Roberta Pennati

Tunicates, including ascidians, are recognized as the true ‘sister group’ of vertebrates and are emerging as models to study larval and post-larval development, including degeneration of central nervous system (CNS), in chordates. Ascidian larvae have the typical chordate body plan that includes a dorsal neural tube. During their metamorphosis, a deep tissue reorganization takes place, with some tissues that degenerate while others develop to become functional during the adult life. The larval CNS also degenerates, and most neurons disappear, making room for the formation of adult CNS. The genome of the ascidian Ciona intestinalis has been sequenced and annotated, with several CNS specific genes that have been characterized. These features make ascidian metamorphosis a good model to study the mechanisms underlying physiological CNS degeneration. To shed light on the molecular determinants of C. intestinalis metamorphosis, we analyzed the proteome at three stages of development: swimming larva (SwL, Hotta stage 28), settled larva (SetL, Hotta stage 32) and metamorphosing larva (MetL, Hotta stage 34). A total of 405 modulated proteins were identified by mass spectrometry by comparing the three stages. Enrichment and network analysis showed the involvement of several processes/pathways, including autophagy and mTOR pathways, and actin cytoskeleton organization and remodeling among the most significant ones. This study helps to elucidate the molecular processes underlying ascidian metamorphosis and provides insight into mechanisms of physiological neurodegeneration in ascidians.

Background: Anthropogenic noise is an emerging threat to marine ecosystems, yet its effects on marine invertebrates, particularly zooplanktonic species, remain poorly understood. Despite increasing evidence of behavioral and physiological impacts in invertebrates, the effects of noise on embryonic development and the molecular mechanisms underlying acoustic responses remain largely unexplored. Here, to address this gap, we investigated the impact of high-intensity underwater noise exposure on embryogenesis of the appendicularian tunicate Oikopleura dioica , a cosmopolitan zooplanktonic tunicate that plays important ecological roles in marine trophic webs and carbon cycling. Under lab-controlled conditions, we examined the effects of experimental noise exposure on early embryogenesis at both morphological and transcriptomic levels using RNA-seq in 8-cell (8c) and early tailbud (ETB) stages. Results: Noise exposure produced no significant increase in embryo malformations compared to non-exposed controls, indicating substantial phenotypic resilience under laboratory conditions. Interestingly, transcriptomic analyses revealed a rapid molecular response of 70 differentially expressed genes (DEG) already detectable after only 30 minutes of exposure at the 8-cell stage, which became markedly amplified with 700 DEGs by the ETB stage. Together, differential expression, GO enrichment, and co-expression network analyses identified coordinated regulation of processes associated with membrane homeostasis, pyrimidine/CTP metabolism, extracellular matrix organization, cytoskeletal architecture, RNA regulation, translational control, proteostasis, mitochondrial metabolism, and developmental pathways. Importantly, both developmental stages precede the formation of differentiated mechanosensory structures, suggesting that the observed responses are unlikely to reflect conventional sound perception. Conclusions: These findings provide the first molecular characterization of noise effects during O. dioica embryogenesis and reveal an unexpected molecular sensitivity of O. dioica embryos to underwater noise despite preserved morphological development. The transcriptional signatures support a mechanobiological framework in which acoustic exposure may directly perturb cellular mechanical homeostasis through membrane-and cytoskeleton-associated processes, triggering compensatory stress-adaptation responses involving proteostasis, RNA regulation, and metabolic reprogramming. Together, these findings establish O. dioica as a valuable emerging model for investigating the developmental and evolutionary consequences of acoustic pollution in marine ecosystems.

Tunicates, including ascidians, are recognized as the true ‘sister group’ of vertebrates and are emerging as models to study the development and degeneration of central nervous system (CNS). Ascidian larvae have the typical chordate body plan that includes a dorsal neural tube. During their metamorphosis, a deep tissue reorganization takes place, with some tissues that degenerate while others develop to become functional during the adult life. The larval CNS also degenerates and most neurons disappear, making room for the formation of adult CNS. The genome of the ascidian Ciona intestinalis has been sequenced and annotated, with several CNS specific genes that have been characterized, revealing specification mechanisms shared with humans. These features make ascidian metamorphosis a good model to study the mechanisms underlying physiological CNS degeneration and to compare them to the pathological conditions typical of neurodegenerative diseases. In order to shed light on the molecular determinants of C. intestinalis metamorphosis and neurodegeneration, we analyzed the proteome at three stages of development: swimming larva (SwL, Hotta stage 28), settled larva (SetL, Hotta stage 32) and metamorphosing larva (MetL, Hotta stage 34). A total of 405 modulated proteins were identified by mass spectrometry by comparing the three stages. Enrichment and network analysis showed the involvement of several processes/pathways, including autophagy and mTOR pathway, and actin cytoskeleton organization and remodeling among the most significant ones. This study elucidates molecular pathways underlying ascidian metamorphosis and highlights shared mechanisms between physiological neurodegeneration in ascidians and pathological neurodegeneration in humans.

Crinoids belong to the Echinodermata, marine invertebrates with a highly derived adult pentaradial body plan. As the sister group to all other extant echinoderms, crinoids occupy a key phylogenetic position to explore the evolutionary history of the whole phylum. However, their development remains understudied compared with that of other echinoderms. Therefore, the aim here was to establish the Mediterranean feather star ( Antedon mediterranea ) as an experimental system for developmental biology. We first set up a method for culturing embryos in vitro and defined a standardized staging system for this species. We then optimized protocols to characterize the morphological and molecular development of the main structures of the feather star body plan. Focusing on the nervous system, we showed that the larval apical organ includes serotonergic, GABAergic and glutamatergic neurons, which develop within a conserved anterior molecular signature. We described the composition of the early post-metamorphic nervous system and revealed that it has an anterior signature. These results further our knowledge on crinoid development and provide new techniques to investigate feather star embryogenesis. This will pave the way for the inclusion of crinoids in comparative studies addressing the origin of the echinoderm body plan and the evolutionary diversification of deuterostomes.

Photosymbiosis indicates a long-term association between animals and photosynthetic organisms. It has been mainly investigated in photosymbiotic cnidarians, while other photosymbiotic associations have been largely neglected. The acoel Symsagittifera roscoffensis lives in obligatory symbiosis with the microalgal Tetraselmis convolutae and has recently emerged as alternative model to study photosymbiosis. Here, we investigated the effects of Bisphenol A, a common plastic additive, on two pivotal stages of its lifecycle: aposymbiotic juvenile development and photosymbiogenesis. Based on our results, this pollutant altered the development of the worms and their capacity to engulf algae from the environment at concentrations higher than the levels detected in seawater, yet aligning with those documented in sediments of populated areas. Data provide novel information about the effects of pollutants on photosymbiotic associations and prompt the necessity to monitor their concentrations in marine environmental matrices.

Serotonin (5-hydroxytryptamine (5-HT)) is a biogenic monoamine with pleiotropic functions. It exerts its roles by binding to specific 5-HT receptors (5HTRs) classified into different families and subtypes. Homologs of 5HTRs are widely present in invertebrates, but their expression and pharmacological characterization have been scarcely investigated. In particular, 5-HT has been localized in many tunicate species but only a few studies have investigated its physiological functions. Tunicates, including ascidians, are the sister group of vertebrates, and data about the role of 5-HTRs in these organisms are thus important for understanding 5-HT evolution among animals. In the present study, we identified and described 5HTRs in the ascidian Ciona intestinalis. During development, they showed broad expression patterns that appeared consistent with those reported in other species. Then, we investigated 5-HT roles in ascidian embryogenesis exposing C. intestinalis embryos to WAY-100635, an antagonist of the 5HT1A receptor, and explored the affected pathways in neural development and melanogenesis. Our results contribute to unraveling the multifaceted functions of 5-HT, revealing its involvement in sensory cell differentiation in ascidians.

Organic pollutants with disrupting activity on the endocrine system are known as endocrine disrupting compounds (ECDs) and are broadly diffuse on the Planet. By different pathways, they reach the marine environment that is the ultimate collector of these substances. Several researches pointed out acute and chronic effects of these pollutants on the reproduction and development of fish and marine mammals. Fewer studies have been dedicated to explore the effects of ECDs on marine invertebrates though these represent most of marine biodiversity. Researches are hampered by limited knowledge of endocrinology and physiology of several animal groups, which has been studied only for few phyla such as mollusks, annelids and arthropods. The clearest example of endocrine disruption by a chemical compound in marine environment is the pseudo-hermaphroditism caused in females of prosobranch mollusks by the exposure to tributyltin, an antifouling agent used in the paints for ships. This molecule has an adverse effect also on crustaceans in which it inhibits the signaling pathway of the molting hormone. Several studies evidenced that lot of marine invertebrate species are sensible to the action of estrogenic compounds (xeno-estrogens) such as Bisphenol A and Nonylphenol that are used in the manufacturing of the plastic materials. Nevertheless, molecular mechanisms responsible for the observed effects are still uncertain. In my laboratory, we demonstrated that tunicates, that do not have estrogen receptors, are sensible to the action of xeno-estrogens that act by binding to a different group of receptors, the Estrogen Related Receptors. Since these receptors are present also in mammals, our studies could help to clarify pathogenic mechanisms shared with humans.

Monolistra are stygobiont isopods spread in numerous karst areas from the south-eastern parts of Dinaric karst to south-eastern Switzerland. They inhabit different typologies of groundwater habitats from lentic to lotic ones where they can be particularly important in terms of biomass. M. pavani is a steno-endemic species that was reported only for a single cave system in Lombardy (Italy). From October 2018 to March 2022, we performed multiple surveys in the subterranean system and in its surrounding springs recording abundance and distribution of the species. In December 2018 we discovered three spring populations/subpopulations of M. pavani in which pigmented individuals also occurred. We performed UV and infrared spectroscopy to evidence potential differences in the signals related to C-C bonds of the aromatic ring of the melanin molecules between cave and spring individuals. In 2020, we also compared circadian rhythm and responses to light stimuli of cave and spring individuals. Occurrence of M. pavani in springs was regular and associated to specific spring microhabitats; individuals were active both during day and during night. Nor spring and cave individuals avoid light stimuli. Circadian rhythm analyses revealed that individuals from springs are more active than those from cave, showing higher activity during day than during night. The 20% of spring individuals are pigmented, and eumelanin occurs in dark individuals. Our results suggest the possible ongoing adaptation to surface habitats at the border with groundwater in a species considered as a strict stygobiont. Molecular analyses on the population relationships are currently ongoing.

Microplastics are contaminants of global environmental concern. They can be ingested by a variety of organisms when they enter the food web. Several studies have reported trophic transfer of microplastics from low trophic levels to higher ones. Bioaccumulation has been suggested to occur but few studies have demonstrated it for marine environments. In this article, in controlled laboratory conditions, we exposed filter‐feeder ascidian juveniles to microplastics in the presence or in absence of mixotrophic cryptomonad flagellates. Cryptomonads can efficiently ingest microbeads, and their presence significantly increased the concentration of microplastics in the digestive tract of the ascidians. Our results demonstrate the occurrence of microplastic bioaccumulation in the lower levels of the marine trophic chain and suggest that unicellular organisms can be key actors in microplastic trophic transfer at the microscale level.