A core innate immune gene panel derived from germ-free larval zebrafish transcriptomics resolves strain-specific induction by Aeromonas veronii and Lacticaseibacillus rhamnosus GG.

The intestinal microbiota is critical for innate immune maturation in fish, yet candidate probiotics and commensals are often evaluated using narrow inflammatory readouts that may obscure strain-specific immune programs. Here, we developed a compact, mechanistically informative host-response panel to distinguish microbiota-dependent immune induction in germ-free (GF) zebrafish larvae. Whole-larva transcriptomes from GF and conventionally reared (CONR) zebrafish at 7 days post-fertilization (dpf) were analyzed to identify immune-related differentially expressed genes (DEGs) using KEGG and Gene Ontology biological process annotations. Candidates were prioritized by STRING-based protein-protein interaction analysis of human orthologs and four CytoHubba topological algorithms, then validated by qPCR and neutrophil imaging in GF, CONR, and GF larvae mono-associated with resident Aeromonas veronii (A. veronii) or probiotic Lacticaseibacillus rhamnosus (L. rhamnosus) GG. GF rearing altered the expression of 304 genes, including 201 downregulated genes, with enrichment centered on neutrophil chemotaxis, chemokine signaling, and humoral defense. Integrative functional and network filtering identified cxcl8a, cxcl12b, lyn, and mxc as convergent hubs; together with il1b and saa, these genes formed a six-gene panel. Compared with CONR larvae, GF larvae showed reduced panel expression and fewer neutrophils, with 12.0 versus 28.3 neutrophils per larva, respectively. A. veronii upregulated all six genes and increased neutrophil abundance to 38.3 per larva, exceeding the CONR level. In contrast, L. rhamnosus GG upregulated only il1b, saa, and lyn and partially restored neutrophil numbers to 17.8 per larva. This qPCR-compatible panel distinguishes strain-specific innate immune induction beyond single-cytokine assays and provides a practical framework for functional screening of commensal and probiotic candidates in fish models.