Maturation of the medaka immune system depends on reciprocal interactions between the microbiota and the intestinal tract

The interactions between the host immune system and intestinal microorganisms have been studied in many animals, including fish. However, a detailed analysis has not been performed in medaka, an established fish model for biological studies. Here, we investigated the effect of immunodeficiency on the microbiota composition and the effect of gut bacteria on intestinal epithelial development and immune responses in medaka. Chronological analysis of the intestinal microbiota of interleukin 2 receptor subunit gamma (il2rg) mutant medaka showed a gradual decrease in the evenness of operational taxonomic units, mainly caused by the increased abundance of the Aeromonadaceae family. Exposure of wild-type medaka to high doses of an intestine-derived opportunistic bacterium of the Aeromonadaceae family induced an inflammatory response, suggesting a harmful effect on adult il2rg mutants. In addition, we established germ-free conditions in larval medaka and observed large absorptive vacuoles in intestinal epithelial cells, indicating a block in epithelial maturation. Transcriptome analysis revealed a decrease in the expression of genes involved in the defense response, including the antimicrobial peptide gene hepcidin, whose expression is induced by lipopolysaccharide stimulation in normal larvae. These results show that reciprocal interactions between the microbiome and the intestinal tract are required for the maturation of the medaka immune system.


Introduction
The intestine develops initially under sterile conditions and is colonized by microbial communities after birth.The intestinal microbiome affects many aspects of host physiology, including nutrient uptake, energy homeostasis, adaptive immune homeostasis and control of inflammatory signaling (1)(2)(3)(4).The gut microbiome plays a pivotal role in the initiation and progression of several diseases, including cancer (5).
Immune-compromised humans, including those with severe combined immunodeficiency (SCID), are known to have disrupted microbiota (6,7).Defects in INTERLEUKIN 2 RECEPTOR SUBUNIT GAMMA (IL2RG) that encodes common g chain (gc), a type I cytokine receptor, on the X chromosome, cause X-linked immunodeficiency (X-SCID) in humans (8).In zebrafish, a major genetic model fish, it has been shown that gut microbiota composition plays a crucial role in the maintenance of innate immunity (9).Furthermore, a recent study reported that recombination-activating gene 1 (rag1) mutants lacking T and B cells and il2rg mutants, lacking T and natural killer (NK) cells, had a differential intestinal bacterial composition (10,11); however, these studies did not analyze the changes in gut microbiota over time in detail.
Furthermore, the effect of commensal microbiota on the host immune response has been actively studied in zebrafish (12), largely by establishing protocols for growing fish under germ-free and gnotobiotic conditions (13,14).Previous studies using a zebrafish germ-free model have demonstrated that colonization by commensals in newly hatched zebrafish primes neutrophils and induces several genes encoding proinflammatory and antiviral mediators, increasing the resistance of larvae to viral infection; commensal microbe recognition was found to be mediated mainly through a TLR/MyD88 signaling pathway, and professional phagocytes were identified as the source of these immune mediators (15).Moreover, endogenous microbiota establish the normal homeostatic level of neutrophils in the intestine; this process is mediated by lipopolysaccharide (LPS)-induced intestinal alkaline phosphatase through dephosphorylation of LPS and negative feedback of TLR signaling (16).The process of hatching itself changes the phenotype of skin keratinocytes that are responsible for both sensing the hyposmolarity of the aquatic environment and mediating immune effector mechanisms.The transition from the chorion environment to the rearing media is sensed through a transient potential receptor vanilloid 4/Ca 2+ /TGFb-activated kinase 1/NF-kB signaling pathway (17).In addition, some studies have clarified that microbial signaling via Myd88 is important for intestinal epithelial cell proliferation, changes in the intestinal leukocyte subsets, and transcription of antibacterial genes (18,19).
Medaka are freshwater fishes that have been established as model organisms for studies on genetics, developmental biology, and evolutionary biology (20).Inbred medaka strains are beneficial in physiological studies, especially those related to immune responses.Medaka shares genes involved in hematopoiesis and lymphocyte development with zebrafish and mammals (21)(22)(23)(24).Changes in gut microbiota composition in immunocompromised medaka, including the inflammatory cytokine interleukin 17 A/F1 mutant (25), and the effects of defects in its receptor interleukin-17 receptor A1 on the downregulation of intestinal metabolism-related genes have been reported (26).However, the effects of defective adaptive immunity of medaka have not yet been studied.Furthermore, although il2 and il2ra have not yet been identified in medaka, the medaka ortholog of il2rg is expected to be necessary for lymphocyte development, as reported in zebrafish (11,27).In addition, the effects of germ-free conditions on intestinal epithelial development and immune function in medaka have not been studied.
Here, we established il2rg mutant medaka and identified dynamic changes in intestinal bacterial composition using chronological microbiota analysis.Furthermore, we established a germ-free condition for medaka and found the contribution of intestinal bacteria to intestinal epithelium development and the expression of genes in the defense response pathway.These findings indicate the requirement of reciprocal interactions between the microbiome and the intestinal tract for the maturation of the medaka immune system.

Fish lines
The medaka (Oryzias latipes) lines were obtained from the National Bioresource Project (NBRP) Medaka and maintained at Utsunomiya University: inbred strain Hd-rR (strain ID: IB178), standard line OK-cab (strain ID: MT830), and cab-Tg (rag1:egfp), a transgenic medaka expressing egfp under the control of an immature lymphocyte-specific rag1 promoter (strain ID: TG848) (22).Fish were maintained in temperature-controlled tanks (26°C) with a water circulation system under a 14 h light/10 h dark cycle.This study was conducted in accordance with the ethical guidelines of the Utsunomiya University Animal Experimentation Committee, and the experimental protocols were approved by the committee (approval no.A20c-0012).The developmental stages of medaka were designated as previously described (28).

Genome editing
Alt-R CRISPR-Cas9 crRNA targeting exon 3 of il2rg (ENSORLG00000001795), Alt-R CRISPR-Cas9 tracrRNA, and the Cas9 protein were purchased from Integrated DNA Technologies (IDT; Singapore).The sgRNA sequences are listed in Table S1.RNA-protein complexes were prepared according to the manufacturer's instructions.Glass capillaries with 1.0 mm outer diameter were pulled (temperature 62°C, force: two light and two heavy weights provided by the manufacturer) using a micropipette puller PC-10 (Narishige Instruments, Tokyo, Japan).The capillary was filled with crRNA (0.75 mM), tracrRNA (1.5 mM), and 0.25 mg/mL Cas9 protein.Then 1-2 nL of the mixture was injected into 1-cell Cab embryos using a FemtoJet 4i (Eppendorf, Hamburg, Germany).G0 fish were crossed with wild-type (WT) Cab fish to establish the il2rg mutant strains.il2rg del4 mutation was chosen for phenotypic analysis.The primers used for genotyping are shown in Table S1.

Imaging of medaka specimens
The larvae and adult medaka were anesthetized with 0.1% (v/v) 2-phenoxyethanol and immobilized in 3% methylcellulose.Images were captured using a DP73 digital camera (Olympus, Tokyo, Japan) under an MZ16F fluorescence stereo microscope (Leica, Houston, TX, USA).

Reverse transcription-PCR
RNA was extracted using the NucleoSpin RNA kit (Macherey-Nagel, Düren, Germany) with on-column DNase treatment.The RNA was reverse transcribed using a QuantiTect Reverse Transcription Kit (Qiagen).To detect immunoglobulin Mu (igm) and t-cell receptor beta chain (tcrb) cDNA after VDJ recombination, nested PCR was performed using primer sets targeting the variable (V) and constant (C) regions.Two V regions highly expressed in wild-type kidneys were chosen for RT-PCR analysis, respectively (data not shown).Amplification was performed in a thermal cycler using the following program: 1 min at 95°C, cycles of 10 sec at 95°C, 30 sec at 59°C, 30 sec at 72°C, and 5 min at 72°C.The cycle numbers were as follows: actb (actin beta), 25 cycles; igm (igV H -Cm) and tcrb (tcrVb-Cb2), 30 cycles plus nested 30 cycles.The primers used for RT-PCR are listed in Table S1.Q u a n t i t a t i v e P C R ( q P C R ) w a s p e r f o r m e d u s i n g THUNDERBIRD SYBR qPCR Mix (Toyobo, Osaka, Japan) and LightCycler 480 (Roche, Basel, Switzerland).The primers used for qPCR are listed in Table S1.

Metagenome analysis
The WT and il2rg mutants derived from incrosses of heterozygous il2rg mutants were raised together.After genotyping, they were maintained in a water tank separated by a mesh to share the same water environment.Intestinal bacterial DNA was extracted using a QuickGene DNA Tissue Kit (Kurabo, Osaka, Japan) following the method described by Wong et al. (29) with some modifications.Intestines were harvested from WT and il2rg -/-medaka using sterile tweezers, transferred to microfuge tubes with 0.8 g zirconia beads and 200 mL Tissue lysis Buffer MDT, and immediately frozen in liquid nitrogen.Intestines were then quickly thawed at 65°C, 50 mL lysozyme (50 mg/mL) was added, and incubated at 30°C for 45 min.Subsequently, the bacterial lysates were processed following the protocol of Bacterial Genomic DNA Extraction from Stool (DF-1) of the QuickGene DNA tissue kit (Kurabo).
The V3 and V4 regions of the 16S rRNA gene were amplified using primer pairs with Illumina overhang sequences (Table S1).Index tag sequences were added to the amplicons using the Nextera XT Index Kit v2 (Illumina) and sequenced using the MiSeq Reagent Kit v3 (Illumina).The 301-bp paired-end reads were trimmed to remove low-quality ends (quality score < 15) and adapters and merged using the DADA2 plugin of QIIME2 (https://qiime2.org/).The curated sequences were annotated using the QIIME2 q2-featureclassifier plugin and Silva 132 99% OTUs (full-length, seven-level taxonomy) classifier pre-trained to the full-length Silva database to assign taxonomy to all ribosomal sequence variants.
Alpha-and beta-diversity analyses were performed using the q2-diversity plugin wrapped in QIIME2 at a sampling depth of 15,000 sequences.Alpha diversity metrics (Shannon and Chao1 indices) were calculated, and statistical significance was determined using the Kruskal-Wallis test.For Beta diversity analysis, principal coordinate analysis (PCoA) was performed using Bray-Curtis and weighted UniFrac metrics and visualized using a QIIME2 emperor visualizer (ver.2022.2.0).The statistical significances of weighted UniFrac metrics distances were calculated using Permutational Multivariate Analysis Of Variance (PERMANOVA).

Isolation and identification of adult gut-derived bacteria
The intestinal tracts were harvested from adult Hd-rR strain using sterile tweezers.After washing the surface of the intestine with sterile water, it was crushed in sterile phosphate-buffered saline (PBS) using a Power Masher II (Nippi Incorporated, Tokyo, Japan).Diluted intestinal bacteria was aerobically cultured on solid LB media at 28°C overnight, and the colonies were picked up for liquid culture.Bacterial DNA was extracted as described by Wong et al. (29) with some modifications.The bacterial pellet was suspended in 200 mL enzymatic lysis buffer (20 mM Tris-Cl (pH 8.0), 2 mM EDTA (pH 8.0), and 1% Triton X-100) and immediately frozen in liquid nitrogen.Subsequently, the samples were quickly thawed at 65°C, 20 mL 200 mg/mL lysozyme was added, and incubated at 30°C for 45 min.Next, 10 mL proteinase K (10 mg/mL) and 230 mL 40% guanidine hydrochloride were added, and the sample was incubated at 56°C for 30 min.Bacterial lysates were processed using a Mag Extractor-Genome (Toyobo) for DNA extraction.
The bacterial 16S rRNA gene was amplified using primers (16SrRNA 27F and 16SrRNA 1491R).Amplification was performed in a thermal cycler using the following program: 1 min at 95°C, 40 cycles of 10 sec at 95°C, 30 sec at 59°C, 30 sec at 72°C, and 5 min at 72°C.The amplicons were directly sequenced using 16SrRNA 27F, 16SrRNA 1491R and 16SrRNA 907R primers with BigDye Terminator v3.1CyleSequencing Kit (Thermo Fisher Scientific, Waltham, MA, USA) and analyzed using 3500 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA).The primer sequences are listed in Table S1.Bacterial families were determined using the National Center for Biotechnology Information (NCBI) BLAST program (www.ncbi.nlm.nih.gov/cgi-bin/blast).A phylogenetic tree was generated with MegAlign (Lasergene, DNASTAR, Madison, WI) using MUSCLE alignment and a maximum likelihood model with 1000 bootstraps.

Bacterial exposure
One of the type A clones of Aeromonas isolated from the intestine of an adult Hd-rR strain was spread on a solid Brain Heart Infusion (BHI) medium (BD, Franklin Lakes, NJ).A single colony was picked and cultured in liquid BHI medium at 25°C overnight, then 1:100 dilution was further cultured at 25°C for 4 h until the growth phase was reached.Subsequently, the culture was centrifuged at 2000 × g for 10 min, and the supernatant was replaced with PBS.The OD600 of the culture was measured using an Od meter (Thomas Scientific, Swedesboro, NJ, USA).Two months post-fertilization (mpf), WT medaka were exposed to the bacteria at 6.5×10 7 colony forming units (CFU)/mL in filtered water using the bath method.CFU of the bacteria were calculated as the colony numbers of diluted bacteria after overnight culture on the solid BHI medium.Twenty percent of the water-containing bacteria was replaced with filtered water every day.

Flow cytometry
Whole kidney marrow and spleen cells were prepared as previously described (30) with some modifications.Cells were obtained by pipetting the organs into 1 mL ice-cold 1% fetal bovine serum (FBS) in 0.9× PBS.After centrifugation, the pellet was gently mixed with 1 mL distilled water at 20°C by pipetting 4 times to lyse the erythrocytes by osmotic shock to prepare splenocytes, and immediately 1 mL of 1.8× PBS was added.Cells were washed with 1% FBS in 0.9× PBS by centrifugation and then filtered using a 40 mm-stainless mesh.Flow cytometry (FCM) was performed using a FACSLyric flow cytometer (BD Biosciences).Data analysis was performed using Flowjo10.6.2 (BD Biosciences).

Preparation of Aeromonas for gnotobiotic culture
A colony of Aeromonas isolated from the intestine of an adult Hd-rR strain was picked and transferred to liquid LB medium.OD600 values were measured using a UV-Vis Spectrophotometer V-630 (Jasco, Tokyo, Japan).By counting colony numbers after overnight culture on LB agar plate, CFU per OD600 were calculated so that 1× 10 6 CFU/mL of Aeromonas culture was added to larvae.
At 6 days post-fertilization (dpf) (stage 36), embryos were transferred into sterile antibiotics-embryonic culture medium (AB-ECM) (200 mg/mL ampicillin, 5 mg/mL kanamycin, 0.5 mg/mL amphotericin B in ECM, filtered using 0.22 mm filters) and cultured at 28°C for 4 h.The embryos were then transferred to a sterile beaker containing AB-ECM.After washing three times with sterile ECM, embryos were soaked in 0.1% PVP-I solution (0.1% PVP-I in ECM, filtered using a 0.22 mm filter) for 2 min.After washing three times with sterile ECM, the embryos were transferred to another sterile beaker with 0.003% bleach solution (0.003% sodium hypochlorite in ECM, filtered using 0.22 mm filter) and incubated at room temperature for 30 min.For germ-free condition, embryos were washed three times with sterile ECM and transferred into 25 cm 2 flasks with sterile ECM.
At 12 dpf, when all embryos hatched, 200 mL of the culture medium was spread onto the solid culture media to test for bacterial contamination.Larvae were transferred to new 25 cm 2 flasks with sterile ECM (for germ-free), 5 mm-filtered fish system water (for conventional control), or 1 × 10 6 CFU/mL Aeromonas in sterile ECM (for gnotobiotic) with mashed gamma-irradiated powdered food (Otohime S1, Marubeni Nisshin Feed Co., LTD, Tokyo, Japan).
Two days after culture in respective mediums (14 dpf), 200 mL of the culture medium of all groups was spread onto solid culture media to test for bacterial contamination.The larvae were then transferred to new 25 cm 2 flasks containing sterile ECM.Subsequently, two days after culturing (16 dpf), the larvae were used for phenotypic analyses.

Bacteria culture from the larval culture medium
A total of 200 mL germ-free, conventional, or gnotobiotic culture medium containing larvae was spread onto solid LB or trypticase soy agar (TSA; Thermo Fisher Scientific) media for aerobic culture at 28°C for 2 days.In addition, anaerobic culture was performed in a solid TSA medium at 28°C for 4 days.AnaeroPack-Anaero (Mitsubishi Gas Chemical, Tokyo, Japan) was used for anaerobic culture, and the anaerobic status was checked using an Anaero-indicator (Mitsubishi Gas Chemical, Tokyo, Japan).

Histological analysis
Whole larvae were fixed in Bouin's solution overnight.Paraffin sectioning was performed as described in a previous study (31).Coronal sections (5 mm thick) of larval intestines were stained with hematoxylin and eosin.For Periodic acid-Schiff (PAS) staining, the deparaffinized sections were placed in a 0.5% periodic acid solution (Fujifilm Wako, Osaka, Japan) for 5 min and rinsed twice with distilled water.Sections were stained with Schiff's reagent (Fujifilm Wako) for 5 min, washed with a sulfurous acid solution (Fujifilm Wako) three times, rinsed under running tap water, and counterstained with hematoxylin.For acidic mucin staining, deparaffinized sections were placed in 3% acetic acid for 3 min and stained with Alcian blue solution (pH 2.5; Fujifilm Wako) for 30 min.The sections were rinsed under running tap water and counterstained with hematoxylin.Images were captured using a DP72 camera (Olympus) under a BX60 microscope (Olympus).
GO enrichment analysis was performed for the 53 genes downregulated in germ-free larvae (Table S3) using Panther software (version 17.0; http://pantherdb.org/).The analysis was performed using annotation datasets of the complete GO biological processes and the reference list of human genes.Eighteen genes that were uncharacterized or lacked human orthologs were excluded from the analysis.

il2rg mutation affects lymphocyte development
To assess the effects of a defective adaptive immune system on the intestinal microbiota of medaka, immunodeficient medaka (il2rg -/-) were generated by genome editing using the CRISPR/ Cas9 system, targeting exon 3 of il2rg (Figure S1A).A mutant line with a 4 bp deletion in il2rg was established, which was predicted to cause a frameshift and encode only 75 appropriate amino acids, followed by 12 unrelated amino acids and a stop codon (Figure S1B).The resulting protein lacked most of the fibronectin type III domain (FNIII) and the entire transmembrane domain (TM) (Figure S1C) and was considered a null allele.The homozygous form of this frameshift mutant did not exhibit any gross developmental defects.However, crossing the mutant with rag1:egfp transgenic medaka, which expresses EGFP under the control of the immature lymphocyte-specific rag1 promoter (22), revealed a severe reduction in EGFP-expressing cells in the larval thymus, suggesting defects in T cell development (Figure 1A).This severe reduction in T-cells was continuously observed in the adult thymus (Figure 1B).
Next, we investigated the transcripts of rearranged lymphocyte antigen receptors in the kidney, equivalent to the mammalian bone marrow as a site of hematopoiesis.Reverse transcription PCR (RT-PCR) analysis detected VDJ-recombined igm transcripts in B cells but not tcrb transcripts in the il2rg mutant (Figure 1C).qPCR analysis of adult kidney cells revealed a severe reduction in il2rg expression in il2rg mutants (Figure 1D).Under these conditions, the expression of pan B cell marker cd79, early B cell marker ebf1, and relatively mature B cell marker cd22l in the WT and il2rg mutant did not differ significantly (Figure 1D).In contrast, the expression of T cell markers cd8a and cd4-2 was significantly decreased in il2rg mutants (Figure 1D).Among other blood populations, the expression of the neutrophil marker mpx was comparable, but that of the natural cell maker nkl.1 was significantly reduced in il2rg mutants than those in WT medaka (Figure 1D).These results suggest that il2rg mutation affects T and NK cells, as observed in the zebrafish il2rg mutants (11,27); nevertheless, they grew to adult stage in filtered water with a weekly water change (mortality rate at 3 mpf: 0/15), despite being sensitive to lower water quality (mortality rate: 11/14 in a recirculating system sharing water with other medaka tanks).

Dynamic change of frequency of intestinal microbiota in il2rg mutant adult medaka
The analysis of the intestinal bacterial composition of the WT and il2rg mutants at 2, 2.5, and 3 mpf revealed that most OTUs were represented by Proteobacteria, Fusobacteriota, and Firmicutes at the phylum level in both the WT and il2rg mutants at all time points (Figures 2A, 3A).However, the abundance of Proteobacteria was significantly higher in the il2rg mutants than that in WT at 2.5 and 3 mpf.Especially at 3 mpf, more than 90% of the intestinal microbiota of il2rg mutants comprised Proteobacteria.Furthermore, the abundance of Fusobacteriota was inversely correlated with that of Proteobacteria.
At the family level, Aeromonadaceae was abundant in WT adult medaka intestines (Figures 2B, 3B), which was consistent with the previous report (25).The abundance of Aeromonadaceae gradually increased in the il2rg mutants at 2.5 and 3 mpf and was significantly higher than that in the WT (Figures 2B, 3B).This enrichment of Aeromonadaceae was observed repeatedly in independent experiments (Table S2).Other significant changes at the family level included a temporary increase in Shewanellaceae at 2.5 mpf and an increase in Enterobacteriaceae at 3 mpf in il2rg mutants.

Dynamic change of evenness of intestinal microbiota in il2rg mutant adult medaka
To compare the overall composition of intestinal bacteria between the WT and il2rg mutants at each time point, alpha diversity indices were analyzed.Shannon indices, which indicate the evenness of OTUs, showed that the WT was unchanged at all three time points (Figure 4A).Although the evenness of the OTUs in the il2rg mutant was comparable to that of the WT at 2 and 2.5 mpf, it was significantly decreased at 3 mpf (Figure 4A), which mainly reflected the increased abundance of Aeromonadaceae.In contrast, Chao1 indices, which indicate the richness of OTUs, were comparable between WT and il2rg mutants at all time points, although the index of WT medaka at 3 mpf was higher than that at earlier time points (Figure 4B).
Next, we quantified the similarity of bacterial composition among each fish and each group.Analysis of beta-diversity indices using weighted UniFrac and Bray-Curtis plots revealed a stable pattern in WT at all time points.In contrast, the intestinal bacterial composition of il2rg mutants showed dynamic shifts at 2.5 and 3 mpf (Figures 4C,  D).Considering the phylogenetic distances to the datasets, weighted UniFrac indices were chosen for the quantitative measurement of OTUs.The calculation of the distances indicated that the bacterial composition of il2rg mutants was significantly distant from that of the WT at 2.5 and 3 mpf (Figure 4E).

Effect of Aeromonas as a culturable main component of intestinal bacteria on adult medaka
Next, we identified readily culturable bacteria derived from the medaka intestine.A culture of aseptically harvested intestines from adult WT medaka under aerobic conditions revealed uniform shapes and colors of the colonies (Figure S2A).The 16S rRNA gene sequences from the 16 colonies were grouped into three types: type A (13 colonies), type B (two colonies), and type C (one colony).The phylogenetic tree of the 16S rRNA gene sequences of the Aeromonas family showed that all three types were close to Aeromonas and away from Oceanimonas (Figure S2B).Type A and C sequences were the closest to those of A. intestinalis, recently isolated from human feces (35).The type B sequence was identical to the FPC-0866 strain, derived from eels and defined as A. hydrophila (36).However, A. hydrophila strain ATCC 7966 was relatively phylogenetically distant from the FPC-0866 strain, indicating that the resolution of 16S rRNA gene sequencing was not high enough to identify the species of Aeromonas precisely.Nevertheless, all three types are close to Aeromonas; therefore, we considered Aeromonas a representative bacterium of adult medaka.
To examine the effect of intestine-derived Aeromonas on adult medaka, WT medaka were exposed to 6.5 × 10 7 CFU/mL of one of the type A clones.Although this exposure did not cause lethality (14/14), exposed fish commonly showed vasodilation of the liver and an enlarged spleen (Figure S2C), suggesting an inflammatory response.FCM analysis of kidney and spleen cells showed that the exposure did not change the populations of kidney cells; however, the lymphoid population overwhelmed the myeloid population in the spleen of the exposed fish (Figures S2D, S2E), implying lymphocyte involvement in the antibacterial response.

Establishment of germ-free condition of medaka
Next, we sought to establish a method for maintaining medaka larvae under germ-free conditions.To this end, we followed one of the protocols for zebrafish germ-free culture (14) with some modifications for medaka.PVP-I and bleach treatment of eggs at 6 dpf before hatching and culture of eggs and larvae thereafter with sterile AB kept the larvae sterile, which was confirmed by the absence of colonies after the larval culture medium was spread on LB agar medium for aerobic culture and TSA agar medium for aerobic and anaerobic cultures (Figure S3).Gnotobiotic conditions were also achieved by adding 1 × 10 6 CFU/mL Aeromonas derived from adult medaka intestine to a germ-free culture.

Intestinal epithelial cell development of germ-free medaka
Next, to analyze the structural changes in the intestines of wildtype germ-free larvae, we compared the anterior, middle, and posterior intestinal structures (37) under germ-free, conventional, and gnotobiotic conditions.The anterior intestine contained small absorptive vacuoles in its epithelial layer.However, no apparent structural differences were observed between the conventional, germ-free, and gnotobiotic conditions.No structural differences were observed in the posterior intestines (Figure S4).
However, apparent structural differences were observed in the middle intestine.In the germ-free intestine, although the thickness of the intestinal tube itself was comparable to that in the conventional condition (Figures 5A, B), the thickness of the epithelial layers was significantly increased compared to that in the conventional condition (Figures 5A, C).Close observation of the germ-free epithelium revealed enlarged absorptive vacuoles.Although such vacuoles were also present in the conventional intestine, their number was significantly higher in the germ-free intestine (Figures 5A, D).These vacuole structures were not goblet cells because they did not stain with PAS (for neutral mucin) or with Alcian blue (for acidic mucin) (Figure 5E).The increased epithelium thickness and absorptive vacuole enlargement were negated by adding Aeromonas bacteria to the gnotobiotic conditions (Figures 5A, C, D).

Decreased expression of LPSdependent defense response genes in germ-free condition
To investigate the effects of immune-related gene expression in germ-free medaka, RNA-seq analysis of whole bodies of medaka larvae grown under conventional or germ-free conditions was performed.Fifty-three genes were significantly downregulated, and six were significantly upregulated in germ-free larvae (Tables S3, S4).Gene ontology analysis of the downregulated genes showed significant enrichment of defense response (GO:0006952) genes (p = 0.0018; Figure 6A), which included the regulation of intestinal absorption (GO:1904478) genes (hamp and apoa4a) (p = 0.00024).
qPCR analysis of whole bodies of germ-free larvae showed a tendency to decrease the expression of seven genes (grn, epx, hamp, hp, c1q, mrc1, and apoa4), except fasn under germ-free conditions compared to conventional conditions (Figure 6B).Interestingly, the expression of grn, epx, hamp, hp, c1q, and mrc1, excluding apoa4, was partially recovered under gnotobiotic conditions (Figure 6B).qPCR analysis using intestinal RNA samples showed identical expression patterns-the expression of five genes (epx, hamp, hp, c1q, and mrc1), except grn decreased in germ-free conditions and recovered in gnotobiotic conditions (Figure 6C).qPCR analysis of whole bodies of larvae injected with LPS, a toll-like receptor 4 (TLR4) signaling inducer, showed increased hamp, hp, and c1q expression (Figure 6D), suggesting that the expression of these genes is affected by the detection of bacterial components by TLRs.

Discussion
In this study, we showed that reciprocal interactions between the microbiome and the intestinal tract are required for the maturation of the medaka immune system using il2rg mutant and the establishment of germ-free conditions.
In humans, il2rg (gc) is responsible for X-SCID (8).Although receptor complexes utilizing IL2RG and the cytokines serving as these ligands have not been completely identified in teleosts, il2rg is expected to be involved in important cytokine signaling pathways for lymphocyte development and function.A recent study in zebrafish showed that il2rg mutation affects T and NK cell development (11,27); concordantly, medaka il2rg mutants reported in this study showed a similar phenotype to that of the zebrafish mutant (Figure 1).The lack of T cells likely affects B cell activation by T-dependent antigens (38), as observed in mammals, leading to overall defects in adaptive immunity.As il2rg is expressed by pan-B cells which are unaffected by il2rg mutation, as well as T and NK cells in the kidney (data not shown), a lower expression level of il2rg in il2rg mutant does not reflect the depletion of il2rg-expressing cells, but a reduction of il2rg mRNA in each cell, possibly because of nonsense-mediated mRNA decay.
In our chronological analysis of intestinal microbiota, the intestinal bacterial composition in WT medaka was stable at 2-3 mpf (Figures 2, 3), with a predominance of Proteobacteria (46.2 ± 4.7% at 2 mpf), especially Aeromonadaceae family (23.7 ± 1.7%).This result is similar to that of a previous report (25), indicating that the Aeromonadaceae family is the major component of medaka intestinal flora.
To compare the microbiota composition between the WT and il2rg mutants, we cohoused both genotypes.As we observed a highly variable gut microbiota composition among the discrete fish tanks (data not shown), cohousing could overcome this problem by focusing on genotype discrepancies.Furthermore, co-housing organisms, including fish, possessing a wide variety of microorganisms, is a common phenomenon in the natural environment.While changes in the gut microbiota composition have been reported in zebrafish il2rg and rag1 mutants (10,11), the present study using il2rg mutant medaka was carried out in-depth using chronological analysis.Although the gut bacterial composition of the il2rg mutant was similar to that of the wild type at 2 mpf, it showed a dynamic change in the following 1 month with a significant predominance of Proteobacteria, mainly Aeromonadaceae (Figure 3).This increased abundance of Aeromonadaceae led to reduced evenness of OTUs in the mutants, even though richness itself was unchanged (Figure 4).Hence, in contrast to earlier time points, the intestinal bacterial composition pattern in the il2rg mutant was dramatically and significantly changed at 3 mpf, suggesting that a deficiency in T and NK cells affects the intestinal bacterial composition.Although reduced bacterial diversity has also been observed in the zebrafish il2rg mutant (11), the abundance of Aeromonadaceae showed an opposite trend in these two species-zebrafish showed an increased abundance of Firmicutes.This contrast could be because of the physiological differences between them.
We cloned Aeromonas as the main intestinal bacteria (Figure S2).Aeromonas is a genus comprising several gram-negative rodshaped bacteria, including A. hydrophila and A. veronii and is present in the gut as opportunistic bacteria (39,40).Exposure to the A. hydrophila FPC-0866 strain derived from the Japanese eel was reported to cause lethality in an apoptosis-associated speck-like (ASC) protein (a component of the inflammasome) coding gene mutant (36).Phylogenetic analysis of the medaka intestine-derived Aeromonas clones based on 16S rRNA gene sequences indicated a high similarity to FPC-0866 (Figure S2).The inflammatory phenotype of the liver and spleen and an increase in the lymphoid population in the spleen after exposure to type A clones of Aeromonas suggest the harmful effects of this bacterium at high concentrations.It is possible that the overwhelming Aeromonas in immunodeficient il2rg mutants is one of the reasons for lethality in early adulthood.Some fish species have been reported to lack the principal components of adaptive immunity; Atlantic cod does not have CD4 + T cells or their related genes (41) and some anglerfish species engage in permanent sexual parasitism and even lack rag-based adaptive immunity (42).In particular, anglerfish are likely to control environmental microorganisms primarily through innate immunity.In addition, innate immunity is thought to be more important than adaptive immunity in fish (43, 44) and has been shown to control intestinal microbiota composition in zebrafish (9).However, the present study, together with other studies in zebrafish (10,11) showed that the maintenance of healthy gut microbiota also depends on adaptive immunity.T cell development in teleosts occurs in waves.For zebrafish, T cells colonize the intestine as early as 5dpf and increase during inflammation (45); a second wave begins to colonize the larva at around 21 dpf (46,47).In zebrafish rag1 mutants, a different intestinal microbial community has been reported compared to its WT siblings at 14 wpf, represented by enrichment of Vibrio (10).Although microbiota analysis was not done at earlier timepoints, qPCR analysis indicated that enrichment of Vibrio was apparent at 5 wpf.Together, these observations suggest that T cells colonizing the periphery during the first and subsequent waves may have little immediate impact on the composition of the intestinal microbiota.Whether this is because re-organization of the microbiota is a slow process or because subtle changes may escape detection remains to be clarified.In any case, our il2rg mutant medaka showed a change in microbial composition only after 2.5 mpf (Figures 2-4).Reduced expression of lipopolysaccharide (LPS)-sensitive genes in germ-free conditions.(A) List of defense response (GO:0006952) genes whose expression is decreased in whole bodies of germ-free larvae identified using gene ontology analysis compared to that in conventional larvae (Related data shown in Tables S3 and S4).(B, C) Quantitative PCR (qPCR) analysis of defense response genes of 16 days post-fertilization (dpf) larvae in conventional (CV), germ-free (GF), and gnotobiotic (GB) conditions.Whole bodies (CV: n = 3, GF: n = 3, GB: n = 4) (B) and intestines (CV: n = 3, GF: n = 3, GB: n = 2) (C).Results represent one of the two independent experiments with similar results.(D) qPCR analysis of the defense response genes of whole bodies of LPS-or mock-stimulated (-) embryos at 9 dpf (n = 5, respectively).Results represent one of the three independent experiments with similar results.qPCR data represent the mean ± SEM. *, p < 0.05; **, p < 0.01 determined using unpaired two-tailed t-test.medaka has been provided (48), it is possible that the microbiota also changes during waves of T cell development.These findings suggest that the detectable change of composition of the microbiota in fish lacking T and NK cells only at 2.5 mpf could be related to the developmental regulation of these cell lineages.The cellular mechanisms for microbiota regulation, including the timing of second-wave T cell development in medaka, are topics for future work.

Although evidence for T cell colonization of the larval intestine in
We established the method for growing medaka larvae in germfree and gnotobiotic conditions for the first time.Under germ-free conditions, absorptive vacuole intestinal epithelium in the midintestine of the larvae was significantly larger than that of those under the conventional and gnotobiotic conditions (Figure 5).Large absorptive vacuoles are observed in the immature and occasionally pathogenic epithelium in mammals (49,50), suggesting that intestinal epithelium maturation is blocked or delayed in germfree larvae.As germ-free culture was carried out up to 15 dpf in this study, the phenotypic changes in the intestinal epithelium after longer germ-free cultures were unclear.However, this is feasible for future studies.
RNA-Seq analysis showed that the genes downregulated under germ-free conditions were enriched in those involved in defense responses (Figure 6).Three out of eight genes, hamp, hp, and c1q, were downregulated in germ-free conditions; however, their expression was recovered in gnotobiotic conditions in both whole bodies and intestines and was dependent on in vivo LPS stimulation.Consistently, in mammals, these three genes are upregulated by LPS stimulation via TLR4 (51-55).In particular, hamp expression was more than 300 times downregulated in germfree whole bodies and intestines.In addition, LPS stimulation induced hamp expression more than 80 times.These findings are consistent with a previous study in mouse myeloid cells, which showed that hamp is induced by LPS through TLR4 (51).The antimicrobial peptide hepcidin, encoded by hamp, has antiviral activity in medaka (56).In addition, in vitro antiviral, antibacterial, and antitumor activities of marine medaka (O.melastigmus) have been reported (57), suggesting the importance of this antimicrobial peptide upon exposure of medaka larvae to microorganisms.The main function of hp is to bind to hemoglobin released from damaged erythrocytes; nevertheless, hp also promotes immune tolerance to LPS by suppressing proinflammatory cytokines (52,53).c1q, a component of the complement C1 complex, suppresses LPS-induced pro-inflammatory cytokine production in dendritic cells and monocytes in mice (54,55).These results suggest that the reduction in the expression of these genes under germ-free conditions was due to a lack of bacteria-induced TLR signaling.In addition, the downregulated expression of mrc1, a marker of M2 macrophages activated by IL-4 (58) and independent of M1 macrophages through LPS stimulation, was also restored under gnotobiotic conditions, which likely reflects that mrc1 expression was only mildly upregulated (1.9x) by LPS injection.
The germ-free and gnotobiotic conditions of medaka will be useful for further characterization of the effects of intestinal bacteria on larval physiology.Compared to zebrafish, which hatch before the completion of organogenesis, medaka organogenesis is completed during the embryonic stages.Accordingly, the physiological effects of bacterial exposure may differ among species.

1 il2rg
FIGURE 1 il2rg mutation affects T and B cell development.(A) rag1:egfp expression in larval thymocytes at 10 days post-fertilization (dpf).Dorsal (left) and lateral (right) views of the indicated genotypes.Dotted circles indicate the thymus.(B) rag1:egfp expression in adult thymocytes at two months postfertilization (mpf).Lateral views of indicated genotypes with (left) and without (right) visible light transmission.Dotted circles indicate the thymus.Note a few EGFP-positive thymocytes in il2rg mutants.(C) Reverse transcription PCR (RT-PCR) of whole kidney cells of the indicated genotypes using indicated primers to detect VDJ recombined igm and tcrb.Two fish for each genotype were used.actb expression was used as standard.(−); water control.(D) Quantitative PCR (qPCR) analysis of whole kidney cells of the indicated genotypes (n = 4, respectively).The average expression level in il2rg +/+ is normalized as 1.Data represent the mean ± standard error of the mean (SEM).*, p < 0.05; **, p < 0.01 determined using unpaired two-tailed t-test.Results represent one of the two independent experiments with similar results.

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FIGURE 2 Microbiota analysis of intestines from adult wild type and il2rg mutant medaka.(A, B) The intestinal bacterial composition of adult wild-type (WT) and il2rg mutant (Mut) medaka at 2, 2.5, and 3 months post-fertilization (M).The number of fish in each group (n) is indicated.(A) The 100% stacked bar charts indicate the phyla of all bacteria.(B) The 100% stacked bar charts indicate the family of all bacteria.

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FIGURE 4 Dynamic change of intestinal bacterial composition in il2rg mutant.(A, B) Alpha diversity analysis of intestinal bacteria composition of wild-type (WT) and il2rg mutant (Mut) medaka at the indicated age (months post-fertilization, M) using Shannon (A) and Chao1 indices (B).The number of fish in each group (n) is indicated.(C, D) Beta diversity analysis of intestinal bacteria composition of WT and il2rg mutants at the indicated age using weighted UniFrac (C) and Bray-Curtis plots (D).The number of fish in each group (n) is identical to (A, B). (E) The statistical significances of weighted UniFrac (C) distances were calculated using PERMANOVA.

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FIGURE 5 Immature intestinal epithelium in germ-free condition.(A) Coronal sections of middle intestines after larval culture in conventional (CV), germ-free (GF), and gnotobiotic (GB) conditions.Enlarged images of the area of yellow squares are shown below.The absorptive vacuoles in the enterocytes in germ-free conditions are enlarged.Scale bars: 50 mm (top) and 20 mm (bottom).(B) Comparable middle intestinal tube areas in each larval culture condition.Data indicates the average of three sections per fish; n = 3, respectively.(C) Increased thickness of middle intestinal epithelium in germfree conditions.Ratios of epithelium area vs. total epithelial tube area are shown.Data indicates the average of three sections per fish; n = 3, respectively.(D) Increased absorptive vacuole sizes in germ-free conditions.Data represent the mean ± SEM.Statistical significance was determined using an unpaired two-tailed t-test.The number of vacuoles measured is as follows; CV: n = 243, GF: n = 143, GB: n = 44.(E) Periodic acid-Schiff (PAS) (top) and Alcian blue (bottom) staining of mid intestines.Arrows indicate stained goblet cells.The absorptive vacuoles are away from goblet cells with PAS-positive neutral mucin and Alcian blue-positive acidic mucin.Scale bar, 20 mm.