic units)  into the adductor muscle of pathogen-free 284 \noysters donors. This protocol allows for pathogen  transmission through the natural infectious 285 \nroute to oysters of interest (recipient oysters). The OsHV-1 µVar donor oyster pool was 286 \ncomposed of  25% of F15 family oysters, 25% of F14 family oysters which are POMS 287 \nsusceptible oysters (de Lorgeril et al. 2018)  and 50% of genetically diversified NSI oysters  288 \n(~50% of susceptibility). The ratio was 1 donor oyster for 1 recipient oyster. Immediately after 289 \nOsHV-1 µVar injection into donors  (adductor muscle) , recipient and donor oysters were 290 \nuniformly distributed in each of the five experimental tanks. After 48 hours of cohabitation, all 291 \ndonor oysters were removed from the tanks. 292 \nIn each tank, one oyster of each population exposed to each condition was sampled just before 293 \nthe beginning of the experimental infection (t=0h infection) and three hours post cohabitation 294 \nwith OsHV -1 µVar donor oysters (t=3h infection) to perform molecular analysis  on whole 295 \ntissue samples. The shell was removed, the  whole flesh flash frozen into liquid nitrogen and 296 \nstored at -80°C until it was grounded in liquid nitrogen (Retsch MM400 mill) to a powder that 297 \nwas then stored at -80°C until DNA and RNA extraction. 298 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted May 17, 2024. ; https://doi.org/10.1101/2024.05.17.594303doi: bioRxiv preprint \n\n12 \n \nThe mortality was recorded daily during eight days. Dead recipient oysters were removed daily 299 \nfrom the tanks. 300 \nDuring the mortality monitoring, 1 mL of water in each tank was sampled every day for the 301 \ndetection and the quantification of OsHV-1 µVar.  302 \n 303 \nVibrio aestuarianus experimental infection by cohabitation 304 \nVibrio aestuarianus experimental infection was performed either on control or microorganisms 305 \nexposed oysters (12 months old ; mean individual weight = 9.42  ± 1.29g) with a cohabitation 306 \nprotocol previously describe d in (Azéma et al. 2017) . A randomized complete block design 307 \ncomposed of five 100L replicate tanks filled with filtered and UV -treated seawater and 308 \nmaintained at 20°C with adequate aeration and without food were used. Each tank contained 309 \n10 oysters of each population exposed to each condition (total: 350 oysters per tank).  The V. 310 \naestuarianus 02/041 strain (Garnier et al. 2008) was grown in Zobell medium at 22°C for 24h 311 \nunder agitation. The bacterial concentration was determined by spectrophotometry at 600nm 312 \nand adjusted to an optical density (OD 600) of 1 representing 5.108 bacteria per mL. V. 313 \naestuarianus donor oysters were injected in the adductor muscle with 100µL of the V. 314 \naestuarianus 02/041 suspension and were then equally distributed among the five tanks. The V. 315 \naestuarianus donor oyster population was composed of  an equi -number of the four oyster 316 \npopulations produced for this project (Brest, La Tremblade, Arcachon and Thau population s). 317 \nImmediately after V. aestuarianus injection into donors, donor oysters were added to the five 318 \ntanks containing the recipient oysters. A ratio of 1 V. aestuarianus donor oyster for 1.5 recipient 319 \noyster was used. After 48 hours of cohabitation, V. aestuarianus donor oysters were removed 320 \nfrom the tanks. 321 \nThe mortality was recorded daily during 15 days, and all the dead oysters were removed from 322 \nthe tanks. During the mortality monitoring, 1 mL of water in each tank was sampled every day 323 \nfor the detection and the quantification of V. aestuarianus. 324 \n  325 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted May 17, 2024. ; https://doi.org/10.1101/2024.05.17.594303doi: bioRxiv preprint \n\n13 \n \nStatistical Analysis of oyster mortality 326 \nOyster mortality rates were compared between the different microorganisms exposure set using 327 \nsurvival analysis performed on R (v 4.2.1) (R Core Team 2022) with the package survminer (v 328 \n0.4.9) (https://cran.r -project.org/web/packages/survminer/index.html). The Kaplan -Meier 329 \nmethod was used to represent the cumulative survival rate and log -rank test to determine the 330 \ndifference between conditions. A multivariate Cox proportional hazards regression model was 331 \nused to compute Hazard-Ratio (HR) with confidence intervals of 95%. 332 \n 333 \nOysters and water Genomic DNA extraction and sequencing 334 \nDNA extraction from larvae (pool of 10000 to 20000 individuals) collected during 335 \nmicroorganisms exposure was extracted with the DNA from the tissue Macherey -Nagel kit 336 \naccording to the manufacturer’s protocol. Prior to 90 min of proteinase K lysis, an additional 337 \nmechanical lysis was performed by vortexing samples with zirconia/silica beads (BioSpec). 338 \nDNA from individual juvenile oyster tissues collected just before and during experimental 339 \ninfection was extracted from oyster powder  with the DNA from tissue Macherey -Nagel kit 340 \naccording to the manufacturer’s protocol. Prior to 90 min of proteinase K lysis, an additional 341 \n12-min mechanical lysis (Retsch MM400 mill) was performed with zirconia/silica beads 342 \n(BioSpec). DNA extraction from water collected dur ing microorganisms exposure  and 343 \nexperimental infections was extracted with the DNA from tissue Macherey-Nagel tissue kit 344 \nfollowing the manufacturer support protocol for genomic DNA and viral DNA from blood 345 \nsample. 346 \nDNA concentration and purity were checked with a Nanodrop ND-1000 spectrometer (Thermo 347 \nScientific).  348 \n 349 \nqPCR analysis 350 \nDetection and quantification of OsHV-1 µVar and V. aestuarianus was performed by real-time 351 \nquantitative PCR . All amplification reactions were performed on Roche LightCycler® 480 352 \nReal-Time thermocycler. Each reaction was carried out in triplicate in a total volume of 10 µL 353 \ncontaining the DNA sample (2.5 µL), 5 µL of Takyon ™ SYBER MasterMix blue dTTP 354 \n(Eurogentec, ref UF-NSMT-B0701) and 1 µL at 500 nM of each primers for OsHV -1 µVar 355 \n(OsHVDPFor5’-ATTGATGATGTGGATAATCTGTG and OsHVDPFor   356 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted May 17, 2024. ; https://doi.org/10.1101/2024.05.17.594303doi: bioRxiv preprint \n\n14 \n \n5’-GGTAAATACCATTGGTCTTGTTCC) (Webb et al. 2007)  and for V. aestuarianus   357 \n(DNAj-F 5′ -GTATGAAATTTTAACTGACCCACAA and DNAj -R  358 \n5′-CAATTTCTTTCGAACAACCAC) (Saulnier et al. 2009). qPCR cycling conditions were as 359 \nfollows: 3 min at 95°C, followed by 45 cycles of amplification at 95°C for 10 s, 60°C for 20 s, 360 \nand 72°C for 30s. After these PCR cycles a melting temperature curve of the amplicon was 361 \ngenerated to verify the specificity of the amplification. The DNA polymerase catalytic subunit 362 \namplification product cloned into the pCR4 -TOPO vector was used as a standard at 10 -fold 363 \ndilutions ranging from 10 3 to 10 10 copies/ml for OsHV-1 µVar quantification and genomic 364 \nDNA from V. aestuarianus  ranging from 10 2 to 10 7 copies/ml for V. aestuarianus  365 \nquantification. Absolute quantification of OsHV-1 µVar or V. aestuarianus was calculated by 366 \ncomparing the observed Cp values to standard curve. 367 \n 368 \n16S rDNA library construction and sequencing 369 \nLibrary construction (with primers 341F 5 ’-CCTAYGGGRBGCASCAG and 806R 5 ’-370 \nGGACTACNNGGGTATCTAAT targeting he V3 -V4 region  of the 16S rRNA gene ) 371 \n(Klindworth et al. 2013) and sequencing on a MiSeq v2 (2x250 bp) were performed by ADNid 372 \n(Montpellier, France).  373 \n 374 \nRNA extraction and sequencing 375 \nRNA was extracted from oyster powder (individual) by using the Direct-Zol RNA miniprep kit 376 \n(Zymo Research) according to the manufacturer’s protocol. RNA concentration and purity were 377 \nchecked using a Nanodrop DN-1000 spectrometer (Thermo Scientific), and their integrity was 378 \nanalysed by capillary electrophoresis on a BioAnalyzer 2100 (Agilent).  379 \n 380 \nRNAseq library construction and sequencing  381 \nRNA-Seq experiments were performed on 3 individuals per condition. RNA-Seq library 382 \nconstruction and sequencing were performed by the Bio -Environment Platform (University of 383 \nPerpignan, France) . Stranded  libraries were constructed  from 500 ng of total RNA  using 384 \nNEBNext UltraII and sequenced on a NextSeq550 instrument (Illumina) in single-end reads of 385 \n75 bp. 386 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted May 17, 2024. ; https://doi.org/10.1101/2024.05.17.594303doi: bioRxiv preprint \n\n15 \n \nBioinformatic pipelines for 16S rRNA gene barcoding analysis 387 \nPreviously published barcoding datasets (de Lorgeril et al. 2018; King et al. 2019b; Clerissi et 388 \nal. 2020, 2022; Fallet et al. 2022)  from 687 POMS-resistant and 664 POMS -sensitive oysters 389 \nwere re-analysed in this study in order to predict bacteria which were potentially associated 390 \nwith oyster POMS resistant phenotypes. Datasets used for these analyses are in Supplementary 391 \nFile 1, Table S3. These datasets were individually analysed under the Toulouse Galaxy instance 392 \n(https://vm-galaxy-prod.toulouse.inra.fr/) (Goecks et al. 2010) with the Find Rapidly OTU with 393 \nGalaxy Solution (FROGS) pipeline (Escudié et al. 2018) . In brief, paired reads were merged 394 \nusing FLASH (Magoč and Salzberg 2011). After denoising and primer/ adapter removal with 395 \ncutadapt (Martin 2011), clustering was performed using SWARM (Mahé et al. 2014) , which 396 \nuses a novel clustering algorithm with a threshold (distance = 3) corresponding to the maximum 397 \nnumber of differences between two OTUs. Chimeras were removed using VSEARCH (Rognes 398 \net al. 2016). We filtered out the data set for singletons and performed an affiliation using Blast 399 \nagainst the Silva 16S rDNA database (release 132) to produce an OTU and affiliation tables. In 400 \norder to identify bacterial taxa which were significantly overrepresented in the microbial 401 \ncommunity associated to POMS resistant  compared to POMS sensitive oysters, the “LDA 402 \nEffect Size” (LEfSe) method (Segata et al. 2011)  was used with a normalized relative 403 \nabundance matrix. This method uses a Kruskal-Wallis followed by Wilcoxon tests (pval ≤ 0.05) 404 \nand then performs a linear discriminant analysis (LDA) and evaluate the effect size. The taxa 405 \nwith a LDA score greater than 2 were considered as significantly enriched in POMS resistant 406 \ncompared to sensitive oysters. 407 \nSequencing data obtained on the samples from this study were processed with the SAMBA (v 408 \n3.0.2) workflow developed by the SeBiMER (Ifremer’s Bioinformatics Core Facility). Briefly, 409 \nAmplicon Sequence Variants (ASV) were constructed with DADA2 (Callahan et al. 2016) and 410 \nthe QIIME2 dbOTU3 (v 2020.2) tools (Bolyen et al. 2019), then, contaminations were removed 411 \nwith microDecon (v 1.0.2) (McKnight et al. 2019) . Taxonomic assignment of ASVs was 412 \nperformed using a Bayesian classifier trained with the Silva database v.138 using the QIIME 413 \nfeature classifier (Wang et al. 2007). Finally, community analysis and statistics were performed 414 \non R (R version 4.2.1) (R Core Team 2022) using the packages phyloseq (v 1.40.0) (McMurdie 415 \nand Holmes 2013) and Vegan (v 2.6-4) (Oksanen et al. 2022). Unique and overlapping ASVs 416 \nof each sample group were plotted using the UpsetR package (v 1.4.0) (Conway et al. 2017) . 417 \nFor beta-diversity, the ASVs counts were preliminary normalized with the “rarefy_even_depth” 418 \nfunction (rngseed = 711) from the package phyloseq (v 1.40.0)(McMurdie and Holmes 2013). 419 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted May 17, 2024. ; https://doi.org/10.1101/2024.05.17.594303doi: bioRxiv preprint \n\n16 \n \nPrincipal Coordinates Analysis (PcoA) were computed to represent dissimilarities between the 420 \nsamples using the Bray -Curtis distance matrix. Differences between groups were assessed by 421 \nstatistical analyses (Permutational Multivariate Analysis of Variance) using the adonis2 422 \nfunction implemented in vegan (Oksanen et al. 2022).  423 \nIn order to follow the long-term installation (or not) of each of the bacteria used in the multi -424 \nstrain bacterial mixes in the oyster microbiota, 16S rRNA genes sequences obtained during the 425 \nidentification of each of the bacteria composing the multi-strain bacterial mixes were used as a 426 \nquery for a similarity BLASTn search against all the ASVs sequence from the dataset (Altschul 427 \net al. 1990) . A mock community composed of equal amount s of DNA from the bacteria 428 \ncomposing the multi-strain bacterial mixes were also used as a positive control to validate our 429 \nsearch method. ASVs sequences with a percentage of identity higher than 99% were considered 430 \npresent in the tested samples.  431 \n 432 \nBioinformatic pipeline for RNA-Seq analysis 433 \nAll data treatments were carried out under a local galaxy instance ( http://bioinfo.univ-perp.fr) 434 \n(Goecks et al. 2010). Reads quality was checked with FastQC (Babraham Bioinformatics) with 435 \ndefault parameters (Galaxy Version 0.72). Adapters were removed using Trim Galore (Galaxy 436 \nVersion 0.6.3) (Babraham Bioinformatics). Reads were mapped on C. gigas genome (assembly 437 \ncgigas_uk_roslin_v1) using RNA STAR (Galaxy Version 2.7.8a)  (Supplementary File 3: 438 \nRNAseq Mapping result s) and HTSeq -count (Anders et al. 2015)  was used to count the 439 \nnumber of reads overlapping annotated genes (mode Union) (Galaxy Version 0.9.1). The 440 \ndifferential gene expression levels were analysed with the DESeq2 R package (v 1.36.0) (Love 441 \net al. 2014). Finally, Rank-based Gene Ontology Analysis (GO_MWU package) was performed 442 \nusing adaptive clustering and a rank-based statistical test (Mann–Whitney U-test combined with 443 \nadaptive clustering) with the following parameters: largest = 0.5; smallest = 10; 444 \nclusterCutHeight = 0.25. The signed “-Log(adj pval)” (obtained from the DESeq2 analysis) was 445 \nused as an input for the GO_MWU analysis. The R and Perl scripts used can be downloaded 446 \n[https://github.com/z0on/GO_MWU] (Wright et al. 2015).   447 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted May 17, 2024. ; https://doi.org/10.1101/2024.05.17.594303doi: bioRxiv preprint \n\n17 \n \nResults: 448 \n23 bacterial strains with potential beneficial effects were selected to generate the multi-449 \nstrain bacterial mixes.  450 \nTo isolate bacteria with potential beneficial effects against oyster infectious disease, we 451 \nhypothesised that bacteria should be isolated from disease resistant oysters. For this purpose, 452 \nwild oysters aged between 12 and 18 months were sampled closed to farming area s. Oysters 453 \nlocated in these areas  are submitted to high pathogen pressure and have been shown to be 454 \nresistant to POMS disease  (Gawra et al. 2023) . To maximise the biodiversity of the bacterial 455 \ncollection, oysters were sampled from 4 geographical French sites at two different seasons. A 456 \ntotal of 334 bacteria were isolated (Supplementary File 1, Table S4); from which 166 bacteria 457 \nwere obtained from the February 2020 sampling campaign, and 168 bacteria from the 458 \nNovember 2020 sampling campaign. This corresponded to 97, 144, 56, and 67 bacteria isolated 459 \nfrom Brest, La Tremblade, Arcachon, and Thau sites, respectively. They were named according 460 \nto the sampling site (“ARG” for Brest bay, “LTB” for La Tremblade in Marennes Oleron bay, 461 \n“ARC” for Arcachon bay and “THAU” for Thau lagoon) followed by the number of the isolate. 462 \nThe 16S rRNA gene sequence was obtained for 293 strains. The identified bacteria were divided 463 \ninto the following phyla: Proteobacteria (62.8%), Firmicutes (15.3%), Bacteroidetes (12.3%) 464 \nand Actinobacteria (9.6%) (Figure 2). The three major genera were Vibrio, Bacillus and 465 \nShewanella (Figure 2). The majority of the isolated species were found in all sites.  466 \n 467 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted May 17, 2024. ; https://doi.org/10.1101/2024.05.17.594303doi: bioRxiv preprint \n\n18 \n \n 468 \nFigure 2: 293 strains were identified in the bacterial collection sampled from POMS-469 \nresistant oysters. 470 \nPhylogenetic tree of the 293 identified bacteria composing the collection of bacteria isolated 471 \nfrom POMS -resistant oyster s sampled in the Brest bay (pink), La Tremblade in  Marennes-472 \nOleron bay (yellow), the Arcachon bay (brown) and the Thau lagoon (grey)  based on the V1-473 \nV5 loop alignment of bacterial 16S rDNA by a Maximum likelihood method with the Tamura-474 \nNei parameter model in MEGA X (301 sequences) and 1000 bootstrap replicates. The collection 475 \nis composed by 62.8% of Proteobacteria (different shades of blue), 15.3% of Firmicutes 476 \n(orange), 12.3% of Bacteroidetes (green) and 9.6% of Actinobacteria (salmon).  477 \n 478 \nIn parallel, in silico  correlation analysis was performed to predict bacteria preferentially 479 \nassociated with resistant or sensitive oysters. This L efSE analysis  (Segata et al. 2011)  was 480 \nperformed based on previously published 16S rRNA genes barcoding datasets which describes 481 \nthe bacterial part of the bacterial microbiota community isolated from 687 POMS-resistant and 482 \n664 POMS-sensitive oysters (Supplementary File 1, Table S3). Based on this analysis, 118 483 \nbacterial genera were shown as preferentially associated with POMS -resistant oysters 484 \n(Supplementary File 1, Table S5). By combining the data obtained from this predictive in 485 \nsilico analysis and data from the scientific literature about bacteria shown to be beneficial in an 486 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted May 17, 2024. ; https://doi.org/10.1101/2024.05.17.594303doi: bioRxiv preprint \n\n19 \n \naquaculture context (Rengpipat et al. 2000; Zhang et al. 2009; Kesarcodi -Watson et al. 2012; 487 \nTouraki et al. 2012; Sun et al. 2013; Guzmán-Villanueva et al. 2014; Yan et al. 2014; Reda and 488 \nSelim 2015; Tan et al. 2016; Chauhan et al. 2017; Makled et al. 2017; Lv et al. 2019) , we 489 \nselected 12, 17, 10 and 8 bacteria for the Brest , La Tremblade, Arcachon and Thau site s 490 \nrespectively (Table 1). These bacterial strains were then tested for their cytotoxic effects on 2 491 \ndays old larvae. The most cytotoxic bacteria were discarded.  Based on these results, we kept 492 \nfive, seven, five and five site -specific bacteria to produce the Brest, La Tremblade, Arcachon 493 \nand Thau multi -strain bacterial mix es respectively (Table 1). A fifth multi -site bacterial mix 494 \nwas produced from bacteria isolated from oysters sampled on all sites. For this purpose, seven 495 \ndifferent bacteria were chosen because they display ed the least cytotoxic effects on larvae 496 \n(Table 1). 497 \nIn summary, we collected bacteria from disease -resistant oysters. We then combined our 498 \nfindings with existing literature and utilized in silico predictive analysis. This allowed us to 499 \ncreate four site-specific and one multi -site multi-strain bacterial mixes, all of which have the 500 \npotential to benefit oyster health.  501 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted May 17, 2024. ; https://doi.org/10.1101/2024.05.17.594303doi: bioRxiv preprint \n\n20 \n \nTable 1: Composition of the 5 multi -strain bacterial mixes produced according to their 502 \npredictive beneficial properties. 503 \n 504 \nEnvironment \n Collection of bacteria  Nb. of \ngenera \nselected \nfor \ncytotoxic \nassay on \nlarvae  \n Nb. of \nbacteria \nselected \nafter \ncytotoxical \nassay  \n Multi-strain bacterial mixes \n Nb. of \nbacteria in \nthe \ncollection \nNb. of \ngenera  \n   \nNames Strains  \n    \nBrest   97 40   12   5   Brest Mix \nShewanella sp. ARG21 \nMarinibacterium sp. ARG39 \nShewanella sp. ARG89 \nShewanella sp. ARG96 \nShewanella sp. ARG129 \nLa Tremblade   144 45   17   8   La Tremblade Mix \nHalomonas sp. LTB66 \nNeptunomonas sp. LTB74 \nPsychrobacter sp. LTB83 \nParacoccus sp. LTB95 \nHalomonas sp. LTB102 \nCobetia sp. LTB109 \nSulfitobacter sp. LTB127 \nArcachon   56 26   10   5   Arcachon Mix \nShewanella sp. ARC21 \nBacillus sp. ARC34 \nColwellia sp. ARC55 \nNeptunomonas sp. ARC59 \nTenacibaculum sp. ARC64 \nThau    67 18   8   5   Thau Mix \nShewanella sp. THAU5 \nParacoccus sp. THAU19 \nRuegeria sp. THAU28 \nShewanella sp. THAU34 \nParacoccus sp. THAU46 \n                  Multi-site Mix \nMarinibacterium sp. ARG39 \nShewanella sp. ARG89 \nHalomonas sp. LTB57 \nCobetia sp. LTB109 \nNeptunomonas sp. ARC59 \nParacoccus sp. THAU19 \n          Paracoccus sp. THAU46 \n 505 \n 506 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted May 17, 2024. ; https://doi.org/10.1101/2024.05.17.594303doi: bioRxiv preprint \n\n21 \n \nMicroorganisms exposure during larval rearing induces long term protection against 507 \nPOMS and Vibriosis which relies on bacterial mix composition and oyster origin.  508 \nThe multi-strain bacterial mixes were added to four oyster populations during the larval rearing. 509 \nThe four populations were the sympatric oysters from which the bacteria were isolated ( i.e., 510 \nBrest, La Tremblade, Arcachon and Thau). An exposure with a whole microbiota community 511 \ncoming from healthy hatchery donor oysters was also performed (ME seawater D0-D14). This 512 \noysters were shown to be devoid of the three main pathogens (V. coralliilyticus, OsHV-1 µVar 513 \nand Haplosporidium costale ) of C. gigas  from larvae to juveniles (Azéma et al. 2017; 514 \nDégremont et al. 2021) . Oyster’s larvae were exposed to bacterial mixes either from blastula 515 \n(3h post-fertilization (pf)) to pediveliger stage (14 days pf) (D0 to D14) or from veliger stage 516 \n(seven days pf) to pediveliger stage (14 days pf) (D7 to D14)  (Figure 1). Overall, these 517 \nmicroorganisms exposures during larval rearing stages displayed from moderate to strong effect 518 \non larval survival. These effects rely on oyster origins and, also, on the bacterial content of the 519 \nmicroorganism exposure (Supplementary File 4 Effect of bacterial mixes on oyster larvae  520 \nand Supplementary File 2, Figure S2). 521 \n 522 \nSubsequently, each oyster population (exposed and control) were challenged with OsHV -1 523 \nµVar infection during juvenile stages or V. aestuarianus during adult stages. The success of the 524 \nexperimental infection was verified by quantifying the viral or Vibrio  DNA concentration in 525 \nthe sea water of the experimental tanks (Supplementary File 1, Table S6 and Table S7). 526 \nIn response to OsHV -1 µVar infection, a  significant reduction of the mortality risk of 21% 527 \n(Log-rank test: pval = 0.038), 25% (Log-rank test: pval = 0.009), and 28% (Log-rank test: pval 528 \n= 0.008) was observed in the oysters (all populations combined) exposed to the Arcachon D0-529 \nD14, La Tremblade D7-D14 and D0-D14 ME seawater mixes, respectively (Figure 3). We 530 \nobserved that the mortality start ed 3 days after the POMS disease induction, and differences 531 \nbetween the control and exposed sample s appeared as soon as mortality start ed for oysters 532 \nexposed to the Arcachon D0-D14, La Tremblade D7-D14 and, ME seawater D0-D14 oysters 533 \n(Supplementary File 2, Figure S3).  534 \nIn response to vibriosis, a significant reduction of the mortality risk of 28% (Log-rank test: pval 535 \n= 0.006) was observed for the ME seawater D0 -D14 exposed oysters ( Figure 4) 536 \n(Supplementary File 2, Figure S4). Other exposures did not lead to reduction of mortality.  537 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted May 17, 2024. ; https://doi.org/10.1101/2024.05.17.594303doi: bioRxiv preprint \n\n22 \n \nFor both Vibriosis and viral infection, the beneficial effect in response to each of the mixes 538 \ndepended on the oyster origin ( Supplementary File 2, Figure S 3). Oysters originating from 539 \nArcachon showed the best reduction in mortality in response to both infections regardless of 540 \nthe bacterial exposure conditions during the larval stages. The effect of the microorganisms 541 \nexposure was intermediate on oysters from La Tremblade and less pronounced on oysters from 542 \nBrest and Thau (Supplementary File 2, Figure S3).  543 \nIn summary, larval exposure to bacterial mixes or Microorganism -Enriched seawater (ME 544 \nseawater) conferred a beneficial effect on the survival of the oysters against the POMS disease 545 \nin juvenile oysters while only Microorganism-Enriched seawater (ME seawater) conferred a 546 \nbeneficial effect against Vibriosis. No preferential beneficial effect was nevertheless observed 547 \nwhen the oysters were exposed to their sympatric compared to allopatric strains. 548 \n 549 \nFigure 3: Bacterial mixes and ME -seawater exposure during larval rearing reduce the 550 \nmortality risk induced by POMS 551 \nForest plot representing the Hazard -Ratio value of mortality risk during the OsHV -1 µVar 552 \nexperimental infection for oysters ( all populations co mbined) exposed to microorganisms 553 \ncompared to control oysters.  The numbers in to brackets under the different conditions 554 \ncorrespond to the number of oysters used during the experimental infection. The Hazard-Ratio 555 \nvalue is indicated to the right of the conditions, except for the control condition, which is 556 \nindicated as reference. The p-value of the log rank test is indicated on the right -hand side of 557 \neach row. 558 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted May 17, 2024. ; https://doi.org/10.1101/2024.05.17.594303doi: bioRxiv preprint \n\n23 \n \n 559 \nFigure 4: ME-seawater exposure during oyster larval rearing can reduce the mortality 560 \nrisk induced by V. aestuarianus. 561 \nForest plot representing the Hazard -Ratio value of mortality risk during the V. aestuarianus 562 \nexperimental infection for oysters (All populations confounded) exposed to microorganisms 563 \ncompared to control oysters. The numbers in to brackets under the different conditions 564 \ncorrespond to the number of oysters used during the experimental infection. The Hazard-Ratio 565 \nvalue is indicated to the right of the conditions, except for the control condition, which is 566 \nindicated as reference. The p-value is indicated on the right-hand side of each row. 567 \n 568 \nMicroorganism exposure during larval rearing induce d long term c hanges of the 569 \nmicrobiota composition. 570 \nTo test the immediate and long -term effect of the  microorganism exposure on the oyster 571 \nmicrobiota composition, we analysed the bacterial communities by 16S rRNA gene sequencing 572 \nduring the larval stage after seven days of exposure and during the juvenile stage seven months 573 \nafter the exposure . We focused our study on the three condition s of bacterial exposure that 574 \nconferred significant increase on the survival of oyster s during OsHV -1 µVar and V. 575 \naestuarianus experimental infection. 576 \nSequencing of the V3 -V4 hypervariable region of the 16S r RNA gene resulted in a total of 577 \n10,868,202 clusters. After quality check (deleting primers and low-quality sequences, merging, 578 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted May 17, 2024. ; https://doi.org/10.1101/2024.05.17.594303doi: bioRxiv preprint \n\n24 \n \nand removing chimeras) and ASV clustering, 5,322,399 reads (49%) with an average of 35,962 579 \nreads per sample were retained for downstream analyses.  580 \nA higher species richness was observed seven days after the exposure for ME-seawater exposed 581 \nlarvae but not after exposure to bacterial mixes (Figure 5 A,B). This difference was not 582 \nmaintained at juvenile stage  (Figure 5 C). Dissimilarity analysis, based on the Bray -Curtis 583 \nindex, showed that the larvae microbiota composition differed between conditions after seven 584 \ndays of microorganism exposure, whatever the condition (Table 2). This difference remained 585 \nstatistically significant at juvenile stage for ME seawater D0-D14 and La Tremblade D7-D14 586 \nconditions (Table 2).  587 \nWe additionally checked for the presence of the added bacteria, during the larval stage, after 588 \nseven days of exposure with the last addition of bacteria done 48 hours before sampling, and at 589 \njuvenile stage seven months post -exposure. Two bacterial strains out of the 5 added in larvae 590 \nexposed to Arcachon D0-D14 were retrieved and represented 3.3 to 25.9 % of the total bacterial 591 \ncommunity (Supplementary File 2, Figure S5A). ASVs associated with the added bacteria of 592 \nthe La Tremblade D7-D14 ranged from 0.09 to 0.96 % in the  corresponding larvae samples 593 \n(Supplementary File 2, Figure S5C). None of the ASVs corresponding to bacteria used for 594 \nthe exposure could be detected at the juvenile stages seven months post-exposure 595 \n(Supplementary File 2,  Figure S5B,D). Furthermore, either for larvae or juvenile oysters, 596 \nbacterial strains did not show a preference for implantation in their sympatric host  population 597 \n(Supplementary File 2, Figure S5 ). Using this pipeline of detection, we were able to detect 598 \nthese ASVs on a mock control contain ing an artificial mix of bacteria in the same proportion 599 \nexcept for Paracococcus sp. LTB95 and Psychrobacter sp. LTB83 (Supplementary File 2, 600 \nFigure S6). This indicated that the lack of detection of the ASVs in exposed oyster is due to an 601 \nabsence of the bacteria rather than a technical shortcoming in our detection pipeline, except for 602 \nParacococcus sp. LTB95 and Psychrobacter sp. LTB83.  603 \nIn summary, a few proportions of the different bacteria that were added during the larval rearing 604 \nwere detected in the oyster microbiota 48h after the last addition of bacteria, and none of them 605 \nwere maintained on a long-term basis. Despite this lack of bacterial colonization, the overall 606 \ncomposition of the microbiota was modified in response to the bacterial exposure and these 607 \nchanges remained up to the juvenile stages. 608 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted May 17, 2024. ; https://doi.org/10.1101/2024.05.17.594303doi: bioRxiv preprint \n\n25 \n \n 609 \nFigure 5: The richness of oyster microbiota is transiently increased after larval exposure 610 \nto Microorganism-Enriched seawater. 611 \nThe alpha-diversity indexes (observed species richness) of larvae microbiota after seven days 612 \nof exposure (A,B), or juvenile microbiota seven month s after the exposure (C)  are indicated. 613 \nFor larval stages (A) and (B), analys es were performed on all oyster population s confounded 614 \nwhich represent eight pools of 10000 -20000 larvae sampled in eight independent tanks for 615 \nexposure to ME D0-D14 and Arcachon D0-D14 (A) and on four pools of 10000-20000 larvae 616 \nsampled in four independent tanks for exposure to La Tremblade D7 -D14 (B). For juvenile 617 \nstages (C), analyses were performed on all oyster population confounded which represent 68 618 \nindividuals sampled in five independent tanks. Significant changes are indicated by their p -619 \nvalue and \"ns\" stands for “not significant”.  620 \n 621 \nTable 2: Long -lasting modifications in C. gigas microbiota composition occurred 622 \nfollowing microorganisms exposure.  623 \nPermanova (adonis2) on the Bray-Curtiss dissimilarity matrix showing the effects of microbial 624 \nexposure on microbiota community compared to control condition for larvae after seven days 625 \nof microbial exposure and for juveniles seven months after the microbial exposure. For larvae, 626 \nanalyses were performed on all oyster populations confounded which represent eight pools of 627 \n10000-20000 larvae sampled in eight independent tanks for exposure to ME D0 -D14 and 628 \nArcachon D0-D14 and on four pools of 10000-20000 larvae sampled in four independent tanks 629 \nfor exposure to La Tremblade D7 -D14. For juvenile stages, analys es were performed on all 630 \noyster population confounded which represent 6 8 individuals sampled in  five independent 631 \ntanks. 632 \n 633 \n    Larvae (after 7 days of exposure)   Juvenile (7 months)     \nConditions \n(Compared to \ncontrol)   Dum Sq R² F p   Dum Sq R² F p \n                      \nME D0-D14   0.75 0.18 4.46 0.001   0.19 0.04 1.45 0.026 \nArcachon D0-D14   0.85 0.19 3.30 0.001   0.08 0.02 0.75 0.908 \nLa Tremblade D7-D14   0.43 0.29 2.98 0.036   0.17 0.04 1.55 0.033 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted May 17, 2024. ; https://doi.org/10.1101/2024.05.17.594303doi: bioRxiv preprint \n\n26 \n \nMicroorganisms exposure during larval rearing induced long-term changes in oyster 634 \nimmunity. 635 \nThe long-term impact of the microorganisms exposure on oyster gene expression was analysed 636 \nby RNA-seq on juvenile oysters before and during POMS challenge. In total, RNA sequencing 637 \nproduces between 15.1 and 36.6 million reads per sample (mean number of reads = 26 millions). 638 \nAmong these reads, 67.28% to 77.52% were mapped on C. gigas reference genome (assembly 639 \ncgigas_uk_roslin_v1) (Supplementary File 3: RNAseq Mapping result). 640 \nFor each of the four oyster population s, the number of differentially expressed genes ( DEGs) 641 \nin oysters exposed to bacterial mixes or to ME seawater compared to control oysters, was higher 642 \nbefore the infection than 3h after the beginning of the infection except for the condition where 643 \nBrest oysters were exposed to  ME seawater (Figure 6). Furthermore, each oyster population 644 \ndisplayed a specific transcriptomic response, which strongly varied according to the 645 \nmicroorganism exposure. (Figure 7). 646 \n 647 \n 648 \nFigure 6: Long-lasting changes in gene expression was observed in juvenile oysters seven 649 \nmonths after larval exposure. 650 \nHistogram of differentially expressed genes (DEGs) in oysters exposed to Arcachon D0-D14, 651 \nLa Tremblade D7-D14 or ME seawater D0-D14 compared to control oysters for the four oyster 652 \npopulations (Brest, Arcachon, La Tremblade and Thau) prior to OsHV-1 µVar infection (green) 653 \nand 3h post infection (blue). n=3 individuals per condition. 654 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted May 17, 2024. ; https://doi.org/10.1101/2024.05.17.594303doi: bioRxiv preprint \n\n27 \n \n 655 \nFigure 7: Specific gene expression profiles were observed in response to each 656 \nmicroorganism exposure. 657 \nHeatmap of differentially expressed genes (DEGs) in oysters exposed to Arcachon D0-D14, La 658 \nTremblade D7-D14 or ME seawater D0-D14 compared to control oysters for the four oyster 659 \npopulations (Brest, Arcachon, La Tremblade and Thau) (A) prior to OsHV -1 µVar infection 660 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted May 17, 2024. ; https://doi.org/10.1101/2024.05.17.594303doi: bioRxiv preprint \n\n28 \n \nand (B) 3h post OsHV-1 µVar infection. The intensity of DEG ratios is represented by the Log2 661 \nFold Changes (Log2FC) for over expressed DEGs (in red) and under expressed DEGs (in blue). 662 \nn=3 individuals per condition. 663 \n 664 \nTo identify which biological processes were affected by the microbial exposure, we conducted 665 \na Rang -Based Gene Ontology Analysis ( GO_MWU) (Wright et al. 2015) . The range of 666 \nbiological process enriched in DEGs (microorganisms exposed vs control) before and during 667 \nthe onset of the POMS disease included many GO terms such as, metabolism, RNA and DNA 668 \nprocess, protein processing, signal transduction, transport, and immune functions. We then  669 \nfocused on the enriched immune functions in oysters exposed to microorganisms compared to 670 \nthe control oysters ( Figure 8). The most significantly enriched functions related to immunity 671 \nacross all oyster populations and all treatments were general functions of immunity (defence 672 \nresponse, immune system process), functions related to the response to organisms (response to 673 \nbacterium, response to virus), a function related to the positive regulation of response to 674 \nstimulus and a function related to G-protein signalling pathway (Figure 8). As the oysters from 675 \nArcachon showed the greatest reduction in mortality risk in the face of viral infection and V. 676 \naestuarianus, with all the microbial exposures, we then analysed, for these oysters only, the 677 \nindividual DEGs for the main enriched functions linked to immunity described in (Figure 8). 678 \nThis analysis revealed that before the infection (t=0), gene coding for Pattern Recognition 679 \nReceptor (PRRs) (C -type lectins, C1q domain containing protein), innate immune pathways 680 \n(toll-interleukin receptor (TIR), Complement pathway), interaction with bacteria (Bactericidal 681 \npermeability-increasing protein) and antiviral pathways (RNA and DNA Helicases, RNA -682 \ndependent RNA polymerase) were found to be over-represented in microbial exposed oysters 683 \ncompared to control oysters (Figure 9) (Supplementary File 5 List of DEGs). 684 \nIn summary, long-lasting changes in gene expression were observed in juvenile oysters seven 685 \nmonths after they ha d been exposed to bacterial mixes or Microorganisms Enriched seawater 686 \nduring larval stages. The long-lasting transcriptional responsiveness was found to be influenced 687 \nby the host's origin , was specific to the type of treatment and significantly impacts the host 688 \nimmune response.  689 \n 690 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted May 17, 2024. ; https://doi.org/10.1101/2024.05.17.594303doi: bioRxiv preprint \n\n29 \n \n 691 \nFigure 8: GO term enrichment analysis revealed important immune pathways modified 692 \nin response to the microorganism exposure. 693 \nDot plot showing the overrepresented GO terms (FDR <0.1) of biological process (BP) related 694 \nto immune function identified using GO_MWU for the four oyster populations (Brest, 695 \nArcachon, La Tremblade and Thau) exposed to Arcachon D0-D14, La Tremblade D7-D14 or 696 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted May 17, 2024. ; https://doi.org/10.1101/2024.05.17.594303doi: bioRxiv preprint \n\n30 \n \nME seawater D0-D14 compared to control oysters at t=0 and t=3h OsHV-1 µVar infection. The 697 \ndot size is proportional to the number of differentially expressed genes (DEG) in the biological 698 \nprocess compared to the control condition, and the colour of the dot shows the significance. 699 \n 700 \n 701 \n 702 \n 703 \nFigure 9: Detailed immune -related gene expression revealed key genes modified in 704 \nArcachon oysters in response to microorganism exposure. 705 \nTranscriptomic response of  immune related genes for  oysters of the Arcachon population  706 \nexposed to Arcachon D0 -D14, La Tremblade D7 -D14 or ME seawater D0-D14 compared to 707 \ncontrol condition before OsHV-1 µVar experimental infection. Heatmap of DEGs associated 708 \nwith immune processes. Only DEGs found under at least two conditions of exposure to micro-709 \norganisms were shown. The intensity of DEG  ratios is expressed in Log2 Fold changes 710 \n(Log2FC) for over expressed DEGs (in red) and under expressed DEGs (in blue). 711 \n 712 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted May 17, 2024. ; https://doi.org/10.1101/2024.05.17.594303doi: bioRxiv preprint \n\n31 \n \nDiscussion: 713 \nOsHV-1 µVar, a threatening pathogen for oyster production, has spread not only in Europe 714 \n(Segarra et al. 2010; Petton et al. 2021)  but also to the United States (Friedman et al. 2005) , 715 \nJapan (Shimahara et al. 2012) , Australia (Paul-Pont et al. 2013) , China (Bai et al. 2015)  and 716 \nNew-Zealand (Delisle et al. 2022). On the other hand, the pathogenic bacterium V. aestuarianus 717 \nhas been observed to spread across Europe (Mesnil et al. 2022) . Innovative research and 718 \nconcerted efforts are currently being explored for safeguarding C. gigas  and ensuring the 719 \nsustainability of oyster farming on a global scale  (Green and Montagnani 2013; Dégremont et 720 \nal. 2015, 2020; Lafont et al. 2017). One promising avenue of research involves education of the 721 \noyster immune system through proper setting of the microbiota during early life. Similar to the 722 \nway early microbial colonization impacts human health  (Gensollen et al. 2016; Renz et al. 723 \n2017), introducing specific microorganisms to oyster larvae can potentially educate their innate 724 \nimmune systems and improve disease resistance  (Galindo-Villegas et al. 2012; Fallet et al. 725 \n2022). The immune system in oysters is set up early during the development since the existence 726 \nof a primitive immune system has been detected in the trochophore larva (Tirapé et al. 2007; 727 \nLiu et al. 2015). This microbial education plan is a promising strategy as it is easy to implement, 728 \nnot costly and, can be performed on numerous animals (several hundred million of larvae) at 729 \nthe same time by bath or on their diet. However, a challenge arises in the form of current 730 \nhatchery practices, which aim to minimize the introduction of both non -pathogenic and 731 \npathogenic microorganisms into larval tanks  (Bourne et al. 1989; Helm et al. 2004; Eljaddi et 732 \nal. 2021; Cordier et al. 2021) . Mortality issues, particularly during larval rearing, have led to 733 \nthe use of antibiotics in hatcheries. Therefore, finding a balance between educating the immune 734 \nsystem and addressing concerns about uncontrolled microbiota transfer is crucial. Here, our 735 \nstudy explored the feasibility of microbial education in oyster larvae while considering and 736 \nmitigating the risks associated with uncontrolled transfer of hazardous microorganisms. 737 \nFor this purpose, we investigated the long-term protection conferred by a larval exposure to a 738 \ncontrolled non-pathogenic whole microbiota transferred from donor oysters. The donor oysters 739 \nused in this study were always kept in biosecured facilities. In this way, the oysters were shown 740 \nto be devoid of the three main pathogens of C. gigas from larvae to juveniles (Azéma et al. 741 \n2017; Dégremont et al. 2021) . In parallel, we performed the same a ssay using  a reduced , 742 \nsynthetic bacterial community composed of cultivable bacteria. The cultivable bacteria were 743 \nisolated from POMS-resistant oysters and selected according to their predictive beneficial effect 744 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted May 17, 2024. ; https://doi.org/10.1101/2024.05.17.594303doi: bioRxiv preprint \n\n32 \n \non POMS disease based on robust correlation analysis . The microbial exposures w ere 745 \nperformed on 4 different oyster populations  each exposed to either a sympatric or allopatric  746 \nmulti-strain bacterial mixes. We showed that larval exposure to a whole microbiota from donor 747 \noysters provided protection against both the POMS disease and V. aestuarianus infection. In a 748 \ndifferent way, larvae exposed to multi-strains bacterial mixes showed improved survival against 749 \nOsHV-1 µVar but no protection against V. aestuarianus  infection. The host origin was 750 \nidentified as a critical factor for the protection conferred and no preferential effect was observed 751 \nwhen sympatric multi -strains mixes were used.  This work demonstrates the potential of 752 \nleveraging the oyster microbiota to enhance long -term disease resistance in oyster s and sheds 753 \nlight on the importance of considering the host origin in such protective mechanisms.  754 \n 755 \nTargeting early developmental stages as a strategic window for probiotic application to induce 756 \nlong-term protection has been proposed and explored in various animal models  such as 757 \nmammals or humans (see review by Hashemi et al. 2016), but also those relevant to livestock 758 \nproduction (Wang et al. 2022; Villumsen et al. 2023) . Introducing beneficial microorganisms 759 \nduring these stages can influence both the host's microbiota composition and immune system 760 \ndevelopment, potentially leading to long-term beneficial immunomodulation. Our results are in 761 \nline with these findings since we observe d a shift in both the transcr iptional pattern  and 762 \nmicrobiota composition of oysters exposed to  beneficial microorganisms compared to their 763 \nnon-exposed counterparts , even  seven months after the exposure. The long-lasting 764 \ntranscriptional responsiveness was found to be influenced by the host's origin and was specific 765 \nto the type of microbial treatment administered. A significant portion of the differentially 766 \nexpressed genes in exposed oysters were associated with immune functions, with a particular 767 \nemphasis on pattern recognition receptors (PRRs). Intriguingly, the observed difference in 768 \nphenotype between oysters stimulated with the whole microbiota and those stimulated with 769 \nmulti-strain bacterial mixes could not be fully explained through a thorough analysis of the 770 \ndifferentially expressed genes. This suggests that additional factors or intricate interactions 771 \nwithin the oyster's immune system and microbiota may contribute to the differential response.  772 \nFurthermore, when the oysters were challenged with OsHV-1 µVar three hours after exposure, 773 \nchanges in the transcriptional pattern were still evident in oysters exposed to beneficial 774 \nmicroorganisms compared to their non -exposed counterparts, albeit to a lesser extent  than 775 \nbefore the infectious challenge . This indicates a dynamic interplay between the immune 776 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted May 17, 2024. ; https://doi.org/10.1101/2024.05.17.594303doi: bioRxiv preprint \n\n33 \n \nresponse against the virus and the prior microbial stimulation, with the virus potentially exerting 777 \na more pronounced effect on the transcriptional response. 778 \nOur findings further indicates that exposure to either bacterial mixes or whole microbiota, leads 779 \nto changes in the microbiota composition. This was observed during the exposure but also on a 780 \nlong-term basis as previously observed in other studies (Padeniya et al. 2022; Villumsen et al. 781 \n2023; Takyi et al. 2024) . Interestingly, the bacteria added as part of the bacterial mixes were 782 \nnot detected using the employed method. This suggests that the added bacteria did not 783 \neffectively integrate the oyster microbiota, even shortly after the start of the exposure. Similar 784 \nstudies indicate that administered bacteria fail to establish and only persist temporarily in the 785 \nmicrobiota of exposed animals.  For instance, the Aeromonas sp. strain administered to oyster 786 \nlarvae was undetectable 72 hours after addition (Gibson et al. 1998) . Similarly, exposing the 787 \nEuropean abalone (Haliotis tuberculata) to the Pseudoalteromonas sp. hCg-6 exogenous strain 788 \nresulted in a transient establishment of the probiotic strain in the haemolymph rather than a 789 \nsustained interaction (Offret et al. 2018) . Additionally, Arctic Char ( Salvelinus alpinus ) 790 \nexposed to various probiotic strains did not show detectable levels of the administered strains 791 \nfour weeks after probiotics administration (Knobloch et al. 2022) . The change in microbiota 792 \ncomposition observed on long term basis might thus be linked to ongoing interactions between 793 \nthe microbiota and the immune system, leading to a continuous reshaping of both elements and 794 \nexplaining also the observed long-term transcriptional changes.  795 \n 796 \nConclusion: 797 \nOur study successfully investigated methods which aimed at exposing oysters to specific 798 \nbeneficial microorganisms during larval rearing to educate their immune system. We took into 799 \naccount the potential risks associated to this microbial exposure while ensuring that the oysters' 800 \ninnate immune system was primed for improved disease resistance. We demonstrated the 801 \npotential of leveraging this microbial education  to enhance disease resistance to two major 802 \noyster pathogens, OsHV-1 µVar and V. aestuarianus, which are current critical threat for oyster 803 \nfarming worldwide. Additionally, our findings emphasize the potential of using controlled 804 \nwhole microbiota transfers as the best strategy to safeguard oyster health in aquaculture settings.  805 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted May 17, 2024. ; https://doi.org/10.1101/2024.05.17.594303doi: bioRxiv preprint \n\n34 \n \nAdditional optimizations will be required to identify the most effective settings for enhancing 806 \nthe beneficial impact of microbial education. The timing, duration of exposure, and rearing 807 \nconditions are essential factors for the practical application of this approach in aquaculture 808 \nenvironments. Exploring combinations with other strategies, such as selecting oysters with 809 \ngenetic backgrounds that are more receptive to microbial education, is another avenue that 810 \ncertainly deserves further investigation. 811 \n 812 \n 813 \nList of abbreviations 814 \nASV: Amplicon Sequence Variants 815 \nCFU: Colony‑forming unit 816 \nDEG: Differentially expressed gene 817 \nHR: Hazard-Ratio 818 \nLEfSe: Linear discriminant analysis (LDA) Effect Size 819 \nMB: Marine Broth 820 \nNSI: Naissains Standardisés Ifremer or standardised Ifremer spats 821 \nOsHV-1 µVar: Ostreid Herpes Virus 1 µVar 822 \nOTU: Operational Taxonomic Unit 823 \npf: post-fertilization 824 \nPOMS: Pacific Oyster Mortality Syndrome 825 \nRNA‑Seq: Sequencing of the polyadenylated ribonucleic acids 826 \n 827 \nCompeting interests 828 \nThe authors declare that they have no competing interests. 829 \n  830 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted May 17, 2024. ; https://doi.org/10.1101/2024.05.17.594303doi: bioRxiv preprint \n\n35 \n \nAcknowledgments 831 \nThe authors warmly thank the staff of the Ifremer stations of Argenton, La Tremblade and 832 \nArcachon for their help and hospitality during the various oyster sampling campaigns. We are 833 \ngrateful to Leo Duperret , Emily Kunselman , Nicole Faury, Cyrielle Lecadet and Delphine 834 \nTourbiez for their help during the oyster experimental infections and Abdellah Benabdelmouna 835 \nand Christophe Ledu for their help during the larval rearing.  We are grateful to Jean -François 836 \nAllienne, Margot Doberva and Michèle Laudié from the Bio -Environment platform (UPVD, 837 \nRégion Occitanie, CPER 2007 -2013 Technoviv, CPER 2015 -2020 Technoviv2) for technical 838 \nsupport in library preparation and sequencing . We are grateful to the BIO2MAR platform 839 \n(http://bio2mar.obs-banyuls.fr) for access to instrumentation.  840 \n 841 \nAuthors' contributions 842 \nLDa, LDé, BM, BP, EM, GC and JVD contributed to oysters sampling. LDa, PC, RL and LI 843 \ncontributed to bacteria collection. LDa, PC, LDé, BM, BP, MM, EM, JVD, ET and CC 844 \nperformed oyster experiments. LDa, PC, JFA, CG, OR, JVD, ET and CC prepared samples and 845 \nperformed DNA and RNA extraction on oysters samples for analyses. LDa, MAT, JFA, OR 846 \nand JP performed qPCR analyses. LDa, ET and CC performed microbiota analyses. LDa, JVD, 847 \nET and CC performed RNAseq analyses. LDa, LDé, BM, MAT, BP, MM, YG, JVD, ET and 848 \nCC conceptualized and designed the experiments. LDa, LDé, BM, MAT, BP, YG, JVD, ET 849 \nand CC wrote the original draft. LDa, YG, JVD, ET and CC involved in funds acquisition. All 850 \nauthors read and approved the final manuscript. 851 \n 852 \nFunding  853 \nThe present study was supported by the Ifremer project GT -huitre and by the Fond Européen 854 \npour les Affaires Maritimes et la Pêche (FEAMP, GESTINNOV project 855 \nn°PFEA470020FA1000007), the project “Microval” of the Bonus Qualité Recherche program 856 \nof the University of Perpignan, the project “gigantimic 1” from the federation de recherche of 857 \nthe university of Perpignan, the project “gigantimic 2” from the kim food and health foundation 858 \nof MUSE and the project ANR DECICOMP (ANR-19-CE20-0004). This study is set within 859 \nthe framework of the \"Laboratoires d'Excellences (LABEX)\": TULIP (ANR‐10‐LABX‐41) and 860 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted May 17, 2024. ; https://doi.org/10.1101/2024.05.17.594303doi: bioRxiv preprint \n\n36 \n \n“CeMEB” (ANR-10-LABX-04-01). Luc Dantan is a recipient of a PhD grant from the Region 861 \nOccitanie (Probiomic project) and the University of Perpignan Via Domitia graduate school 862 \nED305. 863 \n 864 \nAvailability of data and materials 865 \nRaw sequence data for RNA-seq and 16S sequencing for metabarcoding analysis have been 866 \nmade available through the SRA database (BioProject accession number PRJNA1078733). 867 \nR script for survival, DEseq2 and microbiota analyses are available by using the following link: 868 \nhttps://zenodo.org/records/11200726. 869 \n 870 \nEthical approval  871 \nThe animal (oyster Crassostrea gigas) testing followed all european regulations concerning 872 \nanimal experimentation. 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