The in vitro anti-oomycetes activity of linalool against S. parasitica CQT2. A) Inhibition rate (IR) of EOs and their major components against S. parasitica CQT2. B) Determination of MIC of linalool against S. parasitica spores, employing malachite green (2.5 mg/mL) as a positive control (PC) and Tween 20 as a negative control (NC). C) Effects of linalool on mycelium growth inhibition over a 60h period at varying concentrations. D) Examination of the percentage inhibition of radial growth (PIRG %) in mycelium treated with linalool after 60 hours. E) Effects of linalool on the viability of S. parasitica CQT2 through a PI staining assay was conducted under 3 conditions: Control (no linalool), 1×MIC (0.05%) linalool treatment, and 2×MIC (0.1%) linalool treatment. F) Effects of different concentrations of linalool on mycelium growth on PDA plates.

Effects of linalool on the cell membrane integrity of S. parasitica CQT2. A) Observation of the effect of linalool at 6×MIC (0.3%) on the cell membrane of S. parasitica CQT2 using the FM4-64 and SYTO 9 staining assay. B-C) SEM images of S. parasitica CQT2 mycelium without (no linalool, B) and with 1×MIC (0.05%, C) linalool treatment. D-G) TEM images of S. parasitica CQT2 mycelium without (no linalool, D) and with 1×MIC (0.05%, E-G) linalool treatment. CM: cell membrane; CW: cell wall; ER: rough endoplasmic reticulum; MI: mitochondria; VA: vacuoles. H) The GO classification of differentially expressed genes (DEGs). I) 4 different functions classes (linked to ribosome function, involved in DNA/RNA function, connected to signal transduction and regulation, and pertaining to cell metabolism) of differentially expressed genes (DEGs) involved in intrinsic component of membrane.

The in vitro anti-oomycete mechanisms revealed by transcriptome analysis. A-B) The GO enrichment of up and down regulated DEGs. C) The KEGG enrichment of DEGs. Significant enrichment was labeled as “*”. P < 0.05, P < 0.01, and P < 0.001 were labeled as “*”, “**”, and “***”, respectively. D) Comparison of ribosome biogenesis in eukaryotes pathway between the linalool treated mycelium and the control group. E) Comparison of RNA polymerase pathway between the linalool treated mycelium and the control group. F) The tertiary structure of NOP1 was displayed using a cartoon representation. G) Molecular docking of linalool with NOP1 was performed by enlarging the regions binding to the NOP1 activation pocket to showcase the detailed amino acid structures, which were presented using a surface model, while the small molecule was displayed with a ball-and-stick representation. H) Comparison of ABC transporters pathway between the linalool treated mycelium and the control group. The red squares represented up-regulated genes, and the blue squares represented down-regulated genes. I) Visual analysis of metabolic pathways with iPath3.0. The figure represented gene set annotated pathways, red and green represented pathways annotated by genes in different gene sets, respectively, and blue represented pathways co-annotated by genes in two gene sets.

Protective effect of linalool on grass carp infected with S. parasitica. A) The experimental design. Grass carp were raised for 2 weeks without feeding, fish without infection and linalool (negative control group, NC), fish infected with S. parasitica (positive control group, PC), and 10 fish uninfected soaked water containing linalool for 2 days and then 1×106 spores /mL secondary zoospores were added (linalool prophylactic group, LP), and fish infected with S. parasitica soaked for 7 days in water containing linalool (linalool therapeutic group, LT). B) The symptoms of S. parasitica infection in grass carps of different groups. C) The survival rates of grass carp infected with S. parasitica of different groups. D-F) Alkaline phosphatase (AKP), lysozyme (LYZ), and superoxide dismutase (SOD) activities in serum of grass carp of different groups. The results were presented as the means ± SD and were analyzed using one-way ANOVA followed by post hoc tests (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). G) Histopathological analysis grass carp tissues in different groups. The arrows of different colors indicated: inflammatory cell infiltration in the kidney (blue arrow), cytoplasmic pyknosis (red arrow), nuclei displaced toward one side (yellow arrow), the red and white pulp was poorly demarcated, and a larger volume of melano-macrophage centers (black area), critical damage to the epithelium (green arrow) and myofiber (white arrow).

Global transcriptomic analysis after S. parasitica infection and linalool treatment in grass carp and in-depth analysis of crucial KEGG pathway and differentially expressed genes (DEGs). A-B) The GO enrichment of up and down regulated differentially expressed genes (DEGs) in linalool therapeutic (LT) and linalool prophylactic (LP) groups. C-D) The KEGG enrichment of up and down regulated differentially expressed genes (DEGs) in linalool therapeutic (LT) and linalool prophylactic (LP) groups. E) Complement and coagulation cascades pathway between S. parasitica infection and linalool treatment in the spleens of grass carp. F) 31 differentially expressed genes (DEGs) related to Toll-like receptor signaling pathway. G) 29 differentially expressed genes (DEGs) related to chemokine signaling pathway. The red squares represented up-regulated genes, and the blue squares represented down-regulated genes. H) Expression levels of cfh, masp2, c1r, c3, cfb, ap-1, il-1β, and il-6 in the spleen revealed variations among the PC, LP, and LT groups. For RT-qPCR, the results were presented as the means ± SD and were analyzed using independent t-tests (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

The effect of linalool on regulating gut microbiota of grass carp infected with S. parasitica and correlation analysis. A-D) The α diversity index comparison among prophylactic (LP), and linalool therapeutic (LT) groups. F) PCoA using Bray–Curtis distance revealed variations among the positive control (PC), linalool prophylactic (LP), and linalool therapeutic (LT) groups (ANOSIM R = 0.3086, P = 0.061). G) Relative abundance of the top 10 species in the gut from the different groups (phylum and genus levels). H) Column chart of LDA value distribution. Discriminative biomarkers identified by linear discriminant analysis effect size (LEfSe) with logarithmic LDA score greater than 3.0. I) Heat map of the differences in predicted functional metabolisms within gut bacterial KEGG pathways. J) The correlation layout of potential related immune genes, specific microbial species, and physical characteristics. Pairwise comparisons of characterizations are presented, with a color gradient and block size denoting Pearson’s correlation coefficients.

Model diagram of the mode of action of linalool on S. parasitica and grass carp.

In vitro, (1) linalool influenced DNA transcription, tRNA transport, rRNA processing and maturation (5.8S, 18S, and 25S), and the biogenesis and assembly of ribosome subunits (40S and 60S) in the cell, which might lead to the reduction of S. parasitica growth; (2) Linalool disrupted the cell membrane, and the upregulation of damage; (3) ABC transporters contributed to metabolic resistance by pumping linalool out of the cell. In vivo, (1) Linalool enhanced the complement and coagulation system which in turn activated host immune defense and lysate S. parasitica cells; (2) Linalool promoted wound healing, tissue repair, and phagocytosis to cope with S. parasitica infection; (3) Linalool positively modulated the immune response by increasing the abundance of beneficial Actinobacteriota; (4) Linalool stimulated the production of inflammatory cytokines (il-1β and il-6) and chemokines (ccl19 and ccl5) to lyse S. parasitica cells.

Primers for qPCR were used in this study.