In vitro evaluation of the potential allelopathic and ichthyotoxic effect of the raphidophyte Heterosigma akashiwo and the dinoflagellate Alexandrium catenella

https://doi.org/10.1016/j.marenvres.2022.105800Get rights and content

Highlights

  • Allelopathic compounds exuded appear to correlate with ichthyotoxicity in Alexandrium catenella.

  • Competition (co-cultivation) generates an increase in the production of allelochemicals.

  • Saxitoxin did not inhibit competing microalga nor produce in vitro ichthyotoxicity.

Abstract

Ichthyotoxic algal blooms cause economic losses throughout the world. However, the mechanisms and molecules proposed so far fail to explain the massiveness of these events. In this research, the allelopathic effect of two bloom-forming species (the raphidophyte Heterosigma akashiwo and dinoflagellate Alexandrium catenella) was evaluated between them and with Rhodomonas salina bioassay. Mono- and co-cultures were carried out with the aim of providing evidence of the relation between allelopathy and ichthyotoxicity. The allelopathic inhibitory effect of the A. catenella's supernatant was significantly enhanced when supernatants were obtained from co-cultures with direct contact between these species. We could not observe any allelopathic response provoked by H. akashiwo. On the other hand, A. catenella was able to decrease the cell concentration of H. akashiwo and R. salina. Besides, allelopathy and ichthyotoxicity were found for A. catenella's supernant, being the allelopathic effect not related to saxitoxin. These results reinforce the hypothesis that the allelopathic effect being regulated by the presence of other microalgae and could be responsible for ichthyotoxicity.

Introduction

Harmful Algal Blooms (HABs) are of particular concern because they are responsible for economic losses worldwide, including million dollars for the aquaculture industry in different countries including Chile (Andersen et al., 2015; Dorantes-Aranda et al., 2015; Mardones et al., 2015). Despite efforts to control, monitor, and mitigate these events, they still cannot be predicted nor totally avoided (Gallardo-Rodríguez et al., 2018). At least 70–80 species of phytoplankton are classified as ichthyotoxic, including raphidophytes, dinoflagellates, dictyophytes and some haptophytes (Anderson et al., 2012). In Chile, these events have been mainly associated with the raphidophyte Heterosigma akashiwo, and the dinoflagellates Karenia selliformis and Alexandrium catenella (Mardones et al., 2016). Also, the co-existence of H. akashiwo and A. catenella has been suggested by other authors (Yamasaki et al., 2018). The toxicological mechanisms responsible for the ichthyotoxic properties of H. akashiwo remain under debate but three main mechanisms have been proposed: (i) high production of reactive oxygen species (ROS) (Marshall et al., 2005; Twiner and Dixon, 2001); (ii) a high free fatty acids (FFA) content, which has been associated with hemolytic activity in fish blood cells (Ling and Trick, 2010; Marshall et al., 2003); and (iii) production of neurotoxins or cardiotoxins (Arakawa et al., 1997; Astuya et al., 2015); however these ichthyotoxicity-responsible mechanisms have not been consistently expressed in all fish-mortality events. The ichthyotoxic effect of Chilean strains of A. catenella has been suggested to be caused by the synergistic effect of ROS (superoxide anion), DHA (docosahexaenoic acid) and potentially other polyunsaturated fatty acids (PUFA) and toxins (saxitoxins) (Dorantes-Aranda et al., 2015; Mardones et al., 2015).

Global warming and increased eutrophication of coastal waters will increase HABs in the next future (Griffith and Gobler, 2020). Ichthyotoxic species involved in these events are known, however, the exact mode of action or the chemical nature of the ichthyotoxins required further study. So far, the proposed mechanisms failed to explain the massiveness of these events.

Controlled-conditions studies have been generally limited to microalgae-abiotic factor interactions (temperature, salinity) or the exposure to pure toxins (standard) using in vivo or in vitro fish cell models, however, HABs are complex events resulting from dynamic balances where different species or strains of microalgae, toxic and non-toxic, adjust their populations to biotic and abiotic conditions (availability of light, nutrients, and turbulence). Besides, some species may also cause adverse or beneficial effects on others. Allelopathy is defined as any direct or indirect inhibitory or stimulating effect from one organism to another through the production of chemical secretions (Yamasaki et al., 2009). Several bloom-forming species are known to influence the growth of others by an allelopathic effect (Granéli and Hansen, 2006; Wang et al., 2020). Studies on allelopathic interactions are important to understand the phytoplankton communities’ dynamics and the mechanisms that lead to the formation (Fernández-Herrera et al., 2016) and dynamics (Wang et al., 2020) of harmful algal blooms. For instance, Alexandrium spp. has been reported to cause cell lysis, inhibition of metabolism, and loss of mobility in competing organisms (Fistarol et al., 2004; Long et al., 2018; Tillmann and John, 2002). Fistarol et al. (2004) suggested that the allelopathic effect could be related to the icththyotoxic and hemolytic properties previously described in this dinoflagellate (Fistarol et al., 2004). Some allelochemicals act specifically on the cell surface, causing irreparable damage to the plasma membrane or inducing the formation of temporary cysts (Fernández-Herrera et al., 2016), while others show effects on cell growth and inhibition of photosynthesis (Hakanen et al., 2014). Allelopathic compounds such as extracellular peptides, hydroxamate chelators, polyunsaturated aldehydes, and toxins have been described (Granéli et al., 2008); notwithstanding, the allelopathic compound could be a toxin. For example, KmTx3 toxin from Karlodinium veneficum, may be responsible for both allelopathy and ichthyotoxicity (Wang et al., 2020). However, in most cases, the chemical nature of these compounds, or even their biological function, remains unknown.

Moreover, a collateral effect of this chemical warfare between phytoplankton species, is the ichthyotoxicity (Granéli and Hansen, 2006), which has been associated with the production in microalgae of secondary metabolites with lytic capacity affecting other species’ cell membranes (Fernández-Herrera et al., 2016; Granéli and Hansen, 2006; Wang et al., 2020). Thus, molecules causing ichthyotoxicity would be released by microalgae to control competing organisms. Besides, for fish in cages (reared at high density), it is not possible to escape from the toxic environment.

The aim of this study was to evaluate the allelopathic effect of exudates of two bloom-forming microalgae (H. akashiwo and A. catenella) grow in mono- and co-cultures, using an in vitro model of ichthyotoxicity.

Section snippets

Algal species and culture conditions

Three microalgae species were used in this research: Heterosigma akashiwo (CCMP302, New Zealand) from the Provasoli-Guillard marine phytoplankton culture center located in Maine, USA, Alexandrium catenella (PFB45) and Rhodomonas salina (CCM-UDEC 153) obtained from the COPAS SUR austral stock, maintained by FICOLAB (Universidad de Concepción, Chile). The three strains were kept in 250 mL Erlenmeyer flasks using sterile filtered (0.2 μm) seawater with a salinity of 35 PSU cultivated with L1

Kinetic and growth parameters

The cell growth kinetics of the three species evaluated in this research are shown in Fig. 2. According to previous research, the optimal temperature for culturing H. akashiwo is 20 °C (Wang et al., 2017). Since co-cultures with temperature-sensitive species were carried out, H. akashiwo was acclimatized to 15 °C. Fig. 2C shows the effect of temperature on the growth of H. akashiwo. The culture grown at 20 °C (Fig. 2C) reached the steady-state phase at a cell density of 248,000 cells mL−1 while

Discussion

Alexandrium and Heterosigma species are known for their allelopathic effects (Hakanen et al., 2014; Ma et al., 2009, 2011, 2009; Yamasaki et al., 2009, 2018). For instance, a previous research reported cell-lysis effect associated with allelochemical compounds of the dinoflagellate A. tamarense on the cryptophyte R. salina in a short period of time (minutes) (Ma et al., 2011). Experimental evidence has demonstrated the effect of allelopathy among microalgal species and suggest that secondary

Conclusion

Based on the discussed results, it can be suggested that A. catenella's allelopathic effect is similar for the two assayed species (H. akashiwo and R. salina). The allelopathic inhibitory effect of the A. catenella's supernatant (for both species) was significantly enhanced when supernatants were obtained from co-cultures, i.e., direct contact between these species. At the same time, the greater allelopathic effect was observed by direct interaction since both R. salina and H. akashiwo almost

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.

6. Acknowledgments

This research was funded by the project Fondecyt 1200845 (ANID, Chile) and COPAS-COASTAL ANID FB210021.

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