The Nile tilapia (Oreochromis niloticus) NT1 strain was generously provided by Prof. Hong-Yi Gong (Department of Aquaculture, National Taiwan Ocean University). The procedures for sampling tissues from healthy fish are described by Trung and Lee (21).

A total of 80 fish (average weight 45.8 ± 6.8 g) were acclimatized in four 90 L glass tanks (n = 20 each) that were aerated and supplied with dechlorinated tap water (28 ± 2°C). The photoperiod for each tank was maintained at 12 h light/12 h dark. A. hydrophila and Streptococcus agalactiae, which had been isolated from diseased tilapia, were cultured on tryptic soy agar (TSA, Difco) at 28°C overnight. Cells were washed off with sterile phosphate-buffered saline (PBS, pH 7.4) and adjusted to the desired concentration. The experimental fish were weighed and injected intraperitoneally with PBS (control group), A. hydrophila (4 × 10⁵ colony forming units (CFU)/g of fish body weight), or S. agalactiae (1.3 × 10⁴ CFU/g of fish body weight) (n = 20 per group) (21). Tissues were collected (five fish per group) at 4, 8, 24, and 72 h post injection (hpi) and preserved in RNAlater solution (Invitrogen, CA, US). Fish handling and experiments were conducted following the guidelines of the Institutional Animal Care and Use Committee, which is approved by National Taiwan Ocean University (code: 109042).

Total RNA was extracted using a TRI Reagent (Sigma-Aldrich, US) according to the manufacturers’ instructions. RNA quantity and purity were determined using a SpectraMax QuickDrop Spectrophotometer (Molecular Devices, US). Reverse transcription was conducted with 5 μg of total RNA using RevertAid™ reverse transcriptase (200 U/µl, Thermo Fisher Scientific, US). cDNA was diluted with 40 μl of RNase-free distilled water for gene cloning and stored at -20°C (21). The iScript™ gDNA Clear cDNA Synthesis Kit (BioRad, US) was used for cDNA synthesis for qRT-PCR analysis. The elongation factor-1α (EF-1α) (a reference gene) was used for normalization. Gene expression of OnSARM1, OnMyD88, OnTIRAP, OnTRIF, interleukin (IL)-1β, IL-8, IL-12a, IL-12b, tilapia piscidin (TP) 2, TP3, TP4, hepicidine, tumor necrosis factor (TNF)-α, major histocompatibility complex class Ia (MHC Ia), and type I interferon (IFN)d2.8 and IFN2.13 were measured using a StepOnePlus Real-Time PCR instrument (Thermo Fisher Scientific) using the primers listed in Supplementary Table 1. The qRT-PCR reaction mixture (20 μl) was composed of 4 μl of cDNA template, 2 μl of specific primers, 4 μl of PCR-grade water, and 10 μl of RealQ Plus 2 × Master Mix Green (Ampliqon, Denmark). Reaction mixtures were incubated for 10 min at 95°C, followed by 40 cycles of 15 s at 95°C, and finally 1 min at 60°C. A melting curve was generated by increasing the temperature from 60°C to 95°C with 0.3°C increments.

To clone Nile tilapia SARM1, a set of primers was designed based on the complete coding region of the OnSARM1 sequence (Supplementary Table 1). The PCR reactions were conducted using MyTaq Red Mix 2× (Bioline, UK). The PCR thermal cycling was performed via an initial denaturation at 95°C for 1 min, followed by 35 cycles at 95°C for 15 s, 53°C for 30 s, and 72°C for 1.5 min, and a final extension at 72°C for 10 min. The obtained PCR product was cloned into the pGEM-T Easy Vector (Promega, US) and sequenced using Genomics Ltd.

The OnSARM1-GFP and OnSARM1-3×FLAG expression plasmids were prepared using primers GFP-NheI-OnSARM1-F and GFP-HindIII-OnSARM1-R, and FLAG-HindIII-OnSARM1-FL-F and FLAG-XbaI-OnSARM1-FL-R, respectively (Supplementary Table 1). Q5^® High-Fidelity DNA Polymerase (New England Biolabs, US) was used, and the PCR reactions were set up as follows: one cycle at 98°C for 30 s, followed by 35 cycles at 98°C for 15 s, 62°C for 30 s, 72°C for 90 s, and terminated with one cycle of 72°C for 10 min and 95°C for 60 s. The purified PCR products and empty pTurbo-GFP-N (Evrogen, Russia) or p3×FLAG-CMV-14 vector (Sigma-Aldrich) were ligated together after restriction enzyme digestion. Ligation products were transformed into ECOS™ 101 Competent Cells (DH5α) (Yeastern Biotech, Taiwan). Positive colonies were screened using vector-specific primers (Supplementary Table 1), and colonies with correct insert size were grown in LB broth with ampicillin (100 µg/ml) (for p3×FLAG-CMV-14 vector) or kanamycin (50 µg/ml) (for pTurboGFP-N vector) for plasmid preparation.

Homologous sequences were searched through the BLAST algorithm (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The nucleotide sequence of OnSARM1 was translated using the ExPASy translate tool (http://web.expasy.org/translate/). The molecular weight of the protein was calculated using the Compute pI/Mw tool (http://web.expasy.org/compute_pi/). A phylogenetic tree was constructed using the neighbor-joining method in Molecular Evolution Genetics Analysis software version 7 (MEGA7), supported by 10,000 bootstrap repetitions with the Poisson model for amino acid substitution and pairwise deletion for gap treatment (22). EMBOSS Needle online software (https://www.ebi.ac.uk/Tools/psa/emboss_needle/) was used to identify the sequence identity and similarity between vertebrates OnSARM1 (23). Synteny analysis was performed via the Genomics v100.01 program (24). Simple Modular Architecture Research Tool (SMART) (http://smart.embl-heidelberg.de/) was used to predict the deduced amino acid sequence of OnSARM1 (25). Multiple alignments were performed using the ClustalW program (https://www.ebi.ac.uk/Tools/msa/clustalo/).

A tilapia head kidney (THK) cell line (26) was cultured at 27°C in Leibovitz medium (L-15, Gibco) containing 10% Fetal Bovine Serum (FBS, Corning) and 1% penicillin (100 U/ml)/streptomycin (100 μg/ml) (P/S, Gibco). Human embryonic kidney (HEK) 293 cells were provided by Prof. Pin-Wen Chiou (Department of Aquaculture, National Taiwan Ocean University) and were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, GE Healthcare Life Sciences) containing 10% FBS and 1% P/S at 37°C with 5% CO₂ in a humidified incubator. When the cells reached 70-80% confluency, they were passaged or used for seeding.

HEK 293 and THK cells (1 × 10⁵ cells per well) were seeded onto glass coverslips in 24-well culture plates with 1 ml culture medium 16 h before transfection. The following day, 1,000 ng of pTurbo-OnSARM1-GFP plasmids in serum-free medium was mixed with TurboFect Transfection Reagent (Thermo Fisher Scientific) and incubated for 20 min at room temperature following the manufacturer’s instructions before being added to the cells. Forty-eight hours after transfection, the cells were washed thrice with 500 μl of Hank’s Balanced Salt Solution (HBSS; Thermo Fisher Scientific) and fixed with 500 μl of 4% paraformaldehyde (PFA) at room temperature for 15 min. Subsequently, cells were washed thrice with HBSS and incubated for 30 min with HBSS containing Alexa Fluor 546 phalloidin (Thermo Fisher Scientific) in a darkened room. After cells were washed thrice with HBSS again, cells fixed on the coverslip were stained with VECTASHIELD Antifade Mounting Medium containing 4’, 6-diamidino-2-phenylindole (DAPI; Vector Laboratorie, US) on a microscope slide, and the coverslip was sealed with nail polish. Finally, the sealed coverslips were observed using a confocal microscope (Zeiss LSM 880 with Airyscan, Germany).

For the colocalization studies, THK cells (4 × 10⁴ cells per well) were seeded onto glass coverslips in 24-well culture plates, as mentioned above; this was followed by co-transfection with OnSARM1-3×FLAG and HA-tagged OnTRIF, OnTIRAP (27) or OnMyD88 (21) expression plasmids, respectively. The cells were fixed 48 h after transfection; this was followed by permeabilizing with PBS containing 1% (v/v) triton-X100 at room temperature for 5 min and blocking with PBS containing 10% bovine serum albumin (BSA) for 30 min at 37°C. Subsequently, the cells were incubated with PBS containing 3% BSA and monoclonal anti-FLAG M2 mouse antibodies (1:200, Sigma-Aldrich) or anti-HA rabbit antibodies (1:200, clone C29F4, Cell Signaling) for 2 h at 37°C. Alexa Flour™ 488 goat anti-mouse IgG (1:1000, Invitrogen) and Alexa Flour™ 568 goat Anti-rabbit IgG (1:1000, Invitrogen) were used in dark to visualize FLAG tagged (green) or HA tagged proteins (red), respectively. The cells were then stained with VECTASHIELD Antifade Mounting Medium containing DAPI to visualize the nucleus (blue). The images were captured using confocal microscopy (Zeiss LSM 880 with Airyscan). The colocalization efficiency was quantified by observing the overlap of the fluorescence intensity peaks along the white line by line scan analysis profiles (ZEISS ZEN 2.6 (blue edition) software).

Western blotting was conducted according to procedures described in previous research (28). HEK 293 cells were seeded in 24-well plates at a density of 1 × 10⁵ cells per well the day before transfection. The TurboFect Transfection Reagent was used to transfect cells with the indicated plasmids or left untreated as a negative control. Plasmids encoding Nile tilapia MyD88, TRIF, and TIRAP were prepared previously (21, 27). Cell lysates from transfected cells were separated by gradient SDS-PAGE (Bionovas, Canada) before being transferred to a PVDF membrane (Millipore, US). The membrane was immunoblotted with the anti-TurboGFP (d) antibody (1:1000) (Evrogen) or ANTI-FLAG M2 antibody (1:1000) (Sigma-Aldrich). The membrane was then incubated with peroxidase-conjugated AffiniPure goat anti-rabbit or goat anti-mouse IgG (H+L) antibodies (1:5000, Jackson ImmunoResearch Laboratories, US). LuminolPen (Visual Protein) was used to mark the pre-stained molecular weight standard markers, and a SuperLight Chemiluminescent HRP Kit (Bionovas) was used for protein detection. The membrane was visualized using the UVP GelStudio PLUS Touch Imaging System (Analytik Jena, Germany).

To investigate the luciferase activity induced by OnMyD88, OnTRIF, OnTIRAP, and OnSARM1 expressing plasmids, THK cells (5 × 10⁴ cells per well) were seeded in 48-well culture plates with 500 μl of culture medium 16 h before transfection. The cells were then transfected with 250 ng of each expression plasmid or a corresponding empty vector (pcDNA3.0-HA for OnMyD88, OnTRIF, and OnTIRAP and p3×FLAG-CMV-14 vector for OnSARM1), with 250 ng of pNF-κB-Luc (Clontech). Forty-eight hours post transfection, luciferase activity was determined using the luciferase reporter assay system (GeneCopoeia, US) according to the manufacturer’s instructions. Each experiment was performed in triplicate.

To verify the influence of OnSARM1 on the NF-κB activity mediated by OnMyD88, OnTRIF, and OnTIRAP, THK cells were transfected with 125 ng of OnMyD88, OnTRIF, or OnTIRAP plasmids with or without 125 ng of OnSARM1-3×FLAG expression plasmids, with 250 ng of pNF-κB-Luc. Luciferase activity was determined 48 h post transfection.

To study the effects of overexpressing OnSARM1 during antibacterial responses, THK cells were transfected with a pNF-κB-Luc reporter (250 ng) with empty vector or OnSARM1-3×FLAG expression plasmids (250 ng). Forty-eight hours post transfection, the cells were treated with lysates from A. hydrophila (10 μg protein/ml culture medium) and S. agalactiae (10 μg protein/ml culture medium) for 16 h, followed by luciferase assay analysis.

THK cells (2.5 × 10⁵ cells per well in 12 well plate) were seeded the night before transfection. The next day, the cells were transfected with an empty vector (2000 ng per well) or OnSARM1-3×FLAG expression plasmids mixed with an empty vector (1000 ng each). The cells were then stimulated for 4 h with lysates from A. hydrophila (10 μg protein/ml culture medium) 48 h post transfection; this was followed by RNA extraction, cDNA synthesis and gene expression analysis, as described above.

SPSS 25 software (IBM, US) was used to analyze all quantitative real-time PCR data. The expression levels of the OnSARM1 were first normalized to the housekeeping gene EF-1α, and log₂ transformed (29). An unpaired-sample t-test was performed to analyze the significant differences between the treatment and control groups in gene expression levels at each time point. One-way ANOVA (analysis of variance) and Tukey’s test were conducted to analyze the differences in each group in the luciferase assay. P-value < 0.05 was considered significantly different.

The complete coding sequence (CDS) of OnSARM1 was cloned, and the nucleotide and deduced amino acid sequences of OnSARM1 are shown in Supplementary Figure 1. The cloned OnSARM1 cDNA sequence was 2206 bp in length, including a partial 34 bp of 5′-UTR and a partial 36 bp of 3′-UTR. The open reading frame (ORF) of OnSARM1 was 2136 bp, encoding for 711 amino acids.

To investigate the evolutionary relationships of OnSARM1, a phylogenetic tree consisting of 33 SARM1 amino acid sequences from selected vertebrate species was constructed (Figure 1; accession numbers are in Supplementary Table 2). The putative SARM1 of Nile tilapia clustered with other fishes, forming a clade separate from the clades of avian, amphibian, and mammalian sequences. OnSARM1 was closer to flier cichlid, Astatotilapia burtoni, yellow perch, eastern river bream, and zebra mbuna, which all fall within the Actinopterygii class.

Figure 2 shows the genomic structure of SARM1s from Nile tilapia and other selected vertebrates. Genomic structure analysis revealed that except for Ctenopharyngodon idella and Homo sapiens, all of the selected teleost SARM1 shared the same structure, which consisted of eight exons and seven introns. Ctenopharyngodon idella SARM1(CiSARM1) consisted of seven exons and six introns, while Homo sapiens SARM1 consisted of nine exons and eight introns (Figures 2B, F).

Protein identities and similarities of the SARM1s were determined among selected vertebrates (Supplementary Table 3). The results showed that OnSARM1 shared high homology with other vertebrate SARM1s (56.9–91.7% identity and 70.6–95.9% similarity), with the highest homology to the SARM1 from Lates calcarifer (91.7% identity and 95.9% similarity) and the lowest homology to the SARM1 from Mus musculus (56.9% identity and 70.6% similarity).

The characterized domains of OnSARM1 are shown in Figure 3. OnSARM1, similar to other SARM1s, contains two N-terminal armadillo/beta-catenin-like repeats (ARM) domains, two central sterile alpha motifs (SAM), and a C-terminal TIR domain.

Figure 4 presents a comparison of the genome segments containing SARM1 from various fishes and humans. The genes flanking SARM1 were conserved among vertebrates analyzed. Specifically, the gene block containing OnSARM1 was similar to that of Danio rerio, with SLC46A1 (Solute Carrier Family 46 Member 1) and FOXN1 (Forkhead box protein N1) located downstream, and VTNA (vitronectin a), SCARF1 (Scavenger Receptor Class F Member 1), RILP (Rab-interacting lysosomal protein), and PRPF8 (Pre-mRNA-processing-splicing factor 8) located upstream of SARM1.

The gene expression of OnSARM1 in the tissues from healthy Nile tilapia is shown in Figure 5. The expression of OnSARM1 was highly expressed in the trunk kidney (64.2-fold) followed by the muscle (54.1-fold), when compared with the liver that exhibited the lowest expression (set as 1), and it was expressed moderately in the intestine, spleen, gills, skin, and head kidney.

An expression plasmid (pTurbo-OnSARM1-GFP), which encoded for OnSARM1 tagged with GFP at the C-terminus of the TIR domain, was constructed to study the cellular location of OnSARM1. Successful expression of the fusion protein was confirmed by the Western blotting of lysates from pTurbo-OnSARM1-GFP transfected HEK 293 and THK cells (Supplementary Figure 2). The molecular weight of the OnSARM1-GFP fusion protein was approximately 106 kDa (26 kDa Turbo-GFP plus 80 kDa for SARM1), while the pTurbo-GFP vector transfected cells produced a 26 kDa GFP protein. OnSARM1-GFP fusion protein was detected only in the cytoplasm of HEK 293 and THK cells (Figure 6).

The expression profiles of OnSARM1, OnMyD88, OnTIRAP, OnTRIF, IL-1β, IL-8, IL-12b, IFNd2.8 and IFNd2.13 were determined in the head kidney after a challenge with Gram-positive bacteria (S. agalactiae) (Figure 7). The expression levels of OnSARM1 (Figure 7A) and IFNd2.8 (Figure 7H) were not significantly altered at any of the time points examined, while the transcript levels of OnTIRAP (Figure 7C) and OnTRIF (Figure 7D) increased significantly in the head kidney at 3 and 4 time points after being challenged, and OnMyD88 was upregulated at 24 h (Figure 7B). Significant upregulation of IL-1β (Figure 7E) and IL-8 (Figure 7F) was observed at all of the time points examined post S. agalactiae infection. Elevated gene expression of IL-12b and IFNd2.13 was noticed at 8 h and 72 h, and 4 h and 24 h, respectively in the head kidney tissue from the S. agalactiae challenged fish.

In the fish challenged with Gram-negative bacteria (A. hydrophila), the transcript levels of OnSARM1 (Figure 8A) were promoted at 24 h and 72 h, while OnMyD88 was downregulated in the head kidney after the challenge injection (Figure 7B). The expressions of OnTIRAP (Figure 8C) and OnTRIF (Figure 8D) were upregulated at 4 and 3 time points post A. hydrophila infection. Similarly, the expression of IL-1β (Figure 8E) and IL-8 (Figure 8F) reached the highest level at 8 h, and decreased gradually at later time points. The expression of IL-12b (Figure 8G) and IFNd2.8 (Figure 8H) remained almost at the basal level, but IFNd2.13 (Figure 8I) was upregulated at 4 h and 8 h in the head kidney post A. hydrophila infection.

To further study the regulatory role of OnSARM1 in immune cells, we adopted a cell line derived from tilapia head kidney with characteristics of melanomacrophages (26). Enhanced NF-κB promoter activity was seen in the empty vector transfected THK cells after treatment of the lysates from A. hydrophila (8.17 ± 2.05 fold) and S. agalactiae (2.42 ± 0.16 fold). Interestingly, A. hydrophila-mediated induction of NF-κB promoter activity was significantly suppressed in the OnSARM1 overexpressed THK cells (1.93 ± 0.21 fold) (Figure 9A). Although insignificant, this inhibitory effect was also seen in the S. agalactiae treated THK cells that overexpressed OnSARM1 (1.99 ± 0.31 fold).

In order to investigate the interaction among OnSARM1 and other TLR adaptors, the corresponding expressing plasmids were transfected into THK cells in combination with NF-κB reporter plasmids. As shown in Figure 9B, overexpression of OnMyD88 and OnTIRAP alone, but not OnTRIF and OnSARM1, activated NF-κB activity in THK cells, but the co-expression of OnSARM1 with three other adaptors, namely OnMyD88, OnTRIF and OnTIRAP, impaired the activation of the NF-κB reporter (Figures 9C–E).

Studies have shown that SARM1 could physically interact with other TLR adaptors by using co-immunoprecipitation and Western blotting (20), but whether OnSARM1 could co-localize with other TLR adaptors still needs to be determined. To this end, THK cells were transfected with OnSARM1-3×FLAG together with HA-tagged OnTRIF, OnTIRAP or OnMyD88 expression plasmids respectively, and the cells were stained with corresponding antibodies 48 h post-transfection. As shown in Figure 10, OnSARM1 were detected not only scattered in the cytoplasmic region but also formed in discrete foci in the cytosolic region that were found to be partially co-localized with OnTRIF (Figure 10A) or OnMyD88 (Figure 10C), which are present as small speckle-like condensed granules in plasmid transfected THK cells. OnTIRAP was found to be more evenly distributed in cytosol and co-localized with OnSARM1 (Figure 10B). Colocalization of OnSARM1 with the three TLR adaptors was visible as yellow coloring in the merged images and was further confirmed by the correspondence of the peak fluorescence intensities of the indicated proteins in the line scan graphs (Figure 10, bottom row).

We next examined the effect of OnSARM1 on anti-A. hydrophila dependent gene induction in more detail by comparing the mRNA expression level of immune genes in empty vector or OnSARM1 expressing plasmid transfected THK cells after stimulation with lysate from A. hydrophila. As shown in Figure 11, expression of MyD88, TRIF and TIRAP (TLR adaptors), tilapia piscidin 2 (TP2) and TP3 (antimicrobial peptide), major histocompatibility complex class Ia (MHC Ia), interferon (IFN)d2.8 and IFNd2.13, and IL-10 remained at a similar level compared to the empty vector transfected group. In contrast, hepcidin (antimicrobial peptide) was upregulated in the empty vector group to nearly 14-fold (13.94 ± 3.89 fold) after stimulation with lysate from A. hydrophila, but was suppressed in the cells overexpressed OnSARM1 (3.05 ± 3.15 fold) (Figure 11E). The suppressive effects were further exemplified by evidence of the downregulation of proinflammatory cytokine genes such as IL-1β (8.64 ± 2.56 fold versus 3.34 ± 0.84 fold), IL-8 (6.60 ± 0.80 fold versus 4.64 ± 0.47 fold), TNF-α (9.88 ± 2.81 fold versus 5.04 ± 1.56 fold), IL-12a (1.25 ± 0.11 fold versus 0.82 ± 0.19 fold), IL-12b (6.85 ± 1.19 fold versus 3.03 ± 0.96 fold) between the vector or OnSARM1 plasmid transfected THK cells after stimulation (Figure 11).