Algicidal effects of Zostera marina L. and Zostera noltii Hornem. extracts on the neuro-toxic bloom-forming dinoflagellate Alexandrium catenella

Highlights

• We investigated the inhibitory effect of extracts from Zostera marina and Zostera noltii on the growth of Alexandrium catenella.

• Methanolic and aqueous extracts were prepared from fresh and detrital leaves of Zostera species.

• All the extracts exhibited dose-dependent significant inhibition of Alexandrium growth.

• Phenolic concentrations in extracts were found to correlate negatively with EC₅₀ values.

• Phenolic compounds might be responsible of the observed algicidal effect.

Abstract

The inhibitory effects of crude extracts of Zostera marina L. and Zostera noltii Hornemann on the growth of the toxic red tide dinoflagellate Alexandrium catenella were investigated through bio-assays. Methanolic and aqueous extracts were prepared from fresh and detrital leaves of Z. marina and Z. noltii collected in the Thau lagoon and Arcachon bay (France). All the extracts exhibited significant inhibition of A. catenella growth, whatever the species and without the need of continuous addition of extracts. The effective concentrations (EC₅₀) varied in the range 0.036–0.199 g L⁻¹ for Z. noltii and 0.036–0.239 g L⁻¹ for Z. marina. Methanolic extracts prepared from fresh plant tissues were the most potent, with EC₅₀ of 0.036–0.039 and 0.036–0.045 g L⁻¹, for Z. noltii and Z. marina, respectively. After exposure to the extracts, Alexandrium cells exhibited severe morphological anomalies. Hence many cells exhibited a lytic membrane and became necrotic. Cellular degradation appeared dose- and time-dependent. The observed algicidal activities demonstrated the existence of bioactive molecules in the tissues of Zostera leaves, which were stable in the culture medium. The chemical contents of the crude extracts were determined by NMR, LC/MS, and quantitative HPLC. Results showed the predominance of flavonoids and phenolic acids. The total phenolics concentrations correlated negatively with the EC₅₀ values, suggesting that these secondary metabolites might be responsible for the observed algicidal effects.

Introduction

In recent years, the control of harmful algal blooms (HABs) has become an important issue for coastal ecosystem protection (Anderson et al., 2001, Kim, 2006). Aquatic macrophytes have long been suspected of limiting phytoplankton growth through the production and excretion of chemical substances (Gross et al., 2007). There is now strong evidence that chemical interferences occur in freshwater ecosystems with many organisms secreting inhibitory compounds (allelochemicals) against microalgae and cyanobacteria (Nakai et al., 2000, Thomas et al., 2000, Gross et al., 2007). Allelopathy refers to any direct or indirect, harmful or beneficial effect produced by plants, protists (e.g. microalgae, ciliates), bacteria, or viruses on another through the production of chemical compounds that leak into the environment (Rice, 1984). It has been suggested that growth inhibition of phytoplankton by submerged macrophytes may confer an advantage to the former in the competition for light, carbon, and nutrients (Gross, 2003, Gross et al., 2007). In the last decades, studies have shown that chemical interactions could play an important role in marine ecosystems in regulating the diversity, structure and seasonal variations of phytoplankton communities (Granéli and Hansen, 2006, Ianora et al., 2006, Granéli et al., 2008). The allelopathic effects of macroalgae against red tide microalgae have been reported (Jeong et al., 2000, Wang et al., 2007, Tang and Gobler, 2011), involving in particular the impacts of different species of Ulva on A. tamarense populations (Jin and Dong, 2003, Nan et al., 2008). In contrast, non-nutrient interactions between seagrass beds and HAB microalgae have never been reported.

Seagrass beds are thought to have the ability to improve water clarity in their local environment by promoting particle settlement, reducing resuspension and picking up nutrients (Kemp et al., 1984, Ward et al., 1984, Fonseca, 1996). It has also been suggested that the chemical content of Zostera marina leaves might negatively affect the growth and photosynthetic carbon uptake of epiphytic diatoms (Harrison, 1982, Harrison and Durance, 1985). Water-soluble extracts of Z. marina from British Columbia have been shown to inhibit the growth of microalgae and marine bacteria and to control amphipod grazing (Harrison and Chan, 1980). Harrison (1982) suggested that water-soluble inhibitors may explain the low biomass of epiphytes on actively growing leaves, but other bioactive compounds may also play a role. Both European Zostera species contain bioactive substances, among which zosteric acid (Todd et al., 1993, Achamlale et al., 2009a, Achamlale et al., 2009b), which may prevent the settlement of some marine bacteria, algae, barnacles and tube worms.

Across Europe, seagrass beds have declined, but at the same time the frequency of HAB events has increased in the last decades (Glibert et al., 2005). Many Alexandrium species produce potent neurotoxins that may potentially affect aquatic organisms and human consumers of shellfish which bioaccumulate these toxins (May et al., 2010, Anderson et al., 2012). In the Thau lagoon (French Mediterranean lagoon), the first major bloom of the toxic Alexandrium catenella (85 000 cells mL⁻¹) occurred in 1998 (Abadie et al., 1999, Lilly et al., 2002). Since then, recurrent episodes were reported during spring and fall reaching high cell concentrations (3–14 × 10⁶ cells L⁻¹). Toxin contaminations in bivalves frequently exceeded the sanitary threshold and have induced frequent closing of the aquaculture zones with resulting economical damages. Alexandrium outbreaks initiated and developed exclusively in the Crique de l’Angle, a small embayment northeast of Thau lagoon, where the highest concentrations were usually registered (Genovesi et al., 2010, Chambouvet et al., 2011, Laabir et al., 2011). Depending on hydrological conditions, Alexandrium cells may further spread throughout the lagoon (Genovesi et al., 2013).

Interestingly, since 1990 the Crique de l’Angle has suffered an important regression of the Zostera bed which virtually disappeared from this area whereas it persisted in the periphery of the rest of the lagoon (Deslous-Paoli et al., 1998, Plus et al., 2001, Plus et al., 2003a, Plus et al., 2003b, Plus et al., 2005), where blooms did not develop. Coastal ecosystems are very complex, and many controlling factors are involved in the occurrence of HABs (Anderson et al., 2012). Their initiation, duration and intensity may be related to a number of biological, chemical, and physical factors, although, many of these complex relationships have not yet been fully identified (Glibert et al., 2005, Collos et al., 2007, Genovesi et al., 2010, Chambouvet et al., 2011, Laabir et al., 2011, Anderson et al., 2012, Genovesi et al., 2013). In our case, among the possible causes of Alexandrium proliferations, the question arises if it is possible to draw conclusions from the chronology of events observed in the Thau lagoon, and to hypothesize that the disappearance of Zostera beds may have facilitated the settlement of Alexandrium blooms in the Crique de l’Angle. This hypothesis was supported by a cross-analysis of the French REPHY (phytoplankton and phycotoxins) and REBENT (benthic organisms) monitoring network databases that revealed low occurrences of Alexandrium blooms in the vicinity of extensive Zostera beds. In particular, in the Arcachon bay (French Atlantic Coast), a tidal ecosystem sheltering an important Zostera bed, low concentrations of Alexandrium spp. are observed and blooms never occurred (Auby et al., 2011).

In order to test this hypothesis, we have studied the effects of crude extracts of Zostera species on the growth of A. catenella to evaluate their potential negative impact. The main objectives of our study were (i) to assess algicidal activity of the metabolites produced by Z. marina and Zostera noltii, (ii) to compare the inhibitory effects of extracts from Zostera species collected in Mediterranean (Thau lagoon) and Atlantic marine waters (Arcachon bay), and (iii) to identify the causative bioactive substances.