Mucosal tissue specificity of PAMP-related PRR gene expression in Scophthalmus maximus under different modes of Vibrio anguillarum stimulation.

Abstract

To investigate the immune responses of pathogen-associated molecular patterns (PAMPs) recognition-receptor genes in Scophthalmus maximus stimulated by the Gram-negative bacterium live/inactivated Vibrio anguillarum, and lipopolysaccharide (LPS), skin, gill, and intestinal tissues of fish at 0-96 h post-invasion were collected. mRNA was extracted from these mucosal tissues, and the gene expression of the following 7 PAMP recognition receptors was quantitatively analyzed using real-time fluorescence quantitative PCR: LEC, SAP, TLR2, TLR5, TLR14, PGRP2, and LIMP-2. Expression of SmTLR2, SmLEC, SmSAP, and SmPGRP2 were ubiquitously induced in mucosal tissues through invasion by V. anguillarum and related bacterial components. Under V. anguillarum stimulation, SmTLR2, SmTLR14, SmSAP, and SmPGRP2 exhibited the fastest expression in the skin. Under LPS stimulation, SmLEC showed the most rapid upregulation in the gills. Under inactivated V. anguillarum stimulation, the earliest expression of SmLIMP-2 occurred in the intestine. Peak expression under live/inactivated V. anguillarum, and LPS stimulation occurred at 24-48 h, 12-96 h, and 12-96 h post-immunization, reaching values that were 198.91-1.49-fold, 2.3-202.46-fold, and 2.27-96.10-fold higher than those of the control group, respectively. These results demonstrated that pattern recognition receptor (PRR) genes in turbot mucosal tissues were rapidly and abundantly expressed to mediate immune responses post-immunostimulation. We applied Genotype plus Genotype × Environment Interaction (GGE) biplot analysis to comprehensively evaluate immunostimulants and PRR genes regulation and then used the results to establish an optimal stimulation strategy for selective breeding against bacterial diseases. SmTLR14, SmPGRP2, SmLEC, and SmTLR2 were identified as optimal indicator genes in mucosal tissues following stimulation by live/inactivated V. anguillarum or LPS.