Isolation, Structure Elucidation, Relative LC-MS Response, and in Vitro Toxicity of Azaspiracids from the Dinoflagellate <i>Azadinium spinosum</i>

We identified three new azaspiracids (AZAs) with molecular weights of 715, 815, and 829 (AZA33 (<b>3</b>), AZA34 (<b>4</b>), and AZA35, respectively) in mussels, seawater, and <i>Azadinium spinosum</i> culture. Approximately 700 μg of <b>3</b> and 250 μg of <b>4</b> were isolated from a bulk culture of <i>A. spinosum</i>, and their structures determined by MS and NMR spectroscopy. These compounds differ significantly at the carboxyl end of the molecule from known AZA analogues and therefore provide valuable information on structure–activity relationships. Initial toxicological assessment was performed using an in vitro model system based on Jurkat T lymphocyte cytotoxicity, and the potencies of <b>3</b> and <b>4</b> were found to be 0.22- and 5.5-fold that of AZA1 (<b>1</b>), respectively. Thus, major changes in the carboxyl end of <b>1</b> resulted in significant changes in toxicity. In mussel extracts, <b>3</b> was detected at low levels, whereas <b>4</b> and AZA35 were detected only at extremely low levels or not at all. The structures of <b>3</b> and <b>4</b> are consistent with AZAs being biosynthetically assembled from the amino end

Reported allelopathic effects of <i>Ulva</i> spp. and <i>Zostera</i> spp. on harmful algal blooms dinoflagellate species in various marine ecosystems.

<p>Reported allelopathic effects of <i>Ulva</i> spp. and <i>Zostera</i> spp. on harmful algal blooms dinoflagellate species in various marine ecosystems.</p

Light microscope observations of morphological damages of vegetative cells of the targeted dinoflagellate species.

<p>Photographs of <i>Alexandrium pacificum</i> cells cultured with <i>Cymodocea nodosa</i> (a-e) and <i>Ulva rigida</i> (f-j); and of <i>Ostreopsis</i> cf. <i>ovata</i> cultured with <i>Ulva rigida</i> (k-o). a,f,k = control cells; b-e, g-j, l-o = cells under increasing macrophyte weights. Scale bars, 10 μm.</p

Phytochemicals associated with <i>Ulva rigida</i>, <i>Zostera noltei</i> and <i>Cymodocea nodosa</i> species with their reported biological activity.

<p>Phytochemicals associated with <i>Ulva rigida</i>, <i>Zostera noltei</i> and <i>Cymodocea nodosa</i> species with their reported biological activity.</p

Cellular toxin contents (pg.cell^-1) at the end of the experiments (after 10 days) of <i>Ostreopsis</i> cf. <i>ovata</i> (<i>O</i>. cf. <i>ovata</i>) and <i>Prorocentrum lima</i> (<i>P</i>. <i>lima</i>) in presence of the leaves/thalli of <i>Cymodocea nodosa</i> (<i>C</i>. <i>nodosa</i>), <i>Zostera noltei</i> (<i>Z</i>. <i>noltei</i>) and <i>Ulva rigida</i> (<i>U</i>. <i>rigida</i>), and of <i>Alexandrium pacificum</i> (<i>A</i>. <i>pacificum</i>) in presence of <i>C</i>. <i>nodosa</i> leaves.

<p>OVTX-a: Ovatoxin-a; OVTX-b: Ovatoxin-b; OA: Okadaic Acid; DTX-1: Dinophysistoxin-1; Neo-STX, GTX1, GTX3 and GTX4: Carbamoyl toxins; C1 and C2: N-sulfocarbamoyl toxins. ‘< LoD’ and ‘< LoQ’ indicate ‘< Limit of Detection’ and ‘< Limit of Quantification’, respectively. Error bars correspond to the standard deviation (N = 3 replicates, except for control (<i>O</i>. cf. <i>ovata</i> and <i>P</i>. <i>lima</i>) for which the controls of the three experiments have been pooled, N varying between 3 and 9 depending on the considered toxin). When only one among the three triplicates of each treatment was above LoD or LoQ, standard deviation was not calculable, and there is thus no error bar in such cases.</p

Macrophyte collection sites (North of Tunisia, Southern Mediterranean Sea).

<p>Circle: Menzel Jemil station; Triangle: Menzel Bourguiba station.</p

Light (a₁,a₁’,b₁,b₁’), epifluorescence (a₂,a₂’,b₂,b₂’) and superposed light-epifluorescence (a₃,a₃’,b₃,b₃’) microscope photographs of dinoflagellate vegetative cells cultured with <i>Ulva rigida</i> thalli.

<p><b>A</b>: <i>Alexandrium pacificum</i> cells (a₁-a₂-a_{3 =} control, a₁’-a₂’-a₃’ ₌ cell exposed to 0.16g (FW) of <i>Ulva rigida</i> after 3 days of co-culture). <b>B</b>: <i>Ostreopsis</i> cf. <i>ovata</i> cells (b₁-b₂-b_{3 =} control; b₁’-b₂’-b₃’ ₌ cell exposed to 1g FW of <i>Ulva rigida</i> after 10 days of co-culture). Scale bars, 10 μm.</p