METHODOLOGY

T. testudinum Collection and Maintenance

Thalassia testudinum shoots were collected from Boca Ciega Bay and Lassing Park in Tampa, Florida (27.7493° N, 82.6300° W; 27.7927° N, 82.7657° W) on February 16th, 2024. Harvested seagrass ramets included the blades, sheath, and at least a 5 cm section of horizontal rhizome, and all plants collected appeared healthy through visual assessments, lacking indications of stress or lesions. The seagrass ramets were cleaned of epiphytes, transplanted within 24 hours of collection, and maintained in saltwater aquaria. Salinity was maintained at ~32, with diel temperatures of 26-28˚C, and light intensity of 110-112 μmol/m²/s, under a 12:12 hour L:D graduated photoperiod (Aqueon® OptiBright® LED Lights) and constant flow. These growing parameters have been previously described as ideal for T. testudinum maintenance in aquaria and reflect natural conditions of the collection site (Bishop et al., 2017). The substrate consisted of sediment collected from the harvest site, supplemented with aquaria live sand (CaribSea® Aragonite); terracotta pots and plastic garden planters were used to house the seagrass within the aquaria.

Labyrinthula sp. Culture and Infection Method

Cultures of Labyrinthula sp. “E” isolate 8B, a known pathogenic strain, were utilized for all infection trials. This strain has been in culture in our laboratory for over 18 years and its morphology and pathogenicity has been well documented (Bishop et al., 2017; Martin et al., 2016; Trevathan-Tackett et al., 2015). Labyrinthula sp. was maintained in serum-seawater agar (SSA) media with autoclaved T. testudinum blade clippings to encourage growth. The SSA media consisted of 500 mL of 0.22 μm filtered seawater, salinity of 25 from Instant Ocean Sea Salt, supplemented with 6 g agar, 0.05 g each of peptone and nutritional yeast, and 0.5 g dextrose, 5 mL horse serum, and 12.5 mL antibiotic solution containing 1.25% of both streptomycin and penicillin. All chemicals were sourced via Sigma-Aldrich (St. Louis, MO, USA).

Seagrass infection vectors, of 1 cm in length, were boiled or autoclaved to surface sterilize and placed onto agar growth plates. Once the Labyrinthula sp. growth completely encompassed the vector (typically 5-7 days), it was used in infection experiments. Infections proceeded with a healthy 4 cm, fresh weighed blade onto which an infection vector was carefully attached using a Tygon tubing clamp. The full length of the infection vector was placed in direct contact with the healthy blade to encourage Labyrinthula sp. transfer and infection. Sterile seagrass “mock vectors” of 1 cm in length were attached in the same manner to the healthy blades in all control samples. All samples were maintained in individual transparent 50 mL high clarity conical falcon tubes (Thermo Fisher Scientific™, Waltham, MA, USA), containing 45 mL of aquaria water, 26-28˚C, under a light intensity of 100 μmol/m²/s on a 12:12 L:D cycle (GLO® Linear Fluorescent Lighting System) and pulled for assessment at their respective timepoints of interest (24-hrs, 48-hrs, or 72-hrs). Our preliminary experiments showed that most of the physiological and biochemical changes occurred in the host within the first 72-hrs post infection. Thus, a 72-hr time limit was selected for the majority of experiments reported herein. Infected blades that did not require immediate processing were stored at -80˚C for later analysis.

Previously frozen time-course sample blades were placed in liquid nitrogen and ground to a powder using a FastPrep-24® Tissue Homogenizer (Irvine, CA, USA). At first, each sample was placed in a 2 mL lysing matrix tube containing two steel beads without buffer and homogenized at a speed of 6.0 m/s for 60 s. Then, 300 μL of 1x phosphate buffered saline solution (4 mM, pH 7.6) was added to each sample, and the homogenization step was repeated twice until the mixture exhibited a texture of fine paste. Samples were subsequently centrifuged at 17,000 x g for 10 min, and the resulting supernatants were used for hydrogen peroxide and capase-3 assays and normalized using a BCA protein assay kit (Thermo Fisher Scientific^TM) according to manufacturer’s instructions.

A colorimetric hydrogen peroxide assay was conducted on the seagrass Thalassia testudinum to assess in planta levels of a reactive oxygen species, indicating changes in host oxidative metabolism following Labyrinthula sp. infection. The Peroxide Assay Kit was utilized according to the manufacturer’s protocol (Sigma-Aldrich, 2019). and the absorbance at 585 nm was subsequently measured for both standards and samples. The assay uses the chromogenic Fe3+-xylenol orange reaction (FOX), in which a purple complex is formed when Fe2+ is oxidized to Fe3+ by peroxides present in the sample, generating a colorimetric (585 nm) result, proportional to the level of peroxide present (Sigma-Aldrich, 2019; from Peroxide Assay Kit (MAK311) Technical Bulletin).

Hydrogen peroxide (H₂O₂) standards (labeled A–H) were prepared at known micromolar concentrations of 0, 3, 6, 9, 12, 18, 24, and 30 μM using ultrapure water and 3% hydrogen peroxide standard. Fresh hydrogen peroxide standard was prepared on the day of the assay, following kit dilution instructions and using Sigma-Aldrich kit reagents to generate the final 30 μM H₂O₂standard. The labeling scheme for the time series unique sample blades, where n = 5 for each group, is as follows: 24-hr control (samples 1-5), 24-hr infected (samples 6-10), 48-hr control (samples 11-15), 48-hr infected (samples 16-20), 72-hr control (samples 21-25), and 72-hr infected (samples 26-30). Sample extracts were treated and put into a 96-well plate for analysis with a Biotek Synergy Plate Reader.

To determine protein content, a Thermo Scientific™ BCA Protein Assay Kit (Product No. 23225) was used according to manufacturer instructions. The kit outlines a dilution of bovine serum albumin (BSA), which generated a nine-point linear regression corresponding to standard protein concentrations of 0-2000 ug/mL. The plate was read at 562 nm absorbance, and the standard curve was utilized to determine the protein concentrations of all seagrass samples.