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Allelopathy is an efficient strategy by which some microalgae can outcompete other species. Allelochemicals from the toxic dinoflagellate Alexandrium minutum have deleterious effects on diatoms, inhibiting metabolism and photosynthesis and therefore give a competitive advantage to the dinoflagellate. The precise mechanisms of allelochemical interactions and the molecular target of allelochemicals remain however unknown. To understand the mechanisms, the short-term effects of A. minutum allelochemicals on the physiology of the diatom Chaetoceros muelleri were investigated. The effects of a culture filtrate were measured on the diatom cytoplasmic membrane integrity (polarity and permeability) using flow-cytometry and on the photosynthetic performance using fluorescence and absorption spectroscopy. Within 10 min, the unknown allelochemicals induced a depolarization of the cytoplasmic membranes and an impairment of photosynthesis through the inhibition of the plastoquinone-mediated electron transfer between photosystem II and cytochrome b6f. At longer time of exposure, the cytoplasmic membranes were permeable and the integrity of photosystems I, II and cytochrome b6f was compromised. Our demonstration of the essential role of membranes in this allelochemical interaction provides new insights for the elucidation of the nature of the allelochemicals. The relationship between cytoplasmic membranes and the inhibition of the photosynthetic electron transfer remains however unclear and warrants further investigation.

Various species of Alexandrium can produce a number of bioactive compounds, e.g., paralytic shellfish toxins (PSTs), spirolides, gymnodimines, goniodomins, and also uncharacterised bioactive extracellular compounds (BECs). The latter metabolites are released into the environment and affect a large range of organisms (from protists to fishes and mammalian cell lines). These compounds mediate allelochemical interactions, have anti-grazing and anti-parasitic activities, and have a potentially strong structuring role for the dynamic of Alexandrium blooms. In many studies evaluating the effects of Alexandrium on marine organisms, only the classical toxins were reported and the involvement of BECs was not considered. A lack of information on the presence/absence of BECs in experimental strains is likely the cause of contrasting results in the literature that render impossible a distinction between PSTs and BECs effects. We review the knowledge on Alexandrium BEC, (i.e., producing species, target cells, physiological effects, detection methods and molecular candidates). Overall, we highlight the need to identify the nature of Alexandrium BECs and urge further research on the chemical interactions according to their ecological importance in the planktonic chemical warfare and due to their potential collateral damage to a wide range of organisms.

The dinoflagellate Alexandrium minutum produces toxic compounds, including paralytic shellfish toxins, but also some unknown extracellular toxins. Although copper (Cu) is an essential element, it can impair microalgal physiology and increase their toxic potency. This study investigated the effect of different concentrations of dissolved Cu (7 nM, 79 nM and 164 nM) on A. minutum allelochemical potency, here defined as negative effects of a protist on competing protists through the release of chemicals. This was studied in relation to its physiology. The effects of Cu were assessed on A. minutum growth, reactive oxygen species level, photosynthesis proxies, lipid metabolism, exudation of dissolved organic compounds, allelochemical potency and on the associate free bacterial community of A. minutum. Only the highest Cu exposure (164 nM) inhibited and delayed the growth of A. minutum, and only in this treatment did the allelochemical potency significantly increase, when the dissolved Cu concentration was still toxic. Within the first 7 days of the high Cu treatment, the physiology of A. minutum was severely impaired with decreased growth and photosynthesis, and increased stress responses and free bacterial density per algal cell. After 15 days, A. minutum partially recovered from Cu stress as highlighted by the growth rate, reactive oxygen species level and photosystem II yields. This recovery could be attributed to the apparent decrease in background dissolved Cu concentration to a non-toxic level, suggesting that the release of exudates may have partially decreased the bioavailable Cu fraction. Overall, A. minutum appeared quite tolerant to Cu, and this work suggests that the modifications in the physiology and in the exudates help the algae to cope with Cu exposure. Moreover, this study shows the complex interplay between abiotic and biotic factors that can influence the dynamic of A. minutum blooms. Modulation in allelochemical potency of A. minutum by Cu may have e

Allelochemical interactions are likely to be a contributing factor explaining the success of large blooms of the harmful marine dinoflagellate Alexandrium, however, the physiological mechanisms of allelochemical interactions remain poorly described. Here we investigated the sub-lethal effects (on an hourly scale) of a filtrate containing allelochemicals from Alexandrium minutum on the physiology of the common diatom Chaetoceros muelleri. The filtrate induced deleterious effects to the diatom physiology within only 30 min of exposure. Esterase activity and photosynthesis were drastically inhibited, with up to 34% of the population being metabolically inactive and up to 30% reduction in photosystem II quantum yield when exposed to the filtrate. In addition, intracellular reactive oxygen species increased by 26% in response to allelochemical exposure. C. muelleri pigment and lipid analyses indicated that the photosystem II was inhibited, with photoinhibition-like responses (activation of xanthophyll cycles, and changes in associated lipids) upregulated to mitigate the toxic effects of allelochemicals. Changes in the proportions of membrane lipid classes and increased membrane fatty acids saturation by 9% may be an attempt to maintain membrane integrity and associated enzyme activity, or could be the result of deleterious effects on membranes. An 8% decrease in cellular storage lipids (triglycerides) revealed a mobilization of energy suggesting an energetic cost for the diatom to counteract the allelochemical effects. We hypothesize that the rapid alteration of physiological functions such as photosynthesis and some enzymatic activities may result from direct damage on external membranes. Overall this study describes the sub-lethal mechanisms and provides useful biomarkers to understand the role of allelochemical interactions and associated ecological processes in structuring plankton communities.

Harmful microalgal blooms are a threat to aquatic organisms, ecosystems and human health. Toxic dinoflagellates of the genus Alexandrium are known to produce paralytic shellfish toxins and to release bioactive extracellular compounds (BECs) with potent cytotoxic, hemolytic, ichtyotoxic and allelopathic activity. Negative allelochemical interactions refer to the chemicals that are released by the genus Alexandrium and that induce adverse effects on the physiology of co-occurring protists and predators. Releasing BECs gives the donor a competitive advantage that may help to form dense toxic blooms of phytoplankton. However BECs released by Alexandrium minutum are uncharacterized and it is impossible to quantify them using classical chemical methods. Allelochemical interactions are usually quantified through population growth inhibition or lytic-activity based bioassays using a secondary target organism. However these bioassays require time (for growth or microalgal counts) and/or are based on lethal effects. The use of pulse amplitude modulation (PAM) fluorometry has been widely used to assess the impact of environmental stressors on phytoplankton but rarely for allelochemical interactions. Here we evaluated the use of PAM and propose a rapid chlorophyll fluorescence based bioassay to quantify allelochemical BECs released from Alexandrium minutum. We used the ubiquitous diatom Chaetoceros muelleri as a target species. The bioassay, based on sub-lethal effects, quantifies allelochemical activity from different samples (filtrates, extracts in seawater) within a short period of time (2 h). This rapid bioassay will help investigate the role of allelochemical interactions in Alexandrium bloom establishment. It will also further our understanding of the potential relationship between allelochemical activities and other cytotoxic activities from BECs. While this bioassay was developed for the species A. minutum, it may be applicable to other species producing allelochemicals

Understanding the factors that favor the establishment and the persistance of harmful algal blooms (HABs) is essential for predicting them, and subsequently mitigating their negative consequences on the marine environment, human health and the economy. Allelochemical potency, i.e. the release of compounds that inhibit competitors, is hypothesized to favor the organisms that produce them and indeed shapes plankton community. The toxic dinoflagellates from the genus Alexandrium produce allelochemicals, however, associated interactions are poorly understood. The objective of this thesis is to contribute to our understanding by studying the mechanisms behind the allelochemical interactions between the dinoflagellate A. minutum and the diatom Chaetoceros muelleri. The results of this work have highlighted that A. minutum allelochemicals disrupted membrane functioning of the diatoms within minutes. The cascade of physiological events following the allelochemical interaction that was described in this PhD included permeabilisation of cell membranes, inhibition of photosynthesis, production of reactive oxygen species and modifications of the biochemical composition of membranes. Additionally, this project has highlighted that the allelochemical potency of A. minutum can be significantly modulated by environmental parameters, as allelochemical potency of A. minutum increased 4-5 times when exposed to toxic concentrations of Cu. Finally, a bioassay developed during this PhD eased the partial isolation of the allelochemicals through guided-fractionation. Although we did not totally purify the allelochemical(s) of A. minutum, we isolated a group of hydrophobic candidates whose activity has to be further investigated following their isolation. These results provide a better understanding of the physiological and cellular mechanisms underlying these allelochemical interactions. This PhD clearly highlighted the complexity of allelochemical interactions, but also offers new tools a