A Study of Gambierdiscus-Associated Toxins: Toxin Characterization, Toxicological Properties, and Microbial Effects

岡比亞藻毒素的研究︰毒素表徵,毒理學效應及微生物作用

Student thesis: Doctoral Thesis

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Award date3 May 2024

Abstract

Gambierdiscus is a genus of marine autotrophic epi-benthic dinoflagellate that thrives on the surfaces of macroalgae, corals, and sand grains. This genus is the primary producer of ladder polycyclic ether toxins, including ciguatoxins (CTXs), maitotoxins (MTXs), gambierones, gambierol, gambieric acids, and gambieroxide. Most of these toxins can be transferred into higher trophic levels through food webs. Besides, they have the potential to be transformed into more toxic compounds through oxidation processes within animal tissues. The consumption of seafood contaminated with these toxins may cause Ciguatera Food Poisoning (CFP), which is a prevalent non-microbial foodborne illness occurring worldwide. To date, the frequency of the CFP events has been steadily increasing annually posing a significant risk to both human health and fishery economy. In addition to influencing human health, previous studies have shown that Gambierdiscus toxins, for example, P-CTX-1, P-CTX-3C, and C-CTX-1 had detrimental effects on fish physiology and behaviors. To address the problems caused by Gambierdiscus, more studies should focus on toxin detection and new toxin discovery, toxin bioactivities, and mechanisms of toxin production.

Currently, HPLC-MS/MS is the most reliable method for toxin detection and quantification. However, the target analysis heavily relies on toxin standards, resulting in many unknown toxins escaping from detection. On the other hand, non-target analysis needs expertise in discerning toxins from all secondary metabolites based on analyzing ion fragments. Besides, this process is also time-consuming and demands significant labor hood. Therefore, there is an urgent need to develop new methodologies to detect and identify these toxins effectively. Gambierdiscus toxins displayed various bioactivities, including voltage-gated ion channel activation, cell firing, and antifungal activities. Among these toxins, CTXs and MTXs are recognized as major contributors to CFP. Gambierone and 44-methylgambierone exhibit the most widely distribution in different Gambierdiscus species. However, their roles in CFP were not clear. Toxicological studies have shown that CTXs can induce behavioral and morphological alterations in fish, making them susceptible to natural selection pressures. The toxicological study of gambierones on marine fish remains limited. 44-Methylgambierone served as representative hydrophobic toxins produced by Gambierdiscus species. Investigating its toxicological properties and transformation mechanisms will significantly contribute to our comprehension of the impact of Gambierdiscus on marine animals. In addition to chemical analysis and toxicological study, factors that govern algal proliferation and toxin production should also be investigated in this research field. For the biotic factors, previous studies have been mainly confined to the bacteria associated with Gambierdiscus and their involvement in algal toxin production. However, the heterotrophic heterokonts as an important type of eukaryotic microorganism coexisting with Gambierdiscus in nature, their influence on Gambierdiscus has not been documented yet. Considering these research gaps, four objectives were proposed in this study, including (1) the development of the method for toxin detection and discovery, (2) the characterization of new toxins, (3) the investigation of toxin bioactivities, and (4) the factors influencing toxin production.

Firstly, we established an automatic MS/MS data mining strategy for high-throughput screening of polyether compounds from crude extracts of Gambiediscus species. Our results demonstrated a high confidence level in identifying known Gambierdiscus toxins, enabled automatic extraction, and visualized the polyether compound features, facilitating the search for new toxins. By using this method, we comprehensively investigated the toxin profile of four Gambierdiscus strains. Our result showed that three G. caribaeus strains produced gambierone and 44-methylgambierone, with a higher level of the former compound. Besides, G. caribaeus GCBG02 and G. caribaeus S4 also produced 12,13-dihydro-44-methylgambierone. G. balechii has been shown to produce several gambierones with a confidence level above level 3, including gambierone, 44-methylgambierone, 12,13-dihydro-44-methylgambierone, 38-dehydroxy-44-methylgambierone, and desulfo-hydroxyl-gambierone. Furthermore, three out of the four investigated strains showed potential production of P-CTXs. Further structural elucidation was necessary through MS2 fragmentation and NMR analyses.

Secondly, four new and two known gambierones were characterized from G. balechii 1123M1M10 through HPLC/ESI-MS2 analysis, and their fragmentation behaviors and pathways were studied. 44-Methylgambierone and 12,13-dihydro-44-methylgambierone were the major toxins in this strain.

Thirdly, we used the medaka fish model to investigate the toxicity of 44-methylgambierone. The results revealed that 44-methylgambierone can be absorbed by the yolk sac and distributed throughout the entire body of the fish. Furthermore, exposure to 44-methylgambierone impaired larval locomotion with a U-shaped dose-response trend. Specifically, exposure to 0.34 ± 0.03 pg of 44-methylgambierone significantly inhibits heartbeats of medaka larvae. The enriched gene ontology (GO) biological processes and Kyoto Encyclopedia of Gene and Genomes (KEGG) analysis shed light on the alterations in pathways associated with the eye visual system, neural system, heart, hormone regulation, and reproduction.

Lastly, we identified a heterotrophic heterokont from a contaminated Gambierdiscus culture. Then a co-culture experiment was conducted to investigate the interaction between the heterotrophic heterokont and Gambierdiscus. The results showed that the infection by Pseudobodo sp. inhibited the algal proliferation and decreased the cardiotoxicity of algal extracts in the zebrafish model. The systematic chemical analysis of G. balechii discovered three types of toxins, including gambierones, maitotoxin (MTX)-like mega-molecules, and polyol-polyene super-carbon chain compounds. The cellular production of gambierones and MTX-like mega-molecules was reduced by the infection of Pseudobodo sp., which accounts for the detoxification effect on the highly toxic algae, whereas the polyol-polyene super-carbon chain compounds showed a more complicated variation in their cellular production. In addition, Pseudobodo sp. altered the microbiome abundance in the algal culture. The detailed relationships among Pseudobodo sp., G. balechii 1123M1M10, and bacterial community need further study. Taken together, our findings shed light on the complex interactions surrounding dinoflagellates in nature.

In conclusion, this study provided a strategy for the discovery of marine polyether compounds. Additionally, the investigation of the toxin profile of Gambierdiscus species, characterization of new toxins, exploration of the toxicity of representative toxins, and examination of the factors influencing toxin production will serve as a foundation for further research on Gambierdiscus toxins.

    Research areas

  • Gambierdiscus, Polyether compounds, 44-Methylgambierone, Marine medaka, Transcriptome, Pseudobodo sp.