cJUN dependent innate immunity controls microbiome through selective phagocytosis in Nematostella

cJUN dependent innate immunity controls microbiome through selective phagocytosis in Nematostella | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article cJUN dependent innate immunity controls microbiome through selective phagocytosis in Nematostella Sebastian Fraune, Nida Kaya, Mohammad Abukhalaf, Gabriela Fuentes Reyes, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7020203/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Innate immunity, traditionally viewed as non-specific, is increasingly recognized for its capacity to regulate microbial communities with precision. In the sea anemone Nematostella vectensis , we uncover a form of selective immunity mediated by nematosomes—motile immune cell clusters that preferentially phagocytose foreign Vibrio isolates while sparing native bacteria. We identify the transcription factor cJUN as essential for this process: CRISPR/Cas9-mediated knockout of cJUN impairs nematosome proliferation, reduces lysosomal activation, and alters microbiome composition by allowing colonization of non-native strains. These results link immune gene function to microbial selectivity and demonstrate that even early-diverging animals exhibit immune discrimination. Our findings challenge the classical dichotomy between innate and adaptive immunity and reveal that immune specificity may be evolutionarily ancient. This work establishes Nematostella as a model for studying microbiome-induced innate immune training and highlights conserved mechanisms that maintain host-microbe homeostasis. Biological sciences/Immunology/Innate immunity Biological sciences/Microbiology/Microbial communities/Microbiome Metaorganism homeostasis Symbiont recognition innate immunity regulation Nematostella vectensis Nematosomes Phagocytosis Vibrio cJUN/AP-1 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction The maintenance of a healthy functioning metaorganism, consisting of the host and its associated microorganisms, relies on an efficient host immune system. The immune system is based on a complex interplay of mechanisms protecting the host from pathogenic microorganisms, like bacteria 1 , 2 , fungi 3 and viruses 4 , while simultaneously maintaining beneficial microbial relationships 5 , 6 . As part of the innate immune system, phagocytosis is a conserved mechanism found in invertebrates and vertebrates. Most likely it evolved as a mechanism of nutrient uptake in unicellular eukaryotes 7 and evolved during metazoan evolution 8 to a mechanism of specialized immune cells to eliminate pathogens. Thereby, phagocytosis of foreign particles consists of target recognition, internalization into phagosome and digestion of targets via phagolysosome formation 9 , 10 , 11 , 12 , 13 . While the cellular mechanisms of phagocytosis in invertebrates are similar to those in vertebrates 14 , the evolutionary conservation of specific gene functions remains to be determined 10 . The widespread assumption is that the innate immunity provides an immediate, non-specific defense, while the adaptive immunity enables a targeted, antigen-specific response with an immunological memory 15 . However, recent research has challenged the traditional view that invertebrate immune systems lack specificity. Studies have shown that phagocytosis in invertebrates can exhibit a high degree of specificity 9 , 16 . In woodlice, hemocytes demonstrated increased phagocytosis of previously encountered bacterial strains, suggesting the ability to differentiate between closely related bacteria 9 . In the squid-Vibrio symbiosis haemocytes are able to differentiate between the squid’s preferred bacterial symbiont Vibrio fisheri and other bacteria of the Vibrio genus 17 , 18 . In addition, in a number of host-microbe interactions, it has been shown, that phagocytes can both shape the microbiota and be influenced by specific members of the microbiome. In tse-tse flies, for example, hemocyte proliferation depends on colonization by Wigglesworthia 19 . A similar effect is observed in pea aphids, where the presence of some symbionts affects hemocyte abundance and the proportion of granulocytes in the hemocyte population 20 . We therefore hypothesize that bacterial differentiation by the innate immune system at the level of phagocytosis evolved already at the base of animal evolution and enabled already the common ancestor of metazoans to distinguish between different microorganisms. In the following, we used the sea anemone Nematostella vectensis ( N. vectensis ) to study the degree of specificity of phagocytosis and its effects on interactions with its microbiome in an early branching metazoan. N. vectensis belongs to the cnidarians, a sister group of all Bilateria, and exhibits a high genetic complexity characterized by similar signaling pathways for development and innate immunity compared to bilaterian animals 21 , 22 . The microbiome of N. vectensis shows a specific succession during host development and robust adaptations to environmental variations 23 , with a remarkable genotype-environment interaction influencing the variability of the microbiome 24 , 25 . The microbiome-mediated plasticity 24 has been functionally linked to thermal adaptation in N. vectensis 25 . The establishment of the microbiome strongly depends on host mechanisms and transcriptome data support the hypothesis, that phagocytosis is a central mechanism for controlling the initial colonization processes 26 . In N. vectensis , it is shown that nematosomes (Fig. 1 A), small motile multicellular bodies in the gastric cavity, are capable of phagocytosis and may act as immune bodies 27 , 28 . They originate from cnidoglandular tracts, the mesenteries, and consist of nematocytes and a second, not well characterized cell type, that shows phagocytic activity 27 . While nematosomes can immobilize prey with the help of their nematocytes (Fig. 1 B), the second cell type presumably serves the phagocytosis of bacteria (Fig. 1 C), indicating a potential dual function in nutrition and immunity 27 , 28 . In addition, nematosomes are equipped with two types of cilia surrounding the cell complex 27 ( Fig. 1 B ) , which facilitate their active mobility within the fluid of the gastric cavity ( Video S1 ). A further indication for the active contribution to immunity is the fact that nematosomes co-express components of the TLR signaling pathway, such as TLR and NF-κB 29 . During oogenesis, the nematosomes are packed into the egg clutches by the female polyps, which are then expelled by the female polyps with a gelatinous matrix mucus. It is likely that the nematosomes thereby also take over a maternal protection of the embryos during early development (Fig. 1 D). However, there is no direct evidence that nematosomes are able to differentiate bacteria at the level of phagocytosis. Here, we could show, that nematosomes exhibit selective phagocytosis, efficiently ingesting foreign Vibrio isolates and degrading them in the lysosome while sparing native Vibrio isolates. This selective phagocytosis is correlated with the ability of the bacteria to colonize N. vectensis adult polyps. Proteomic analyses revealed distinct protein enrichment patterns linked to phagosomal pathway in response to foreign bacteria, highlighting cJUN’s role in immune-related trafficking. CRISPR/Cas9-mediated cJUN knockout ( cJUN −/− ) resulted in a significantly reduction in nematosomes proliferation and impaired lysosomal activation after engulfment of Vibrio cells. These cJUN −/− polyps are colonized by an altered microbiome and accumulate foreign Vibrio -isolates, demonstrating a causal relationship between the composition of the microbiome and the selective phagocytosis of the nematosomes. Our data provide strong evidences that cJUN is a key regulator for nematosome proliferation and lysosome maturation in N. vectensis and therefore fulfils a central role in microbiome regulation. Methods Nematostella vectensis culture All experimental setups were conducted with adult clonal female Nematostella vectensis polyps, originally collected from the Rhode River in Maryland, United States 30 , 31 . The polyps were fed daily with freshly hatched Artemia nauplii and kept in dark. Culture tanks containing the animals, organized by genotype and gender, were connected to an aquatic system where the medium was flushed out and replaced with fresh Nematostella Medium (NM) with a salinity of 16‰ (Red Sea Salt® and Millipore H2O) at 18°C every other day. Every two weeks, the culture boxes were manually cleaned to remove biofilm and feeding debris. Phagocytosis assay Experimental setups regarding bacterial challenges were performed working with the same bacterial isolates used for mono-associations. Native (NJ1, NJ33 and NA11) 26 and non-native (Hal025 and Hal281 32 ) bacterial isolates were grown at 30°C and 220 rpm overnight in liquid Marine Broth (MB) Medium before diluting the isolates to an OD600 of 0.1/mL. 1 mL of bacteria was centrifuged and diluted with sterile 16‰ NM prior staining with BacLight (Thermo Fisher) for 15 min in dark and room temperature. After incubation, bacteria was centrifuged for one wash step with 16‰ NM before diluting them to OD600 of 0.001 for the bacterial challenge on the nematosomes ex vivo. Bacterial treatment was performed in the dark for 1.5 h at 18°C. During bacterial staining, nematosomes from 5 clonal female polyps were extracted, by pinching a hole in the food region of the polyps and pipetting the discharged nematosomes, and placed into a chamber slide (Thermo Scientific™ Nunc™ Lab-Tek™ II Chamber Slide™ System). After letting the cells stick to the bottom of the slide, they were washed once with NM to get rid of debris. The nematosomes were treated with stained bacteria from an OD600 of 0.001 and incubated for 2 h in the dark. After bacterial treatment, nematosomes were washed 2 times with NM. After that nematosomes are stained with LysoTracker (15 nM, Thermo Fisher) and Hoechst (10 nM, Thermo Fisher) for 45 min at room temperature. Staining solution was washed out with NM after staining. Cells were than fixed with 3% PFA diluted in NM for 15 min on RT and washed out once with NM after treatment. Nematosomes were mounted on slide using ProLong™ Diamond (Thermo Fisher). Images from samples were taken with the confocal microscope Olympus FV3000 Confocal Laserscanning Microscope and later analyzed using ImageJ 33 . Generation of germfree polyps and mono-association experiment Antibiotic treatment (AB treatment) approaches were adapted from the established protocol for generating germfree Hydra polyps 34 and further adapted for Nematostella 35 . Adult clonal cJUN +/+ and cJUN −/− lines were exposed to a combination of five antibiotics: Ampicillin, Neomycin, Streptomycin, Spectinomycin, and Rifampicin, each at a concentration of 50 µg/mL. This treatment was conducted over a period of 2 weeks without any food supply, with the medium being refreshed every day and a replacement of plates every second day. For each treatment condition, five biological replicates were utilized, along with an additional five biological replicates serving as germfree (GF) and wildtype (WT) controls. Following the 2-week AB treatment, polyps were washed in sterile, filtered 16‰ NM before homogenization. A 1:10 dilution of the lysate was plated on Marine Broth (MB) agar plates to confirm sterility, in which GF plates should remain clear without bacterial growth. The remaining lysate was centrifuged, and the pellet was processed for DNA isolation using the Qiagen Blood and Tissue Kit for subsequent molecular analyses, including PCR and quantitative PCR (qPCR). After sterility confirmation remaining polyps were prepared for mono-association with chosen bacterial isolates. After 2 weeks of AB Treatment, polyps remained in sterile and filtered NM prior recolonization. Bacterial isolates were grown at 30°C in liquid MB media overnight. Bacteria was grown to an OD600 of 0,1, were diluted to a final OD600 of 0.001 and exposed to the sterile polyps. We used 5 polyps for each isolate and genotype of polyps with GF and WT control, respectively. The isolates we chose were NJ1, NJ33 and NA11 as native colonizers for Nematostella and Hal025 and Hal281 as non-native, foreign, colonizers, obtained from Halichondria panicea . After recolonizing polyps with single isolates, sampling took place after 2- and 7- days post recolonization (2 dpr and 7 dpr). Polyps were washed three times with 16‰ NM before getting homogenized and plated on MB plates. After an incubation time of 2 days on room temperature, colony forming units (CFU) were counted manually to determine the colonization succession of the single isolates on the polyp. Proteomic analysis Bacterial culture was prepared with an OD600 of 0.001 (see Phagocytosis assay) prior nematosomes extraction from adult polyps. All nematosomes from five biological replicates for each treatment was prepared. We chose the isolated NJ1 as a native colonizer and Hal281 as a non-native isolate and a control group without bacterial challenge. Extracted nematosomes were treated for 2 h at 18°C with the isolates and were washed afterwards with 16‰ NM once. Nematosomes were transferred into a PCR tube and centrifuges by 5000 rpm for 5 min at 4°C to the bottom of the tube. NM was discarded and replaced with 25 µl Lysis Buffer (5 mol/L Urea, 1% Tritonx100, 1xcOmpete EDTA-free, 5 mmol/L DTT). Nematosomes were incubated for 45 min at 37°C with vortexing in between every 15 min. After incubation nematosomes were snapped-freezed at -80°C for further analysis. Samples were then digested according to SP3 protocol 36 with some modifications as follows. After thawing, lysates were mixed each with 25 µL Alkylation buffer (50 mM borate buffer and 25 mM IAA) for 50 min at room temperature (RT). Then, 5 µL of resuspended SP3 beads (20 µg/µL A:B 1:1 mixture) were added to each sample followed by 150 µL ACN and mixed for 30 min at 800 rpm, RT. Then, beads were washed with 300 µL 70% EtOH, followed by 150 µL ACN. A 10 µL digestion buffer (4 ng/µL trypsin/Lys-C, 25 mM borate buffer and 0.01% DDOPM) was added to each sample followed by mixing on a shaker for 10 min at 800 rpm RT. Samples were mixed each by pipetting up and down and kept back on the shaker overnight. Next day, samples were centrifuged at 20,000 x g for 2 min and supernatant (ca. 10 µL) was transferred to LC-MS vials containing 1 µL of 5% FA. LC-MS Proteomics and Data analysis Chromatographic separation was performed on a Dionex U3000 nanoHPLC system equipped with an Acclaim pepmap100 C18 column (2 µm particle size, 75 µm × 500 mm) coupled online to a mass spectrometer. The eluents used were; eluent A: 0.05% formic acid (FA), eluent B: 80% ACN + 0.04% FA. The separation was performed over a programmed 120 minutes run. Initial chromatographic conditions were 4% B for 2 minutes followed by linear gradients from 4–50% A over 90 minutes then 50 to 90% A over 5 minute, and 10 minutes at 90% A. Following this, an inter-run equilibration of the column was achieved by 16 minutes at 4% A. A constant flow rate of 300 nl/min was employed. Data acquisition following separation was performed on an QExactive Plus. Full scan MS acquisition was performed (350–1400 m/z, resolution 70,000). Subsequent data dependent MS/MS scans were collected for the 15 most intense ions (Top15) via HCD activation at NCE 27.5 (resolution 17,500); dynamic exclusion was enabled (20 sec duration). Triplicate measurements were performed for all the samples. Raw data were analyzed against Nematostella vectensis Uniprot database (20.05.2022) (24,497 sequences) plus common contaminants (cRAP). The search was performed on Proteome discoverer 2.5 using a SequestHT search engine with 10 ppm and 0.02 Da precursor and fragment ions tolerances, respectively. Digestion with trypsin with a max of 2 missed cleavages were applied. Strict parsimony criteria have been applied filtering peptides and proteins at 1% FDR. INFERYs rescoring algorithm was applied. Label-free quantification method based on the intensities of the precursor ions was used. Proteins were filtered to have “High” FDR combined confidence and at least 2 identified peptides. Data was further analyzed by Excel and Perseus v 1.6.15.0 37 . Protein intensities were averaged for technical replicates. To perform differential quantitative analysis of proteins, raw protein intensities were extracted, averaged between technical replicates, one outlier replicate per group “Control 5, Native 1 and Non-Native 2” were excluded, then median based normalization was applied to the data. Log2 transformed intensities were grouped in 3 groups depending on the Vibrio treatment (each with 5 replicates). Proteins with at least 4 intensity values in one group were used for further analysis. Missing values were imputed from a normal distribution separately for each replicate (Width 0.3, Downshift 1.8). Statistical analysis was done using ANOVA, permutation-based FDR of 0.01. Gene enrichment analysis was performed on The Database for Annotation, Visualization, and Integrated Discovery (DAVID) 38 , 39 using the functional annotation tool. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium ( http://proteomecentral.proteomexchange.org ) via the PRIDE partner repository 40 with the dataset identifier PXDxxxxxx. CRISPR/Cas 9 mediated knock-out generation CRISPR/Cas9-mediated transgenic lines were generated following the protocol published before 41 . The gene cJUN (NVE21090) was selected based on transcriptomic analysis after recolonization experiments 26 and tissue specificity 27 . Four guide sequences (sgNVE21090E1, sgNVE21090E2, sgNVE21090E3, and sgNVE21090E4) were designed using the web tool CRISPOR.org 42 , with an implemented Nematostella genome. First, the guide oligonucleotides were mixed in equal amounts, annealed for 5 min at 95°C, and then incubated at room temperature for 2–3 h. The annealed oligos were cloned into the gRNA expression vector pDR274 (42250, Addgene). After successful integration of the guides into the vector, the guide sequences were amplified, transcribed in vitro using the MEGAscript™ T7 kit (Thermo Fisher), and purified with the MEGAclear RNA cleanup kit (Thermo Fisher) prior to injection. The injection mix consisted of Cas9 enzyme (1 mg/ml stock) (TrueCut™ Cas9 Protein v2, Thermo Fisher), sgRNAs (450 ng), Alexa fluorescent dye (1.1 M in KCl), and RNase-free water. To obtain fertilized eggs, animals were incubated at 25°C for 11 h to induce gamete production. Egg packages were incubated in sperm media from males for 15 min before dejellying the fertilized egg packages with 4% cysteine (pH 7.4), followed by five washing steps in 16‰ NM. The injection mix was incubated at 37°C for 5 min before injection. The injection setup was conducted according to the microinjection protocol for mRNA and Morpholinos previously described 43 . Injected eggs were raised in the dark at 20°C, with the medium (NM) being exchanged daily, and food introduction after 10–12 days. To confirm the successful integration of CRISPR/Cas9-mediated mutagenesis into the polyps’ genome, we performed crossbreeding, High Resolution Melting Curve Analysis (HRMC) and genotyping. Genomic DNA was isolated from injected juvenile polyps using the DNeasy Blood & Tissue kit (Qiagen). In HRMC, short DNA fragments at the targeted locus are amplified, and changes in these fragments are detected through shifts in the melting curves. Following confirmation of successful mutations, the mutant animals were crossed with wildtype polyps to generate the F1 generation. The same procedure was applied as for the previous F0 generation. Heterozygous animals were further analyzed by Sanger sequencing to determine the precise mutation pattern. Polyps with the same mutation pattern were crossed to generate the F2 generation, which included homozygous mutants ( cJUN −/− ), homozygote wildtype ( cJUN +/+ ) and heterozygote ( cJUN +/− ) offspring, which are used for experimental set ups. Electron microscopy with nematosomes Scanning electron microscopy (SEM) were taken with the Zeiss REM Supra 55VP. Nematosomes were extracted from the polyps and placed on well with Poly-L-Lysine coated cover glasses. All fixation, washing, dehydration were performed as former published 44 . Images were later analyzed using ImageJ. 16S rRNA analysis For 16S rRNA analysis the bioinformatics were performed using Qiime 2 2021.11 45 . First, raw sequences were demultiplexed and quality filtered using the q2-demux plugin followed by denoising with DADA2 46 . The amplicon sequence variants (ASVs) were aligned with mafft and for constructing the phylogeny fasttree2 was conducted 47 , 48 . All samples were rarefied to 900 sequences per samples prior estimation for Alpha-diversity metrics (observed features and Faith’s Phylogenetic diversity 49 ), beta-diversity metrics (Bray-Curtis dissimilarity, Jaccard distance and Unifrac (weighted and unweighted 50 , 51 )) and Principle Coordinate Analysis (PCoA). Taxonomy was assigned to ASVs using q2-feature-classifier classify-sklearn naïve Bayes taxonomy classifier against Greengenes 13_8 99% data set as reference 52 . Further analysis, statistical analysis and plot visualization was conducted with OriginPro (Version 2021. OriginLab Corporation, Northampton, MA, USA.). Results Nematosomes phagocytose Vibrio strains with varying efficiency To test if nematosomes are phagocytosing bacteria differentially, we established a phagocytosis assay that allows to quantify on one hand lysosomal activity and on the other hand bacterial engulfment (Fig. 2 A). Therefore, nematosomes are extracted from the gastric cavity and incubated in a bacterial suspension with defined bacterial concentration. For the assay we chose different Vibrio strains, as they are common marine bacteria and are among the main colonizers of N. vectensis. As native isolates we selected the Vibrio isolates NJ1, NJ33 and NA11 that were cultivated from N. vectensis (Table S1) 26 . We analyzed the phagocytosis rate of these native isolates and compared it with the rate of phagocytosis of foreign Vibrio isolates (Hal025 and Hal281 32 ) derived from the sponge Halichondria panicea (Table S1) . The results revealed that native Vibrio strains were phagocytosed at significantly lower rates, while the foreign isolates Hal025 and Hal281 were engulfed at substantially higher rates ( Fig. 2 A, B ) . The increased phagocytosis of foreign isolates correlated with significant increase in lysosomal activity within nematosomes ( Fig. 2 A, C ) . In contrast, nematosomes confronted with native Vibrio strains did not increase their lysosomal activity (Fig. 2 A, C). In addition to the phagocytosis rate for each isolate, we also compared the colonization efficiency of each isolate in mono-association experiments (Fig. 2 D, E). This recolonization approach revealed that the three native Vibrios , namely NJ1, NJ33 and NA11, colonized in significantly higher rates on the polyp 7 days post recolonization (dpr), compared to the two foreign isolates Hal025 and Hal281 (Fig. 2 E). This trend was already seen after 2 dpr ( Figure S1 ). These results correlate with the observations of elevated phagocytosis rates (Fig. 2 B) and lysosomal actives (Fig. 2 C) of the nematosomes upon foreign Vibrio engulfment suggesting a potential link between nematosome phagocytosis and colonization success. Foreign and native Vibrio isolates cause diverging proteome responses in nematosomes To characterize the differential response of nematosomes to native and foreign Vibrio strains, we performed proteome analysis with extracted nematosomes. Specifically, we assessed the responses of nematosomes after confronting them with NJ1 and Hal281, and compared it to a control treatment without bacterial challenge. The proteome analysis revealed significantly abundant proteins when comparing nematosomes challenged with native and foreign bacterial isolates (Fig. 3 , Table S2, Figure S2 ). A total of 2676 proteins were detected in the proteomic analysis of nematosomes treated with bacterial isolates NJ1 and Hal281, as well as in untreated controls. To extract proteins that were uniquely differently abundant in either NJ1 or Hal281 treatment, we generated five clusters using k -means clustering (Fig. 3 A). Out of the total proteins identified, 157 proteins were detected uniquely in NJ1-treated samples and 104 in Hal281-treated nematosomes ( Fig. 3 B ) . Thereby, cluster 1 represents proteins which emerged exclusively following NJ1 treatment, while cluster 4 contain proteins, which are higher in abundance after confrontation with Hal281. A KEGG enrichment analysis revealed that cluster 1, which contains proteins that are significantly more abundant in NJ1-treated nematosomes, contains proteins related to carbon and nitrogen metabolism (Table S4) . These findings suggest that interaction with native bacteria may promote the host metabolisms, reflecting potential symbiotic interactions. In contrast, that treatment with Hal281 elevated the abundance of proteins belonging to the phagosomal pathway ( Table S3, S4 ). Especially proteins belonging to the cytoskeleton formation of phagosomal formation, like Dynein and Tubulin beta (TUBB) as well as F-actin are increased (Fig. 3 C, Table S3 ). Interestingly, the V-ATPase shows an increase in abundance in both bacterial treatments, potentially linking it to default lysosomal activity (Fig. 2 C). The proteomic analysis revealed an elevated abundance in the phagosomal pathway upon foreign bacterial treatment, indicating an active response of nematosomes in immune response, particularly in recognizing foreign isolate phagocytosis and degradation. To further explore the molecular mechanisms driving these responses, we aimed to manipulate the regulatory pathways involved. cJUN mutation reduces nematosome proliferation In a next step we aimed to alter the function of nematosomes by CRISPR/Cas9 genome editing. Therefore, we screened for potential transcription factors (TF) that are potentially involved in the proliferation and/or immune function of nematosomes. We identified cJUN orthologs, which is highly expressed in nematosomes (NVE21090) (Fig. 4 A), while a second ortholog is mainly expressed in the tentacle region (NVE16876) (Fig. 4 B) of the polyp 26 , 27 , 56 . Interestingly, the cJUN ortholog NVE21090 is also upregulated upon bacterial recolonization 26 . cJUN is an evolutionarily conserved transcription factor that activates immune genes, regulates phagocytosis, and is involved in cell proliferation 57 , 58 , 59 . As central hub, cJUN integrates signaling information of various pathways, including ERK and JNK signaling 60 . In the cnidarian Hydra TLR signaling via MyD88 activates JNK signaling following immune stimulation 61 and in vertebrates cJUN regulates macrophage activation 62 , 63 . Therefore, we selected the cJUN ortholog NVE21090 for CRISPR/Cas9 genome editing 41 , to be able to functionally investigate the role of nematosomes in regulating microbiome composition. We generated deletions in the first exon of NVE21090, which ultimately led to a variation of mutations in animals of the F1 generations (Fig. 4 C). For the subsequent breeding, we selected the male strain M8 and the female strain M11, both carrying the same heterozygous mutation leading to a stop codon in the first exon of the gene. This mutation results in incomplete translation of the gene, leading to the loss of essential domains within the protein (Fig. 4 E ) . All subsequent approaches were conducted on F2 polyps with homozygous mutation named cJUN −/− (Fig. 4 D). Morphological comparisons between cJUN +/+ and cJUN −/− polyps revealed no significant differences in polyp length (Fig. 4 D, F ) . Simultaneously, cJUN −/− polyps showed no significant differences in area size, indicating that cJUN −/− mutation in nematosomes leads to no significant changes in polyps’ body (Fig. 4 D, G). However, a significant reduction in nematosome numbers per polyps was observed (Fig. 4 H). While adult cJUN +/+ polyps harbor around 1100 nematosomes, cJUN −/− polyps only exhibit around 100 per polyp. Interestingly, this lower number seems to be slightly compensated by size, as cJUN −/− nematosomes display a bigger size compared to cJUN +/+ nematosomes (Fig. 4 I). cJUN mutation affects nematosome phagocytosis and bacterial colonization To approach the effect of cJUN on nematosome phagocytosis, we performed the newly established phagocytosis assay, confronting cJUN −/− and cJUN +/+ polyps with the same bacterial isolates (Fig. 5 A). cJUN +/+ revealed similar lysosomal activity and rates of phagocytosis as observed in wt animals after confrontation with foreign and native Vibrio strains (Fig. 2 B, C and Fig. 5 A, B, C ) . However, while cJUN +/+ nematosomes adjust their lysosomal activity in response to different isolates, cJUN −/− nematosomes maintain a similar default lysosomal activation independent of bacterial treatment (Fig. 5 C). Being confronted with the foreign isolates Hal025 and Hal281 was not resulting in the activation of lysosomal activity in the cJUN −/− nematosomes. Analyzing the number of bacteria engulfed in nematosomes revealed an increase of the isolates NA11 and Hal281 (Fig. 5 B) in cJUN −/− nematosomes, most likely by an accumulation of bacterial cells in the phagosome. As the lysosome is not activated in cJUN −/− nematosomes, degradation of bacteria in phagosome is most likely impaired, resulting in arrested phagocytosis. The impaired phagocytosis of foreign Vibrio isolates resulted in an increased recolonization rate in mono-association experiments (Fig. 5 D, E) in adult cJUN −/− polyps compared to cJUN +/+ polyps (Fig. 5 E). Interestingly, the native isolates NJ1 and NJ33 recolonized cJUN −/− polyps significantly lower compared to cJUN +/+ polyps, suggesting even supporting effects of nematosomes for some native colonizers. Bacterial dysbiosis in cJUN -/- polyps Discovering significant differences in the nematosome phenotype of cJUN −/− polyps, we proceeded to analyze its associated microbiome. 16S rRNA gene sequencing revealed significant differences between the microbiome of cJUN −/− and cJUN +/+ polyps ( Figure S3, Table S5 ), with a significantly lower alpha diversity ( Figure S3C ) and evenness ( Figure S3D ) compared to cJUN +/+ animals. These differences in bacterial colonization in cJUN −/− and to cJUN +/+ polyps support previous results indicating that host mechanisms are involved in the control of bacterial establishment in N. vectensis 24 . To test this hypothesis, we recolonized germfree adult cJUN +/+ and cJUN −/− polyps with the bacterial consortia of adult polyps and followed the succession of bacterial establishment over the period of 28 days by 16S rRNA gene sequencing. Over the whole course of the experiment the cJUN genotype has significant effects on microbial community structure (Table 1 , Fig. 6 C), while also the time point after recolonization (days post recolonization, dpr) accounted for differences in microbial colonization ( Table 1 ) . Table 1 Statistical summary of ADONIS and ANOSIM tests on Bray-Curtis, Jaccard, Weighted UniFrac, and Unweighted UniFrac distance matrices, comparing microbial community dissimilarities between cJUN +/+ and cJUN −/− animals. The parameter column indicates whether the analysis was performed at the genotype level or at the dpr level. Adonis R 2 values represent the proportion of variance explained by genotype or dpr while ANOSIM R values indicate the degree of separation between groups. Significant differences are indicated by p-values, with higher R values reflecting stronger microbial dissimilarities between groups. Parameter Metric Adonis R² Adonis p ANOSIM R ANOSIM p genotype Bray-Curtis 0.097 0.005 0.17 0.002 Jaccard 0.099 < 0.001 0.31 < 0.001 Weighted UniFrac 0.073 0.031 0.14 0.007 Unweighted UniFrac 0.101 < 0.001 0.23 < 0.001 dpr Bray-Curtis 0.174 < 0.001 0.42 < 0.001 Jaccard 0.111 < 0.001 0.40 < 0.001 Weighted UniFrac 0.282 < 0.001 0.49 < 0.001 Unweighted UniFrac 0.110 < 0.001 0.28 < 0.001 During the early timepoints (2 and 7 dpr) cJUN +/+ and cJUN −/− polyps exhibit similar patterns in microbial richness and evenness (Fig. 6 A, B). After 14 dpr, the microbiome of cJUN +/+ polyps show significantly higher bacterial alpha diversity and evenness compared to cJUN −/− polyps ( Fig. 6 A, B ) . The increase in Weighted UniFrac distances between the microbiome of the different genotypes reveal that genotype-related differences in the composition of the microbiome become more pronounced over time, as the microbial communities develop differently between cJUN +/+ and cJUN −/− animals (Fig. 6 C). The significant differences in specific microbial taxa observed between the genotypes over time (Fig. 6 D, Figure S4 ), including the dominance of some Alphaproteobacteria taxa ( Figure S4A, B ) and the reduced colonization success of specific Gammaproteobacteria ( Figure S4D ) in cJUN −/− polyps highlight the selective force of nematosomes on specific taxa. Discussion Nematosomes exhibit characteristics of ancient immune cells Our study provides strong evidence that nematosomes are key immune cells in N. vectensis , playing a critical role in microbial selection through selective phagocytosis. The phagocytosis assay demonstrated that nematosomes exhibit a clear preference for engulfing foreign Vibrio isolates while sparing native ones, indicating that the distinguishing of bacteria acts most likely on the level of recognition. Phagocytosis is a fundamental immune defense mechanism in invertebrates, often mediated by circulating cells called immunocytes. These cells fulfill various functions of the innate immune system, including the recognition of pathogens, phagocytosis and the synthesis of antimicrobial proteins. Thereby, the innate response triggered by microbial-associated molecular patterns (MAMPs) is based on the activation of pattern recognition receptors. In vertebrates, macrophages use pattern recognition receptors (PRRs) such as Toll-like receptors (TLRs) and scavenger receptors to recognize MAMPs and discriminate between self and non-self by activating NF-κB 64 , 65 . In nematosomes of N. vectensis the TLR signaling pathway is also highly expressed and has been shown to activate the NF-κB pathway in response to bacterial pathogens, mirroring the innate immune responses seen in vertebrates 29 , 66 . Experimental MyD88 knockdowns in Hydra showed that TLR signaling does not act unidirectionally via the transcription factor NF-κB but is also linked to cJUN, resulting in altered microbiome composition and impaired bacterial recognition 61 . This suggests that nematosomes may use TLR-mediated pathways to recognize bacterial cells and regulate strain-specific immune responses through evolutionarily conserved signaling cascades. Furthermore, the observed correlation between phagocytosis efficiency and bacterial colonization efficiency supports the hypothesis that nematosomes act as selective gatekeepers of microbial establishment in N. vectensis . Native Vibrio isolates that were less frequently phagocytosed colonized the polyps at higher rates, while foreign isolates that were preferentially engulfed and degraded exhibited poor colonization efficiency. The colonization by foreign isolates increased in cJUN −/− polyps, demonstrating that nematosomes play a direct role in shaping the microbiome by selectively removing non-native bacteria before they can establish themselves in the host. The proteomic analysis further supports this immune function, revealing distinct protein patterns in nematosomes upon bacterial exposure. Proteins involved in phagocytosis, including cytoskeletal components such as actin and dynein, were upregulated in response to foreign bacteria, supporting the notion that nematosomes are actively involved in immune surveillance and microbial selection, and emphasizing the functional importance of nematosomes in host-microbe interactions. cJUN - a regulator of nematosome phagocytosis and proliferation Our results highlight cJUN as a crucial regulator of innate immunity in N. vectensis . CRISPR/Cas9-mediated knockout of cJUN resulted in a marked impairment in their ability to mount a selective immune response. In cJUN −/− mutants, nematosomes exhibited a failure to activate lysosomal degradation pathways following bacterial engulfment, leading to an accumulation of both native and foreign bacteria within the phagosome. Similarly, macrophages, disrupted in cJUN, TLRs, or NF-κB, can still engulf pathogens but fail to complete their degradation 67 , 68 , 69 , resulting in “arrested phagocytosis”, where bacteria remain trapped within the phagosomes but are not effectively digested. Certain intracellular pathogens, exploit these host weaknesses by preventing phagosome-lysosome fusion or modifying host signaling pathways to survive within immune cells 70 , 71 , 72 . These evasion tactics allow pathogens to manipulate key stages of the phagocytosis process, including phagosome formation, maturation, and acidification 72 . By interfering with these crucial immune defense mechanisms, bacteria can avoid degradation and establish intracellular infections. However, also symbionts rely on the mechanisms of arrested phagocytosis to persist within host tissue. In sponges, ankyrin-repeat proteins from bacterial symbionts can modulate phagocytosis by interfering with phagosome development, potentially allowing symbionts to escape digestion 73 , 74 . Similarly, in deep-sea mussels, the regulation of mTORC1 signaling helps retain symbionts in gill cells by preventing phagosome digestion 75 . In coral-dinoflagellate symbiosis, the symbiosome is hypothesized to be an early arrested phagosome, with transient gene expression changes occurring during symbiont uptake 76 . Recent results indicate, that anthozoan hosts indiscriminately phagocytose various microalgae, but non-symbiotic species are expelled through vomocytosis. Successful symbionts suppress the host's innate immune response, preventing expulsion and promoting niche formation 77 . These studies highlight the importance of arrested phagocytosis in various symbiotic relationships across different marine organisms. In N. vectensis cJUN −/ − nematosomes exhibited a similar phenotype, where they successfully internalized bacteria but failed to activate lysosomal responses necessary for degradation. The accumulation of engulfed but undegraded bacteria in mutant nematosomes suggests that cJUN plays a crucial role in regulating lysosomal maturation and phagosomal acidification. This parallels its function in vertebrates, where cJUN is involved in the transcriptional regulation of immune effectors, including lysosomal enzymes and phagosome maturation factors 62 . The absence in cJUN-dependent signaling in N. vectensis may thus impair the degradation of foreign Vibrio strains, contributing to microbial persistence and altered colonization patterns. This suggests that cJUN is essential for orchestrating the cytoskeletal and phagosomal dynamics required for effective microbial clearance. In addition, cJUN regulates also nematosome proliferation, as demonstrated by the significant reduction in nematosome numbers in cJUN −/ − mutants. It is well known that cJUN positively regulates cell proliferation by repressing tumor suppressor genes and inducing cyclin D1 transcription in invertebrates 78 , 79 and vertebrates 59 . Thereby, cJUN negatively regulates p53 expression by binding to its promoter, thereby promoting cell cycle progression and proliferation 80 . Nematosomes are budding from distinct regions of the mesenteries into the gastric cavity. In addition to the proliferation of nematosomes, the mesenteries of N. vectensis play a crucial role in endomesodermal patterning and germ cell development 81 , 82 , demonstrating the highly proliferative properties of this tissue. Our results show that cJUN is essential for controlling the expansion of nematosomes, potentially by regulating genes involved in cell division and differentiation, suggesting that cJUN not only governs phagocytosis but also orchestrates the development and maintenance of nematosomes as functional immune units. The loss of cJUN function ultimately resulted in microbial dysbiosis, with mutant polyps displaying altered microbiome compositions dominated by non-native bacterial species. These findings establish a direct link between cJUN-mediated immune regulation and microbiome homeostasis in N. vectensis . The transcription factor cJUN plays a crucial role in immune regulation and homeostasis. In Drosophila, JNK signaling, which activates c-Jun, is essential for innate immunity and development 83 . In mammals, epidermal JunB regulates cutaneous immune cell-microbiota interactions, with its absence leading to atopic dermatitis-like symptoms and spontaneous S. aureus colonization 84 . c-JUN/AP-1 is particularly important in CD8 T cell responses to acute infection, participating in productive immune responses 85 . These studies highlight the complex interplay between cJUN-mediated signaling, immune regulation, and microbiome homeostasis in various organisms and contexts. Recent findings in sponges further support an evolutionarily conserved role in cJUN/AP-1 in symbiont recognition. In Amphimedon queenslandica , symbiotic bacteria induce a rapid transcriptional response involving cJUN/AP-1, NF-κB and IRF, whereas foreign bacteria do not trigger these responses and instead elicit xenobiotic metabolism 86 . This mirrors our observations in N. vectensis and suggests that ancient metazoans already employed AP-1 family transcription factors to distinguish beneficial symbionts from potentially harmful bacteria, maintaining holobiont stability through selective immune regulation. Innate immune specificity and its implications for host-microbe interactions The strong evidence for innate immune specificity in N. vectensis has significant implications for our understanding of the evolution of host-microbe interactions. Traditionally, innate immunity has been viewed as a broad, non-specific defense mechanism, whereas adaptive immunity is considered the primary driver of immune specificity. However, our findings challenge this dichotomy by demonstrating that even early-branching metazoans like cnidarians possess selective innate immune mechanisms. The ability of nematosomes to distinguish between closely related bacterial strains and selectively regulate microbiome composition suggests that innate immune specificity is an ancient and fundamental feature of metazoan immunity. This aligns with recent studies in other invertebrates, which have also demonstrated a surprising degree of innate immune selectivity, further supporting the notion that immune specificity predates the evolution of adaptive immunity 32 , 87 , 88 . In several studies it was shown that invertebrates can differentiate between pathogens at the species and even strain level 89 , 90 . This specificity is particularly evident in the phenomenon of immune priming, where initial exposure to a pathogen provides protection against subsequent encounters. Immune priming in invertebrates is a phenomenon where an initial pathogenic exposure enhances immune defenses against subsequent infections. This adaptive-like immunity has been observed in various invertebrates 91 , 92 , such as in woodlice 9 , where hemocytes show increased phagocytosis of previously encountered bacterial strains. In the oyster Crassostrea gigas , hemocytes exhibit differential phagocytic responses to various bacterial species, demonstrating that invertebrate immune cells can selectively recognize and respond to different microbes 93 , 94 . Similarly, in the squid-Vibrio symbiosis, host immune cells differentiate between preferred symbionts and other closely related bacteria 17 . In addition, immune priming can be enhanced by protective symbionts 95 . Potential mechanisms involved are a sustained immune responses, epigenetic modifications, and metabolic reprogramming, though the underlying mechanisms are not fully understood 96 . However, C-type lectin-like domain (CTLD) proteins have been identified as potential contributors to this specificity, with their extreme gene diversification observed in various invertebrate genomes 97 . Drosophila exhibits a specific primed immune response against certain pathogens based on phagocytosis 98 that requires phagocytes and the Toll pathway. This priming involves exposure to dead or sublethal doses of microbes, eliciting an initial response that enhances protection against subsequent infections 99 . While the mechanisms underlying this specificity and memory are not fully understood, proposed explanations include elevated levels of phagocytosis. These examples provide additional support for the concept that innate immune specificity is an ancient and widespread phenomenon across diverse metazoans. The evolutionary advantage of innate immune specificity likely lies in its ability to balance microbial diversity while preventing colonization by potentially harmful bacteria. In the case of N. vectensis , the selective phagocytosis of foreign bacteria by nematosomes ensures that the microbiome remains stable and beneficial to the host. This mechanism is particularly crucial for organisms with simple immune architectures, where adaptive immune responses are absent. By employing a finely tuned innate immune response, N. vectensis can maintain a dynamic but controlled microbiome, allowing for environmental adaptability without compromising immune defenses. This study, therefore, positions cnidarians as valuable models for exploring the evolutionary origins of immune-microbe interactions and provides insights into how early metazoans may have developed mechanisms for microbial regulation in the absence of adaptive immunity. Conclusion In conclusion, our study provides compelling evidence that N. vectensis employs a selective innate immune system to regulate its microbiome, challenging the traditional perception of innate immunity as a non-specific defense. The role of nematosomes in selectively phagocytosing foreign bacteria, and the involvement of cJUN in orchestrating this process, highlights the molecular complexity of immune regulation in early metazoans. Our findings reinforce the idea that innate immune specificity is evolutionarily ancient and widespread among invertebrates, playing a crucial role in maintaining host-microbe homeostasis. By demonstrating the impact of selective immune responses on microbiome composition, our work contributes to a broader understanding of host-microbe interactions and their evolutionary significance. Future research should further explore the molecular pathways underlying nematosome-mediated immunity and examine how these mechanisms have influenced the evolution of immune systems across metazoans. Declarations Competing Interest The authors declare no competing interests. Funding This work was supported by DFG CRC grant 1182 “Origin and Function of Metaorganisms” (Project B1, Z3). NGS was carried out at the Competence Centre for Genomic Analysis (Kiel) within the CRC 1182 project Z3. Author contributions NHK and SF conceived the study. NHK performed the majority of the experiments and data analysis. MA contributed to proteomic analysis. GF assisted with mono-associations on wildtype polyps. SF supervised the project and provided conceptual input throughout. All authors contributed to data interpretation. NHK wrote the manuscript with input from all authors. All authors read and approved the final manuscript. Acknowledgements We thank Katja Cloppenborg-Schmidt for preparing the 16S rRNA gene library. We would like to acknowledge the Center for Advanced Imaging (CAi) at Heinrich-Heine-University Düsseldorf for providing access to the Olympus FV3000 Confocal Laserscanning Microscope and Zeiss REM Supra 55VP. Especially Dr. Sebastian Hänsch and Dr. Miriam Bäumers for supporting during sample preparation and imaging. 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Supplementary Files KayaetalSupplNatComm.xlsx Table S1, Table S2, Table S3, Table S4, Table S5 KayaetalSupplNatComm.docx Figure S1, Figure S2, Figure S3, Figure S4 KayaetalSupplVideo.mp4 Video S1 Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7020203","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":483523559,"identity":"2062a179-3c5e-48b7-b157-492a7a9b019e","order_by":0,"name":"Sebastian Fraune","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAoElEQVRIiWNgGAWjYBAC9gYwZZNgACQliNLCcwBMpZGu5TApWqQPP5MuqDifZy6RwHjjA1Fa+NLMpGecuV1sOSOB2XIGMVrseRjMpHnbbiduuJHAJs1DlC087N+kef+dg2j5Q5wWHqAtDQcgWojRAdJSbM1zLDlxw5mHzZY9RDps422eGrvEDceTD974QZQ1CMDYQKKGUTAKRsEoGAU4AQB7kS5zvVeKSAAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-6940-9571","institution":"Heinrich-Heine University Düsseldorf","correspondingAuthor":true,"prefix":"","firstName":"Sebastian","middleName":"","lastName":"Fraune","suffix":""},{"id":483523560,"identity":"1f5469db-e11b-4212-a8d9-e495837538e8","order_by":1,"name":"Nida Kaya","email":"","orcid":"","institution":"Heinrich-Heine University Düsseldorf","correspondingAuthor":false,"prefix":"","firstName":"Nida","middleName":"","lastName":"Kaya","suffix":""},{"id":483523561,"identity":"524e0132-489c-4594-9120-46de01480592","order_by":2,"name":"Mohammad Abukhalaf","email":"","orcid":"","institution":"Systematic Proteome Research \u0026 Bioanalytics","correspondingAuthor":false,"prefix":"","firstName":"Mohammad","middleName":"","lastName":"Abukhalaf","suffix":""},{"id":483523562,"identity":"32ac49ab-fa09-440d-98f2-4425863ef540","order_by":3,"name":"Gabriela Fuentes Reyes","email":"","orcid":"","institution":"Heinrich-Heine University Düsseldorf","correspondingAuthor":false,"prefix":"","firstName":"Gabriela","middleName":"Fuentes","lastName":"Reyes","suffix":""},{"id":483523563,"identity":"19d1916c-0113-489f-8031-e81dfc631808","order_by":4,"name":"Ute Hentschel","email":"","orcid":"https://orcid.org/0000-0003-0596-790X","institution":"GEOMAR Helmholtz Centre for Ocean Research Kiel","correspondingAuthor":false,"prefix":"","firstName":"Ute","middleName":"","lastName":"Hentschel","suffix":""},{"id":483523564,"identity":"21378f1f-a0c8-4930-9828-c0f55e73960c","order_by":5,"name":"Andreas Tholey","email":"","orcid":"https://orcid.org/0000-0002-8687-6817","institution":"Christian-Albrechts-Universität Kiel","correspondingAuthor":false,"prefix":"","firstName":"Andreas","middleName":"","lastName":"Tholey","suffix":""}],"badges":[],"createdAt":"2025-07-01 12:20:25","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7020203/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7020203/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":86490533,"identity":"77b756c2-b897-43a3-90fa-3a5b0e275318","added_by":"auto","created_at":"2025-07-11 09:02:44","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":875058,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNematosomes in and outside the polyp\u003c/strong\u003e. \u003cstrong\u003eA)\u003c/strong\u003e Foot region of adult N. vectensis polyp with nematosomes swimming inside the body cavity (red arrows) and nematosomes resting on the body wall of the polyp (white arrows). Scalebar represent 2 mm \u003cstrong\u003eB)\u003c/strong\u003e SEM image of a single nematosome with two extending cnidocytes and discharged tubules (red arrows) and cilia highlighted with blue arrow (Type 1) and with a white arrow (Type 2)\u003csup\u003e27\u003c/sup\u003e. \u003cstrong\u003eC)\u003c/strong\u003e Confocal image of a nematosome stained with Hoechst (blue) and LysoTracker (red). \u003cstrong\u003eD)\u003c/strong\u003e Egg package with eggs representing different cleavage status (white arrows) and nematosomes (red arrows) in between the eggs covered in the matrix. All scalebars represent 10 µm unless otherwise indicated.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7020203/v1/64e42711a56f497f4bcc045c.png"},{"id":86490534,"identity":"d3e91c76-6409-4906-9ed5-4c906d83f1f1","added_by":"auto","created_at":"2025-07-11 09:02:44","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":204504,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNematosomes selectively phagocytose different Vibrio strains. A)\u003c/strong\u003e Confocal images of nematosomes challenged with NJ1 (native colonizer) and Hal281 (foreign colonizer). Vibrios are stained with BacLight and lysosomes with LysoTracker revealing that nematosomes are phagocytosing Hal281 more efficiently in comparison to native colonizer treatment and no bacterial challenge (control). White arrows indicate the overlay of bacterial and lysosomal signal, representing bacterial degradation. Scalebars represent 10 µm unless otherwise indicated. \u003cstrong\u003eB)\u003c/strong\u003e Quantification of bacterial load per nematosome. The percentage of bacteria relative to the nematosome area shows significantly higher bacterial presence in nematosomes exposed to the foreign strains Hal025 and Hal281 compared to native strains and control. N= 10 - 22, Kruskal-Wallis ANOVA, * p\u0026lt;=0,05 ** p\u0026lt;=0,01 *** p\u0026lt;=0,001. \u003cstrong\u003eC) \u003c/strong\u003eLysosomal area relative to total nematosome area following bacterial challenge. Nematosomes exposed to the foreign strains exhibit a significantly larger lysosomal area compared to those treated with native strains and the control, indicating enhanced lysosomal activity. N= 10 - 22, Kruskal-Wallis ANOVA, * p\u0026lt;=0,05 ** p\u0026lt;=0,01 *** p\u0026lt;=0,001. \u003cstrong\u003eD)\u003c/strong\u003e Schematic representation of the experimental setup. Adult N. vectensis polyps underwent a two-week antibiotic (AB) treatment to deplete resident microbiota, followed by mono-association with either native or foreign Vibrio strains. Colonization was assessed after seven days post-recolonization (dpr). \u003cstrong\u003eE) \u003c/strong\u003eQuantification\u003cstrong\u003e \u003c/strong\u003eof Vibriocolonization levels in adult polyps 7 dpr. Colony-forming units (CFU) per polyp are significantly lower for foreign strains compared to native colonizers, indicating reduced colonization efficiency of the foreign strains. N=5 polyps each, Two-way ANOVA revealed significant differences between the groups native (blue) and foreign (red) isolates, * p\u0026lt;=0,05 ** p\u0026lt;=0,01 *** p\u0026lt;=0,001.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7020203/v1/9f2a22227a14a4c0a1ab4334.png"},{"id":86491355,"identity":"b4558026-4c92-49b7-9513-7e8846a19980","added_by":"auto","created_at":"2025-07-11 09:10:44","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":264874,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDifferentially abundant proteins in native vs foreign treatment in nematosomes. \u003c/strong\u003eThis study was conducted with five biological replicates per group: a control group without bacterial treatment, a group treated with a native isolate (NJ1) and a group treated with a foreign isolate (Hal281). A total of 2676 proteins were detected, 257 ,204 and 77 proteins with higher abundance in the native isolate-treated group in the foreign isolate-treated group, and in the untreated control group, respectively. \u003cstrong\u003eA)\u003c/strong\u003e Heatmap showing clustering of differentially abundant proteins across three conditions: nematosomes treated with NJ1, Hal281, and untreated control. Five distinct protein clusters are identified base on functional enrichment. Cluster 1: oxidative phosphorylation and carbon metabolism, Cluster 2 amino acid metabolism, Cluster 3 amino acid degradation and metabolic pathways, Cluster 4 Phagosome, and Cluster 5 Ribosome. Protein abundance is represented as Z-scores, with red indicating higher and green lower abundance. (Permutation-based FDR ANOVA, p\u0026lt;0.01, n= 826) \u003cstrong\u003eB)\u003c/strong\u003e Venn diagram illustrating the proteins identified uniquely in NJ-treated (157 proteins) and uniquely in Hal281-treated (104 proteins), and 100 proteins shared between NJ1 and Hal281 treatments. \u003cstrong\u003eC) \u003c/strong\u003eSchematic representation of the phagosome-lysosome pathway in nematosomes following bacterial exposure. The diagram shows the proposed trafficking routes from bacterial uptake, early phagosome formation, lysosomal fusion, and subsequent degradation\u003csup\u003e53, 54, 55\u003c/sup\u003e. Proteins significantly enriched in NJ1-treated (blue) and Hal281-treated (red) nematosomes are mapped onto corresponding pathway components, suggesting differential regulation of lysosomal processing depending on the bacterial isolate.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7020203/v1/31729c6f5d74bf7d34f0a969.png"},{"id":86490541,"identity":"a8b90283-7f19-416c-939b-347a56c935f7","added_by":"auto","created_at":"2025-07-11 09:02:44","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":252769,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNVE21090 mutations leads to stop codon in the upstream region of exon.\u003c/strong\u003e \u003cstrong\u003eA)\u003c/strong\u003e NVE21090 (cJUN) expression on mesenteries, nematosomes, tentacles and whole polyp\u003csup\u003e26, 27\u003c/sup\u003e. \u003cstrong\u003eB)\u003c/strong\u003e NVE16876 (cJUN) expression across the same tissues. Median ratio normalized read counts are represented, showing tissue specific expression patterns\u003csup\u003e26, 27\u003c/sup\u003e. \u003cstrong\u003eC)\u003c/strong\u003e Schematic illustration of NVE21090 (cJUN) locus with guide recognition sites (UTR in bright grey and exon in dark grey). The F1 generation displays a range of mutations, including deletions (red), insertions (blue), and the PAM sequence (green). The guide sequence is highlighted in purple. F2 with highlighted homozygous mutation leading to a stop codon (bold). Additional predicted SMART domains missing in F2 mutant animals upon confirmed mutation pattern. \u003cstrong\u003eD) \u003c/strong\u003eF2 adult polyps. Left cJUN\u003csup\u003e+/+\u003c/sup\u003e and right cJUN\u003csup\u003e-/-\u003c/sup\u003e. Scalebar represents 2 mm. \u003cstrong\u003eE) \u003c/strong\u003eScheme of protein structure of wildtype and cJUN mutant F2 animals with essential domains missing in cJUN\u003csup\u003e-/- \u003c/sup\u003e(generated with SMART). \u003cstrong\u003eF)\u003c/strong\u003e No significant differences were observed in polyp length between cJUN\u003csup\u003e+/+\u003c/sup\u003e and cJUN\u003csup\u003e-/-\u003c/sup\u003e animals. N=20. \u003cstrong\u003eG)\u003c/strong\u003e Polyp area also remained comparable between genotypes. N=20. \u003cstrong\u003eH)\u003c/strong\u003e The number of nematosomes per polyp was significantly reduced in mutant animals. N=20 polyps, two-sample t-test, * p\u0026lt;=0,05 ** p\u0026lt;=0,01 *** p\u0026lt;=0,001. \u003cstrong\u003eI)\u003c/strong\u003e The area of nematosomes in cJUN\u003csup\u003e-/-\u003c/sup\u003e polyps was slightly larger compared to wildtype nematosomes. N=69-257, two-sample t-test, * p\u0026lt;=0,05 ** p\u0026lt;=0,01 *** p\u0026lt;=0,001.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7020203/v1/c07cf3bf528a4619d7980991.png"},{"id":86491942,"identity":"11d6e8ec-e7e2-44a5-8e99-e3697a99e143","added_by":"auto","created_at":"2025-07-11 09:18:44","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":212970,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ecJUN depleted nematosomes are not able to recognize Vibrio strains. A)\u003c/strong\u003e Confocal images of cJUN depleted and not depleted nematosomes challenged with NJ1 and Hal281. Vibrio staining with BacLight and lysosome staining with LysoTracker reveal that nematosomes are phagocytosing Hal281 more efficiently in comparison to no bacterial challenge and native colonizer treatment. Scalebars represent 10 µm unless otherwise indicated. \u003cstrong\u003eB) \u003c/strong\u003eQuantification of bacterial engulfment per nematosome. The percentage of bacteria relative to nematosome area reveals significantly higher bacterial presence in cJUN\u003csup\u003e-/-\u003c/sup\u003e upon Hal281 treatment compared to cJUN\u003csup\u003e+/+\u003c/sup\u003e nematosomes and native isolate treatment. N= 15-43, Kruskal-Wallis ANOVA, * p\u0026lt;=0,05 ** p\u0026lt;=0,01 *** p\u0026lt;=0,001. \u003cstrong\u003eC)\u003c/strong\u003e Relative lysosomal activity per nematosome following bacterial challenge. Lysosomal area is significantly higher in cJUN\u003csup\u003e+/+ \u003c/sup\u003enematosomes exposed to Hal281 compared to cJUN\u003csup\u003e-/-\u003c/sup\u003e nematosomes and native isolate treatments. N= 15-43, Kruskal-Wallis ANOVA, * p\u0026lt;=0,05 ** p\u0026lt;=0,01 *** p\u0026lt;=0,001. \u003cstrong\u003eD)\u003c/strong\u003e Simplified illustration of AB treatment before mono-association with native and foreign Vibrio strains. \u003cstrong\u003eE) \u003c/strong\u003eMono-association of native and foreign colonizers on adult polyps after 7 days post recolonization. N=5 polyps each, Kruskal-Wallis ANOVA, * p\u0026lt;=0,05 ** p\u0026lt;=0,01 *** p\u0026lt;=0,001.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7020203/v1/549d70e95b0862eb9f112460.png"},{"id":86490539,"identity":"885be521-5405-48d3-9932-3537e8949b06","added_by":"auto","created_at":"2025-07-11 09:02:44","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":127314,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMicrobiome comparison between cJUN\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+/+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e and cJUN\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e-/-\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e after recolonization\u003c/strong\u003e. \u003cstrong\u003e(A, B)\u003c/strong\u003e Alpha diversity metrices for cJUN\u003csup\u003e+/+\u003c/sup\u003e and cJUN\u003csup\u003e-/-\u003c/sup\u003e microbiome comparison over a time course of 1 month (2,7,14,28 dpr). Both genotypes display similar values for richness (A) and evenness (B) after 2 and 7 dpr and separate after 14 dpr. cJUN\u003csup\u003e+/+\u003c/sup\u003e animals show a stable and rich microbiome composition while cJUN\u003csup\u003e-/-\u003c/sup\u003e reveal low level in Faith’s PD and evenness. N=5, Kruskal-Wallis ANOVA, * p\u0026lt;=0,05 ** p\u0026lt;=0,01 *** p\u0026lt;=0,001. \u003cstrong\u003eC) \u003c/strong\u003eWeighted UniFrac distance comparisons between microbial communities of cJUN\u003csup\u003e+/+\u003c/sup\u003e and cJUN\u003csup\u003e-/-\u003c/sup\u003e polyps over time, Kruskal-Wallis ANOVA, * p\u0026lt;=0,05 ** p\u0026lt;=0,01 *** p\u0026lt;=0,001. \u003cstrong\u003eD)\u003c/strong\u003e Bar plots representing the relative abundance of bacterial taxa in cJUN\u003csup\u003e+/+\u003c/sup\u003e and cJUN\u003csup\u003e-/- \u003c/sup\u003eanimals over time. Each bar shows the mean relative abundance of taxa across samples for each genotype, highlighting differences in the microbial community composition among the two groups and in the timepoints after recolonization.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7020203/v1/699088587c7925ee79db73c5.png"},{"id":86493217,"identity":"47aff83e-44b9-4c53-8e75-3553942765ed","added_by":"auto","created_at":"2025-07-11 09:34:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3500921,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7020203/v1/e634acc6-5078-470d-8bcd-5deae3b5da31.pdf"},{"id":86491358,"identity":"86a9fa94-980a-47c5-b197-adc469131d20","added_by":"auto","created_at":"2025-07-11 09:10:44","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":136639,"visible":true,"origin":"","legend":"Table S1, Table S2, Table S3, Table S4, Table S5","description":"","filename":"KayaetalSupplNatComm.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7020203/v1/d986fd1664c9fa810a0e5c53.xlsx"},{"id":86490536,"identity":"c6239be4-6480-498e-8592-736651342863","added_by":"auto","created_at":"2025-07-11 09:02:44","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":232999,"visible":true,"origin":"","legend":"Figure S1, Figure S2, Figure S3, Figure S4","description":"","filename":"KayaetalSupplNatComm.docx","url":"https://assets-eu.researchsquare.com/files/rs-7020203/v1/9691c397b7594ca95b336fe4.docx"},{"id":86490556,"identity":"d08d96af-637c-48a6-86e5-fe6bad25c057","added_by":"auto","created_at":"2025-07-11 09:02:44","extension":"mp4","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":9792340,"visible":true,"origin":"","legend":"Video S1","description":"","filename":"KayaetalSupplVideo.mp4","url":"https://assets-eu.researchsquare.com/files/rs-7020203/v1/b8f75820982ba81ee161e7e2.mp4"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"cJUN dependent innate immunity controls microbiome through selective phagocytosis in \u003ci\u003eNematostella\u003c/i\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe maintenance of a healthy functioning metaorganism, consisting of the host and its associated microorganisms, relies on an efficient host immune system. The immune system is based on a complex interplay of mechanisms protecting the host from pathogenic microorganisms, like bacteria\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e, fungi\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e and viruses\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e, while simultaneously maintaining beneficial microbial relationships\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. As part of the innate immune system, phagocytosis is a conserved mechanism found in invertebrates and vertebrates. Most likely it evolved as a mechanism of nutrient uptake in unicellular eukaryotes\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e and evolved during metazoan evolution\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e to a mechanism of specialized immune cells to eliminate pathogens. Thereby, phagocytosis of foreign particles consists of target recognition, internalization into phagosome and digestion of targets via phagolysosome formation\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. While the cellular mechanisms of phagocytosis in invertebrates are similar to those in vertebrates\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e, the evolutionary conservation of specific gene functions remains to be determined\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe widespread assumption is that the innate immunity provides an immediate, non-specific defense, while the adaptive immunity enables a targeted, antigen-specific response with an immunological memory\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. However, recent research has challenged the traditional view that invertebrate immune systems lack specificity. Studies have shown that phagocytosis in invertebrates can exhibit a high degree of specificity\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. In woodlice, hemocytes demonstrated increased phagocytosis of previously encountered bacterial strains, suggesting the ability to differentiate between closely related bacteria\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. In the squid-Vibrio symbiosis haemocytes are able to differentiate between the squid’s preferred bacterial symbiont \u003cem\u003eVibrio fisheri\u003c/em\u003e and other bacteria of the \u003cem\u003eVibrio\u003c/em\u003e genus\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. In addition, in a number of host-microbe interactions, it has been shown, that phagocytes can both shape the microbiota and be influenced by specific members of the microbiome. In tse-tse flies, for example, hemocyte proliferation depends on colonization by \u003cem\u003eWigglesworthia\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. A similar effect is observed in pea aphids, where the presence of some symbionts affects hemocyte abundance and the proportion of granulocytes in the hemocyte population \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. We therefore hypothesize that bacterial differentiation by the innate immune system at the level of phagocytosis evolved already at the base of animal evolution and enabled already the common ancestor of metazoans to distinguish between different microorganisms.\u003c/p\u003e\u003cp\u003eIn the following, we used the sea anemone \u003cem\u003eNematostella vectensis\u003c/em\u003e (\u003cem\u003eN. vectensis\u003c/em\u003e) to study the degree of specificity of phagocytosis and its effects on interactions with its microbiome in an early branching metazoan. \u003cem\u003eN. vectensis\u003c/em\u003e belongs to the cnidarians, a sister group of all Bilateria, and exhibits a high genetic complexity characterized by similar signaling pathways for development and innate immunity compared to bilaterian animals\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. The microbiome of \u003cem\u003eN. vectensis\u003c/em\u003e shows a specific succession during host development and robust adaptations to environmental variations\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e, with a remarkable genotype-environment interaction influencing the variability of the microbiome\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. The microbiome-mediated plasticity\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e has been functionally linked to thermal adaptation in \u003cem\u003eN. vectensis\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe establishment of the microbiome strongly depends on host mechanisms and transcriptome data support the hypothesis, that phagocytosis is a central mechanism for controlling the initial colonization processes\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. In \u003cem\u003eN. vectensis\u003c/em\u003e, it is shown that nematosomes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA), small motile multicellular bodies in the gastric cavity, are capable of phagocytosis and may act as immune bodies\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. They originate from cnidoglandular tracts, the mesenteries, and consist of nematocytes and a second, not well characterized cell type, that shows phagocytic activity\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eWhile nematosomes can immobilize prey with the help of their nematocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB), the second cell type presumably serves the phagocytosis of bacteria (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC), indicating a potential dual function in nutrition and immunity\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. In addition, nematosomes are equipped with two types of cilia surrounding the cell complex\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e, which facilitate their active mobility within the fluid of the gastric cavity (\u003cb\u003eVideo S1\u003c/b\u003e). A further indication for the active contribution to immunity is the fact that nematosomes co-express components of the TLR signaling pathway, such as TLR and NF-κB\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. During oogenesis, the nematosomes are packed into the egg clutches by the female polyps, which are then expelled by the female polyps with a gelatinous matrix mucus. It is likely that the nematosomes thereby also take over a maternal protection of the embryos during early development (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). However, there is no direct evidence that nematosomes are able to differentiate bacteria at the level of phagocytosis.\u003c/p\u003e\u003cp\u003eHere, we could show, that nematosomes exhibit selective phagocytosis, efficiently ingesting foreign \u003cem\u003eVibrio\u003c/em\u003e isolates and degrading them in the lysosome while sparing native \u003cem\u003eVibrio\u003c/em\u003e isolates. This selective phagocytosis is correlated with the ability of the bacteria to colonize \u003cem\u003eN. vectensis\u003c/em\u003e adult polyps. Proteomic analyses revealed distinct protein enrichment patterns linked to phagosomal pathway in response to foreign bacteria, highlighting cJUN’s role in immune-related trafficking. CRISPR/Cas9-mediated \u003cem\u003ecJUN\u003c/em\u003e knockout (\u003cem\u003ecJUN\u003c/em\u003e\u003csup\u003e\u003cem\u003e−/−\u003c/em\u003e\u003c/sup\u003e) resulted in a significantly reduction in nematosomes proliferation and impaired lysosomal activation after engulfment of \u003cem\u003eVibrio\u003c/em\u003e cells. These \u003cem\u003ecJUN\u003c/em\u003e\u003csup\u003e\u003cem\u003e−/−\u003c/em\u003e\u003c/sup\u003e polyps are colonized by an altered microbiome and accumulate foreign \u003cem\u003eVibrio\u003c/em\u003e-isolates, demonstrating a causal relationship between the composition of the microbiome and the selective phagocytosis of the nematosomes. Our data provide strong evidences that cJUN is a key regulator for nematosome proliferation and lysosome maturation in \u003cem\u003eN. vectensis\u003c/em\u003e and therefore fulfils a central role in microbiome regulation.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cb\u003eNematostella vectensis\u003c/b\u003e \u003cb\u003eculture\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAll experimental setups were conducted with adult clonal female \u003cem\u003eNematostella vectensis\u003c/em\u003e polyps, originally collected from the Rhode River in Maryland, United States\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. The polyps were fed daily with freshly hatched \u003cem\u003eArtemia nauplii\u003c/em\u003e and kept in dark. Culture tanks containing the animals, organized by genotype and gender, were connected to an aquatic system where the medium was flushed out and replaced with fresh \u003cem\u003eNematostella\u003c/em\u003e Medium (NM) with a salinity of 16‰ (Red Sea Salt® and Millipore H2O) at 18°C every other day. Every two weeks, the culture boxes were manually cleaned to remove biofilm and feeding debris.\u003c/p\u003e\u003cp\u003e\u003cb\u003ePhagocytosis assay\u003c/b\u003e\u003c/p\u003e\u003cp\u003eExperimental setups regarding bacterial challenges were performed working with the same bacterial isolates used for mono-associations. Native (NJ1, NJ33 and NA11)\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e and non-native (Hal025 and Hal281\u003csup\u003e32\u003c/sup\u003e) bacterial isolates were grown at 30°C and 220 rpm overnight in liquid Marine Broth (MB) Medium before diluting the isolates to an OD600 of 0.1/mL. 1 mL of bacteria was centrifuged and diluted with sterile 16‰ NM prior staining with BacLight (Thermo Fisher) for 15 min in dark and room temperature. After incubation, bacteria was centrifuged for one wash step with 16‰ NM before diluting them to OD600 of 0.001 for the bacterial challenge on the nematosomes ex vivo. Bacterial treatment was performed in the dark for 1.5 h at 18°C.\u003c/p\u003e\u003cp\u003eDuring bacterial staining, nematosomes from 5 clonal female polyps were extracted, by pinching a hole in the food region of the polyps and pipetting the discharged nematosomes, and placed into a chamber slide (Thermo Scientific™ Nunc™ Lab-Tek™ II Chamber Slide™ System). After letting the cells stick to the bottom of the slide, they were washed once with NM to get rid of debris. The nematosomes were treated with stained bacteria from an OD600 of 0.001 and incubated for 2 h in the dark. After bacterial treatment, nematosomes were washed 2 times with NM. After that nematosomes are stained with LysoTracker (15 nM, Thermo Fisher) and Hoechst (10 nM, Thermo Fisher) for 45 min at room temperature. Staining solution was washed out with NM after staining. Cells were than fixed with 3% PFA diluted in NM for 15 min on RT and washed out once with NM after treatment. Nematosomes were mounted on slide using ProLong™ Diamond (Thermo Fisher). Images from samples were taken with the confocal microscope Olympus FV3000 Confocal Laserscanning Microscope and later analyzed using ImageJ\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003cb\u003eGeneration of germfree polyps and mono-association experiment\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAntibiotic treatment (AB treatment) approaches were adapted from the established protocol for generating germfree \u003cem\u003eHydra\u003c/em\u003e polyps\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e and further adapted for \u003cem\u003eNematostella\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Adult clonal \u003cem\u003ecJUN\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003ecJUN\u003c/em\u003e\u003csup\u003e\u003cem\u003e−/−\u003c/em\u003e\u003c/sup\u003e lines were exposed to a combination of five antibiotics: Ampicillin, Neomycin, Streptomycin, Spectinomycin, and Rifampicin, each at a concentration of 50 µg/mL. This treatment was conducted over a period of 2 weeks without any food supply, with the medium being refreshed every day and a replacement of plates every second day. For each treatment condition, five biological replicates were utilized, along with an additional five biological replicates serving as germfree (GF) and wildtype (WT) controls. Following the 2-week AB treatment, polyps were washed in sterile, filtered 16‰ NM before homogenization. A 1:10 dilution of the lysate was plated on Marine Broth (MB) agar plates to confirm sterility, in which GF plates should remain clear without bacterial growth. The remaining lysate was centrifuged, and the pellet was processed for DNA isolation using the Qiagen Blood and Tissue Kit for subsequent molecular analyses, including PCR and quantitative PCR (qPCR). After sterility confirmation remaining polyps were prepared for mono-association with chosen bacterial isolates. After 2 weeks of AB Treatment, polyps remained in sterile and filtered NM prior recolonization. Bacterial isolates were grown at 30°C in liquid MB media overnight. Bacteria was grown to an OD600 of 0,1, were diluted to a final OD600 of 0.001 and exposed to the sterile polyps. We used 5 polyps for each isolate and genotype of polyps with GF and WT control, respectively. The isolates we chose were NJ1, NJ33 and NA11 as native colonizers for \u003cem\u003eNematostella\u003c/em\u003e and Hal025 and Hal281 as non-native, foreign, colonizers, obtained from \u003cem\u003eHalichondria panicea\u003c/em\u003e. After recolonizing polyps with single isolates, sampling took place after 2- and 7- days post recolonization (2 dpr and 7 dpr). Polyps were washed three times with 16‰ NM before getting homogenized and plated on MB plates. After an incubation time of 2 days on room temperature, colony forming units (CFU) were counted manually to determine the colonization succession of the single isolates on the polyp.\u003c/p\u003e\u003cp\u003e\u003cb\u003eProteomic analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eBacterial culture was prepared with an OD600 of 0.001 (see Phagocytosis assay) prior nematosomes extraction from adult polyps. All nematosomes from five biological replicates for each treatment was prepared. We chose the isolated NJ1 as a native colonizer and Hal281 as a non-native isolate and a control group without bacterial challenge. Extracted nematosomes were treated for 2 h at 18°C with the isolates and were washed afterwards with 16‰ NM once. Nematosomes were transferred into a PCR tube and centrifuges by 5000 rpm for 5 min at 4°C to the bottom of the tube. NM was discarded and replaced with 25 µl Lysis Buffer (5 mol/L Urea, 1% Tritonx100, 1xcOmpete EDTA-free, 5 mmol/L DTT). Nematosomes were incubated for 45 min at 37°C with vortexing in between every 15 min. After incubation nematosomes were snapped-freezed at -80°C for further analysis. Samples were then digested according to SP3 protocol\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e with some modifications as follows. After thawing, lysates were mixed each with 25 µL Alkylation buffer (50 mM borate buffer and 25 mM IAA) for 50 min at room temperature (RT). Then, 5 µL of resuspended SP3 beads (20 µg/µL A:B 1:1 mixture) were added to each sample followed by 150 µL ACN and mixed for 30 min at 800 rpm, RT. Then, beads were washed with 300 µL 70% EtOH, followed by 150 µL ACN. A 10 µL digestion buffer (4 ng/µL trypsin/Lys-C, 25 mM borate buffer and 0.01% DDOPM) was added to each sample followed by mixing on a shaker for 10 min at 800 rpm RT. Samples were mixed each by pipetting up and down and kept back on the shaker overnight. Next day, samples were centrifuged at 20,000 x g for 2 min and supernatant (ca. 10 µL) was transferred to LC-MS vials containing 1 µL of 5% FA.\u003c/p\u003e\u003cp\u003e\u003cb\u003eLC-MS Proteomics and Data analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eChromatographic separation was performed on a Dionex U3000 nanoHPLC system equipped with an Acclaim pepmap100 C18 column (2 µm particle size, 75 µm × 500 mm) coupled online to a mass spectrometer. The eluents used were; eluent A: 0.05% formic acid (FA), eluent B: 80% ACN + 0.04% FA. The separation was performed over a programmed 120 minutes run. Initial chromatographic conditions were 4% B for 2 minutes followed by linear gradients from 4–50% A over 90 minutes then 50 to 90% A over 5 minute, and 10 minutes at 90% A. Following this, an inter-run equilibration of the column was achieved by 16 minutes at 4% A. A constant flow rate of 300 nl/min was employed. Data acquisition following separation was performed on an QExactive Plus. Full scan MS acquisition was performed (350–1400 m/z, resolution 70,000). Subsequent data dependent MS/MS scans were collected for the 15 most intense ions (Top15) via HCD activation at NCE 27.5 (resolution 17,500); dynamic exclusion was enabled (20 sec duration). Triplicate measurements were performed for all the samples.\u003c/p\u003e\u003cp\u003eRaw data were analyzed against \u003cem\u003eNematostella vectensis\u003c/em\u003e Uniprot database (20.05.2022) (24,497 sequences) plus common contaminants (cRAP). The search was performed on Proteome discoverer 2.5 using a SequestHT search engine with 10 ppm and 0.02 Da precursor and fragment ions tolerances, respectively. Digestion with trypsin with a max of 2 missed cleavages were applied. Strict parsimony criteria have been applied filtering peptides and proteins at 1% FDR. INFERYs rescoring algorithm was applied. Label-free quantification method based on the intensities of the precursor ions was used. Proteins were filtered to have “High” FDR combined confidence and at least 2 identified peptides. Data was further analyzed by Excel and Perseus v 1.6.15.0\u003csup\u003e37\u003c/sup\u003e. Protein intensities were averaged for technical replicates. To perform differential quantitative analysis of proteins, raw protein intensities were extracted, averaged between technical replicates, one outlier replicate per group “Control 5, Native 1 and Non-Native 2” were excluded, then median based normalization was applied to the data. Log2 transformed intensities were grouped in 3 groups depending on the \u003cem\u003eVibrio\u003c/em\u003e treatment (each with 5 replicates). Proteins with at least 4 intensity values in one group were used for further analysis. Missing values were imputed from a normal distribution separately for each replicate (Width 0.3, Downshift 1.8). Statistical analysis was done using ANOVA, permutation-based FDR of 0.01. Gene enrichment analysis was performed on The Database for Annotation, Visualization, and Integrated Discovery (DAVID)\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e using the functional annotation tool.\u003c/p\u003e\u003cp\u003eThe mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://proteomecentral.proteomexchange.org\u003c/span\u003e\u003cspan address=\"http://proteomecentral.proteomexchange.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) via the PRIDE partner repository \u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e with the dataset identifier PXDxxxxxx.\u003c/p\u003e\u003cp\u003e\u003cb\u003eCRISPR/Cas 9 mediated knock-out generation\u003c/b\u003e\u003c/p\u003e\u003cp\u003eCRISPR/Cas9-mediated transgenic lines were generated following the protocol published before\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. The gene \u003cem\u003ecJUN\u003c/em\u003e (NVE21090) was selected based on transcriptomic analysis after recolonization experiments\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e and tissue specificity\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Four guide sequences (sgNVE21090E1, sgNVE21090E2, sgNVE21090E3, and sgNVE21090E4) were designed using the web tool CRISPOR.org\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e, with an implemented \u003cem\u003eNematostella\u003c/em\u003e genome. First, the guide oligonucleotides were mixed in equal amounts, annealed for 5 min at 95°C, and then incubated at room temperature for 2–3 h. The annealed oligos were cloned into the gRNA expression vector pDR274 (42250, Addgene). After successful integration of the guides into the vector, the guide sequences were amplified, transcribed \u003cem\u003ein vitro\u003c/em\u003e using the MEGAscript™ T7 kit (Thermo Fisher), and purified with the MEGAclear RNA cleanup kit (Thermo Fisher) prior to injection. The injection mix consisted of Cas9 enzyme (1 mg/ml stock) (TrueCut™ Cas9 Protein v2, Thermo Fisher), sgRNAs (450 ng), Alexa fluorescent dye (1.1 M in KCl), and RNase-free water. To obtain fertilized eggs, animals were incubated at 25°C for 11 h to induce gamete production. Egg packages were incubated in sperm media from males for 15 min before dejellying the fertilized egg packages with 4% cysteine (pH 7.4), followed by five washing steps in 16‰ NM. The injection mix was incubated at 37°C for 5 min before injection. The injection setup was conducted according to the microinjection protocol for mRNA and Morpholinos previously described\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. Injected eggs were raised in the dark at 20°C, with the medium (NM) being exchanged daily, and food introduction after 10–12 days.\u003c/p\u003e\u003cp\u003eTo confirm the successful integration of CRISPR/Cas9-mediated mutagenesis into the polyps’ genome, we performed crossbreeding, High Resolution Melting Curve Analysis (HRMC) and genotyping. Genomic DNA was isolated from injected juvenile polyps using the DNeasy Blood \u0026amp; Tissue kit (Qiagen). In HRMC, short DNA fragments at the targeted locus are amplified, and changes in these fragments are detected through shifts in the melting curves. Following confirmation of successful mutations, the mutant animals were crossed with wildtype polyps to generate the F1 generation. The same procedure was applied as for the previous F0 generation. Heterozygous animals were further analyzed by Sanger sequencing to determine the precise mutation pattern. Polyps with the same mutation pattern were crossed to generate the F2 generation, which included homozygous mutants (\u003cem\u003ecJUN\u003c/em\u003e\u003csup\u003e\u003cem\u003e−/−\u003c/em\u003e\u003c/sup\u003e), homozygote wildtype (\u003cem\u003ecJUN\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e) and heterozygote (\u003cem\u003ecJUN\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/−\u003c/em\u003e\u003c/sup\u003e) offspring, which are used for experimental set ups.\u003c/p\u003e\u003cp\u003e\u003cb\u003eElectron microscopy with nematosomes\u003c/b\u003e\u003c/p\u003e\u003cp\u003eScanning electron microscopy (SEM) were taken with the Zeiss REM Supra 55VP. Nematosomes were extracted from the polyps and placed on well with Poly-L-Lysine coated cover glasses. All fixation, washing, dehydration were performed as former published\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. Images were later analyzed using ImageJ.\u003c/p\u003e\u003cp\u003e\u003cb\u003e16S rRNA analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFor 16S rRNA analysis the bioinformatics were performed using Qiime 2 2021.11\u003csup\u003e45\u003c/sup\u003e. First, raw sequences were demultiplexed and quality filtered using the q2-demux plugin followed by denoising with DADA2\u003csup\u003e46\u003c/sup\u003e. The amplicon sequence variants (ASVs) were aligned with mafft and for constructing the phylogeny fasttree2 was conducted\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. All samples were rarefied to 900 sequences per samples prior estimation for Alpha-diversity metrics (observed features and Faith’s Phylogenetic diversity\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e), beta-diversity metrics (Bray-Curtis dissimilarity, Jaccard distance and Unifrac (weighted and unweighted\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e)) and Principle Coordinate Analysis (PCoA). Taxonomy was assigned to ASVs using q2-feature-classifier classify-sklearn naïve Bayes taxonomy classifier against Greengenes 13_8 99% data set as reference\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. Further analysis, statistical analysis and plot visualization was conducted with OriginPro (Version 2021. OriginLab Corporation, Northampton, MA, USA.).\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eNematosomes phagocytose\u003c/b\u003e \u003cb\u003eVibrio\u003c/b\u003e \u003cb\u003estrains with varying efficiency\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo test if nematosomes are phagocytosing bacteria differentially, we established a phagocytosis assay that allows to quantify on one hand lysosomal activity and on the other hand bacterial engulfment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTherefore, nematosomes are extracted from the gastric cavity and incubated in a bacterial suspension with defined bacterial concentration. For the assay we chose different \u003cem\u003eVibrio\u003c/em\u003e strains, as they are common marine bacteria and are among the main colonizers of \u003cem\u003eN. vectensis.\u003c/em\u003e As native isolates we selected the \u003cem\u003eVibrio\u003c/em\u003e isolates NJ1, NJ33 and NA11 that were cultivated from \u003cem\u003eN. vectensis\u003c/em\u003e \u003cb\u003e(Table S1)\u003c/b\u003e\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. We analyzed the phagocytosis rate of these native isolates and compared it with the rate of phagocytosis of foreign \u003cem\u003eVibrio\u003c/em\u003e isolates (Hal025 and Hal281\u003csup\u003e32\u003c/sup\u003e) derived from the sponge \u003cem\u003eHalichondria panicea\u003c/em\u003e \u003cb\u003e(Table S1)\u003c/b\u003e. The results revealed that native \u003cem\u003eVibrio\u003c/em\u003e strains were phagocytosed at significantly lower rates, while the foreign isolates Hal025 and Hal281 were engulfed at substantially higher rates \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, B\u003cb\u003e)\u003c/b\u003e. The increased phagocytosis of foreign isolates correlated with significant increase in lysosomal activity within nematosomes \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, C\u003cb\u003e)\u003c/b\u003e. In contrast, nematosomes confronted with native \u003cem\u003eVibrio\u003c/em\u003e strains did not increase their lysosomal activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, C).\u003c/p\u003e\u003cp\u003eIn addition to the phagocytosis rate for each isolate, we also compared the colonization efficiency of each isolate in mono-association experiments (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD, E). This recolonization approach revealed that the three native \u003cem\u003eVibrios\u003c/em\u003e, namely NJ1, NJ33 and NA11, colonized in significantly higher rates on the polyp 7 days post recolonization (dpr), compared to the two foreign isolates Hal025 and Hal281 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). This trend was already seen after 2 dpr (\u003cb\u003eFigure S1\u003c/b\u003e). These results correlate with the observations of elevated phagocytosis rates (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB) and lysosomal actives (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC) of the nematosomes upon foreign \u003cem\u003eVibrio\u003c/em\u003e engulfment suggesting a potential link between nematosome phagocytosis and colonization success.\u003c/p\u003e\u003cp\u003e\u003cb\u003eForeign and native\u003c/b\u003e \u003cb\u003eVibrio\u003c/b\u003e \u003cb\u003eisolates cause diverging proteome responses in nematosomes\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo characterize the differential response of nematosomes to native and foreign \u003cem\u003eVibrio\u003c/em\u003e strains, we performed proteome analysis with extracted nematosomes. Specifically, we assessed the responses of nematosomes after confronting them with NJ1 and Hal281, and compared it to a control treatment without bacterial challenge. The proteome analysis revealed significantly abundant proteins when comparing nematosomes challenged with native and foreign bacterial isolates (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, \u003cb\u003eTable S2, Figure S2\u003c/b\u003e). A total of 2676 proteins were detected in the proteomic analysis of nematosomes treated with bacterial isolates NJ1 and Hal281, as well as in untreated controls. To extract proteins that were uniquely differently abundant in either NJ1 or Hal281 treatment, we generated five clusters using \u003cem\u003ek\u003c/em\u003e-means clustering (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Out of the total proteins identified, 157 proteins were detected uniquely in NJ1-treated samples and 104 in Hal281-treated nematosomes \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e. Thereby, cluster 1 represents proteins which emerged exclusively following NJ1 treatment, while cluster 4 contain proteins, which are higher in abundance after confrontation with Hal281. A KEGG enrichment analysis revealed that cluster 1, which contains proteins that are significantly more abundant in NJ1-treated nematosomes, contains proteins related to carbon and nitrogen metabolism \u003cb\u003e(Table S4)\u003c/b\u003e. These findings suggest that interaction with native bacteria may promote the host metabolisms, reflecting potential symbiotic interactions. In contrast, that treatment with Hal281 elevated the abundance of proteins belonging to the phagosomal pathway (\u003cb\u003eTable S3, S4\u003c/b\u003e). Especially proteins belonging to the cytoskeleton formation of phagosomal formation, like Dynein and Tubulin beta (TUBB) as well as F-actin are increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, \u003cb\u003eTable S3\u003c/b\u003e). Interestingly, the V-ATPase shows an increase in abundance in both bacterial treatments, potentially linking it to default lysosomal activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe proteomic analysis revealed an elevated abundance in the phagosomal pathway upon foreign bacterial treatment, indicating an active response of nematosomes in immune response, particularly in recognizing foreign isolate phagocytosis and degradation. To further explore the molecular mechanisms driving these responses, we aimed to manipulate the regulatory pathways involved.\u003c/p\u003e\u003cp\u003e\u003cb\u003ecJUN\u003c/b\u003e \u003cb\u003emutation reduces nematosome proliferation\u003c/b\u003e\u003c/p\u003e\u003cp\u003eIn a next step we aimed to alter the function of nematosomes by CRISPR/Cas9 genome editing. Therefore, we screened for potential transcription factors (TF) that are potentially involved in the proliferation and/or immune function of nematosomes. We identified \u003cem\u003ecJUN\u003c/em\u003e orthologs, which is highly expressed in nematosomes (NVE21090) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA), while a second ortholog is mainly expressed in the tentacle region (NVE16876) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB) of the polyp\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. Interestingly, the \u003cem\u003ecJUN\u003c/em\u003e ortholog NVE21090 is also upregulated upon bacterial recolonization\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. cJUN is an evolutionarily conserved transcription factor that activates immune genes, regulates phagocytosis, and is involved in cell proliferation\u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e. As central hub, cJUN integrates signaling information of various pathways, including ERK and JNK signaling\u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e. In the cnidarian Hydra TLR signaling via MyD88 activates JNK signaling following immune stimulation\u003csup\u003e\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e and in vertebrates cJUN regulates macrophage activation\u003csup\u003e\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eTherefore, we selected the \u003cem\u003ecJUN\u003c/em\u003e ortholog NVE21090 for CRISPR/Cas9 genome editing\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e, to be able to functionally investigate the role of nematosomes in regulating microbiome composition. We generated deletions in the first exon of NVE21090, which ultimately led to a variation of mutations in animals of the F1 generations (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). For the subsequent breeding, we selected the male strain M8 and the female strain M11, both carrying the same heterozygous mutation leading to a stop codon in the first exon of the gene. This mutation results in incomplete translation of the gene, leading to the loss of essential domains within the protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE\u003cb\u003e)\u003c/b\u003e. All subsequent approaches were conducted on F2 polyps with homozygous mutation named \u003cem\u003ecJUN\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eMorphological comparisons between \u003cem\u003ecJUN\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003ecJUN\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e polyps revealed no significant differences in polyp length (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD, F\u003cb\u003e)\u003c/b\u003e. Simultaneously, \u003cem\u003ecJUN\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e polyps showed no significant differences in area size, indicating that cJUN\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mutation in nematosomes leads to no significant changes in polyps\u0026rsquo; body (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD, G). However, a significant reduction in nematosome numbers per polyps was observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH). While adult \u003cem\u003ecJUN\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e polyps harbor around 1100 nematosomes, \u003cem\u003ecJUN\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e polyps only exhibit around 100 per polyp. Interestingly, this lower number seems to be slightly compensated by size, as \u003cem\u003ecJUN\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e nematosomes display a bigger size compared to \u003cem\u003ecJUN\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e nematosomes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI).\u003c/p\u003e\u003cp\u003e\u003cb\u003ecJUN\u003c/b\u003e \u003cb\u003emutation affects nematosome phagocytosis and bacterial colonization\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo approach the effect of \u003cem\u003ecJUN\u003c/em\u003e on nematosome phagocytosis, we performed the newly established phagocytosis assay, confronting \u003cem\u003ecJUN\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003ecJUN\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e polyps with the same bacterial isolates (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). \u003cem\u003ecJUN\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e revealed similar lysosomal activity and rates of phagocytosis as observed in wt animals after confrontation with foreign and native \u003cem\u003eVibrio\u003c/em\u003e strains (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, C \u003cb\u003eand\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, B, C\u003cb\u003e)\u003c/b\u003e. However, while \u003cem\u003ecJUN\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e nematosomes adjust their lysosomal activity in response to different isolates, \u003cem\u003ecJUN\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e nematosomes maintain a similar default lysosomal activation independent of bacterial treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Being confronted with the foreign isolates Hal025 and Hal281 was not resulting in the activation of lysosomal activity in the \u003cem\u003ecJUN\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e nematosomes. Analyzing the number of bacteria engulfed in nematosomes revealed an increase of the isolates NA11 and Hal281 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB) in \u003cem\u003ecJUN\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e nematosomes, most likely by an accumulation of bacterial cells in the phagosome. As the lysosome is not activated in \u003cem\u003ecJUN\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e nematosomes, degradation of bacteria in phagosome is most likely impaired, resulting in arrested phagocytosis.\u003c/p\u003e\u003cp\u003eThe impaired phagocytosis of foreign \u003cem\u003eVibrio\u003c/em\u003e isolates resulted in an increased recolonization rate in mono-association experiments (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD, E) in adult \u003cem\u003ecJUN\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e polyps compared to \u003cem\u003ecJUN\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e polyps (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). Interestingly, the native isolates NJ1 and NJ33 recolonized \u003cem\u003ecJUN\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e polyps significantly lower compared to \u003cem\u003ecJUN\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e polyps, suggesting even supporting effects of nematosomes for some native colonizers.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eBacterial dysbiosis in\u003c/b\u003e \u003cb\u003ecJUN\u003c/b\u003e\u003csup\u003e\u003cb\u003e-/-\u003c/b\u003e\u003c/sup\u003e \u003cb\u003epolyps\u003c/b\u003e\u003c/p\u003e\u003cp\u003eDiscovering significant differences in the nematosome phenotype of \u003cem\u003ecJUN\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e polyps, we proceeded to analyze its associated microbiome. 16S rRNA gene sequencing revealed significant differences between the microbiome of \u003cem\u003ecJUN\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003ecJUN\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e polyps (\u003cb\u003eFigure S3, Table S5\u003c/b\u003e), with a significantly lower alpha diversity (\u003cb\u003eFigure S3C\u003c/b\u003e) and evenness (\u003cb\u003eFigure S3D\u003c/b\u003e) compared to \u003cem\u003ecJUN\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e animals.\u003c/p\u003e\u003cp\u003eThese differences in bacterial colonization in \u003cem\u003ecJUN\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e and to \u003cem\u003ecJUN\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e polyps support previous results indicating that host mechanisms are involved in the control of bacterial establishment in \u003cem\u003eN. vectensis\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. To test this hypothesis, we recolonized germfree adult \u003cem\u003ecJUN\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003ecJUN\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e polyps with the bacterial consortia of adult polyps and followed the succession of bacterial establishment over the period of 28 days by 16S rRNA gene sequencing. Over the whole course of the experiment the cJUN genotype has significant effects on microbial community structure (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC), while also the time point after recolonization (days post recolonization, dpr) accounted for differences in microbial colonization \u003cb\u003e(\u003c/b\u003eTable\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eStatistical summary of ADONIS and ANOSIM tests on Bray-Curtis, Jaccard, Weighted UniFrac, and Unweighted UniFrac distance matrices, comparing microbial community dissimilarities between \u003cem\u003ecJUN\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003ecJUN\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e animals. The parameter column indicates whether the analysis was performed at the genotype level or at the dpr level. Adonis R\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e values represent the proportion of variance explained by genotype or dpr while ANOSIM R values indicate the degree of separation between groups. Significant differences are indicated by p-values, with higher R values reflecting stronger microbial dissimilarities between groups.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"9\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eParameter\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMetric\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003eAdonis R\u0026sup2;\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u003cp\u003eAdonis p\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e\u003cp\u003eANOSIM R\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\"\u003e\u003cp\u003eANOSIM p\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003egenotype\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003eBray-Curtis\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u003cp\u003e0.097\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.005\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e\u003cp\u003e0.002\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003eJaccard\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u003cp\u003e0.099\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003eWeighted UniFrac\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u003cp\u003e0.073\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.031\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e\u003cp\u003e0.007\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003eUnweighted UniFrac\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u003cp\u003e0.101\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003edpr\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003eBray-Curtis\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u003cp\u003e0.174\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003eJaccard\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u003cp\u003e0.111\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003eWeighted UniFrac\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u003cp\u003e0.282\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.49\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003eUnweighted UniFrac\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u003cp\u003e0.110\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eDuring the early timepoints (2 and 7 dpr) \u003cem\u003ecJUN\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003ecJUN\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e polyps exhibit similar patterns in microbial richness and evenness (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, B).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAfter 14 dpr, the microbiome of \u003cem\u003ecJUN\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e polyps show significantly higher bacterial alpha diversity and evenness compared to \u003cem\u003ecJUN\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e polyps \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, B\u003cb\u003e)\u003c/b\u003e. The increase in Weighted UniFrac distances between the microbiome of the different genotypes reveal that genotype-related differences in the composition of the microbiome become more pronounced over time, as the microbial communities develop differently between \u003cem\u003ecJUN\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003ecJUN\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e animals (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). The significant differences in specific microbial taxa observed between the genotypes over time (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD, \u003cb\u003eFigure S4\u003c/b\u003e), including the dominance of some Alphaproteobacteria taxa (\u003cb\u003eFigure S4A, B\u003c/b\u003e) and the reduced colonization success of specific Gammaproteobacteria (\u003cb\u003eFigure S4D\u003c/b\u003e) in \u003cem\u003ecJUN\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e polyps highlight the selective force of nematosomes on specific taxa.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003e\u003cb\u003eNematosomes exhibit characteristics of ancient immune cells\u003c/b\u003e\u003c/p\u003e\u003cp\u003eOur study provides strong evidence that nematosomes are key immune cells in \u003cem\u003eN. vectensis\u003c/em\u003e, playing a critical role in microbial selection through selective phagocytosis. The phagocytosis assay demonstrated that nematosomes exhibit a clear preference for engulfing foreign \u003cem\u003eVibrio\u003c/em\u003e isolates while sparing native ones, indicating that the distinguishing of bacteria acts most likely on the level of recognition. Phagocytosis is a fundamental immune defense mechanism in invertebrates, often mediated by circulating cells called immunocytes. These cells fulfill various functions of the innate immune system, including the recognition of pathogens, phagocytosis and the synthesis of antimicrobial proteins. Thereby, the innate response triggered by microbial-associated molecular patterns (MAMPs) is based on the activation of pattern recognition receptors. In vertebrates, macrophages use pattern recognition receptors (PRRs) such as Toll-like receptors (TLRs) and scavenger receptors to recognize MAMPs and discriminate between self and non-self by activating NF-κB\u003csup\u003e\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e, \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e. In nematosomes of \u003cem\u003eN. vectensis\u003c/em\u003e the TLR signaling pathway is also highly expressed and has been shown to activate the NF-κB pathway in response to bacterial pathogens, mirroring the innate immune responses seen in vertebrates\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u003c/sup\u003e. Experimental MyD88 knockdowns in \u003cem\u003eHydra\u003c/em\u003e showed that TLR signaling does not act unidirectionally via the transcription factor NF-κB but is also linked to cJUN, resulting in altered microbiome composition and impaired bacterial recognition\u003csup\u003e\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e. This suggests that nematosomes may use TLR-mediated pathways to recognize bacterial cells and regulate strain-specific immune responses through evolutionarily conserved signaling cascades.\u003c/p\u003e\u003cp\u003eFurthermore, the observed correlation between phagocytosis efficiency and bacterial colonization efficiency supports the hypothesis that nematosomes act as selective gatekeepers of microbial establishment in \u003cem\u003eN. vectensis\u003c/em\u003e. Native \u003cem\u003eVibrio\u003c/em\u003e isolates that were less frequently phagocytosed colonized the polyps at higher rates, while foreign isolates that were preferentially engulfed and degraded exhibited poor colonization efficiency. The colonization by foreign isolates increased in \u003cem\u003ecJUN\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e polyps, demonstrating that nematosomes play a direct role in shaping the microbiome by selectively removing non-native bacteria before they can establish themselves in the host. The proteomic analysis further supports this immune function, revealing distinct protein patterns in nematosomes upon bacterial exposure. Proteins involved in phagocytosis, including cytoskeletal components such as actin and dynein, were upregulated in response to foreign bacteria, supporting the notion that nematosomes are actively involved in immune surveillance and microbial selection, and emphasizing the functional importance of nematosomes in host-microbe interactions.\u003c/p\u003e\u003cp\u003e\u003cb\u003ecJUN - a regulator of nematosome phagocytosis and proliferation\u003c/b\u003e\u003c/p\u003e\u003cp\u003eOur results highlight cJUN as a crucial regulator of innate immunity in \u003cem\u003eN. vectensis\u003c/em\u003e. CRISPR/Cas9-mediated knockout of \u003cem\u003ecJUN\u003c/em\u003e resulted in a marked impairment in their ability to mount a selective immune response. In \u003cem\u003ecJUN\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mutants, nematosomes exhibited a failure to activate lysosomal degradation pathways following bacterial engulfment, leading to an accumulation of both native and foreign bacteria within the phagosome. Similarly, macrophages, disrupted in cJUN, TLRs, or NF-κB, can still engulf pathogens but fail to complete their degradation\u003csup\u003e\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e, \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e, \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e\u003c/sup\u003e, resulting in \u0026ldquo;arrested phagocytosis\u0026rdquo;, where bacteria remain trapped within the phagosomes but are not effectively digested. Certain intracellular pathogens, exploit these host weaknesses by preventing phagosome-lysosome fusion or modifying host signaling pathways to survive within immune cells\u003csup\u003e\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e, \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e, \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e\u003c/sup\u003e. These evasion tactics allow pathogens to manipulate key stages of the phagocytosis process, including phagosome formation, maturation, and acidification\u003csup\u003e\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e\u003c/sup\u003e. By interfering with these crucial immune defense mechanisms, bacteria can avoid degradation and establish intracellular infections. However, also symbionts rely on the mechanisms of arrested phagocytosis to persist within host tissue. In sponges, ankyrin-repeat proteins from bacterial symbionts can modulate phagocytosis by interfering with phagosome development, potentially allowing symbionts to escape digestion\u003csup\u003e\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e, \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e\u003c/sup\u003e. Similarly, in deep-sea mussels, the regulation of mTORC1 signaling helps retain symbionts in gill cells by preventing phagosome digestion\u003csup\u003e\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e\u003c/sup\u003e. In coral-dinoflagellate symbiosis, the symbiosome is hypothesized to be an early arrested phagosome, with transient gene expression changes occurring during symbiont uptake\u003csup\u003e\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e\u003c/sup\u003e. Recent results indicate, that anthozoan hosts indiscriminately phagocytose various microalgae, but non-symbiotic species are expelled through vomocytosis. Successful symbionts suppress the host's innate immune response, preventing expulsion and promoting niche formation\u003csup\u003e\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e\u003c/sup\u003e. These studies highlight the importance of arrested phagocytosis in various symbiotic relationships across different marine organisms.\u003c/p\u003e\u003cp\u003eIn \u003cem\u003eN. vectensis cJUN\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u003c/em\u003e\u0026minus;\u003c/sup\u003e nematosomes exhibited a similar phenotype, where they successfully internalized bacteria but failed to activate lysosomal responses necessary for degradation. The accumulation of engulfed but undegraded bacteria in mutant nematosomes suggests that cJUN plays a crucial role in regulating lysosomal maturation and phagosomal acidification. This parallels its function in vertebrates, where cJUN is involved in the transcriptional regulation of immune effectors, including lysosomal enzymes and phagosome maturation factors\u003csup\u003e\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e. The absence in cJUN-dependent signaling in \u003cem\u003eN. vectensis\u003c/em\u003e may thus impair the degradation of foreign \u003cem\u003eVibrio\u003c/em\u003e strains, contributing to microbial persistence and altered colonization patterns. This suggests that cJUN is essential for orchestrating the cytoskeletal and phagosomal dynamics required for effective microbial clearance.\u003c/p\u003e\u003cp\u003eIn addition, cJUN regulates also nematosome proliferation, as demonstrated by the significant reduction in nematosome numbers in \u003cem\u003ecJUN\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u003c/em\u003e\u0026minus;\u003c/sup\u003e mutants. It is well known that cJUN positively regulates cell proliferation by repressing tumor suppressor genes and inducing cyclin D1 transcription in invertebrates\u003csup\u003e\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e, \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e\u003c/sup\u003e and vertebrates\u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e. Thereby, cJUN negatively regulates p53 expression by binding to its promoter, thereby promoting cell cycle progression and proliferation\u003csup\u003e\u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e\u003c/sup\u003e. Nematosomes are budding from distinct regions of the mesenteries into the gastric cavity. In addition to the proliferation of nematosomes, the mesenteries of \u003cem\u003eN. vectensis\u003c/em\u003e play a crucial role in endomesodermal patterning and germ cell development\u003csup\u003e\u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e, \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e\u003c/sup\u003e, demonstrating the highly proliferative properties of this tissue. Our results show that cJUN is essential for controlling the expansion of nematosomes, potentially by regulating genes involved in cell division and differentiation, suggesting that cJUN not only governs phagocytosis but also orchestrates the development and maintenance of nematosomes as functional immune units.\u003c/p\u003e\u003cp\u003eThe loss of cJUN function ultimately resulted in microbial dysbiosis, with mutant polyps displaying altered microbiome compositions dominated by non-native bacterial species. These findings establish a direct link between cJUN-mediated immune regulation and microbiome homeostasis in \u003cem\u003eN. vectensis\u003c/em\u003e. The transcription factor cJUN plays a crucial role in immune regulation and homeostasis. In Drosophila, JNK signaling, which activates c-Jun, is essential for innate immunity and development\u003csup\u003e\u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e\u003c/sup\u003e. In mammals, epidermal JunB regulates cutaneous immune cell-microbiota interactions, with its absence leading to atopic dermatitis-like symptoms and spontaneous \u003cem\u003eS. aureus\u003c/em\u003e colonization\u003csup\u003e\u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e\u003c/sup\u003e. c-JUN/AP-1 is particularly important in CD8 T cell responses to acute infection, participating in productive immune responses\u003csup\u003e\u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e85\u003c/span\u003e\u003c/sup\u003e. These studies highlight the complex interplay between cJUN-mediated signaling, immune regulation, and microbiome homeostasis in various organisms and contexts. Recent findings in sponges further support an evolutionarily conserved role in cJUN/AP-1 in symbiont recognition. In \u003cem\u003eAmphimedon queenslandica\u003c/em\u003e, symbiotic bacteria induce a rapid transcriptional response involving cJUN/AP-1, NF-κB and IRF, whereas foreign bacteria do not trigger these responses and instead elicit xenobiotic metabolism\u003csup\u003e\u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e86\u003c/span\u003e\u003c/sup\u003e. This mirrors our observations in \u003cem\u003eN. vectensis\u003c/em\u003e and suggests that ancient metazoans already employed AP-1 family transcription factors to distinguish beneficial symbionts from potentially harmful bacteria, maintaining holobiont stability through selective immune regulation.\u003c/p\u003e\u003cp\u003e\u003cb\u003eInnate immune specificity and its implications for host-microbe interactions\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe strong evidence for innate immune specificity in \u003cem\u003eN. vectensis\u003c/em\u003e has significant implications for our understanding of the evolution of host-microbe interactions. Traditionally, innate immunity has been viewed as a broad, non-specific defense mechanism, whereas adaptive immunity is considered the primary driver of immune specificity. However, our findings challenge this dichotomy by demonstrating that even early-branching metazoans like cnidarians possess selective innate immune mechanisms. The ability of nematosomes to distinguish between closely related bacterial strains and selectively regulate microbiome composition suggests that innate immune specificity is an ancient and fundamental feature of metazoan immunity. This aligns with recent studies in other invertebrates, which have also demonstrated a surprising degree of innate immune selectivity, further supporting the notion that immune specificity predates the evolution of adaptive immunity\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e87\u003c/span\u003e, \u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e88\u003c/span\u003e\u003c/sup\u003e. In several studies it was shown that invertebrates can differentiate between pathogens at the species and even strain level\u003csup\u003e\u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e89\u003c/span\u003e, \u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e90\u003c/span\u003e\u003c/sup\u003e. This specificity is particularly evident in the phenomenon of immune priming, where initial exposure to a pathogen provides protection against subsequent encounters. Immune priming in invertebrates is a phenomenon where an initial pathogenic exposure enhances immune defenses against subsequent infections. This adaptive-like immunity has been observed in various invertebrates\u003csup\u003e\u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e91\u003c/span\u003e, \u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e92\u003c/span\u003e\u003c/sup\u003e, such as in woodlice\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e, where hemocytes show increased phagocytosis of previously encountered bacterial strains. In the oyster \u003cem\u003eCrassostrea gigas\u003c/em\u003e, hemocytes exhibit differential phagocytic responses to various bacterial species, demonstrating that invertebrate immune cells can selectively recognize and respond to different microbes\u003csup\u003e\u003cspan citationid=\"CR93\" class=\"CitationRef\"\u003e93\u003c/span\u003e, \u003cspan citationid=\"CR94\" class=\"CitationRef\"\u003e94\u003c/span\u003e\u003c/sup\u003e. Similarly, in the squid-Vibrio symbiosis, host immune cells differentiate between preferred symbionts and other closely related bacteria\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. In addition, immune priming can be enhanced by protective symbionts\u003csup\u003e\u003cspan citationid=\"CR95\" class=\"CitationRef\"\u003e95\u003c/span\u003e\u003c/sup\u003e. Potential mechanisms involved are a sustained immune responses, epigenetic modifications, and metabolic reprogramming, though the underlying mechanisms are not fully understood\u003csup\u003e\u003cspan citationid=\"CR96\" class=\"CitationRef\"\u003e96\u003c/span\u003e\u003c/sup\u003e. However, C-type lectin-like domain (CTLD) proteins have been identified as potential contributors to this specificity, with their extreme gene diversification observed in various invertebrate genomes\u003csup\u003e\u003cspan citationid=\"CR97\" class=\"CitationRef\"\u003e97\u003c/span\u003e\u003c/sup\u003e. Drosophila exhibits a specific primed immune response against certain pathogens based on phagocytosis \u003csup\u003e\u003cspan citationid=\"CR98\" class=\"CitationRef\"\u003e98\u003c/span\u003e\u003c/sup\u003e that requires phagocytes and the Toll pathway. This priming involves exposure to dead or sublethal doses of microbes, eliciting an initial response that enhances protection against subsequent infections\u003csup\u003e\u003cspan citationid=\"CR99\" class=\"CitationRef\"\u003e99\u003c/span\u003e\u003c/sup\u003e. While the mechanisms underlying this specificity and memory are not fully understood, proposed explanations include elevated levels of phagocytosis. These examples provide additional support for the concept that innate immune specificity is an ancient and widespread phenomenon across diverse metazoans.\u003c/p\u003e\u003cp\u003eThe evolutionary advantage of innate immune specificity likely lies in its ability to balance microbial diversity while preventing colonization by potentially harmful bacteria. In the case of \u003cem\u003eN. vectensis\u003c/em\u003e, the selective phagocytosis of foreign bacteria by nematosomes ensures that the microbiome remains stable and beneficial to the host. This mechanism is particularly crucial for organisms with simple immune architectures, where adaptive immune responses are absent. By employing a finely tuned innate immune response, \u003cem\u003eN. vectensis\u003c/em\u003e can maintain a dynamic but controlled microbiome, allowing for environmental adaptability without compromising immune defenses. This study, therefore, positions cnidarians as valuable models for exploring the evolutionary origins of immune-microbe interactions and provides insights into how early metazoans may have developed mechanisms for microbial regulation in the absence of adaptive immunity.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, our study provides compelling evidence that \u003cem\u003eN. vectensis\u003c/em\u003e employs a selective innate immune system to regulate its microbiome, challenging the traditional perception of innate immunity as a non-specific defense. The role of nematosomes in selectively phagocytosing foreign bacteria, and the involvement of cJUN in orchestrating this process, highlights the molecular complexity of immune regulation in early metazoans. Our findings reinforce the idea that innate immune specificity is evolutionarily ancient and widespread among invertebrates, playing a crucial role in maintaining host-microbe homeostasis. By demonstrating the impact of selective immune responses on microbiome composition, our work contributes to a broader understanding of host-microbe interactions and their evolutionary significance. Future research should further explore the molecular pathways underlying nematosome-mediated immunity and examine how these mechanisms have influenced the evolution of immune systems across metazoans.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eCompeting Interest\u003c/h2\u003e\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThis work was supported by DFG CRC grant 1182 \u0026ldquo;Origin and Function of Metaorganisms\u0026rdquo; (Project B1, Z3). NGS was carried out at the Competence Centre for Genomic Analysis (Kiel) within the CRC 1182 project Z3.\u003c/p\u003e\u003ch2\u003eAuthor contributions\u003c/h2\u003e\u003cp\u003eNHK and SF conceived the study. NHK performed the majority of the experiments and data analysis. MA contributed to proteomic analysis. GF assisted with mono-associations on wildtype polyps. SF supervised the project and provided conceptual input throughout. All authors contributed to data interpretation. NHK wrote the manuscript with input from all authors. All authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e\u003cp\u003eWe thank Katja Cloppenborg-Schmidt for preparing the 16S rRNA gene library. We would like to acknowledge the Center for Advanced Imaging (CAi) at Heinrich-Heine-University D\u0026uuml;sseldorf for providing access to the Olympus FV3000 Confocal Laserscanning Microscope and Zeiss REM Supra 55VP. Especially Dr. Sebastian H\u0026auml;nsch and Dr. Miriam B\u0026auml;umers for supporting during sample preparation and imaging. We would also thank Alexander Knauss producing the Video S1.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eKlas Karre HGL, Gerald Piontek \u0026amp; Rolf Kiessling. Selective rejection of H-2-deficient lymphoma variants suggests alternative immune defence strategy. \u003cem\u003eNature\u003c/em\u003e \u003cstrong\u003e319\u003c/strong\u003e, (1986).\u003c/li\u003e\n\u003cli\u003eVivier E, Tomasello E, Baratin M, Walzer T, Ugolini S. 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Natural insect host-parasite systems show immune priming and specificity: puzzles to be solved. \u003cem\u003eBioessays\u003c/em\u003e \u003cstrong\u003e27\u003c/strong\u003e, 1026-1034 (2005).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Metaorganism homeostasis, Symbiont recognition, innate immunity regulation, Nematostella vectensis, Nematosomes, Phagocytosis, Vibrio, cJUN/AP-1 ","lastPublishedDoi":"10.21203/rs.3.rs-7020203/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7020203/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eInnate immunity, traditionally viewed as non-specific, is increasingly recognized for its capacity to regulate microbial communities with precision. In the sea anemone \u003cem\u003eNematostella vectensis\u003c/em\u003e, we uncover a form of selective immunity mediated by nematosomes—motile immune cell clusters that preferentially phagocytose foreign \u003cem\u003eVibrio\u003c/em\u003e isolates while sparing native bacteria. We identify the transcription factor \u003cstrong\u003ecJUN \u003c/strong\u003eas essential for this process: CRISPR/Cas9-mediated knockout of \u003cem\u003ecJUN\u003c/em\u003eimpairs nematosome proliferation, reduces lysosomal activation, and alters microbiome composition by allowing colonization of non-native strains. These results link immune gene function to microbial selectivity and demonstrate that even early-diverging animals exhibit immune discrimination. Our findings challenge the classical dichotomy between innate and adaptive immunity and reveal that immune specificity may be evolutionarily ancient. This work establishes \u003cem\u003eNematostella\u003c/em\u003e as a model for studying microbiome-induced innate immune training and highlights conserved mechanisms that maintain host-microbe homeostasis.\u003c/p\u003e","manuscriptTitle":"cJUN dependent innate immunity controls microbiome through selective phagocytosis in Nematostella","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-11 09:02:39","doi":"10.21203/rs.3.rs-7020203/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"c8217c3f-73e6-41c0-b821-bd75a5dbd88c","owner":[],"postedDate":"July 11th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":51341187,"name":"Biological sciences/Immunology/Innate immunity"},{"id":51341188,"name":"Biological sciences/Microbiology/Microbial communities/Microbiome"}],"tags":[],"updatedAt":"2025-08-20T13:41:58+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-11 09:02:39","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7020203","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7020203","identity":"rs-7020203","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}