Immune system alterations in naxd−/− larvae. (A) Neutral red stain (5 μg/mL for 24 h) of WT and naxd−/− larvae at 7 dpf. Stained (microglia/phagocytic) cells were quantified manually with ImageJ. The area used for phagocytic cell number determination is indicated by a dotted line. The scale bars represent 100 μm. Data are shown as individual values, each dot representing one larva (n = 9–10 per genotype); horizontal lines represent the means. (B, C) mRNA expression levels of microglia/macrophage markers and cytokines in 7 and 10 dpf naxd−/− larvae relative to WT siblings, based on qPCR analysis on RNA extracted from whole larvae. actb1 was used as a reference gene. Data are means ± SDs from four to five biological replicates, each replicate consisting of a batch of 30 or 10 larvae at 7 and 10 dpf, respectively. For panels (A–C), statistical significance was determined using unpaired t‐tests (*p ≤ 0.05, **p ≤ 0.01, and ****p ≤ 0.0001). (D) Volcano plot highlighting DEGs (naxd−/− vs. WT) with FC ≥ 1 and Q value ≤ 0.05. Significantly upregulated and downregulated genes are shown as red and blue dots, respectively; gray dots represent genes with nonsignificantly changed expression. (E) Rich factor plot of the GOp enrichment analysis performed on the DEGs. The genes in the blue box correspond to significantly downregulated genes assigned to the viral response pathway, and the gene in the pink box corresponds to a significantly upregulated gene in the same pathway. The mxb and mxc genes are also attributed to the seven subsequent pathways listed on the y‐axis.
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