Structural characteristics of Igldcp and orthology analysis. A) Domain composition of Igldcp proteins from different fish species determined with SMART software. B) Schematic representation of the spatial organization of zebrafish Igldcp. C) Predicted 3D structure of zebrafish Igldcp using the I-TASSER server. The model with the best C-score was selected. D) Fish-enriched chordate orthology analysis of Igldcp constructed with OrthoFinder. The igldcp genes, represented in purple, were found to be absent outside the subclass Neopterygii. Salmonids and cyprinids contained two copies of the igldcp gene, except for zebrafish, which contained only one copy. The presence of two copies in salmonids and cyprinid species may be due to the additional genome duplication events in these fish families. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Synteny conservation analysis of the zebrafish igldcp gene and phylogenetic analysis. A) Synteny analysis of igldcp (lgals17) in some fish species with the gene properly identified in the genome. B) Phylogenetic tree constructed with proteins annotated as Lgals17 (Igldcp) in the NCBI database and different B7 family members that showed greater similarity to Igldcp in mammalian species. The tree was rooted with a canonical galectin domain (CDR) from D. rerio and constructed with iTOL v6.

Expression patterns of the zebrafish igldcp gene. Basal expression of igldcp in zebrafish A) during ontogeny and B) in different tissues from healthy adult zebrafish. C) Modulation of igldcp expression in zebrafish larvae (3 dpf) infected with SVCV, IPNV or RGNNV at 24, 48 and 72 hpi. D) Induction of igldcp and isg15 expression in zebrafish larvae at 72 h after the microinjection of the expression plasmids pcDNA3.1-ifnphi1, pcDNA3.1-ifnphi2 and pcDNA3.1-ifnphi3. The expression level of igldcp was normalized to the expression of the 18S rRNA gene. The fold change in expression was calculated by standardizing the normalized data to the control group. The graphs present the means ± SEMs of 3–5 independent biological replicates. Statistically significant differences are displayed as follows: ∗∗∗ (0.0001 < p < 0.001), ∗∗ (0.001 < p < 0.01) or ∗ (0.01 < p < 0.05).

In vitro and in vivo evaluations of the antiviral activity of Igldcp. A) qPCR detection of SVCV, IPNV and RGNNV in the ZF4, RTG-2 and SSN-1 cell lines, respectively, transfected with the expression plasmid pcDNA3.1-igldcp or the corresponding empty control plasmid. The expression levels of the target viral genes were normalized to the expression of the 18S rRNA gene corresponding to the origin of the cell line. B) Effect of the overexpression of igldcp on zebrafish larvae. Zebrafish embryos were microinjected with the plasmid pcDNA3.1-igldcp or pcDNA3.1, and at 3 dpf, the larvae were infected or not infected with RGNNV. The expression of igldcp in the different groups was evaluated by qPCR at 24 hpi and normalized to the expression of the 18S rRNA gene. The survival rate after the challenge was determined over a period of 11 days and is presented in Kaplan‒Meier survival curves. C) Effect of igldcp knockdown on zebrafish larvae. Zebrafish embryos were microinjected with Mo-igldcp or Mo-C, and at 3 dpf, the larvae were infected or not infected with RGNNV. The replication of RGNNV was determined 24 hpi by qPCR detection of the capsid protein (CP)-encoding gene, which was normalized to the expression of the zebrafish 18S rRNA gene. The survival rate after the challenge was determined over a period of 11 days and is presented in Kaplan‒Meier survival curves. The qPCR data are presented as the means ± SEMs of 3–5 independent biological replicates. Statistically significant differences are displayed as follows: ∗∗∗ (0.0001 < p < 0.001), ∗∗ (0.001 < p < 0.01) or ∗ (0.01 < p < 0.05).

Overall analysis of the transcriptome of zebrafish larvae inoculated with pcDNA3.1-igldcp or the empty control plasmid pcDNA3.1 in the absence or presence of RGNNV infection. A) Stacked column chart representing the number and intensity (in log2 of the fold-change) of the DEGs in different comparisons. (B) Principal component analysis (PCA) plot constructed with the TMP values of the entire repertoire of zebrafish genes. C) Venn diagram constructed with the DEGs obtained for the comparisons of pcDNA3.1-igldcp vs. pcDNA3.1 and pcDNA3.1 RGNNV vs. pcDNA3.1, revealing the common and exclusive genes affected by Igldcp overexpression and RGNNV infection. D) Venn diagram constructed with the DEGs obtained for the comparisons pcDNA3.1-igldcp vs. pcDNA3.1 and pcDNA3.1-igldcp RGNNV vs. pcDNA3.1 RGNNV, which allows the identification of DEGs modulated by Igldcp both in the absence and presence of infection and DEGs modulated by Igldcp only in the absence of infection or only in the presence of infection. E) Venn diagram constructed with the DEGs obtained for the comparisons pcDNA3.1-igldcp RGNNV vs. pcDNA3.1 and pcDNA3.1- RGNNV vs. pcDNA3.1, showing the number of common and exclusive DEGs modulated by infection in both groups of larvae (overexpressing or not overexpressing igldcp).

Igldcp modulates a variety of processes, including the immune response. A) GO enrichment analyses of the DEGs between zebrafish larvae (4 dpf) inoculated with pcDNA3.1-igldcp or pcDNA3.1 plasmid at the one-cell stage embryo. Different biological process terms directly involved in the immune response were observed. B) Heatmap representing the TPM values of the immunity-related genes induced by Igldcp in zebrafish larvae.

Heatmaps representing the expression of genes involved in different processes modulated by Igldcp in zebrafish larvae and the modulation of caspase a activity. The heatmaps were constructed with the TPM values of the DEGs A) with homology to NACHT, LRR and PYD domain-containing proteins (NLRP3/12-like), B) annotated as zinc-finger proteins, C) involved in cell‒cell adhesion and the extracellular matrix, and D) with a role in the cell cycle/chromatin/cytoskeleton. E) Measurement of caspase a activity in zebrafish larvae at 3 dpf after the injection of pcDNA3.1-igldcp or pcDNA3.1 at the one-cell embryo stage and incubated in the absence or presence of the caspase-1-specific inhibitor Ac-YVAD-CHO. The graphs represent the means ± SEMs of 5 independent biological replicates. Statistically significant differences are displayed as ∗ (0.01 < p < 0.05).

Igldcp modulates the expression of immune-related genes that are also affected by RGNNV infection. Representation of the TPM values of immune-related genes that are commonly differentially expressed between pcDNA3.1-igldcp vs. pcDNA3.1 and pcDNA3.1 RGNNV vs. pcDNA3.1. The graphs present the means ± SEMs of the biological replicates for the four experimental groups.

Igldcp modulates the expression of genes that are also affected by RGNNV infection and encode A) NLRP3/12-like proteins, B) zinc-finger proteins, C) cell‒cell adhesion or extracellular matrix proteins, and D) cell cycle/chromatin/cytoskeleton-related proteins. Representation of the TPM values of genes that are commonly differentially expressed between pcDNA3.1-igldcp vs. pcDNA3.1 and pcDNA3.1 RGNNV vs. pcDNA3.1. The graphs present the means ± SEMs of the biological replicates for the four experimental groups.

The expression profile of immune-related genes not directly modulated by Igldcp but differentially expressed after RGNNV challenge between larvae previously inoculated with pcDNA3.1-igldcp or pcDNA3.1. Heatmap presenting the mean TPM values of the immunity-related genes in the different experimental groups.

Expression profile of genes involved in different metabolic processes not directly modulated by Igldcp but differentially expressed after RGNNV challenge between larvae previously inoculated with pcDNA3.1-igldcp or pcDNA3.1. Heatmaps presenting the mean TPM values of the genes involved in A) the kynurenine pathway, B) retinol metabolism and C) carbohydrate and lipid metabolism in the different experimental groups.