Growth, physiological responses and microcystin-production/-release dynamics of Microcystis aeruginosa exposed to various luteolin doses
Introduction
Toxic cyanobacterial blooms (TCBs) frequently occur in freshwaters worldwide, and deteriorate water quality by causing hypoxia and cyanotoxins pollution (Graham et al., 2010; Paerl and Paul, 2012; Rigosi et al., 2014). Microcystis is dominant TCB-forming cyanobacterium that can release microcystins (MCs), the most pervasive cyanotoxins threatening animals and humans (Carmichael, 1992; Harke et al., 2016; Li et al., 2011, 2017). The methods involving salvage, coagulation and copper-based algaecides cannot be widely applied for Microcystis inhibition, due to low efficiency, high cost and secondary pollution (Jančula and Maršálek, 2011; Hu et al., 2014). It is urgently needful to seek a low-cost and eco-friendly strategy to control Microcystis-dominant TCBs.
Numerous plants produce special metabolites with target-specificity and low toxicity (Xiao et al., 2010). Flavonoids are a group of polyphenolic compounds produced by many terrestrial and aquatic plants (Ververidis et al., 2007; Conrad et al., 2009). Various flavonoids can exert inhibitory effects on harmful algal growth, among which luteolin is commonly distributed in vegetables, fruits and medicinal herbs, and has been identified to inhibit Microcystis growth (Jiang et al., 2012; Huang et al., 2015; Chen et al., 2019). Luteolin also has robust anti-inflammatory effects and induces tumor apoptosis, thus luteolin acts as an edible flavonoid for health-care and anticancer therapy (Li et al., 2007; Imran et al., 2019). From these views, luteolin seemed to emerge as a promising algaecide with high safety for actual application. However, evaluating the eco-safety of luteolin also especially depended on MC-release and -production dynamics while applying it as algaecide, which was scarcely revealed to date. Although only Yu et al. (2019) reported luteolin-affected MC-producing gene transcription without dynamic data, yet directly detecting the dynamic changes of MC-release/-production by Microcystis along luteolin-stress can provide more direct MC-pollution data, and is highly necessary to better assess MCs-risks.
So far, research on Microcystis growth under luteolin-stress is still largely insufficient, especially the few related studies performed test within only 5–7 days (Huang et al., 2015; Chen et al., 2019; Yu et al., 2019), to explore Microcystis growth under prolonged stress has greater ecological implication. Moreover, inhibitory concentration threshold of luteolin is helpful to gain its accurate dose for inhibitory effect, and such threshold may vary with exposure time, but time-dependent threshold values for luteolin inhibiting Microcystis are largely lacking. Besides, during cyanobacterial photosynthesis, phycobiliproteins (PBPs) can capture light in spectral ranges where chlorophylls poorly absorb, and transfer energy to photosynthesis reaction center. PBPs also involve in life-sustaining process under adverse condition (Blankenship, 2014; Zhao et al., 2017). Thus PBPs play crucial roles in photosynthetic and metabolic processes, but how luteolin impacts PBP-synthesis of Microcystis as time elapsed has never been revealed.
Based on above, to explore time-dependent Microcystis-inhibiting concentration of luteolin along a prolonged test is urgently needed, during which the dynamics of algal physiological responses (including PBP-synthesis) and MC-release/production under luteolin-stress should also be revealed to better acquire anti-algal mechanisms and application reasonability of luteolin. Here, MC-producing Microcystis aeruginosa FACHB-915 acted as test Microystis. To fill above gaps, this study firstly determined time-dependent half-maximal inhibitory concentration (IC50) values of luteolin against Microcystis growth during a 14 day-stress, and the luteolin doses for subsequent test were set based on the IC50 values. Secondly, this study explored Microcystis-inhibiting mechanisms of various luteolin doses, which involved the dynamics of chlorophyll-a (CLA) and PBPs syntheses, antioxidant responses and oxidative damage of Microcystis along 14 day-stress; Thirdly, dynamics of MC-production/-release by Microcystis was tested to assess the eco-risk for applying luteolin to inhibit Microcystis. For these goals, cell density, CLA and PBPs (e.g., phycocyanin (PC), allophycocyanin (APC) and phycoerythrin (PE)) contents, antioxidases (e.g., superoxide dismutase (SOD), catalase (CAT)) activities, and glutathione (GSH) and malondialdehyde (MDA) contents, and intracellular/extracellular MCs contents of Microcystis exposed to various luteolin doses were determined. These findings would provide novel insights and beneficial guidance to control Microcystis-dominant TCBs and guarantee aquatic system safety.
Section snippets
Microcystis cultivation and chemicals
M. aeruginosa FACHB-915 strain was supplied by Freshwater Algae Culture Collection at Institute of Hydrobiology, Chinese Academy of Sciences. This strain was pre-cultivated in BG11 medium at 25 ± 1 °C under light-dark alternation of 12 h: 12 h with fluorescent lights of 2000 lux.
Luteolin was purchased from Energy Chemical, Inc. (Shanghai, China). Stock solution of luteolin was prepared in dimethyl sulfoxide (DMSO) and stored at 4 °C before use. Other chemicals were analytical grade except as
Determining time-dependent IC50 for luteolin against Microcystis growth
Preliminary test showed that luteolin at 0.5 mg/L slightly promoted Microcystis growth along test, and luteolin at > 0.5 mg/L more strongly inhibited growth with rising dose. The slight growth stimulation at low luteolin dose could be regarded as a hormesis effect on Microcystis at low toxicity level, which also appeared for Microcystis growth at lower-dose chemicals (e.g., antibiotics, pesticides, industrial pollutants) (Wang et al., 2007; Qiu et al., 2013; de Morais et al., 2014; Liu et al.,
Conclusion
This study revealed 6.5 mg/L as nearly IC50 value of luteolin against MPM growth until day 14. GIR almost elevated with rising luteolin dose, and at each dose GIR elevated firstly and then leveled off or dropped in mid-late stage, thus GIR showed dose- and time-dependence. Correspondingly, its physiological responses and MC-production/-release under luteolin-stress were also dose- and time-dependent:
- (i)
At rising luteolin dose, MPM almost more strongly stimulated CLA, APC, PE, SOD, CAT and GSH to
CRediT authorship contribution statement
Jieming Li: Conceptualization, Formal analysis, Investigation, Resources, Writing - original draft, Writing - review & editing, Supervision, Funding acquisition, Project administration. Jiaqi Hu: Validation, Formal analysis, Writing - original draft. Linrong Cao: Formal analysis, Investigation. Yue Yuan: Formal analysis.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
This research was jointly sponsored by the National Natural Science Foundation of China (No. 31872694) and Specialized Research Fund for the Doctoral Program of Higher Education of China (No. 20130008120026).
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