“Ciona robusta (formerly Ciona intestinalis type A) as model system for ecotoxicological studies”

The invertebrate urochordate Ciona robusta (formerly Ciona intestinalis type A) is a sessile marine benthic organism distributed worldwide that attaches to the surfaces of both natural and artificial substrates, from shallow water to the deep sea. Based on its phylogenetic position as basal chordate, from more than a century Ciona represented an excellent model system for studying developmental biology, thanks to the rapid embryonic and larval development, resemblance to vertebrates, ease of management, low cost, transparent body, low risk of ethical issues and a number of techniques and genomic resources developed in the course of the years. Recently, in light of such interesting features, Ciona has been used to evaluate the embriotoxicity of legacy pollutants (e.g. heavy metals, pesticides, organic compounds). Notably, the genetic, genomic and molecular tools available for Ciona allow a deeper investigation of the molecular mechanisms affected by pollutants and could provide insights on their mode of actions (MoA). Within this thesis, the effects of two classes of contaminants of emerging concerns (CECs) have been tested on the embryogenesis of C. robusta. CECs are a group of natural and synthetic chemicals including nanoscale particles and transformation products, which have been increasingly found at low levels in surface waters. These compounds may pose a risk to aquatic life and, thus, it is fundamental to assess their potential effects on marine organisms. In detail, the effects of two dispersants named as A and B, used for cleaning up the petroleum hydrocarbon contamination in case of accidental oil spills at sea, and polystyrene nanoparticles (PS NPs), as proxy for nanoplastics have been investigated on C. robusta larval development. The four chapters of the thesis report findings on the effects of the two dispersants and PS NPs bearing different surface charges by using two approaches: 1) embryotoxicity, by looking at adverse effects in developing embryos and sub-lethal biological responses on functional proteins and enzymes; 2) mechanisms of action (MoA) at molecular level using different techniques as Real-Time PCR, RNA sequencing and bioinformatics. We demonstrated how embriotoxicity in C. robusta could represent a useful tool to evaluate the impact of dispersants on marine species. The data obtained indicated a different toxicity between dispersants A and B, confirmed also by phenotype alterations. Moreover, the evaluation of the expression of selected genes involved in stress response (SODa, SODb, MnSOD, GPx, HSP60, HSP70), detoxification (Cyp450, GST, GluR) and cell survival (p38, Cas8) indicated dispersant B as teratogen while dispersant A having less impact on C. robusta larvae. Regarding nanoplastic, surface charges seem to play a significant role in the observed embryotoxicity of the amino-modified PS NPs (PS-NH2) in agreement with their behavior in exposure media. No effects were found for carboxyl-modified PS NPs (PS-COOH) on C. robusta embryo development while from mild to severe 2 phenotype alterations were observed upon exposure to PS-NH2, including behavioral traits (e.g. swimming performances). Among those mostly evident, embryos resulted unable to hatch and several abnormal phenotypes were found. In addition, induction of oxidative stress linked to an increase of ROS production and the down-regulation of some representative genes involved in stress response (HSP70, HSP60, MnSOD, cytochrome b, p-38 mapk and caspase 8) were observed. The analysis of transcriptome, through differential RNA-seq, allowed to identify altered pathways affected by PS-NH2. Several genes resulted dysregulated upon the exposure to PS-NH2, while the GO analysis, which classified genes in three different subclasses, revealed that the number of genes affected, belonging to different subclasses, have a dose-response relationship with the concentration tested. Going deeply into the bioinformatic analysis, “glutathione synthesis and recycling pathway”, “neurotransmitter clearance pathway”, “passive transport by aquaporins” and “fructose and mannose metabolism”, “starch and sucrose metabolism” and “glycolysis” pathways resulted affected. The alteration of these pathways could be related to the hypoxic microenvironment due to the dense coating of PS-NH2 around the egg envelopes of Ciona embryos. Similar findings in terms of embryotoxicity and phenotype alterations have been observed in another ascidian species, Phallusia mammillata, exposed in similar conditions to amino-modified PS NPs (PS-NH2). Furthermore, the quantitative analyses of Phallusia phenotype using the software Toxicosis8, revealed the affection of both central and peripheral nervous system. The use of C. robusta embryos as a model to study the effects of dispersants and PS NPs as proxy for nanoplastics proved to be instrumental in shedding light on different aspects of developmental toxicity exerted by those CECs. Moreover, these results will hopefully provide important information useful for higher and more complex chordates.