A stable-isotope mass spectrometry-based metabolic footprinting approach to analyze exudates from phytoplankton

Phytoplankton exudates play an important role in pelagic ecology and biogeochemical cycles of elements. Exuded compounds fuel the microbial food web and often encompass bioactive secondary metabolites like sex pheromones, allelochemicals, antibiotics, or feeding attractants that mediate biological interactions. Despite this importance, little is known about the bioactive compounds present in phytoplankton exudates. We report a stable-isotope metabolic footprinting method to characterise exudates from aquatic autotrophs. Exudates from 13C-enriched alga were concentrated by solid phase extraction and analysed by high-resolution Fourier transform ion cyclotron resonance mass spectrometry. We used the harmful algal bloom forming dinoflagellate Alexandrium tamarense to prove the method. An algorithm was developed to automatically pinpoint just those metabolites with highly 13C-enriched isotope signatures, allowing us to discover algal exudates from the complex seawater background. The stable-isotope pattern (SIP) of the detected metabolites then allowed for more accurate assignment to an empirical formula, a critical first step in their identification. This automated workflow provides an effective way to explore the chemical nature of the solutes exuded from phytoplankton cells and will facilitate the discovery of novel dissolved bioactive compounds

Functional genomics of a non-toxic Alexandrium lusitanicum culture

Thesis (Ph. D.)--Joint Program in Oceanography/Applied Ocean Science and Engineering (Massachusetts Institute of Technology, Dept. of Biology; and the Woods Hole Oceanographic Institution), 2007.Includes bibliographical references.Paralytic shellfish poisoning (PSP) is a human intoxication associated with the consumption of shellfish contaminated with a family of neurotoxins called saxitoxins. Many species in the dinoflagellate genus Alexandrium have been shown to produce these toxins. Here I report the first case of a culture of Alexandrium that has completely lost the ability to produce saxitoxins. The loss of toxicity was accompanied by a reduction in growth capability. A subculture of this isolate maintains the ability to produce toxins and to grow at rates and to cell abundances that were characteristic of the original Alexandrium culture. The growth and toxicity differences in the two isolates were demonstrated to be a property of the dinoflagellate itself and were not dependent on the different bacterial symbionts associated with each culture. The pair of subcultures is a novel experimental system to study gene expression related to toxin production and growth in dinoflagellates. The products of gene expression were analyzed in the two subcultures of Alexandrium grown under the same conditions, but where toxicity and growth differ. At the metabolome level, compounds were identified that were unique to the non-toxic isolate; their emergence may be correlated to a disruption of the biosynthetic pathway for PSP toxins.(cont.) These compounds share some characteristics and potential structural similarities with saxitoxins, though they are not any of the known toxin derivatives. Difference gel electrophoresis (DIGE) identified proteins differentially expressed between the two subcultures. Identification of some of these proteins was possible by searching the expressed sequence tag (EST) database for dinoflagellates. Proteins shown to be down-regulated in the non-toxic, slower growing subculture are all enzymes from the Calvin cycle, which may explain the limited growth of the non-toxic isolate. Other unknown, differentially expressed proteins may relate to the loss of toxicity, but their identity and function remain unresolved.by Claudia A. Martins.Ph.D

Response of the toxic dinoflagellate Alexandrium minutum to exudates of the eelgrass Zostera marina

Biotic interactions are a key factor in the development of harmful algal blooms. Recently, a lower abundance of planktonic dinoflagellates has been reported in areas dominated by seagrass beds, suggesting a negative interaction between both groups of organisms. The interaction between planktonic dinoflagellates and marine phanerogams, as well as the way in which bacteria can affect this interaction, was studied in two experiments using a non-axenic culture of the toxic dinoflagellate Alexandrium minutum exposed to increasing additions of eelgrass (Zostera marina) exudates from old and young leaves and to the presence or absence of antibiotics. In these experiments, A. minutum abundance, growth rate and photosynthetic efficiency (Fv/Fm), as well as bacterial abundance, were measured every 48 h. Toxin concentration per cell was determined at the end of both experiments. Our results demonstrated that Z. marina exudates reduced A. minutum growth rate and, in one of the experiments, also the photosynthetic efficiency. These results are not an indirect effect mediated by the bacteria in the culture, although their growth modify the magnitude of the negative impact on the dinoflagellate growth rate. No clear pattern was observed in the variation of toxin production with the treatmentAgencia Estatal de Investigación | Ref. PID2021-125643OB-C21Agencia Estatal de Investigación | Ref. CTM2017-83362-RAgencia Estatal de Investigación | Ref. FPU20/0011

The role of bacteria in paralytic shellfish poisoning

Historically the production of paralytic shellfish toxins (PST), has been attributed to dinoflagellates. However, in the last decade, increasing evidence has been presented to indicate the involvement of a wide range of bacterial species including cyanobacteria and heterotrophic bacteria (Gallacher et al., 1997). Several studies investigating bacteria capable of PST production, have identified bacteria associated with dinoflagellates are capable of autonomous PST production (Gallacher et al., 1997). However, more recent research has focussed on the effects of these bacteria on toxin production by dinoflagellates, for which the production of bacterial-free (axenic) cultures is essential to identify whether dinoflagellates are capable of autonomous toxin production, in the absence of bacteria. Many different methods to produce axenic algal cultures have been published, including washing methods and the addition of bacteriolytic compounds. However, efforts to generate axenic dinoflagellate cultures, have been hampered not only by difficulties in removing associated bacteria, but also by the lack of effective methods for assessing the presence of certain bacteria. Traditionally, the absence of bacterial growth on marine media was considered acceptable proof for axenic status. However, as the numbers of bacteria determined by culture methods falls short of numbers detected using microscopy (Akagi et al., 1977), culture methods alone have been deemed inadequate to determine the axenic status of algal cultures. In this study, the production of an axenic dinoflagellate culture was vital, firstly, to assess the effect on dinoflagellate toxin production following removal of all associated bacteria, and secondly, to identify whether original toxicity was restored when the microflora was replaced. Methods to assess the axenic nature of cultures combined traditional methods of culturing, with epifluorescence microscopy, the method now frequently relied upon for axenic confirmation. However, molecular techniques were also included, which allowed the axenic status of dinoflagellate cultures to be confidently determined. The availability of molecular techniques also enabled an assessment of the bacterial diversity associated with original dinoflagellate cultures to be conducted, with culture-based and non culture-based identification systems adopted. This investigation indicated that a diverse range of bacteria were associated with cultures, although discrepancies between the two detection methods were noted. Results from the assessment of axenic dinoflagellate cultures confirmed the need for molecular methods, as bacterial DNA was identified in cultures which were considered axenic cultures using media assessment and epifluorescence microscopy. (Abstract shortened by ProQuest.)

Toxic effects of the emerging Alexandrium pseudogonyaulax (Dinophyceae) on multiple trophic levels of the pelagic food web

The dinoflagellate Alexandrium pseudogonyaulax, a harmful algal bloom species, is currently appearing in increasing frequency and abundance across Northern European waters, displacing other Alexandrium species. This mixotrophic alga produces goniodomins (GDs) and bioactive extracellular substances (BECs) that may pose a threat to coastal ecosystems and other marine resources. This study demonstrated the adverse effects of A. pseudogonyaulax on four marine trophic levels, including microalgae (Rhodomonas salina), microzooplankton (Polykrikos kofoidii) and mesozooplankton (Acartia tonsa), as well as fish gill cells (RTgill-W1, Oncorhynchus mykiss), ultimately leading to enhanced mortality and cell lysis. Furthermore, cell-free supernatants collected from A. pseudogonyaulax cultures caused complete loss of metabolic activity in the RTgill-W1 cell line, indicating ichthyotoxic properties, while all tested GDs were much less toxic. In addition, cell-free supernatants of A. pseudogonyaulax led to cell lysis of R. salina, while all tested GDs were non-lytic. Finally, reduced egg hatching rates of A. tonsa eggs exposed to cell-free supernatants of A. pseudogonyaulax and impaired mobility of P. kofoidii and A. tonsa exposed to A. pseudogonyaulax were also observed. Altogether, bioassay results suggest that the toxicity of A. pseudogonyaulax is mainly driven by BECs and not by GDs, although further research into factors modulating the lytic activity of Alexandrium spp. are needed