Exploring the sexual phase of the diatom <i>Pseudo-nitzschia multistriata</i>: from genes to metabolites

Phytoplankton are an important component of the marine ecosystems since they are at the base of marine food webs and are responsible for about 50% of the oxygen production at a global scale. The knowledge of phytoplankton life cycles is important to understand the mechanisms regulating the dynamics of their populations in the natural environment but also to improve the capability to cultivate them and thus exploit their biotechnological potential. The model organisms chosen for my PhD project is a marine planktonic diatom, Pseudo-nitzschia multistriata, known to produce the neuro-toxin domoic acid (DA), the causative agent of Amnesic Shellfish Poisoning.Pseudo-nitzschia multistriata is a pennate heterothallic species whose life cycle has been described and can be controlled in laboratory conditions. Cells of the opposite Mating Types (MTs) can switch from asexual to sexual reproduction after reaching a threshold size and it is very important that this process is finely synchronized and regulated to ensure its success. There is evidence of a chemical cross talk that mediates mating and some of the genes involved in the process were identified.In this thesis, several aspects related to the reciprocal perception of the opposite MTs and the comparison a between a ‘treatment’ in which sexual reproduction was ongoing as compared to the ‘control’ of monocultures of single parental strains were untangled focusing on a set of genes involved in the process.Several experiments illustrated in Chapter 2 were aimed at i) further elucidating the expression pathways of the target genes and ii) identifying a set of genes that could be used as marker genes for future isolation of putative pheromones. The results of the experiments suggest that in P. multistriata there are two constitutive pheromones called MRP1 and 7488 produced by MT+ and MT-, respectively. Among these the gene 7488 was upregulated in MT- exposed to the culture medium in which MT+ was growing; this gene can thus be considered the as the molecular marker for a bio-assay to detect the fraction of MT+ conditioned medium containing the putative pheromone. The experiments carried out in this thesis also suggested that that the constitutive putative pheromone of MT+ perceived by MT- could be MRP1, a gene encoding for a small secreted protein, that is differentially expressed between opposite MTs and is highly induced during sex in the MT+.In Chapter 3, I have illustrated the spatial pattern of a set of genes involved in the sexual phase using the large database of genes from the Tara Oceans expedition. The co-occurrence of MT related genes and one meiotic gene in nine TARA stations suggests that sexual reproduction was occurring in those sites. The spatial distribution of these genes was not uniform and this can be explained with differences in their basal expression levels and/or by the absence of species in which the genes are present.Finally, in Chapter 4 I explored the difference between metabolites produced by the parental strains in vegetative growth in monoculture and by a co-culture of strains of opposite MT undergoing sex. The metabolomics analysis revealed that there were not exclusive metabolites distinguishing the vegetative phase and the sexual phase, but there were a number of mostly unidentified metabolites that increased their quantity in the sexual phase. The results of this thesis add novel information on several aspects concerning the mate perception of a planktonic diatom, elucidating hierarchical activation of genes during the sexual phase. Furthermore, with future identification of the constitutive cue of MT+, MT- gene expression changes in response to the first MT+ signal might be clearly elucidated using RNAseq. Last but not least, this work indicates that genes related to sex could be makers for sexual detection at sea, becoming a useful tool for ecological purposes

Angitola lake sediments: preliminary data and biotic indices

The Angitola lake is an artificial basin located in Calabria Region, in South Italy, part of the “Natural Regional Park of Serre”. Its surface area is 1.96 km2 and it is approximately 3 km away from Sant’Eufemia gulf. The basin was created in 1966 by damming the homonymous river. Four major rivers enter the lake: three in the far southeast, the fourth, smaller and active only during the winter period, in the southwest area. The international Ramsar Convention includes the basin and surroundings; in light of the Rio Convention, its directives have been transposed in the “Birds Directive” (BD) and “Habitats Directive” (HD). The Angitola lake, entrusted to the WWF Calabria, is one of the 2299 Italian SIC (code IT9340086): this area significantly contributes in maintaining and restoring the endangered freshwater habitat listed in HD, in protecting biodiversity of the region and it is part of the Natura 2000 network. The present study is part of the Angitola FISH2O project (European Fisheries Fund/FEP code 02/BA/12) and it aimed to examine the benthic macro-invertebrates community of the southeast part of Angitola lake. The first 15 cm in depths of sediments have a very variable composition, from fine sand to mud (rich in organic matter). This variability can be explained by the different characteristic of the chosen sampling transepts. Fauna sampling, carried out by core drills and plankton nets, shows presence of Diptera and Tricoptera larvae and some Nematoda, Polichaeta and Mollusca. Preliminary conclusion is that the Angitola lake is a diversified environment in which areas with different anthropic interference are present. The benthic macro-invertebrates community might be involved by possible disturbances induced by chemicals (e.g. water pollution) and/or physical variations (e.g. high sedimentation). Medium and long-term investigations are imperative to protect and promote the lake biodiversity, to verify the effects of seasonal fluctuations and how these are related to human activities such as tourism, demographic increase and industrial activities

A Metabolomics Exploration of the Sexual Phase in the Marine Diatom Pseudo-nitzschia multistriata.

Pseudo-nitzschia multistriata is a planktonic marine diatom with a diplontic life cycle comprising a short sexual phase, during which gametes are produced following the encounter of two diploid cells of opposite mating type (MT). Gene expression studies have highlighted the presence of substantial changes occurring at the onset of sexual reproduction. Herein, we have hypothesized that the amount and nature of cellular metabolites varies along the mating process. To capture the metabolome of Pseudo-nitzschia multistriata at different harvesting times in an unbiased manner, we undertook an untargeted metabolomics approach based on liquid chromatography-tandem mass spectrometry. Using three different extraction steps, the method revealed pronounced differences in the metabolic profiles between control cells in the vegetative phase (MT+ and MT-) and mixed strains of opposite MTs (cross) undergoing sexual reproduction. Of the 2408 high-quality features obtained, 70 known metabolites could be identified based on in-house libraries and online databases; additional 46 features could be classified by molecular networking of tandem mass spectra. The reduction of phytol detected in the cross can be linked to the general downregulation of photosynthesis during sexual reproduction observed elsewhere. Moreover, the role of highly regulated compounds such as 7-dehydrodesmosterol, whose changes in abundance were the highest in the experiment, oleamide, ectoine, or trigonelline is discussed

Mate Perception and Gene Networks Regulating the Early Phase of Sex in Pseudo-nitzschia multistriata

Diatoms are photosynthetic microorganisms playing a key role in the functioning of aquatic ecosystems; they are at the base of the food web and are the main drivers of biogeochemical processes. These microalgae have a unique diplontic life cycle in which the vegetative phase entails a cell size reduction that would lead to the extinction of the cell population if the size was not restored, usually by sexual reproduction. The switch from asexual to sexual reproduction needs to be finely synchronized and regulated to ensure its success; to this aim, cells evolved complex chemical crosstalk that mediates mating. We focused our attention on the marine diatom Pseudo-nitzschia multistriata, investigating the reciprocal perception of the opposite mating type (MT) and the genes and signaling molecules putatively involved in the process. From previously available transcriptomic data, we selected a panel of genes deregulated during the early phase of sexual reproduction, confirming for some of them a role during mate perception and establishing a hierarchy governing their behavior. Moreover, we explored the nature of the molecules controlling sexual reproduction in this species, unveiling that the signaling is mediated by the secretion of protein and non-protein cues from the MT&minus; and MT+, respectively. Our results point to a model in which the two MTs stimulate each other, but the stimulation by the MT&minus; is amplified after MT+ perception in a positive feedback manner

Exploring tomato Solanum pennellii introgression lines for residual biomass and enzymatic digestibility traits

Background: Residual biomass production for fuel conversion represents a unique opportunity to avoid concerns about compromising food supply by using dedicated feedstock crops. Developing tomato varieties suitable for both food consumption and fuel conversion requires the establishment of new selection methods. Results: A tomato Solanum pennellii introgression population was assessed for fruit yield, biomass phenotypic diversity, and for saccharification potential. Introgression lines 2-5, 2-6, 6-3, 7-2, 10-2 and 12-4 showed the best combination of fruit and residual biomass production. Lignin, cellulose, hemicellulose content and saccharification rate showed a wide variation in the tested lines. Within hemicellulose, xylose value was high in IL 6-3, IL 7-2 and IL 6-2, whereas arabinose showed a low content in IL 10-2, IL 6-3 and IL 2-6. The latter line showed also the highest ethanol potential production. Alkali pre-treatment resulted in the highest values of saccharification in most of lines tested, suggesting that chemical pretreatment is an important factor for improving biomass processability. Interestingly, extreme genotypes for more than one single trait were found, allowing the identification of better genotypes. Cell wall related genes mapping in genomic regions involved into tomato biomass production and digestibility variation highlighted potential candidate genes. Molecular expression profile of few of them provided useful information about challenged pathways. Conclusions: The screening of S. pennellii introgression population resulted very useful for delving into complex traits such as biomass production and digestibility. The extreme genotypes identified could be fruitfully employed for both genetic studies and breeding