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Stem cell niches are defined as the microenvironments where stem cells home, receiving stimuli defining their fate. In vertebrates, stem cell niches are stable and physically confined compartments.Botryllus schlosseriis an invertebrate colonial chordate where temporary stem cell niches have been identified in adult individuals that are cyclically resorbed and replaced by a new generation of clonal zooids.B. schlosserialso displays remarkable regenerative abilities, being capable of whole-body regeneration, but the cellular source of these processes is still unknown. Here we identified by means of a high-resolution morphological characterization a new putative stem cell niche in the ampullae of the circulatory system acting as a stem cell source during asexual reproduction. Stem cells of the ampullae travel via the circulatory system and contribute to the development of several organs and could explain where stem cells contributing to whole-body regeneration are stored. The ampullae niches are stable during the life cycle and regeneration ofB. schlosseri, while additional niches of the zooid are dynamically established and colonised by circulating stem cells. Our results reveal an unprecedented dynamicity of stem cell niches in highly regenerative invertebrates.

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., This study was supported by the European Cooperation in Science and Technology, COST Action 16203 MARISTEM (Stem Cells of Marine/Aquatic Invertebrates: From Basic Research to Innovative Applications).

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.

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 the 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.

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.

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.

In the present study, we describe the identified and characterized the gene and the transcript of a novel glutathione peroxidase-7 (GPx7) from the solitary ascidian Ciona robusta, an invertebrate chordate widely distributed in temperate shallow seawater. The putative nucleotide and amino acid sequences were compared with those of GPx7 from other metazoans and phylogenetic analysis suggests the presence of a high evolutionary pressure in the contest of neutral evolution. The mRNA of CrGPx7 is located in hemocytes and ovarian follicular cells, as revealed by in situ hybridization. The time course of CrGPx7 mRNA levels in the presence of Cd, Cu and Zn, showed upregulation in the final stages of the experiments, suggesting a role of GPx7 in late protection from oxidative stress. Our in silico analyses of the crgpx7 promoter region revealed putative consensus sequences similar to mammalian metal-responsive elements (MRE) and xenobiotic-responsive elements (XRE), suggesting that the transcription of these genes directly depends on metals. Cell-free extract from C. robusta tissues show the presence of selenium-independent GPx activity that is inhibited by the presence of metals.

As an invertebrate, the compound ascidian Botryllus schlosseri faces nonself only with innate immunity. In this species, we already identified the key components of the lectin and alternative complement activation pathways. In the present work, by mining the transcriptome, we identified a single transcript codifying for a protein, member of the C1q-domain-containing protein family, with a signal peptide followed by two globular C1q (gC1q) domains. It shares a similar domain organisation with C1q/TNF-related proteins 4, the only vertebrate protein family with two gC1q domains. Our gC1q domain-containing protein, called BsC1qDC, is actively transcribed by immunocytes. The transcription is modulated during the Botryllus blastogenetic cycle and is upregulated following the injection of Bacillus clausii cells in the circulation. Furthermore, the injection of bsc1qdc iRNA in the vasculature results in decreased transcription of the gene and a significant impairment of phagocytosis and degranulation, suggesting the involvement of this molecule in immune responses.

International audience; 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.

Colonies of the marine, filter-feeding ascidian Botryllus schlosseri undergo cyclical generation changes or takeovers. These events are characterised by the progressive resorption of adult zooids and their replacement by their buds that grow to adult size, open their siphons and start filtering. During the take-over, tissues of adult zooids undergo extensive apoptosis; circulating, spreading phagocytes enter the effete tissues, ingest dying cells acquiring a giant size and a round morphology. Then, phagocytes re-enter the circulation where they represent a considerable fraction (more than 20%) of circulating haemocytes. In this study, we evidence that most of these circulating phagocytes show morphological and biochemical signs of apoptosis. Accordingly, these phagocytes express transcripts of orthologues of the apoptosis-related genes Bax, AIF1 and PARP1. Electron microscopy shows that giant phagocytes contain apoptotic phagocytes inside their own phagocytic vacuole. The transcript of the orthologues of the anti-apoptotic gene IAP7 was detected only in spreading phagocytes, mostly abundant in phases far from the take-over. Therefore, the presented data suggest that, at take-over, phagocytes undergo phagocytosis-induced apoptosis (PIA). In mammals, PIA is assumed to be a process assuring the killing and the complete elimination of microbes, by promoting the disposal of terminally differentiated phagocytes and the resolution of infection. In B. schlosseri, PIA assumes a so far undescribed role, being required for the control of asexual development and colony homeostasis.

The colonial ascidian Botryllus schlosseri is a cosmopolitan, marine filter feeder, introduced as a laboratory research organism in the 1950s. Currently, it is widely used in many laboratories to investigate a variety of biological questions. Recently, it has become a species of concern, as it is an invasive species in many coastal environments. Here, we review studies on the geographical distribution of the species, sexual and asexual reproduction in the field, tolerance to temperature, salinity and anthropogenic activity, polychromatism, enzymatic polymorphism, and the genetic basis of pigmentation. Studying the relationship between genetic polymorphism and the adaptation of B. schlosseri to environmental stress is a challenge of future research and will improve our understanding of its evolutionary success and invasive potential.

Tunicates or Urochordates are the closest relatives of vertebrates. They are marine filter-feeding animals, found at all the latitudes, and can assume a planktonic or benthic lifestyle. They are, with few exceptions, hermaphroditic animals with an indirect development: almost all the tunicate species have a tadpole swimming larva. Coloniality is widespread and a larval stage followed by metamorphosis is the rule in the class Ascidiacea. Blooms of pelagic tunicates are quite common in warm seasons: their ecological relevance relies on the key role they play in the alimentary chain of open seas.

New biocides such as the organometallic compound zinc pyrithione (ZnP) have been massively introduced by many countries in formulations of antifouling paints following the ban on tributyltin (TBT). The effects of sublethal concentrations (LC50=82.5 μM, i.e., 26.2 mg/l) on cultured haemocytes of the ascidian Botryllus schlosseri have been investigated and compared with TBT. The percentage of haemocytes with amoeboid morphology and containing phagocytised yeast cells were significantly (p

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.

Summary The colonial tunicate Botryllus schlosseri is a widespread filter-feeding ascidian that lives in shallow waters and is easily reared in aquaria. Its peculiar blastogenetic cycle, characterized by the presence of three blastogenetic generations (filtering adults, buds, and budlets) and by recurrent generation changes, has resulted in over 60 years of studies aimed at understanding how sexual and asexual reproduction are coordinated and regulated in the colony. The possibility of using different methodological approaches, from classical genetics to cell transplantation, contributed to the development of this species as a valuable model organism for the study of a variety of biological processes. Here, we review the main studies detailing rearing, staging methods, reproduction and colony growth of this species, emphasizing the asymmetry in sexual and asexual reproduction potential, sexual reproduction in the field and the laboratory, and self- and cross-fertilization. These data, opportunely matched with recent tanscriptomic and genomic outcomes, can give a valuable help to the elucidation of some important steps in chordate evolution. genesis 53:105–120, 2015. © 2014 Wiley Periodicals, Inc.

Background We performed an analysis of the transcriptome during the blastogenesis of the chordate Botryllus schlosseri, focusing in particular on genes involved in cell death by apoptosis. The tunicate B. schlosseri is an ascidian forming colonies characterized by the coexistence of three blastogenetic generations: filter-feeding adults, buds on adults, and budlets on buds. Cyclically, adult tissues undergo apoptosis and are progressively resorbed and replaced by their buds originated by asexual reproduction. This is a feature of colonial tunicates, the only known chordates that can reproduce asexually. Results Thanks to a newly developed web-based platform (http://botryllus.cribi.unipd.it), we compared the transcriptomes of the mid-cycle, the pre-take-over, and the take-over phases of the colonial blastogenetic cycle. The platform is equipped with programs for comparative analysis and allows to select the statistical stringency. We enriched the genome annotation with 11,337 new genes; 581 transcripts were resolved as complete open reading frames, translated in silico into amino acid sequences and then aligned onto the non-redundant sequence database. Significant differentially expressed genes were classified within the gene ontology categories. Among them, we recognized genes involved in apoptosis activation, de-activation, and regulation. Conclusions With the current work, we contributed to the improvement of the first released B. schlosseri genome assembly and offer an overview of the transcriptome changes during the blastogenetic cycle, showing up- and down-regulated genes. These results are important for the comprehension of the events underlying colony growth and regression, cell proliferation, colony homeostasis, and competition among different generations. Electronic supplementary material The online version of this article (doi:10.1186/s12864-016-2598-1) contains supplementary material, which is available to authorized users.

After the widespread ban of TBT, due to its severe impact on coastal biocoenoses, mainly related to its immunosuppressive effects on both invertebrates and vertebrates, alternative biocides such as Cu(I) salts and the triazine Irgarol 1051, the latter previously used in agriculture as a herbicide, have been massively introduced in combined formulations for antifouling paints against a wide spectrum of fouling organisms. Using short-term (60 min) haemocyte cultures of the colonial ascidian Botryllus schlosseri exposed to various sublethal concentrations of copper(I) chloride (LC 50 = 281 μM, i.e., 17.8 mg Cu L −1 ) and Irgarol 1051 (LC 50 > 500 μM, i.e., >127 mg L −1 ), we evaluated their immunotoxic effects through a series of cytochemical assays previously used for organotin compounds. Both compounds can induce dose-dependent immunosuppression, acting on different cellular targets and altering many activities of immunocytes but, unlike TBT, did not have significant effects on cell morphology. Generally, Cu(I) appeared to be more toxic than Irgarol 1051: it significantly ( p −1 ), and affected calcium homeostasis and mitochondrial cytochrome-c oxidase activity at 0.01 μM (∼1 μg L −1 ). Both substances were able to change membrane permeability, induce apoptosis from concentrations of 0.1 μM (∼10 μg L −1 ) and 200 μM (∼50 mg L −1 ) for Cu(I) and Irgarol 1051, respectively, and alter the activity of hydrolases. Both Cu(I) and Irgarol 1051 inhibited the activity of phenoloxidase, but did not show any interactive effect when co-present in the exposure medium, suggesting different mechanisms of action.

Colonies of the compound ascidian Botryllus schlosseri undergo recurrent generation changes during which massive, natural apoptosis occurs in zooid tissues: for this reason the species is emerging as an interesting model of invertebrate chordate, phylogenetically related to vertebrates, for studies of apoptosis during development. In the present work, we carried out a series of morphological, cytofluorimetrical and biochemical analyses, useful for a better characterization of Botryllus apoptosis. Results are consistent with the following viewpoints: (i) both intrinsic and extrinsic pathways, probably connected by the BH3-only protein Bid, are involved in cell death induction; (ii) phagocytes, once loaded with senescent cells, frequently undergo apoptosis, probably as a consequence of oxidative stress caused by prolonged respiratory burst, and (iii) senescent phagocytes are easily recognized and ingested by other phagocytes, responsible for their clearance. In addition, results suggest the conservation of apoptosis induction mechanisms throughout chordate evolution.

Colonies of the compound ascidian Botryllus schiosseri undergo regular generation changes, during which adult zooids are progressively resorbed and replaced by growing buds. The generation change, or take-over, is characterized by massive cell death by apoptosis, as indicated by nuclear condensation, activation of caspases, overexpression of molecules recognized by antibodies against mammalian Bax, Fas, and FasL, changes in the expression of surface molecules by senescent cells of zooid tissues, and recruitment of circulating phagocytes in zooid tissues which ensure the complete clearing of dying cells. The entire process lasts 24-36 h at 20 degrees C and has been subdivided, on the basis of the degree of contraction of old zooids, into four substages. In the present work, we carried out a detailed morphological analysis of the events occurring in zooid tissues during the take-over substages. Results Indicate that traces of apoptosis can be found in the epidermis, peribranchial epithelium, and heart in the late substage but are easily found in the branchial basket 2-4 h after the beginning of the generation change, thus confirming the antero-posterior progression of cell death, at least in the alimentary system.

The supernatant from cultures of haemocytes of the compound ascidian Botryllus schlosseri incubated with zymosan (conditioned medium; CM) can enhance yeast phagocytosis by Botryllus blood cells. It contains molecules recognised by antibodies raised against the mammalian pro-inflammatory cytokines IL-1-alpha and TNF-alpha which appear as a single band of 60 kDa in immunoblot analysis. The effects on phagocytosis are abolished by the presence of sugars, such as galactose and rhamnose, sharing the same hydroxyl group configuration at C2 and C4. The same sugars also inhibit the haemagglutinating activity of the CM, suggesting the presence of lectins with opsonic activity. With immunoblot analysis, we confirmed the presence, in CM, of B. schlosseri rhamnose-binding lectins (BsRBLs), recently identified and characterised by our team, as a single electrophoretic band of 37 kDa. We had already demonstrated that these molecules are synthesised and secreted by activated phagocytes. Since previous studies have demonstrated that cytotoxic morula cells, and not phagocytes, are the haemocytes responsible for the release of molecules recognised by anti-cytokine antibodies, we propose a new scenario in which morula cells act as sentinels, able to sense foreign molecules and release immunomodulatory factors which induce phagocytes to secrete lectins able to enhance phagocytosis by acting as bridges between foreign particles and phagocyte surfaces.

Using short-term hemocyte cultures of the colonial ascidian Botryllus schlosseri exposed to various sublethal concentrations of Diuron (3-(3,4-diclorophenyl)-1,1-dimethylurea) and TCMS pyridine (2,3,5,6-tetrachloro-4-(metylsulphonyl)pyridine), we evaluated their immunotoxic effects through a series of cytochemical assays previously used for organotin compounds. At concentrations higher than 250 micro M and 10 micro M for Diuron and TCMS pyridine, respectively, both biocides exerted immunosuppressant effects on Botryllus hemocytes, causing i) deep changes in the cytoskeleton that irreversibly affect cell morphology and phagocytosis, ii) induction of DNA damage, iii) leakage of oxidative and hydrolytic enzymes due to membrane alteration. Unlike organotin compounds, Diuron and TCMS pyridine do not inhibit cytochrome-c-oxidase, and only TCMS pyridine triggers oxidative stress. When co-present, they exert an antagonistic interaction on cytoskeletal components.

In the present study, we report on haemocyte distribution, determined by a Coulter Counter, in the clam Tapes philippinarum. In addition, cytoskeleton components of haemocytes were examined using specific probes for F-actin and alpha-tubulin. The mean number of circulating haemocytes was 5 (x10(6))cells/ml haemolymph. Two main haemocyte populations were found in the haemolymph: small cells, 2-3microm in diameter and 10-100fl in volume; and large cells, 6-10microm in diameter and 150-400fl in volume. Analysis of the haemocyte cytoskeleton revealed bundles of actin filaments oriented according to the cell major axis, and microtubules radiating from the microtubule-organizing centre in proximity of the nucleus. Interestingly, mitotic spindles were also found radiating from the microtubule-organizing centres, located at the spindle poles (centrosomes) of undifferentiated cells. On the basis of both our previous findings regarding circulating stem cells (Cima, F., Matozzo, V., Marin, M.G., Ballarin, L., 2000. Haemocytes of the clam Tapes philippinarum (Adams & Reeve, 1850): morphofunctional characterisation. Fish Shellfish Immunol 10, 677-693) and new information from the present study, we suggest that haemoblasts are able to divide in the haemolymph of T. philippinarum. To our knowledge, this is the first report of mitotic spindles in circulating haemocytes from a bivalve species.

After the definitive ban on tin-based antifouling substances, new organic compounds have recently been introduced in antifouling paint formulations, as either principal or booster biocides. In most cases, previous risk assessment of these biocides has been inadequate so that their possible effects on aquatic ecosystems is a matter of great concern. We studied the effects of two new organic biocides often associated in paint formulations, Sea-Nine 211 (4,5 dichloro-2-n-octyl-4-isothiazoline-3-one) and chlorothalonil (2,4,5,6-tetrachloroisophthalonitrile), on haemocytes of the compound ascidian Botryllus schlosseri exposed for 60 min to various concentrations (from 0.1 to 10 microM) of the xenobiotics. This species had previously proved to be a good bioindicator of organotin compounds. Both compounds, at concentrations of 1 and 10 microM, altered the morphology of phagocytes, and these changes were closely related to disrupting effects on cytoskeletal components. At the same concentrations, phagocytosis, which requires cytoskeletal modifications for pseudopod formation, was severely hindered. Both compounds were able to induce apoptosis of Botryllus blood cells, probably as a consequence of severe oxidative stress related to the reported decrease of intracellular reduced glutathione (GSH) content. In the case of Sea-Nine 211, a substantial increase in intracellular Ca(2+) and a negative effect on Ca(2+)-ATPase activity may also be involved in the activation of the cell death machinery. Cytochrome-c-oxidase was also significantly inhibited by the two biocides, indicating perturbation of the mitochondrial respiratory chain. Isodynamic mixtures of Sea-Nine 211 and chlorothalonil were used to evaluate the occurrence of interactions between the two compounds. Results suggest the combined action of partial additivity when cell-spreading and cytochrome-c-oxidase activity were considered, and were indicative of antagonism in the case of the GSH depletion. On the whole, our results indicate that short-term in vitro exposure of haemocytes to high concentrations of Sea-Nine 211 and chlorothalonil provokes a marked reduction in haemocyte functionality, higher than or comparable to that of TBT. These assays of acute toxicity stress the immunosuppressive potential of these compounds, which, although counterbalanced by their short half-life in the marine environment, can lead to biocoenosis dismantling through rapid bioaccumulation by filter-feeding non-target benthic organisms.

A recurrent blastogenetic cycle characterizes colonies of the ascidian Botryllus schlosseri. This cycle starts when a new zooid generation opens its siphons and ends with take-over, when adult zooids cease filtering and are progressively resorbed and replaced by a new generation of buds, reaching functional maturity. During the generation change, massive apoptosis occurs in the colony, mainly in the tissues of old zooids. In the present study, we have investigated the behaviour of haemocytes during the colonial blastogenetic cycle, in terms of the occurrence of cell death and the expression of molecules involved in the induction of apoptosis. Our results indicate that, during take-over, caspase-3 activity in haemocyte lysates increases. In addition, about 20%-30% of haemocytes express phosphatidylserine on the outer leaflet of their plasma membrane, show DNA fragmentation and are immunopositive for caspase-3. Senescent cells are quickly ingested by circulating phagocytes that frequently, having once engulfed effete cells, in turn enter apoptosis. Dying cells and corpses are replaced by a new generation of cells that appear in the circulation during the generation change.

We re-investigated the behavior of hemocytes during the non-fusion (rejection) reaction between genetically incompatible colonies of the ascidian Botryllus schlosseri. In the course of the reaction, hemocytes - mainly morula cells - crowd inside the blind ends of marginal vascular vessels (known as ampullae) of the colonial leading edge (LE) facing the foreign colony which suggests the occurrence of chemotactic attraction of circulating hemocytes towards the ampullar lumen. Then, cells migrate, through the ampullar tips, into the partially fused tunics and contribute to the formation of the necrotic spots along the contact borders which characterize the reaction. Studies on histological sections clearly indicate that, although morula cell concentration is always higher in ampullae of the LE than in those of the lateral (L) part of the colony, their frequency significantly increases in LE ampullae of rejecting colonies with respect to LE ampullae of both fusing and isolated colonies. In addition, in vitro chemotaxis experiments demonstrated that blood plasma from incompatible colonies can stimulate morula cell migration through polycarbonate filters and this passage is inhibited by antibodies raised against mammalian pro-inflammatory cytokines. The possible nature and role of molecules recognized by anti-cytokine antibodies in hemocyte migration are discussed.

Phagocytes of the compound ascidian Botryllus schlosseri are capable of constitutive macropinocytosis (MP) at sites of membrane ruffling along the leading edge. This gives rise to the formation of initially irregular vesicles which then move to the inside of the cells and acquire a more regular morphology. Both phagocyte spreading and MP are enhanced by the recognition of molecules containing the sequence Arg-Gly-Asp (RGD): this suggests that, as in mammals, integrin activation is involved in the induction of both cell spreading and endocytosis. The occurrence of MP is associated with increased oxygen consumption and a rise in the production of superoxide anion, as indicated by nitroblue tetrazolium reduction, and ATP, as indicated by increased cytochrome oxidase activity. On the whole, our results indicate the conservation of common mechanisms of MP induction throughout the Chordate phylum.

We studied changes in the morphology of morula cells, a common haemocyte type in botryllid ascidians, during both the rejection reaction (occurring between contacting, genetically incompatible colonies) and fusion (occurring between compatible colonies), and in short-term cultures of haemocytes incubated with heterologous or autologous blood plasma. In both the rejection reaction and haemocyte cultures in the presence of heterologous blood plasma, we observed alterations in morula cells, consistent with a degranulation event, and their expression of molecules recognised by anti-IL-1-alpha- and anti-TNF-alpha-antibodies. Anti-cytokine-antibodies markedly reduced the extent of the in vitro cytotoxicity, when haemocytes were exposed to heterologous blood plasma. In addition, the increase in the production of nitrite ions and the decrease of the in vitro cytotoxicity by the nitric oxide synthase inhibitor N(omega)-nitro-L-arginine methyl ester, suggest the role of nitric oxide in cell death. These results provide new clues to understand the process of rejection reaction in botryllid ascidians.

Haematological features were compared between diploid and triploid specimens of the ray-finned fish Umbrina cirrosa. No significant differences between diploids and triploids were reported in haematocrit and total haemoglobin concentration, but erythrocytes and thrombocytes were significantly greater in size in triploids. Glycaemia was significantly lower in diploids, whereas triploid erythrocytes were more resistant to osmotic stress. In triploids, a greater fraction of leukocytes was positive for alkaline phosphatase activity, when stimulated with Bacillus clausii spores, otherwise no significant increase of oxygen consumption was observed in triploid leukocytes after stimulation, based on assays for superoxide anions. Triploids were characterized by a lower concentration of circulating blood cells with a lower surface/volume ratio when compared with diploids. These features may lead to a general disadvantage of triploids in withstanding stress conditions: a situation that needs to be taken into account in aquaculture practice.

When genetically incompatible colonies of the compound ascidian Botryllus schlosseri contact each other, a rejection reaction occurs, characterised by the appearance of cytotoxic foci along the touching borders. In the course of this reaction, morula cells, a common haemocyte-type in ascidians, release their vacuolar content, mainly phenoloxidase and its polyphenol substrata, upon the recognition of soluble factors diffusing from the alien colony through the partially fused tunic. In a previous paper, we demonstrated the relationship between phenoloxidase and cytotoxicity. Here, we investigated the effects of superoxide dismutase, catalase and sorbitol (scavengers of superoxide anions, peroxides and hydroxyl radicals, respectively) on the cytotoxicity observed in haemocyte cultures incubated with heterologous blood plasma. Although the above compounds have no effects on morula cell degranulation and phenoloxidase activity, they suppress cell death, suggesting that oxidative stress plays a key role in in vitro cytotoxicity. In addition, sorbitol reduces the extent of the cytotoxicity occurring in the rejection reaction between incompatible colonies, which stresses the important role of hydroxyl radicals in this process. The observation of a decrease in total and non-protein thiols in haemocytes previously incubated with heterologous blood plasma fits the hypothesis of oxidative stress as the main cause of phenoloxidase-related cytotoxicity.

The effects of tributyltin (TBT, as the chloride) on circulating cells from the clam Tapes philippinarum were investigated, in order to set up quick and reproducible in vitro bioassays to evaluate TBT toxicity in bivalve molluscs. Haemocytes, collected from the adductor muscle of clams, were exposed for 60 min at 25 °C to sublethal concentrations of TBT (0.01, 0.05, 0.1 μM) and the effects on uptake of the vital dye Neutral Red, and both superoxide dismutase (antioxidant enzyme) and lysozyme (bacteriolytic enzyme) activities were spectrophotometrically evaluated. Exposure of haemocytes to 0.05 μM TBT caused a significant increase (P < 0.05) in Neutral Red dye uptake compared with controls, whereas no differences resulted after exposure to 0.01 and 0.1 μM TBT. Enlarged lysosomes were observed in haemocytes exposed to 0.05 μM TBT. Moreover, in haemocytes treated with 0.05 μM and 0.1 μM TBT, superoxide dismutase activity significantly decreased (P < 0.05 and P < 0.01 respectively) with respect to that of controls. A significant decrease in lysozyme activity was also observed in haemocytes exposed to 0.05 (P < 0.01) and 0.1 μM TBT (P < 0.001). These results suggest a relationship between TBT exposure and alterations in functional responses of haemocytes in T. philippinarum. The proposed assays are sensitive, rapid and reproducible. They may be proposed as biomarkers, although their responsiveness needs to be more fully evaluated in haemocytes collected after clam exposure in both laboratory and field conditions.

Cima, F., Perin, A., Burighel, P. and Ballarin, L. 2001. Morpho-functional characterization of haemocytes of the compound ascidian Botrylloides leachi (Tunicata, Acidiacea). — Acta Zoologica (Stockholm) 82 : 261‐274 A morpho-functional study of the colonial ascidian Botrylloides leachi haemocytes was carried out to propose their classification, relationships and specializations. This characterization was obtained by (i) investigations of both living and aldehydefixed cells by light and electron microscopy; (ii) cytochemical and cytoenzymatic assays; (iii) lectin-affinity assays; (iv) phagocytosis and haemagglutination assays; and (v) anti-CD34 immunocytochemical assay for vertebrate haematopoietic stem cells. Results indicate that the haemoblast is a circulating stem cell and there are at least five haemocyte differentiation pathways, the last two of which have never been described in botryllids: (i) phagocytic line (hyaline amoebocytes and macrophage-like cells) share ultrastructural features, the same hydrolytic enzymes and WGA lectin binding, and are involved in yeast phagocytosis and erythrocyte rosette formation; (ii) cytotoxic line (granular amoebocytes and morula cells) with vacuoles containing oxidative enzymes and polyphenolic substrates; (iii) vacuolated cell line (pigment cells and nephrocytes) involved in catabolite storage; (iv) compartment cell line (compartment amoebocytes and compartment cells) able to agglutinate erythrocytes and characterized by vacuoles with a moderately electron-dense content, positive to arylsulphatase activity and binding DBA, UEA-I, HPA lectins; and (v) granular cell line includes trophic cells, able to infiltrate the gut epithelium, showing a cytoplasm filled of PAS-positive vacuoles with arylsulphatase, chloroacetylesterase and β -glucuronidase activities.

Immunocytochemical methods were used to study the presence and distribution of IL-1-alpha- and TNF-alpha-like molecules in the hemocytes of the colonial ascidian Botryllus schlosseri. Only a few unstimulated hemocytes were positive to both the antibodies used. When the hemocytes were stimulated with either mannan or phorbol 12-mono-myristate, the phagocytes were not significantly changed in their number, staining intensity, or cell morphology. In contrast, stimulated morula cells were intensely labeled, indicating that these cells play an important immunomodulatory role.

article The mechanism of interaction of Cr(VI) with isolated rat liver mitochondria was investigated in this study. The results suggest that Cr(VI) induces the opening of the membrane permeability transition pore (MPT). The phenomenon is cyclosporine-sensitive and is in agreement with the cyclosporine-sensitive apoptosis observed in the cells incubated with this compound. Moreover the action of Cr(III), that is formed in the cells by a reduction of Cr(VI), has been also analysed. The results obtained demonstrated that the Cr(III) does not induce the opening of the MPT in isolated mitochondria, but it has a protective effect in preventing Cr(VI) MPT opening. Therefore, these results suggest that apoptosis is regulated by a balance between Cr(VI) accumulation in the cytoplasm and Cr(III) formation.

In the colonial ascidian Botryllus schlosseri, tributyltin (TBT), a powerful antifouling biocide, acts as immunotoxic xenobiotic since, at a sublethal concentration (10 µM), it causes an irreversible and significant decrease in in vitro yeast phagocytosis, associated with considerable changes in the shape of phagocytes, which withdraw their pseudopodia and become spherical, due to structural damage of cytoskeletal components. The addition of TBT to the culture medium causes a significant decrease in the amoebocytic index, i.e. the percentage of amoeboid-shaped haemocytes, and prolonged washing in sea water never succeeds in restoring amoeboid shape. In these cytoskeletal alterations, F-actin undergoes extensive depolymerisation, resulting in the absence of FITC-phalloidin fluorescence. Microtubules are not recognisable as single filaments with anti-alpha-tubulin immunofluorescence, although the centrosome is not affected. The addition of increasing exogenous calmodulin (CaM) concentrations (from 20 to 120 µM) after incubation in TBT determines a significant increase in the amoebocytic index, although it is not able to bring it to that of controls, suggesting that CaM in the medium in any case externally exerts an influence on haemocytes pretreated with TBT. The copresence of TBT and exogenous CaM at concentrations higher than 80 µg/ml restores the amoebocytic index and cytoskeletal morphology. The latter appears complete for microtubules and partial for microfilaments. Experiments with isodynamic mixtures of TBT and specific CaM inhibitors, i.e. chlorpromazine (CPZ) and N-(6-aminohexyl)-5-chloronaphtalene-1-sulfonamide (W-7), reveal the synergistic effect of antagonism, indicating competition for the same site - a Ca(2+)-CaM hydrophobic region - by both interacting substances and, therefore, the formation of a TBT-CaM complex. Instead, isodynamic mixtures with thapsigargin, an inhibitor of Ca(2+)-ATPase of the endoplasmic reticulum, have an effect of potentiation, suggesting that TBT indirectly interacts with this Ca(2+)-ATPase activity. We hypothesise that the main mechanism of action of TBT in B. schlosseri phagocytes is alteration of Ca(2+) homeostasis by means of direct interaction with endogenous CaM, which induces a conformational change preventing the regulative activity of CaM on Ca(2+)-ATPase. Consequently, an excess of cytosolic Ca(2+) accumulates which, together with the inhibition of CaM-dependent kinases and Ca(2+)-regulated proteins, produces extensive cytoskeletal disorganisation.

Using a polyclonal antibody raised against purified phenoloxidase from the colonial ascidian Botryllus schlossen, we studied its distribution among haemocytes and its intracellular location. The enzyme is present inside granular amoebocytes and morula cells, thus confirming the close relationship between the two cell types, as suggested by previous histochemical and histoenzymatic analysis. Immunocytochemistry in both light and electron microscopy shows that phenoloxidase is located inside the vacuoles of moru‐la cells, known to be the effectors of the rejection reaction.

Early events in apoptosis include chromatin condensation followed by DNA fragmentation as well as translocation of phosphatidylserine (PS) in the outer plasma membrane. Organotin compounds increase intracellular Ca2+ levels and cause apoptosis in mammalian cells. In investigating whether TBT may also induce apoptosis in haemocytes of the ascidian Botryllus schlosseri, we exposed haemocytes to this xenobiotic at the sublethal dose of 10 µM, causing cell shrinkage and inhibition of phagocytosis and respiratory burst. Apoptosis was revealed as (i) chromatin condensation, with Acridine Orange nuclear staining; (ii) DNA fragmentation, with the TUNEL reaction; (iii) PS translocation, with the annexin-V assay; and (iv) loss of membrane permeability with the Trypan Blue diffusion assay. After 1 h of exposure, nuclear changes, i.e. significant collapse and cleavage of chromatin, were observed and cytoplasm blebbing occurred, together with surface alterations triggered by PS exposure. Haemocyte mortality increased significantly only after 2 h. All these apoptotic events may be closely related to a TBT-induced cytosolic calcium increase resulting in activation of endonucleases. Copyright ­© 1999 John Wiley & Sons, Ltd.

A humoral agglutinin from the hemolysate of the colonial ascidian Botryllus schlosseri was purified by affinity chromatography. This agglutinin does not require metal cations for its activity and is specific for derivatives of d -galactose. On SDS-PAGE analysis, it was resolved in two bands, of 17 and 19 kDa in reducing conditions and 15 and 16 kDa in non-reducing conditions. This behavior is due to the establishment of disulfide bridges between the thiols of cysteine, well-represented in the molecule as revealed by amino acid analysis. The latter also indicated high percentages of hydrophilic residues, probably involved in sugar recognition. The lectin is an opsonin, as it increases both the phagocytic index and the number of phagocytized yeast cells. The hypothesis that this Botryllus agglutinin belongs to the galectin family of lectins is discussed.

The aim of this investigation was to quantify the effects of tributyltin (TBT) on the immune reactivity of haemocytes from the cultivated clam Tapes philippinarum (Adams and Reeve, 1850) using a series of in vitro bioassays. It is known that TBT has adverse effects on cellular immune functions like mobility, phagocytosis and lysosomal enzyme activity. As defining TBT-sensitive immunologic biomarkers in sentinel organisms is important in the field of ecotoxicology, the authors propose three indexes, amoebocytic (A.I.), phagocytic (P.I.), and lysosomal activity (L.A.I.), as sensitive and useful biomarkers to assess environmental risks due to TBT contamination.

One of the most harmful groups of coastal pollutants is the organotin compounds (OTCs) which have severe effects on both aquatic organisms and mammals including humans. The immunotoxic effects of OTCs were studied in the cultivated clam Tapes philippinarum by determining the immunosuppressant role on in vitro yeast phagocytosis at low doses (0.01, 0.05, 0.1 microM). The phagocytic index was significantly reduced in an irreversible non-lethal manner depending on concentration and lipophilic affinity. The order of inhibition was TBTor = DBTMBT for butyltins and TPTCTPTAor = TPTH for triphenyltins.

The major thiol-containing molecules involved in controlling the level of intracellular ROS in eukaryotes, acting as a nonenzymatic detoxification system, are metallothioneins (MTs), glutathione (GSH) and phytochelatins (PCs). Both MTs and GSH are well-known in the animal kingdom. PC was considered a prerogative of the plant kingdom but, in 2001, a phytochelatin synthase (PCS) gene was described in the nematode Caenorhabditis elegans; additional genes encoding this enzyme were later described in the earthworm Eisenia fetida and in the parasitic nematode Schistosoma mansoni but scanty data are available, up to now, for Deuterostomes. Here, we describe the molecular characteristics and transcription pattern, in the presence of Cd, of a PCS gene from the invertebrate chordate Ciona intestinalis, a ubiquitous solitary tunicate and demonstrate the presence of PCs in tissue extracts. We also studied mRNA localization by in situ hybridization. In addition, we analyzed the behavior of hemocytes and tunic cells consequent to Cd exposure as well as the transcription pattern of the Ciona orthologous for proliferating cell nuclear antigen (PCNA), usually considered a proliferation marker, and observed that cell proliferation occurs after 96h of Cd treatment. This matches the hypothesis of Cd-induced cell proliferation, as already suggested by previous data on the expression of a metallothionein gene in the same animal.

In the compound ascidian Botryllus schlosseri, phagocytes change their morphology and frequency during the colonial blastogenic cycle. In the regression phase, characterized by intense phagocytosis, zooids of the old generation are progressively resorbed, the frequency of hyaline amoebocytes falls abruptly, and the frequency of macrophage‐like cells increases significantly. Moreover, the hyaline amoebocytes engulf foreign materials, withdraw their cytoplasmic projections, and change their shape from flat and fusiform to spherical or ovoidal: all these processes suggest that hyaline amoebocytes represent the precursors of macrophage‐like cells. During regression, a significant increase in blood levels of hydrogen peroxide and acid phosphatase is observed, in agreement with data obtained in in vitro experiments showing an increase in reactive oxygen metabolite production, nitrite ion release, and acid phosphatase secretion associated with phagocytosis.

This study was designed to investigate the effects of butyltins on yeast phagocytosis in vitro by haemocytes of the colonial ascidian Botryllus schlosseri. This species has been reported to be very sensitive to organotins. Results show that, in analogy to reports on mammalian leukocyte reactivity, butyltins exert inhibitory effects on phagocytosis in a concentration-dependent manner, mainly by influencing cellular calcium homeostasis by interacting with the calcium pump. As the immunosuppressant activity of organotins described in mammals and teleosteans also occurs in an invertebrate marine species, the latter may assume a role as a sensitive biosensor of butyltins as immunotoxins in aqueous ecosystems.

Tunicates are chordate invertebrates, closely related to vertebrates, which represent valuable organisms for the study of a variety of biological processes from an evolutionary point of view. As invertebrates, they rely on innate immunity to cope with foreign, potentially pathogenic material. Among tunicates, the compound ascidian Botryllus schlosseri is emerging as a reliable model organism for the study of innate immune responses. However, there is a general lack of knowledge on the signalling pathways activated during immune responses and, in particular, in phagocytosis. In the present work, we carried out a preliminary investigation of the signalling pathways involved in phagocytosis, with particular reference to MAPK activation. We studied in vitro zymosan phagocytosis in the presence of manumycin A, which inhibit the activation of Ras, PD98059, SP600125 and SB202190, inhibitors of Erk, JNK and p38, respectively, parthenolide, N-acetyl-l-cysteine (NAC) and pyrrolidine dithiocarbamate (PDTC), inhibiting NF-kB activation. In addition we carried out immunoblot and immunocytochemistry analysis with the use of anti-pErk1/2, anti-pp38, anti-pJNK, anti-NF-kB (p50) and anti-pan Ras antibodies. Results demonstrate that the recognition of foreign cells triggers a phosphorylation cascade leading to the activation of Ras-like small GTPases, MAPKs and NF-kB and argue in favour of a conservation, also in ascidians, of the main signalling pathways.