Elsevier

Aquatic Toxicology

Volume 206, January 2019, Pages 203-211
Aquatic Toxicology

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

https://doi.org/10.1016/j.aquatox.2018.11.019Get rights and content

Highlights

  • M. aeruginosa can survive with limited negative impacts to cellular structure and function up to a certain threshold between 7–10 ppt.

  • 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.

  • 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.

  • 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.

Abstract

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.

Introduction

Globally, aquatic ecosystems are being impacted by increased salinity associated with drought, hydrologic alterations, and sea level rise (Nielsen et al., 2005; Kundzewicz et al., 2008; White and Kaplan, 2017). The expansion of the toxigenic cyanobacteria, Microcystis aeruginosa, (conventionally recognized as a freshwater species) into estuarine habitats has been reported throughout North America, Australia, Europe and Japan in response to episodic changes in water movement and the acquisition of halotolerance genes (Lehman et al., 2013; Paerl and Paul, 2012; Tanabe et al., 2018). One such geographic locale that periodically experiences blooms of M. aeruginosa (Aubel et al., 2006-Lakeline) and dynamic saline conditions up to 160 km upstream due to semi-diurnal tidal influences, is the St. Johns River (SJR; Northeast Florida, United States) (Environmental Protection Board, 2017). Blooms of M. aeruginosa are consistently reported in oligohaline (0.5–5 ppt) areas of the SJR, and scums of this species have been identified at the mouth of the river in recent years (Environmental Protection Board, 2017). Additionally, blooms of M. aeruginosa have been documented in mesohaline (5–18 ppt) regions of the SJR. The most notable occurrence was during a multi-HAB event during the summer of 2010, in which the dominance of M. aeruginosa was correlated with elevated river salinity due to extended periods of reverse directional flow (Environmental Protection Board, 2017). Additionally, water testing during the HAB event revealed high concentrations of microcystins (MCs), potent hepatotoxins, that were implicated as a major factor in a mass fish die-off during that time period (Environmental Protection Board, 2017). Depending upon the amplitude of the tidal exchange, habitats in the lower estuary can experience extensive salinity changes which can have profound effects on the health, survival, and toxin production of M. aeruginosa.

Salinity stress is known to disrupt redox homeostasis and lead to an accumulation of reactive oxygen species (ROS) in a wide array of photoautotrophic organisms through the impairment of CO2 reduction within the Calvin cycle, photosynthetic electron transport, or through an increase in photorespiration (Latifi et al., 2009; Abogadallah, 2010; Choudhury et al., 2013). The buildup of ROS, such as superoxide anion (O2), hydrogen peroxide (H2O2) and hydroxyl radicals (radical dotOH), have the capacity to damage multiple intracellular targets, including proteins, lipids and nucleic acids (Halliwell, 2006). Under moderate levels of stress, ROS accumulation can alter cellular redox homeostasis enough to activate selected signaling cascades, including programmed cell death (PCD; Chandra et al., 2000; Schmitt et al., 2014). Although ROS can regulate a series of biological responses in cyanobacteria, such as cellular differentiation and the formation of dispersal cells (i.e. heterocysts, hormogonia, filament polarity) (Rai et al., 1978; Berman-Frank et al., 2004; Adamec et al., 2005), ROS accumulation can also lead to degradation of cellular membranes and consequent toxin release (Ross et al., 2006). Given the ubiquitous nature of ROS and the dynamic set of biological responses generated in cyanobacteria, there continues to be a considerable gap in the understanding of the adaptive mechanisms employed by cyanobacteria in response to salinity-induced ROS production. Furthermore, mesohaline conditions may allow for ROS accumulation, subsequent mass cellular degradation, and associated toxin release of an entire bloom.

The interplay between ROS production and PCD has been well documented in model terrestrial plant systems as well as in select phytoplankton species under adverse abiotic conditions (Pennell and Lamb, 1997; Van Breusegem and Dat, 2006; Bidle, 2016). Salinity stress has also been shown to induce PCD in various cyanobacterial species, though the selective advantages or adaptive roles have not yet been elucidated (Ning et al., 2002; Swapnil et al., 2017). Ross et al. (2006) previously demonstrated that the exogenous application of H2O2 induces PCD in M. aeruginosa, as measured by a metacaspase-like protease assay. This finding prompted the current study which further explored the relationship among salinity-induced oxidative stress, PCD and corresponding impacts, such as toxin release, in M. aeruginosa.

The MCs produced by M. aeruginosa are known to have prolonged, deleterious impacts on aquatic habitats (Paerl et al., 2001; Landsberg, 2002; Miller et al., 2010), local economies (Hudnell and Dortch, 2008) and human and animal health (Mez et al., 1997; Pitosis et al., 2000; Nasri et al., 2008; Miller et al., 2010; Brown et al., 2018). There are over 150 described structural variants of MCs that share primary amino acid groups (d-Ala1-l-X2-d-MeAsp3-l-Y4-Adda5-d-Glu6-MDha7) and monocyclic heptapeptide ring structures, but differ in amino acid composition in the X2 and Y4 positions, as well as in various positions where functional groups have been inserted or lost (Foss and Aubel, 2015). MCs are synthesized non-ribosomally through a mixed polyketide synthase/nonribosomal peptide synthetase gene cluster (genes mcyA-J). The expression of mcyD has been demonstrated to be especially useful in monitoring MC biosynthesis, as it is involved in the formation of the β-amino acid Adda ([2S,3S,8S,9S,4E,6E,]-3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid; Rinehart and Harada, 1988) side chain, present in all congeners of MCs (Kaebernick et al., 2000; Sevilla et al., 2008). The Adda moiety is integral to MC’s toxicosis as it binds to, and inhibits, phosphatases 1 and 2 (Goldberg et al., 1995; MacKintosh et al., 1995) and the lack of the myc-D gene prevents MC biosynthesis (Kaebernick et al., 2000; Tillet et al., 2000; Sevilla et al., 2008).

While there is an increased awareness of harmful cyanobacterial blooms (CyanoHABs) occurring in brackish waters (Sneed et al., 2017), the biological responses of one of the most toxigenic and ubiquitous species of cyanobacteria, M. aeruginosa, under saline conditions continues to remain understudied. It is generally believed that elevated salinity has a negative impact on M. aeruginosa viability (Preece et al., 2017). However, there is still a lack of detailed understanding of how increases in salinity affect the physiology (i.e. growth, oxidative stress response, PCD) of M. aeruginosa and in turn, how this regulates toxin production and/or release into the surrounding environment. In an effort to better understand this relationship, a series of controlled experiments were designed to test the effects of mesohaline conditions on M. aeruginosa tolerance, viability, and physiological responses.

Section snippets

General culture maintenance and experimental design

A unialgal, non-axenic strain of Microcystis aeruginosa (strain LB 2385) was obtained from the UTEX Culture Collection of Algae at the University of Texas at Austin. LB 2385 represents a well-studied model strain that has been previously used in a number of reports (Wilson et al., 2005; Lee et al., 2013; Bista et al., 2014; Pineda-Mendoza et al., 2016). Stock cultures were maintained in BG-11 medium at 23 °C under a 12:12 light:dark (L:D) cycle (irradiance of 53 μmol photons m−2 s-1). Cultures

Cellular abundance, chl-a content and photochemical efficiency

Over the 11 day time period, both salinity and time had a significant effect on M. aeruginosa abundance (Fig. 1A). Furthermore, there was a significant interaction between salinity and time. A summary of the statistical results is provided in Supplemental Tables S1 and S2. Starting cell concentrations for all treatments had mean values of 9.35 × 103 cells μL−1. Through day 4, there was no significant difference in cell abundance among treatments. However, by day 7, the control and 3 ppt

Discussion

While M. aeruginosa is widely considered a freshwater species, findings from these experiments are in general agreement with other reports suggesting that selected strains can have a notable tolerance for increased saline conditions (Robson and Hamilton, 2003; Orr et al., 2004; Verspagen et al., 2006; Tonk et al., 2007; Tanabe et al., 2018). However, once hyperosmotic thresholds are surpassed, salt stress can have a potentiating effect on ROS production, subsequently yielding a decline in cell

Acknowledgements

We thank Dr. Eric Johnson for his assistance with statistical analysis. This work was supported by a University of North Florida Academic Affair’s Faculty Scholarship Development Grant awarded to C.R.

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