A broad-taxa approach as an important concept in ecotoxicological studies and pollution monitoring.

Aquatic invertebrates play a pivotal role in (eco)toxicological assessments because they offer ethical, cost-effective and repeatable testing options. Additionally, their significance in the food chain and their ability to represent diverse aquatic ecosystems make them valuable subjects for (eco)toxicological studies. To ensure consistency and comparability across studies, international (eco)toxicology guidelines have been used to establish standardised methods and protocols for data collection, analysis and interpretation. However, the current standardised protocols primarily focus on a limited number of aquatic invertebrate species, mainly from Arthropoda, Mollusca and Annelida. These protocols are suitable for basic toxicity screening, effectively assessing the immediate and severe effects of toxic substances on organisms. For more comprehensive and ecologically relevant assessments, particularly those addressing long-term effects and ecosystem-wide impacts, we recommended the use of a broader diversity of species, since the present choice of taxa exacerbates the limited scope of basic ecotoxicological studies. This review provides a comprehensive overview of (eco)toxicological studies, focusing on major aquatic invertebrate taxa and how they are used to assess the impact of chemicals in diverse aquatic environments. The present work supports the use of a broad-taxa approach in basic environmental assessments, as it better represents the natural populations inhabiting various ecosystems. Advances in omics and other biochemical and computational techniques make the broad-taxa approach more feasible, enabling mechanistic studies on non-model organisms. By combining these approaches with in vitro techniques together with the broad-taxa approach, researchers can gain insights into less-explored impacts of pollution, such as changes in population diversity, the development of tolerance and transgenerational inheritance of pollution responses, the impact on organism phenotypic plasticity, biological invasion outcomes, social behaviour changes, metabolome changes, regeneration phenomena, disease susceptibility and tissue pathologies. This review also emphasises the need for harmonised data-reporting standards and minimum annotation checklists to ensure that research results are findable, accessible, interoperable and reusable (FAIR), maximising the use and reusability of data. The ultimate goal is to encourage integrated and holistic problem-focused collaboration between diverse scientific disciplines, international standardisation organisations and decision-making bodies, with a focus on transdisciplinary knowledge co-production for the One-Health approach.

The stress granule component TIAR during the non-embryonic development of the colonial ascidian Botryllusschlosseri.

TIAR, is a nucleic acid binding protein involved in the formation of cytoplasmic foci known as stress granules, in which mRNA translation is temporarily blocked in response to stressful conditions. TIAR is used as stress granules molecular marker in vertebrates, but it is not so deeply investigated in invertebrates, especially in marine organisms. In the present work, we investigated the role of TIAR in the colonial ascidian Botryllus schlosseri during its non-embryonic development, featured by the cyclical renewal of the colony. We studied the extent of transcription during the colonial blastogenetic cycle and the location of the transcripts in Botryllus tissues. Using an anti-TIAR antibody specific for ascidians, by immunocytochemistry and immunohistochemistry assays, we studied the expression of the protein in haemolymph cells and body tissues and by transmission electron microscopy we identified its subcellular localisation. The anti-TIAR antibody was also microinjected in the circulatory system of B. schlosseri to study its effect on non-embryonic development and immune responses. Results indicate a delay in the progression of the blastogenetic cycle in injected colonies. In addition, degranulation of circulating cytotoxic cells and phagocytosis by professional, circulating phagocytes, two fundamental processes of innate immunity, were also negatively affected.

New Data on the Rhamnose-Binding Lectin from the Colonial Ascidian Botryllus schlosseri: Subcellular Distribution, Secretion Mode and Effects on the Cyclical Generation Change.

Botryllus schlosseri in a cosmopolitan ascidian, considered a reliable model organism for studies on the evolution of the immune system. B. schlosseri rhamnose-binding lectin (BsRBL) is synthesised by circulating phagocytes and behaves as an opsonin by interacting with foreign cells or particles and acting as a molecular bridge between them and the phagocyte surface. Although described in previous works, many aspects and roles of this lectin in Botryllus biology remain unknown. Here, we studied the subcellular distribution of BsRBL during immune responses using light and electron microscopy. In addition, following the hints from extant data, suggesting a possible role of BsRBL in the process of cyclical generation change or takeover, we investigated the effects of interfering with this protein, by injecting a specific antibody in the colonial circulation, starting one day before the generation change. Results confirm the requirement of the lectin for a correct generation change and open new queries on the roles of this lectin in Botryllus biology.

Botryllin, a Novel Antimicrobial Peptide from the Colonial Ascidian Botryllus schlosseri.

By mining the transcriptome of the colonial ascidian Botryllus schlosseri, we identified a transcript for a novel styelin-like antimicrobial peptide, which we named botryllin. The gene is constitutively transcribed by circulating cytotoxic morula cells (MCs) as a pre-propeptide that is then cleaved to mature peptide. The synthetic peptide, obtained from in silico translation of the transcript, shows robust killing activity of bacterial and unicellular yeast cells, causing breakages of both the plasma membrane and the cell wall. Specific monoclonal antibodies were raised against the epitopes of the putative amino acid sequence of the propeptide and the mature peptide; in both cases, they label the MC granular content. Upon MC degranulation induced by the presence of nonself, the antibodies recognise the extracellular nets with entrapped bacteria nearby MC remains. The obtained results suggest that the botryllin gene carries the information for the synthesis of an AMP involved in the protection of B. schlosseri from invading foreign cells.

Biomaterials and Bioactive Natural Products from Marine Invertebrates: From Basic Research to Innovative Applications.

Aquatic invertebrates are a major source of biomaterials and bioactive natural products that can find applications as pharmaceutics, nutraceutics, cosmetics, antibiotics, antifouling products and biomaterials. Symbiotic microorganisms are often the real producers of many secondary metabolites initially isolated from marine invertebrates; however, a certain number of them are actually synthesized by the macro-organisms. In this review, we analysed the literature of the years 2010-2019 on natural products (bioactive molecules and biomaterials) from the main phyla of marine invertebrates explored so far, including sponges, cnidarians, molluscs, echinoderms and ascidians, and present relevant examples of natural products of interest to public and private stakeholders. We also describe omics tools that have been more relevant in identifying and understanding mechanisms and processes underlying the biosynthesis of secondary metabolites in marine invertebrates. Since there is increasing attention on finding new solutions for a sustainable large-scale supply of bioactive compounds, we propose that a possible improvement in the biodiscovery pipeline might also come from the study and utilization of aquatic invertebrate stem cells.

Studying Regeneration in Ascidians: An Historical Overview.

Ascidians are sessile tunicates, that is, marine animals belonging to the phylum Chordata and considered the sister group of vertebrates. They are widespread in all the seas, constituting abundant communities in various ecosystems. Among chordates, only tunicates are able to reproduce asexually, forming colonies. The high regenerative potentialities enabling tunicates to regenerate damaged body parts, or the whole body, represent a peculiarity of this taxon. Here we review the methodological approaches used in more than a century of biological studies to induce regeneration in both solitary and colonial species. For solitary species, we refer to the regeneration of single organs or body parts (e.g., siphon, brain, gonad, tunic, viscera). For colonial species, we review a plethora of experiments regarding the surgical manipulation of colonies, the regeneration of isolated colonial entities, such as single buds in the tunic, or part of tunic and its circulatory system.

A pan-metazoan concept for adult stem cells: the wobbling Penrose landscape.

Adult stem cells (ASCs) in vertebrates and model invertebrates (e.g. Drosophila melanogaster) are typically long-lived, lineage-restricted, clonogenic and quiescent cells with somatic descendants and tissue/organ-restricted activities. Such ASCs are mostly rare, morphologically undifferentiated, and undergo asymmetric cell division. Characterized by 'stemness' gene expression, they can regulate tissue/organ homeostasis, repair and regeneration. By contrast, analysis of other animal phyla shows that ASCs emerge at different life stages, present both differentiated and undifferentiated phenotypes, and may possess amoeboid movement. Usually pluri/totipotent, they may express germ-cell markers, but often lack germ-line sequestering, and typically do not reside in discrete niches. ASCs may constitute up to 40% of animal cells, and participate in a range of biological phenomena, from whole-body regeneration, dormancy, and agametic asexual reproduction, to indeterminate growth. They are considered legitimate units of selection. Conceptualizing this divergence, we present an alternative stemness metaphor to the Waddington landscape: the 'wobbling Penrose' landscape. Here, totipotent ASCs adopt ascending/descending courses of an 'Escherian stairwell', in a lifelong totipotency pathway. ASCs may also travel along lower stemness echelons to reach fully differentiated states. However, from any starting state, cells can change their stemness status, underscoring their dynamic cellular potencies. Thus, vertebrate ASCs may reflect just one metazoan ASC archetype.

Stem Cells and Innate Immunity in Aquatic Invertebrates: Bridging Two Seemingly Disparate Disciplines for New Discoveries in Biology.

The scopes related to the interplay between stem cells and the immune system are broad and range from the basic understanding of organism's physiology and ecology to translational studies, further contributing to (eco)toxicology, biotechnology, and medicine as well as regulatory and ethical aspects. Stem cells originate immune cells through hematopoiesis, and the interplay between the two cell types is required in processes like regeneration. In addition, stem and immune cell anomalies directly affect the organism's functions, its ability to cope with environmental changes and, indirectly, its role in ecosystem services. However, stem cells and immune cells continue to be considered parts of two branches of biological research with few interconnections between them. This review aims to bridge these two seemingly disparate disciplines towards much more integrative and transformative approaches with examples deriving mainly from aquatic invertebrates. We discuss the current understanding of cross-disciplinary collaborative and emerging issues, raising novel hypotheses and comments. We also discuss the problems and perspectives of the two disciplines and how to integrate their conceptual frameworks to address basic equations in biology in a new, innovative way.

Ancient Immunity. Phylogenetic Emergence of Recognition-Defense Mechanisms.

Although still scarcely considered by the majority of the biomedical world, invertebrates have greatly contributed to the elucidation of fundamental biological problems [...].

Stem cells of aquatic invertebrates as an advanced tool for assessing ecotoxicological impacts.

Environmental stressors are assessed through methods that quantify their impacts on a wide range of metrics including species density, growth rates, reproduction, behaviour and physiology, as on host-pathogen interactions and immunocompetence. Environmental stress may induce additional sublethal effects, like mutations and epigenetic signatures affecting offspring via germline mediated transgenerational inheritance, shaping phenotypic plasticity, increasing disease susceptibility, tissue pathologies, changes in social behaviour and biological invasions. The growing diversity of pollutants released into aquatic environments requires the development of a reliable, standardised and 3R (replacement, reduction and refinement of animals in research) compliant in vitro toolbox. The tools have to be in line with REACH regulation 1907/2006/EC, aiming to improve strategies for potential ecotoxicological risks assessment and monitoring of chemicals threatening human health and aquatic environments. Aquatic invertebrates' adult stem cells (ASCs) are numerous and can be pluripotent, as illustrated by high regeneration ability documented in many of these taxa. This is of further importance as in many aquatic invertebrate taxa, ASCs are able to differentiate into germ cells. Here we propose that ASCs from key aquatic invertebrates may be harnessed for applicable and standardised new tests in ecotoxicology. As part of this approach, a battery of modern techniques and endpoints are proposed to be tested for their ability to correctly identify environmental stresses posed by emerging contaminants in aquatic environments. Consequently, we briefly describe the current status of the available toxicity testing and biota-based monitoring strategies in aquatic environmental ecotoxicology and highlight some of the associated open issues such as replicability, consistency and reliability in the outcomes, for understanding and assessing the impacts of various chemicals on organisms and on the entire aquatic environment. Following this, we describe the benefits of aquatic invertebrate ASC-based tools for better addressing ecotoxicological questions, along with the current obstacles and possible overhaul approaches.

Typical 2-Cys Peroxiredoxins as a Defense Mechanism against Metal-Induced Oxidative Stress in the Solitary Ascidian Ciona robusta.

Typical 2-Cys peroxiredoxins (2-Cys Prdxs) are proteins with antioxidant properties belonging to the thioredoxin peroxidase family. With their peroxidase activity, they contribute to the homeostatic control of reactive oxygen species (ROS) and, therefore, participate in various physiological functions, such as cell proliferation, differentiation, and apoptosis. Although Prdxs have been shown to be potential biomarkers for monitoring aquatic environments, minimal scientific attention has been devoted to describing their molecular architecture and function in marine invertebrates. Our study aims to clarify the protective role against stress induced by exposure to metals (Cu, Zn, and Cd) of three Prdxs (Prdx2, Prdx3, and Prdx4) in the solitary ascidian Ciona robusta, an invertebrate chordate. Here, we report a detailed pre- and post-translational regulation of the three Prdx isoforms. Data on intestinal mRNA expression, provided by qRT-PCR analyses, show a generalized increase for Prdx2, -3, and -4, which is correlated to metal accumulation. Furthermore, the increase in tissue enzyme activity observed after Zn exposure is slower than that observed with Cu and Cd. The obtained results increase our knowledge of the evolution of anti-stress proteins in invertebrates and emphasize the importance of the synthesis of Prdxs as an efficient way to face adverse environmental conditions.

Beyond Adult Stem Cells: Dedifferentiation as a Unifying Mechanism Underlying Regeneration in Invertebrate Deuterostomes.

The diversity of regenerative phenomena seen in adult metazoans, as well as their underlying mechanistic bases, are still far from being comprehensively understood. Reviewing both ultrastructural and molecular data, the present work aims to showcase the increasing relevance of invertebrate deuterostomes, i.e., echinoderms, hemichordates, cephalochordates and tunicates, as invaluable models to study cellular aspects of adult regeneration. Our comparative approach suggests a fundamental contribution of local dedifferentiation -rather than mobilization of resident undifferentiated stem cells- as an important cellular mechanism contributing to regeneration in these groups. Thus, elucidating the cellular origins, recruitment and fate of cells, as well as the molecular signals underpinning tissue regrowth in regeneration-competent deuterostomes, will provide the foundation for future research in tackling the relatively limited regenerative abilities of vertebrates, with clear applications in regenerative medicine.

Insights into the Complement System of Tunicates: C3a/C5aR of the Colonial Ascidian Botryllus schlosseri.

As an evolutionary ancient component of the metazoan immune defense toolkit, the complement system can modulate cells and humoral responses of both innate and (in jawed vertebrates) adaptive immunity. All the three known complement-activation pathways converge on the cleavage of C3 to C3a and C3b. The anaphylatoxin C3a behaves as a chemokine in inflammatory responses, whereas C3b exerts an opsonic role and, ultimately, can activate the lytic pathway. C3aR, one of the mammalian receptors for C3a, is a member of the G-protein-coupled receptor family sharing seven transmembrane alpha helixes. C3aR can act as a chemokine and recruit neutrophils, triggering degranulation and respiratory burst, which initiates an inflammatory reaction. Mining the transcriptome of the colonial ascidian Botryllus schlosseri, we identified a transcript showing homology with both mammalian C3aR and C5aR. The gene (bsc3/c5ar) is actively transcribed in morula cells, the circulating immunocyte triggering the inflammatory reactions in response to the recognition of nonself. Its transcription is modulated during the recurrent cycles of asexual reproduction known as blastogenetic cycles. Moreover, the treatment of hemocytes with C3aR agonist, induces a significant increase in the transcription of BsC3, revealing the presence of an autocrine feedback system able to modulate the expression of C3 in order to obtain a rapid clearance of potentially dangerous nonself cells or particles. The obtained results support the previously proposed role of complement as one of the main humoral components of the immune response in tunicates and stress the importance of morula cells in botryllid ascidian innate immunity.

Complement system and phagocytosis in a colonial protochordate.

In the present work, we investigated, in the colonial ascidian Botryllus schlosseri, the role of complement C3 (BsC3) in phagocytosis. We studied the modulation of BsC3 transcription in the course of the colonial blastogenetic cycle, with particular reference to the takeover, when apoptotic cells in the tissues of old zooids are cleared by circulating phagocytes. In situ hybridisation with BsC3 riboprobes labelled only morula cells, the most abundant haemocytes. Anti-hC3 antibody recognised morula cells and also phagocytes when haemocytes were previously incubated with zymosan. The inhibition of C3 activation prevented the labelling of phagocytes. In phagocytosis assays with haemocytes from colonies injected with anti-hC3 antibody or bsc3 iRNA, the capability to ingest target cells was significantly (p < 0.001) reduced. Therefore, our results strongly support a key role of BsC3 in phagocytosis and open to new investigations on the nature of the receptors of the products of BsC3 activation.

Sixty years of experimental studies on the blastogenesis of the colonial tunicate Botryllus schlosseri.

In the second half of the eighteenth century, Schlosser and Ellis described the colonial ascidian Botryllus schlosseri garnering the interest of scientists around the world. In the 1950's scientists began to study B. schlosseri and soon recognized it as an important model organism for the study of developmental biology and comparative immunology. In this review, we summarize the history of B. schlosseri studies and experiments performed to characterize the colony life cycle and bud development. We describe experiments performed to analyze variations in bud productivity, zooid growth and bilateral asymmetry (i.e., the situs viscerum), and discuss zooid and bud removal experiments that were used to study the cross-talk between consecutive blastogenetic generations and vascular budding. We also summarize experiments that demonstrated that the ability of two distinct colonies to fuse or reject is controlled by a single polymorphic gene locus (BHF) with multiple, codominantly expressed alleles. Finally, we describe how the ability to fuse and create chimeras was used to show that within a chimera somatic and germline stem cells compete to populate niches and regenerate tissue or germline organs. Starting from the results of these 60 years of study, we can now use new technological advances to expand the study of B. schlosseri traits and understand functional relationships between its genome and life history phenotypes.

Functional amyloidogenesis in immunocytes from the colonial ascidian Botryllus schlosseri: Evolutionary perspective.

Cytotoxic morula cells (MCs) and phagocytes are the circulating immunocytes of the colonial ascidian Botryllus schlosseri: Both these cells can synthesise amyloid fibrils, supporting the idea that physiological amyloidogenesis is involved in inflammation and modulation of immune responses. Intriguingly, amyloid of B. schlosseri immunocytes is made of two different proteins. MCs, the first cells to sense non-self and involved in the allorejection reaction between contacting genetically incompatible colonies, use melanin encapsulation as the principal method to fight non-self. They release amyloid fibrils formed by p102 protein that allow the packaging and deposit of melanin and other toxic molecules nearby the invader or in the contact region of incompatible colonies. Phagocytes release amyloid-based extracellular traps when challenged with microbes: their amyloid fibrils harbour BsAPP, an orthologue of the vertebrate amyloidogeneic protein APP. This strategy of immune response, present also in human neutrophils, allows phagocytes to block and engulf bacteria and fungi.

The haemocytes of the colonial aplousobranch ascidian Diplosoma listerianum: Structural, cytochemical and functional analyses.

Diplosoma listerianum is a colonial aplousobranch ascidian of the family Didemnidae that is native to the northeast Atlantic and exhibits a cosmopolitan distribution in temperate waters. It lacks a shared colonial circulation crossing the tunic, and the zooids are connected only by the common tunic. In the present study, the haemocytes of this ascidian were analysed via light and electron microscopy. Their phagocytic and enzymatic activities, staining and immunostaining properties, and lectin affinity were examined with various classical methods reconsidered and modified for small marine invertebrates. Eight morphotypes were identified in reference to corresponding cell types described in other ascidians: undifferentiated cells (haemoblasts), storage cells for nitrogenous catabolites (nephrocytes) and immunocytes. The immunocytes are involved in immune responses, acting as (1) phagocytes, rich in hydrolases and involved in the clearance of both foreign particles and effete cells (hyaline amoebocytes and macrophage-like cells); (2) cytotoxic cells, able to degranulate and induce cytotoxicity through the release of the enzyme phenoloxidase after an immune stimulus (granular amoebocytes and morula cells); and (3) basophilic cells with an affinity for ConA and NPA that contain heparin and histamine and that show sensitivity to the compound 48/80, promoting their degranulation (mast cell-like granulocytes). In addition, a particular cell type showing exceptional development of the Golgi apparatus and large vacuoles containing a filamentous material has been recognised (spherule cell), for which a role in tunic repair and fibrogenesis has been hypothesised.

Immunity in Protochordates: The Tunicate Perspective.

Tunicates are the closest relatives of vertebrates, and their peculiar phylogenetic position explains the increasing interest toward tunicate immunobiology. They are filter-feeding organisms, and this greatly influences their defense strategies. The majority of the studies on tunicate immunity were carried out in ascidians. The tunic acts as a first barrier against pathogens and parasites. In addition, the oral siphon and the pharynx represent two major, highly vascularized, immune organs, where circulating hemocytes can sense non-self material and trigger immune responses that, usually, lead to inflammation and phagocytosis. Inflammation involves the recruitment of circulating cytotoxic, phenoloxidase (PO)-containing cells in the infected area, where they degranulate as a consequence of non-self recognition and release cytokines, complement factors, and the enzyme PO. The latter, acting on polyphenol substrata, produces cytotoxic quinones, which polymerize to melanin, and reactive oxygen species, which induce oxidative stress. Both the alternative and the lectin pathways of complement activation converge to activate C3: C3a and C3b are involved in the recruitment of hemocytes and in the opsonization of foreign materials, respectively. The interaction of circulating professional phagocytes with potentially pathogenic foreign material can be direct or mediated by opsonins, either complement dependent or complement independent. Together with cytotoxic cells, phagocytes are active in the encapsulation of large materials. Cells involved in immune responses, collectively called immunocytes, represent a large fraction of hemocytes, and the presence of a cross talk between cytotoxic cells and phagocytes, mediated by secreted humoral factors, was reported. Lectins play a pivotal role as pattern-recognition receptors and opsonizing agents. In addition, variable region-containing chitin-binding proteins, identified in the solitary ascidian Ciona intestinalis, control the settlement and colonization of bacteria in the gut.

Regulatory cis- and trans-elements of mitochondrial D-loop-driven reporter genes in budding tunicates.

To unveil the underlying mechanism of mitochondrial gene regulation associated with ageing and budding in the tunicate Polyandrocarpa misakiensis, mitochondrial non-coding-region (NCR)-containing reporter genes were constructed. PmNCR2.3K/GFP was expressed spatiotemporally in a pattern quite similar to mitochondrial 16SrRNA. The reporter gene expression was sensitive to high dose of rifampicin similar to mitochondrial genes, suggesting that the transcription indeed occurs in mitochondria. However, the gene expression also occurred in vivo in the cell nucleus and in vitro in the nuclear extracts. Mitochondrial transcription factor A (PmTFAM) enhanced reporter gene expression, depending on the NCR length. A budding-specific polypeptide TC14-3 is an epigenetic histone methylation inducer. It heavily enhanced reporter gene expression that was interfered by histone methylation inhibitors and PmTFAM RNAi. Our results indicate for the first time that the nuclear histone methylation is involved in mitochondrial gene activity via TFAM gene regulation.

Protection from Oxidative Stress in Immunocytes of the Colonial Ascidian Botryllus schlosseri: Transcript Characterization and Expression Studies.

Botryllus schlosseri is a cosmopolitan colonial ascidian that undergoes cyclical generation changes, or take-overs, during which adult zooids are resorbed and replaced by their buds. At take-over, adult tissues undergo diffuse apoptosis and effete cells are massively ingested by circulating phagocytes, with a consequent increase in oxygen consumption and in production of reactive oxygen species (ROS). The latter are responsible for the death of phagocytes involved in the clearance of apoptotic cells and corpses by phagocytosis-induced apoptosis. However, the majority of phagocytes and hemocytes do not die, even if they experience oxidative stress. This fact suggests the presence of detoxification mechanisms assuring their protection. To test this assumption, we searched for transcripts of genes involved in detoxification in the transcriptome of B. schlosseri. We identified and characterized transcripts for Cu/Zn superoxide dismutase (SOD), γ-glutamyl-cysteine ligase modulatory subunit (GCLM), glutathione synthase (GS), and two glutathione peroxidases (i.e., GPx3 and GPx5), all involved in protection from ROS. We also carried out a phylogenetic analysis of the putative amino acid sequences, confirming their similarity to their vertebrate counterparts, and studied the location of their mRNAs by in situ hybridization on hemocyte monolayers. We also analyzed gene transcription during the colonial blastogenetic cycle, which is the interval of time between one take-over and the next, by qRT-PCR. In addition, we investigated the effects of cadmium (Cd), an inducer of oxidative stress, on gene transcription. Our results indicated that i) antioxidant gene expression is modulated in the course of the blastogenetic cycle and upon exposure to Cd, and ii) hemocytes synthesize both enzymatic and nonenzymatic antioxidants, in line with the idea that they represent a major detoxification system for ascidians.