Investigation of biological factors that may contribute to bioactivity in Haliclona (Porifera, Haplosclerida)
Marra, Maria Vittoria
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Sponges (phylum Porifera) are considered the most prolific source of bioactive compounds of marine origin. These molecules have varied chemical nature and have been shown to possess several properties, e.g. antibacterial, antiviral, antifungal, anti-inflammatory, antitumor. For the sponges they play important ecological roles as antipathogenic, antipredatory, antifouling and anti-overgrowth agents. Sponges are also known to host a large array of microbial symbionts such as heterotrophic bacteria, cyanobacteria, archaea, fungi, and microalgae. Based on the amount of microorganisms living within their tissues, sponges have been classified as high microbial abundance (HMA) and low microbial abundance (LMA) species, even though this classification seems to be too simplistic in some cases. For some potent bioactive compounds isolated from sponges, the associated microorganisms (especially heterotrophic and phototrophic bacteria as well as dinoflagellates) have been shown to be the true producers. The general aim of this project was to investigate possible biological mechanisms underlying the bioactivity detected in several Irish and Mediterranean sponge species currently placed into the genus Haliclona (Grant, 1841), i.e. H. oculata, H. mediterranea, H. cinerea, H. fulva, H. mucosa, H. simulans, H. sarai, H. viscosa and H. indistincta. The nine species studied were selected as representative of the main phylogenetic clades within the order Haplosclerida with the idea that all the information gathered could also contribute to solve the challenging taxonomic classification of the species within this order via an integrative taxonomy approach. In this sense, I firstly investigate the bacteria and archaea associated with the five Irish Haliclona species using transmission electron microscopy (TEM) and a next generation sequencing approach. Both methods determined all the sponges are LMA with species-specific microbial associations. However, the 16S rRNA gene data showed a high bacterial diversity that is incongruent with the sparse microbial diversity observed via TEM, suggesting that ecological factors such as diet and substrate contribute in shaping the predominant bacterial communities observed in these sponges. The TEM analysis also showed the presence of putative intercellular fungal spores, thus I have applied a cultivationdependent method to analyse the fungi associated with all target Haliclona species. Also in this case the results obtained suggested a possible environmental origin for the fungi isolated and therefore a 16 doubtful involvement of these microorganisms in the production of any bioactive compounds. I also used TEM to characterise cells with inclusions in the target Haliclona species because these types of cell have been associated with the production and/or storage of bioactive compounds in numerous other sponges. This has given me the opportunity to describe a novel type of cell with inclusions abundant in the tissue of H. indistincta, H. viscosa and H. sarai. These cells show a remarkably similar morphology to mucus-producing cells in higher animals and in fact all three species produce a similar type of thick sticky mucus. The attempts made to isolate these cells and characterise them chemically in order to determine if they contain the bioactive compounds have failed, thus I have chased the putative mucous nature of these cells to obtain a marker that could allow their isolation. For this reason I carried out a proteomic study of H. indistincta aimed at ascertaining the presence of mucins in this species. As comparison, I performed the same analysis on other two target Haliclona species producing mucus with different physical properties, i.e. H. cinerea and H. mucosa. The analysis showed that no true mucins are present in any of the three species, but all of them produce different mucin-like proteins. The further characterisation of these glycoproteins could give additional clues to help isolate the cells with inclusions in H. indistincta, H. viscosa and H. sarai, but also to investigate the possible role played by the mucin-like proteins in shaping the microbial communities hosted by the sponge species considered.