Human-induced salinity changes impact marine organisms and ecosystems.

Climate change is fundamentally altering marine and coastal ecosystems on a global scale. While the effects of ocean warming and acidification on ecology and ecosystem functions and services are being comprehensively researched, less attention is directed toward understanding the impacts of human-driven ocean salinity changes. The global water cycle operates through water fluxes expressed as precipitation, evaporation, and freshwater runoff from land. Changes to these in turn modulate ocean salinity and shape the marine and coastal environment by affecting ocean currents, stratification, oxygen saturation, and sea level rise. Besides the direct impact on ocean physical processes, salinity changes impact ocean biological functions with the ecophysiological consequences are being poorly understood. This is surprising as salinity changes may impact diversity, ecosystem and habitat structure loss, and community shifts including trophic cascades. Climate model future projections (of end of the century salinity changes) indicate magnitudes that lead to modification of open ocean plankton community structure and habitat suitability of coral reef communities. Such salinity changes are also capable of affecting the diversity and metabolic capacity of coastal microorganisms and impairing the photosynthetic capacity of (coastal and open ocean) phytoplankton, macroalgae, and seagrass, with downstream ramifications on global biogeochemical cycling. The scarcity of comprehensive salinity data in dynamic coastal regions warrants additional attention. Such datasets are crucial to quantify salinity-based ecosystem function relationships and project such changes that ultimately link into carbon sequestration and freshwater as well as food availability to human populations around the globe. It is critical to integrate vigorous high-quality salinity data with interacting key environmental parameters (e.g., temperature, nutrients, oxygen) for a comprehensive understanding of anthropogenically induced marine changes and its impact on human health and the global economy.

Diversity and microhabitat associations of Labyrinthula spp. in the Indian River Lagoon System.

Seagrasses create foundational habitats in coastal ecosystems. One contributing factor to their global decline is disease, primarily caused by parasites in the genus Labyrinthula. To explore the relationship between seagrass and Labyrinthula spp. diversity in coastal waters, we examined the diversity and microhabitat association of Labyrinthula spp. in 2 inlets on Florida's Atlantic Coast, the Indian River Lagoon (IRL) and Banana River. We used amplicon-based high throughput sequencing with 2 newly designed primers to amplify Labyrinthula spp. from 5 seagrass species, water, and sediments to determine their spatial distribution and microhabitat associations. The SSU primer set identified 12 Labyrinthula zero-radius operational taxonomic units (ZOTUs), corresponding to at least 8 putative species. The ITS1 primer set identified 2 ZOTUs, corresponding to at least 2 putative species. Based on our phylogenetic analyses, which include sequences from previous studies that assigned seagrass-related pathogenicity to Labyrinthula clades, all but one of the ZOTUs that we recovered with the SSU primers were from non-pathogenic species, while the 2 ZOTUs recovered with the ITS1 primers were from pathogenic species. Some of the ZOTUs were widespread across the sampling sites and microhabitats (e.g. SSU ZOTU_10), and most were present in more than one site. Our results demonstrate that targeted metabarcoding is a useful tool for examining the relationships between seagrass and Labyrinthula diversity in coastal waters.

Mosquito control pesticides and sea surface temperatures have differential effects on the survival and oxidative stress response of coral larvae.

The declining health of coral reefs is intensifying worldwide at an alarming rate due to the combined effects of land-based sources of pollution and climate change. Despite the persistent use of mosquito control pesticides in populated coastal areas, studies examining the survival and physiological impacts of early life-history stages of non-targeted marine organisms are limited. In order to better understand the combined effects of mosquito pesticides and rising sea surface temperatures, we exposed larvae from the coral Porites astreoides to selected concentrations of two major mosquito pesticide ingredients, naled and permethrin, and seawater elevated +3.5 °C. Following 18-20 h of exposure, larvae exposed to naled concentrations of 2.96 µg L(-1) or greater had significantly reduced survivorship compared to controls. These effects were not detected in the presence of permethrin or elevated temperature. Furthermore, larval settlement, post-settlement survival and zooxanthellae density were not impacted by any treatment. To evaluate the sub-lethal stress response of larvae, several oxidative stress endpoints were utilized. Biomarker responses to pesticide exposure were variable and contingent upon pesticide type as well as the specific biomarker being employed. In some cases, such as with protein carbonylation and catalase gene expression, the effects of naled exposure and temperature were interactive. In other cases pesticide exposure failed to induce any sub-lethal stress response. Overall, these results demonstrate that P. astreoides larvae have a moderate degree of resistance against short-term exposure to ecologically relevant concentrations of pesticides even in the presence of elevated temperature. In addition, this work highlights the importance of considering the complexity and differential responses encountered when examining the impacts of combined stressors that occur on varying spatial scales.

Corrigendum: 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.

[This corrects the article DOI: 10.3389/fpls.2021.612947.].

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.

Seagrass wasting disease, caused by protists of the genus Labyrinthula, is an important stressor of the dominant macrophyte in Florida Bay (FB), United States, Thalassia testudinum. FB exhibits countervailing gradients in plant morphology and resource availability. A synoptic picture of the Thalassia-Labyrinthula relationship was obtained by assessing the activity of four immune biomarkers in conjunction with pathogen prevalence and load [via quantitative PCR (qPCR)] at 15 sites across FB. We found downregulated immune status paired with moderate pathogen load among larger-bodied host phenotypes in western FB and upregulated immunity for smaller-bodied phenotypes in eastern FB. Among the highest immune response sites, a distinct inshore-offshore loading pattern was observed, where coastal basins exposed to freshwater runoff and riverine inputs had the highest pathogen loads, while adjacent offshore locations had the lowest. To explain this, we propose a simple, conceptual model that defines a framework for testable hypotheses based on recent advances in resistance-tolerance theory. We suggest that resource availability has the potential to drive not only plant size, but also tolerance to pathogen load by reducing investment in immunity. Where resources are more scarce, plants may adopt a resistance strategy, upregulating immunity; however, when physiologically challenged, this strategy appears to fail, resulting in high pathogen load. While evidence remains correlative, we argue that hyposalinity stress, at one or more temporal scales, may represent one of many potential drivers of disease dynamics in FB. Together, these data highlight the complexity of the wasting disease pathosystem and raise questions about how climate change and ongoing Everglades restoration might impact this foundational seagrass species.

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.

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 qPCR-based 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.

Exposure to the Florida red tide dinoflagellate, Karenia brevis, and its associated brevetoxins induces ecophysiological and proteomic alterations in Porites astreoides.

As reef-building corals are increasingly being exposed to persistent threats that operate on both regional and global scales, there is a pressing need to better understand the complex processes that diminish coral populations. This study investigated the impacts of the Florida red tide dinoflagellate Karenia brevis and associated brevetoxins on selected facets of coral biology using Porites astreoides as a model system. When provided with choice assays, P. astreoides larvae were shown to actively avoid seawater containing red tide (5×105 cells L-1-7.6×106 cells L-1) or purified brevetoxins (0.018 µg mL-1 brevetoxin-2 and 0.0018 µg mL-1 brevetoxin-3). However, forced exposure to similar treatments induced time-dependent physiological and behavioral changes that were captured by PAM fluorometry and settlement and survival assays, respectively. Adult fragments of P. astreoides exposed to red tide or associated brevetoxins displayed signs of proteomic alterations that were characterized by the use of an iTRAQ-based quantitative proteomic analysis. The novel use of this technique with P. astreoides demonstrated that protein regulation was highly contingent upon biological versus chemical treatment (i.e. live K. brevis vs. solely brevetoxin exposure) and that several broad pathways associated with cell stress were affected including redox homeostasis, protein folding, energy metabolism and reactive oxygen species production. The results herein provide new insight into the ecology, behavior and sublethal stress of reef-building corals in response to K. brevis exposure and underscore the importance of recognizing the potential of red tide to act as a regional stressor to these important foundation species.

Phylogenetic and chemical diversity of three chemotypes of bloom-forming lyngbya species (Cyanobacteria: Oscillatoriales) from reefs of southeastern Florida.

The cyanobacterial genus Lyngbya includes free-living, benthic, filamentous cyanobacteria that form periodic nuisance blooms in lagoons, reefs, and estuaries. Lyngbya spp. are prolific producers of biologically active compounds that deter grazers and help blooms persist in the marine environment. Here, our investigations reveal the presence of three distinct Lyngbya species on nearshore reefs in Broward County, FL, sampled in 2006 and 2007. With a combination of morphological measurements, molecular biology techniques, and natural products chemistry, we associated these three Lyngbya species with three distinct Lyngbya chemotypes. One species, identified as Lyngbya cf. confervoides via morphological measurements and 16S rRNA gene sequencing, produces a diverse array of bioactive peptides and depsipeptides. Our results indicate that the other two Lyngbya species produce either microcolins A and B or curacin D and dragonamides C and D. Results from screening for the biosynthetic capacity for curacin production among the three Lyngbya chemotypes in this study correlated that capacity with the presence of curacin D. Our work on these bloom-forming Lyngbya species emphasizes the significant phylogenetic and chemical diversity of the marine cyanobacteria on southern Florida reefs and identifies some of the genetic components of those differences.

Bioassay-guided isolation and identification of desacetylmicrocolin B from Lyngbya cf. polychroa.

Bioassay-guided fractionation of a non-polar extract of Lyngbya cf. polychroa resulted in the isolation of the cytotoxic desacetylmicrocolin B (1) as well as the known compounds microcolins A (2) and B (3). Compound 1 was found to inhibit the growth of HT-29 colorectal adenocarcinoma and IMR-32 neuroblastoma cells with half maximal inhibitory concentration (IC(50)) values of 14 nM for both cancer cell types. Microcolins A and B were found to have little activity against two strains of the marine fungus Dendryphiella salina with LD(50) values above 200 microg/mL. Compounds 1, 2, and 3 were obtained by reverse-phase chromatography and their structures were determined by NMR and MS. In this paper we report the isolation, identification, and biological activity of 1.

Mesohaline conditions represent the threshold for oxidative stress, cell death and toxin release in the cyanobacterium Microcystis aeruginosa.

As aquatic ecosystems become increasingly affected by hydrologic alterations, drought and sea level rise a need exists to better understand the biological effects of elevated salinity on toxigenic cyanobacteria such as Microcystis aeruginosa. This study investigated the impacts of oligohaline/low mesohaline conditions and exposure time on selected physiological and biochemical responses in M. aeruginosa including cell viability, oxidative stress, antioxidant responses, in addition to microcystin synthesis and release into the surrounding environment. M. aeruginosa was able to grow in most test salinity treatments (1.4-10 ppt), as supported by cell abundance data and chlorophyll-a (chl-a) concentrations. Physiological data showed that after certain salinity thresholds (∼7ppt) were surpassed, salt stress had cascading effects, such as increased ROS production and lipid peroxidation, potentiating the decline in cellular viability. Furthermore, elevated salinity induced oxidative stress which was concomitant with a decrease in cell abundance, chl-a concentration and photochemical efficiency in the 7-10 ppt treatments. M. aeruginosa did not synthesize microcystins (MCs) in response to increased saline conditions, and mcy-D expression was not correlated with either salinity treatment or extracellular MC concentrations, indicating that salinity stress could inhibit toxin production and that released toxins were likely synthesized prior to exposure. Additionally, extracellular MC concentrations were not correlated with decreased cellular integrity, as evidenced by SYTOX analyses, suggesting that toxins may be released through mechanisms other than cellular lysis. Results from this study support that M. aeruginosa can survive with limited negative impacts to cellular structure and function up to a certain threshold between 7-10 ppt. However, after these thresholds are surpassed, there is radical decline in cell health and viability leading to toxin release. This work underscores the importance of understanding the balance between ROS production and antioxidant capacities when assessing the fate of M. aeruginosa under mesohaline conditions.

Involvement of reactive oxygen species and reactive nitrogen species in the wound response of Dasycladus vermicularis.

We investigated the signaling events involved in the wound response of the marine macroalga Dasycladus vermicularis, finding nitric oxide (NO) production in relation to injury. The addition of exogenous H2O2 to aliquots of injured algae accelerated the kinetics of NO production in the wounded region. Similarly, the addition of an NO donor caused an increase in detectable H2O2 around the site of injury. By wounding or incubating uninjured algae with an NO donor, peroxidase activity was enhanced. Based on the use of selected pharmacological probes, our results indicate that H2O2 production involves the upstream activation of signaling events similar to those observed in the physiology of higher plants.

Chemical defenses: from compounds to communities.

Marine natural products play critical roles in the chemical defense of many marine organisms and in some cases can influence the community structure of entire ecosystems. Although many marine natural products have been studied for biomedical activity, yielding important information about their biochemical effects and mechanisms of action, much less is known about ecological functions. The way in which marine consumers perceive chemical defenses can influence their health and survival and determine whether some natural products persist through a food chain. This article focuses on selected marine natural products, including okadaic acid, brevetoxins, lyngbyatoxin A, caulerpenyne, bryostatins, and isocyano terpenes, and examines their biosynthesis (sometimes by symbiotic microorganisms), mechanisms of action, and biological and ecological activity. We selected these compounds because their impacts on marine organisms and communities are some of the best-studied among marine natural products. We discuss the effects of these compounds on consumer behavior and physiology, with an emphasis on neuroecology. In addition to mediating a variety of trophic interactions, these compounds may be responsible for community-scale ecological impacts of chemically defended organisms, such as shifts in benthic and pelagic community composition. Our examples include harmful algal blooms; the invasion of the Mediterranean by Caulerpa taxifolia; overgrowth of coral reefs by chemically rich macroalgae and cyanobacteria; and invertebrate chemical defenses, including the role of microbial symbionts in compound production.

Toxin release in response to oxidative stress and programmed cell death in the cyanobacterium Microcystis aeruginosa.

An unprecedented bloom of the cyanobacterium Microcystis aeruginosa Kütz. occurred in the St. Lucie Estuary, FL in the summer of 2005. Samples were analyzed for toxicity by ELISA and by use of the polymerase chain reaction (PCR) with specific oligonucleotide primers for the mcyB gene that has previously been correlated with the biosynthesis of toxic microcystins. Despite the fact that secreted toxin levels were relatively low in dense natural assemblages (3.5 microg l(-1)), detectable toxin levels increased by 90% when M. aeruginosa was stressed by an increase in salinity, physical injury, application of the chemical herbicide paraquat, or UV irradiation. The application of the same stressors caused a three-fold increase in the production of H(2)O(2) when compared to non-stressed cells. The application of micromolar concentrations of H(2)O(2) induced programmed cell death (PCD) as measured by a caspase protease assay. Catalase was capable of inhibiting PCD, implicating H(2)O(2) as the inducing oxidative species. Our results indicate that physical stressors induce oxidative stress, which results in PCD and a concomitant release of toxin into the surrounding media. Remediation strategies that induce cellular stress should be approached with caution since these protocols are capable of releasing elevated levels of microcystins into the environment.

Elevated Temperature and Allelopathy Impact Coral Recruitment.

As climate change continues to alter seawater temperature and chemistry on a global scale, coral reefs show multiple signs of degradation. One natural process that could facilitate the recovery of reef ecosystems is coral recruitment, which can be influenced by the benthic organisms in a local habitat. We experimentally tested both a global stressor (increased seawater temperature) and a local stressor (exposure to microcolin A, a natural product from a common marine benthic cyanobacterium) to determine how these stressors impacted coral larval sublethal stress, survival and settlement. Larvae of Porites astreoides had the same survival and settlement as the controls after exposure to increased temperature alone, but elevated temperature did cause oxidative stress. When exposed to natural concentrations of microcolin A, larval survival and settlement were significantly reduced. When larvae were exposed to these two stressors sequentially there was no interactive effect; but when exposed to both stressors simultaneously, there was a synergistic reduction in larval survival and an increase in oxidative stress more than in either stressor treatment alone. Increased seawater temperatures made larvae more susceptible to a concurrent local stressor disrupting a key process of coral reef recovery and resilience. These results highlight the importance of understanding how interactive stressors of varying spatial scales can impact coral demographics.

Lipopolysaccharides elicit an oxidative burst as a component of the innate immune system in the seagrass Thalassia testudinum.

This study represents the first report characterizing the biological effects of a lipopolysaccharide (LPS) immune modulator on a marine vascular plant. LPS was shown to serve as a strong elicitor of the early defense response in the subtropical seagrass Thalassia testudinum Banks ex König and was capable of inducing an oxidative burst identified at the single cell level. The formation of reactive oxygen species (ROS), detected by a redox-sensitive fluorescent probe and luminol-based chemiluminescence, included a diphenyleneiodonium sensitive response, suggesting the involvement of an NADPH oxidase. A 900 bp cDNA fragment coding for this enzyme was sequenced and found to encode a NAD binding pocket domain with extensive homology to the Arabidopsis thaliana rbohF (respiratory burst oxidase homolog) gene. The triggered release of ROS occurred at 20 min post-elicitation and was dose-dependent, requiring a minimal threshold of 50 µg/mL LPS. Pharmacological dissection of the early events preceding ROS emission indicated that the signal transduction chain of events involved extracellular alkalinization, G-proteins, phospholipase A2, as well as K(+), Ca(2+), and anion channels. Despite exclusively thriving in a marine environment, seagrasses contain ROS-generating machinery and signal transduction components that appear to be evolutionarily conserved with the well-characterized defense response systems found in terrestrial plants.

Climate change and ocean acidification effects on seagrasses and marine macroalgae.

Although seagrasses and marine macroalgae (macro-autotrophs) play critical ecological roles in reef, lagoon, coastal and open-water ecosystems, their response to ocean acidification (OA) and climate change is not well understood. In this review, we examine marine macro-autotroph biochemistry and physiology relevant to their response to elevated dissolved inorganic carbon [DIC], carbon dioxide [CO2 ], and lower carbonate [CO3 (2-) ] and pH. We also explore the effects of increasing temperature under climate change and the interactions of elevated temperature and [CO2 ]. Finally, recommendations are made for future research based on this synthesis. A literature review of >100 species revealed that marine macro-autotroph photosynthesis is overwhelmingly C3 (≥ 85%) with most species capable of utilizing HCO3 (-) ; however, most are not saturated at current ocean [DIC]. These results, and the presence of CO2 -only users, lead us to conclude that photosynthetic and growth rates of marine macro-autotrophs are likely to increase under elevated [CO2 ] similar to terrestrial C3 species. In the tropics, many species live close to their thermal limits and will have to up-regulate stress-response systems to tolerate sublethal temperature exposures with climate change, whereas elevated [CO2 ] effects on thermal acclimation are unknown. Fundamental linkages between elevated [CO2 ] and temperature on photorespiration, enzyme systems, carbohydrate production, and calcification dictate the need to consider these two parameters simultaneously. Relevant to calcifiers, elevated [CO2 ] lowers net calcification and this effect is amplified by high temperature. Although the mechanisms are not clear, OA likely disrupts diffusion and transport systems of H(+) and DIC. These fluxes control micro-environments that promote calcification over dissolution and may be more important than CaCO3 mineralogy in predicting macroalgal responses to OA. Calcareous macroalgae are highly vulnerable to OA, and it is likely that fleshy macroalgae will dominate in a higher CO2 ocean; therefore, it is critical to elucidate the research gaps identified in this review.

Macroalgal extracts induce bacterial assemblage shifts and sublethal tissue stress in Caribbean corals.

Benthic macroalgae can be abundant on present-day coral reefs, especially where rates of herbivory are low and/or dissolved nutrients are high. This study investigated the impact of macroalgal extracts on both coral-associated bacterial assemblages and sublethal stress response of corals. Crude extracts and live algal thalli from common Caribbean macroalgae were applied onto the surface of Montastraea faveolata and Porites astreoides corals on reefs in both Florida and Belize. Denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene amplicons was used to examine changes in the surface mucus layer (SML) bacteria in both coral species. Some of the extracts and live algae induced detectable shifts in coral-associated bacterial assemblages. However, one aqueous extract caused the bacterial assemblages to shift to an entirely new state (Lobophora variegata), whereas other organic extracts had little to no impact (e.g. Dictyota sp.). Macroalgal extracts more frequently induced sublethal stress responses in M. faveolata than in P. astreoides corals, suggesting that cellular integrity can be negatively impacted in selected corals when comparing co-occurring species. As modern reefs experience phase-shifts to a higher abundance of macroalgae with potent chemical defenses, these macroalgae are likely impacting the composition of microbial assemblages associated with corals and affecting overall reef health in unpredicted and unprecedented ways.

Effects of short-term hypersalinity exposure on the susceptibility to wasting disease in the subtropical seagrass Thalassia testudinum.

Seagrass meadows are a vital component of coastal ecosystems and have experienced declines in abundance due to a series of environmental stressors including elevated salinity and incidence of disease. This study evaluated the impacts of short-term hypersalinity stress on the early stages of infection in Thalassia testudinum Banks ex König by assessing changes in cellular physiology and metabolism. Seagrass short shoots were exposed to ambient (30 psu) and elevated (45 psu) salinities for 7 days and subsequently infected for one week by the causative pathogen of wasting disease, Labyrinthula sp. The occurrence of wasting disease was significantly lower in the hypersalinity treatments. Additionally, while exposure to elevated salinity caused a reduction in chlorophyll a and b content, T. testudinum's health, in terms of photochemical efficiency, was not significantly compromised by hypersalinity or infection. In contrast, plant respiratory demand was significantly enhanced as a function of infection. Elevated salinity caused T. testudinum to significantly increase its in vivo H(2)O(2) concentrations to levels that exceeded those which inhibited Labyrinthula growth in a liquid in vitro assay. The results suggest that while short-term exposure to hypersalinity alters selected cellular processes this does not necessarily lead to an immediate increase in wasting disease susceptibility.

Dragonamides C and D, linear lipopeptides from the marine cyanobacterium brown Lyngbya polychroa.

Two new linear lipopeptides, 1 and 2, and a known compound, curacin D, have been isolated from a marine cyanobacterium, brown Lyngbya polychroa, collected from Hollywood Beach, Fort Lauderdale, Florida. Their planar structures were elucidated by 1D and 2D NMR techniques, and absolute configurations were assigned using chiral HPLC. The new compounds were assigned the trivial names dragonamide C (1) and dragonamide D (2), as their peptide moiety is related to previously reported dragonamides A and B.