THE TOXIC EFFECT OF AN OXYLIPINS-CONTAINING MACROALGAE EXTRACT ON SEA URCHIN REPRODUCTION

Abstract

The classical view of food webs, with plants and algae considered as passive participants, has been revaluated in recent years, since it is now clear that plants and algae possess complex and highly evolved chemical defenses against their grazers, including the production of bioactive molecules. Among these, oxylipins play a pivotal role in the systemic defense mechanisms of macroalgae, accumulating in response to pathogens, metals and against mechanical tissue disruption by grazers. Diatoms are known to be able to compromise the hatching success of their grazers, such as copepods and sea urchins, by releasing toxic oxylipins in the environment after cell damage and during grazing. Ericaria brachycarpa, a canopy-forming brown algae belonging to the Sargassaceae family, contribute to form the habitat in the Mediterranean Sea for the sea urchin species Arbacia lixula, whose grazing activity exerts a primary control on the abundance of canopy-forming algae, including E. brachycarpa, and the formation of barren grounds. The aim of this work was to evaluate the effects of an oxylipins-containing extract from E. brachycarpa on the development of A. lixula embryos, evaluating the fertilization success and the embryotoxic activity from fertilization (0 h) to the pluteus stage (72 h). The range of concentrations tested was chosen to cover a full 0-100 % abnormality curve, with doses ranging from 0 to 40 μg/mL. The extract was added at three developmental endpoints: zygote (0 hpf), gastrula (24 hpf) and pluteus (48 hpf) and showed a dose-dependent and developmental stage-specific effect. Gastrulae were the most sensitive to the extract with the lowest EC50 (5.366 μg/mL). At low concentrations we measured the changes of two morphological parameters to compare the ability to correctly grow and calcify in the four-armed pre-feeding echinopluteus stage: the length of the post-oral arm and larval body width. We found a great decrease of these two parameters in exposed embryos compared to controls, that may eventually lead to an impaired ability to feed and thus ultimately reduce the possibility to correctly metamorphose. The highest concentration tested (40 µg/ml) caused 100% mortality of the embryos at all stages. Western Blot experiments showed the modulation of different molecular markers (HSP60, LC3, p62, CHOP and cleaved caspase-7), showing enhanced autophagy at low concentrations and apoptosis at high concentrations. The TUNEL assay confirmed high levels of fragmented DNA in 48 h exposed embryos. These data support the hypothesis that macroalgae may exert a sort of population control against their grazers, releasing in the marine environment toxic compounds, such as oxylipins, following their tissue disruption by sea urchins and other grazers. Sea urchins intense grazing can greatly decrease the abundance of seaweeds, leading to the “sea-urchin barren ground”. Since A. lixula will potentially benefit from warmer temperatures and the population control implemented by E. brachycarpa on its reproductive success may not be sufficient in a climate change scenario, a deeper understanding of the A. lixula-E. brachycarpa interaction will have a key importance for next decades, to avoid the risk of a greater amount of barrens in the sublittoral rocky surfaces of the Mediterranean Sea.