Integrating host immune status, Labyrinthula spp. load and environmental stress in a seagrass pathosystem: Assessing immune markers and scope of a new qPCR primer set

Abstract

Recent trends suggest that marine disease outbreaks caused by opportunistic pathogens are increasing in frequency and severity. One such malady is seagrass wasting disease, caused by pathogens in the genus Labyrinthula. It is suspected that pathogenicity is intimately linked to the ability of the host to initiate defense responses; however, supportive evidence is lacking. To address this, we developed two techniques, including 1) a new qPCRbased pathogen detection method, and 2) an immune profiling panel via four host-biomarker assays (measuring peroxidase, exochitinase, polyphenol oxidase, and lysozyme activities). These techniques were then used to experimentally investigate the impact of environmental stressors (namely, elevated temperature and salinity) on host immunity and how immune status might affect susceptibility to Labyrinthula infection. In the first experiment, we subjected individual turtlegrass (Thalassia testudinum) shoots to short-term (7 d) abiotic stressors alone. In a second experiment, the same abiotic stressor conditions were followed by pathogen exposure (7 additional d), simulating a scenario where we attempt to isolate the impact of environmental stressors on the host seagrass species by removing the stressor as the pathogen is introduced. The qPCR assay successfully quantified the abundance of Labyrinthula spp. cells from both pure cultures and seagrass tissues across a broad range of predominately pathogenic strains, with high sensitivity. Immune enzyme assays revealed that all four biomarkers were constitutively active in turtlegrass individuals, but specific activities were largely unaffected by the chosen abiotic stressor conditions. We also identified positive correlations between pathogen load and two biomarkers (peroxidase, exochitinase), regardless of abiotic stress treatment, further demonstrating the potential utility of these biomarkers in future applications. OPEN ACCESSCitation: Duffin P, Martin DL, Pagenkopp Lohan KM, Ross C (2020) Integrating host immune status, Labyrinthula spp. load and environmental stress in a seagrass pathosystem: Assessing immune markers and scope of a new qPCR primer set. PLoS ONE 15(3): e0230108. https://doi.org/ 10.

Spatial Patterns of Thalassia testudinum Immune Status and Labyrinthula spp. Load Implicate Environmental Quality and History as Modulators of Defense Strategies and Wasting Disease in Florida Bay, United States

“…Our initial specificity test surveyed 37 Labyrinthula sp. isolates, reliably amplifying 100% of putatively pathogenic strains and only 15% of non-pathogenic strains (Duffin et al, 2020). In this current study, we were interested in providing more conclusive evidence that our protocol is indeed strongly biased toward pathogenic Labyrinthula spp.…”

“…Our group recently developed a new qPCR primer set in an effort to accurately quantify Labyrinthula spp. loading in seagrass tissue (Duffin et al, 2020). Notably, these primers were designed to target putatively pathogenic Labyrinthula spp.…”

“…cause wasting disease in the long-lived, climax seagrass species, Thalassia testudinum (turtlegrass; Robblee et al, 1991;Blakesley et al, 2002;Trevathan-Tackett et al, 2013). Recent manipulative work has shown that parasite virulence is a complex function of host competency, physiological stress, and immune status (Brakel et al, 2014(Brakel et al, , 2019Trevathan-Tackett et al, 2015;Bishop et al, 2017;Duffin et al, 2020), making the Thalassia-Labyrinthula pathosystem an attractive model for testing host-parasite interactions across a range of environmental scenarios. Using newly-developed quantitative PCR (qPCR) and immunoassay techniques (Duffin et al, 2020), we examined the relationship between the immune status of T. testudinum and Labyrinthula spp.…”

“…Recent manipulative work has shown that parasite virulence is a complex function of host competency, physiological stress, and immune status (Brakel et al, 2014(Brakel et al, , 2019Trevathan-Tackett et al, 2015;Bishop et al, 2017;Duffin et al, 2020), making the Thalassia-Labyrinthula pathosystem an attractive model for testing host-parasite interactions across a range of environmental scenarios. Using newly-developed quantitative PCR (qPCR) and immunoassay techniques (Duffin et al, 2020), we examined the relationship between the immune status of T. testudinum and Labyrinthula spp. loading along well-documented resource (i.e., phosphorus and sediment depth), morphological (i.e., leaf area and above-ground biomass), and stress (i.e., salinity regime) gradients in Florida Bay (FB), Florida, United States.…”

“…Labyrinthula infection is thought to be widespread in FB and likely factors into lost productivity and mortality that follow periodic reductions in light availability (e.g., sediment resuspension events and algal bloom) and other physiological challenges presented to FB seagrasses (e.g., Robblee et al, 1991;Carlson et al, 1994;Durako and Kuss, 1994;Blakesley et al, 2002;Hall et al, 2016). Such challenges allow opportunistic pathogens, such as Labyrinthula spp., to capitalize on compromised host immunity (Bishop et al, 2017;Duffin et al, 2020). Given current climate predictions of the region (e.g., increased temperatures, altered precipitation, more frequent hurricanes, and sea level rise; Pederson et al, 2012;Koch et al, 2015;Carlson et al, 2018), and the ongoing restoration of the greater Everglades ecosystem (i.e., the Comprehensive Everglades Restoration Plan, CERP; Everglades Forever Act, 1994;Florida Forever Act, 2000;Water Resources Development Act, 2000) aimed at bringing increased freshwater delivery to the upper portions of the FB, it is likely that Thalassia-Labyrinthula interactions will play an important role in the resilience of T. testudinumdominated meadows.…”