FIGURE 3.

Kinetics of the immune response in trout pharynx following I. multifiliis parasites infection. (A) Heat map demonstrated results from qPCR of mRNAs for selected immune markers in I. multifiliis–infected fish versus control fish measured on days 0.5, 1, 4, 7, 14, 21, 28, and 75 postinfection in pharynx of rainbow trout (n = 9). Color value, log₂ (fold change). (B) Venn diagrams of RNA-Seq experiment showed the overlap of genes upregulated or downregulated in the pharynx of rainbow trout at 14 or 28 d postinfection with I. multifiliis versus control fish (n = 9 per group). (C and D) Pie charts showed the percentages of immune and nonimmune genes upregulated or downregulated in the pharynx of rainbow trout at 14 (C) and 28 d (D) postinfection with I. multifiliis versus control fish by RNA-Seq studies. (E and F) Biological processes that were significantly altered of rainbow trout 14 (E) and 28 d (F) postinfection with I. multifiliis versus control fish revealed by RNA-Seq studies. Fold change differences between control and I. multifiliis–infected samples were calculated using cutoff of 2-fold. Significant differential expression at each time point (14 and 28 d) was established by unpaired t tests (p < 0.05). (G and H) Representative innate (G) and adaptive (H) immune genes modulated by I. multifiliis infection on day 14 or 28. (I) Confirmation of RNA-Seq studies by qPCR of 14 and 28 d infection with I. multifiliis. Circle, day 14 after infected with I. multifiliis; triangle, day 28 after infected with I. multifiliis; rectangle with colors, different colors represent different genes. The selected genes were the following: IgT, IgM, pIgR, Complement C1R (C1R), IL-8, IL-1β, integrin β-1 (ITGB1), MHC (MHC class II), CD22, CD3Z, C-type Lectin Domain Family 4 Member E (CLC4E), asporin (ASPN), and TLR-8.