Fig 1.
Workflow associated with seagrass (A) constitutive immunity assessment (Experiment 1) and (B) induced immunity assessment (Experiment 2) described in this study.
Table 1.
Labyrinthula spp. isolates used to test the effectiveness of the qPCR assay.
Twelve (12) putatively seagrass-pathogenic (“P”) and 27 seagrass non-pathogenic (“N”) Labyrinthula spp. isolates of various origins used to test the effectiveness of the qPCR assay. Isolate host/substrate code: ZM = Zostera marina; ZP = Zostera pacifica; PT = Phylospadix torreyi; PO = Posidonia oceanica; CN = Cymodocea nodosa; SI = Syringodium filiforme; HW = Halodule wrightii; TT = Thalassia testudinum; PS = Phylospadix scouleri; ML = detrital mangrove leaves; Px = Phylospadix sp.
Fig 2.
Immune responses in T. testudinum following abiotic stress.
Immune biomarker activity levels (A-D: peroxidase, exochitinase, polyphenol oxidase, and lysozyme, respectively) of T. testudinum individuals from Experiment 1 are shown under various treatments (control/ambient, hypersalinity, hyposalinity, or elevated temperature). Error bars reflect ±1 SEM. Letters represent significant differences in mean activity levels among treatment groups as determined by ANOVA (see S1 Table for details), followed by multiple comparisons LSD post hoc tests (two-tailed, α = 0.05; see S2 Table for details). Sample size n = 5, with the following exceptions: hypersalinity and elevated temperature groups, n = 4 each.
Fig 3.
The number of Labyrinthula sp. “E” isolate 8b cells μL-1 (log2) across a cell dilution series is plotted against threshold (CT) values to calculate extraction efficiency. Each sample was run in triplicate. Error bars represent ±1 SEM.
Fig 4.
Relationship between Labyrinthula sp. “E” isolate 8b cell count values obtained through qPCR and percent leaf area coverage of ‘induced lesions’ as determined through Wasting Index methods.
AICc provided the final logistic model choice (R2 = 0.68). See S3 Table for raw data.
Fig 5.
Immune response in T. testudinum following abiotic stress and Labyrinthula sp. infection.
Immune biomarker activity levels (parts A-D: peroxidase, exochitinase, polyphenol oxidase, and lysozyme, respectively) of T. testudinum individuals from Experiment 2 are shown under various treatments [control/ambient (“-”) Laby.; ambient (“+”) Laby.; hypersalinity (“+”) Laby.; hyposalinity (“+”) Laby.; elevated temperature (“+”) Laby.]. Error bars reflect ±1 SEM. Different letters represent significant differences in mean activity levels among treatment groups as determined by ANOVA (see S4 Table for details), followed by multiple comparisons LSD post hoc tests (two- tailed, α = 0.05; see S5 Table for details). Sample size n = 5, with one exception: EXOC, hyposalinity treatment, n = 4.
Fig 6.
Infection responses in T. testudinum.
Results of qPCR assays measuring pathogen (number of Labyrinthula sp. “E” isolate 8b cells mg tissue -1) in T. testudinum individuals from Experiment 2, under control [no Labyrinthula sp., or (“-”) Laby.] and pathogen-exposed [ambient (“+”) Laby.; hypersalinity (“+”) Laby.; hyposalinity (“+”) Laby.; elevated temperature (“+”) Laby.] conditions. Error bars reflect ±1 SEM. Different letters represent significant differences in mean activity levels among treatment groups as determined by ANOVA, followed by multiple comparisons LSD post hoc tests (two- tailed, α = 0.05). Sample size n = 5, with one exception: Ambient (“-”) Laby. treatment, n = 4.
Fig 7.
Results of correlation analyses between pathogen load (number of Labyrinthula sp. “E” isolate 8b cells mg tissue-1) and immune biomarker activity (parts A-D: Peroxidase, exochitinase, polyphenol oxidase, and lysozyme, respectively) in T. testudinum individuals from Experiment 2.
Significance (p-values) are based on Pearson product-moment correlation coefficients (A-C) or Spearman’s rho correlation coefficients (D). Sample size in parts A, C and D = 24; sample size in part B = 23.
Fig 8.
Association of immune responses and pathogen loading in T. testudinum.
The heat map displays relative values of the five parameters measured (POX, EXOC, PPO, and LYS activity, and pathogen load) in T. testudinum individuals in Experiment 2 [subjected to control/ambient (“–”) Labyrinthula sp., ambient (“+”) Labyrinthula sp., hypersalinity (“+”) Labyrinthula sp., hyposalinity (“+”) Labyrinthula sp., or elevated temperature (“+”) Labyrinthula sp., shaded white, gray, light blue, blue, and dark blue, respectively]. Each column represents the relative pathogen load (Labyrinthula sp. “E” isolate 8b) or activity at each of the four biomarkers. In addition, each row represents one individual from the second stress-response experiment (in ascending order of pathogen loading, from top to bottom). The shade assigned to each individual at a given parameter reflects the Z-score of proportional activity at that parameter scaled by column, with blue, white, and red representing Z-scores of -4, 0, and 4, respectively.