Phagocyte dynamics in the blastogenetic cycle of the colonial ascidian Botryllus schlosseri: Cell senescence, segregation and clearance after efferocytosis.

In the colonial ascidian Botryllus schlosseri, phagocytes are involved in the clearance of apoptotic cells and corpses during the periodical generation changes or takeovers (TOs) that assure the renewal of the colonial zooids. The persistent respiratory burst associated with efferocytosis, leads to the induction of senescence. Indeed, giant, senescent phagocytes are abundant in the colonial circulation at TO, whereas, in the other phases of the colonial blastogenetic cycle, they colonise the ventral islands (VIs), a series of mesenchymal niches located in the lateral lacunae of the mantle, on both sides of the subendostylar sinus.

Immunotoxic effects of exposure to the antifouling copper(I) biocide on target and nontarget bivalve species: a comparative study between and .

Edible bivalves constitute an important bioresource from an economic point of view, and studies on their immune responses to environmental pollutants are crucial for both the preservation of biodiversity and economic reasons. The worldwide diffusion of copper(I)-based antifouling paints has increased copper leaching into coastal environments and its potential impact on both target and nontarget organisms. In this study, immunotoxicity assays were carried out with short-term (60 min) cultures of hemocytes from the bivalves -a mussel dominant in the macrofouling community-and -a clam dominant in the soft-sediment community-exposed to CuCl to compare the toxic effects on their immune responses.

Botryllin, a Novel Antimicrobial Peptide from the Colonial Ascidian .

By mining the transcriptome of the colonial ascidian , 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.

Potential disruptive effects of copper-based antifouling paints on the biodiversity of coastal macrofouling communities.

The expanded use of copper(I)-based antifouling paints (AF) has increased copper leaching into coastal environments, requiring attention and legislative restrictions for potential long-term effects on benthic populations. The ecological succession of macrofouling communities was analysed on wooden and stainless steel panels coated with four copper(I)-based AF (Paints A-D) immersed for 10 months in the Lagoon of Venice. With the exception of Paint B, which contained only copper(I) compounds and was based on hard-matrix technology, the other paints were based on self-polishing matrices and various booster biocides.

Two facets of geotextiles in coastal ecosystems: Anti- or profouling effects?

Nonwoven geotextile fabrics have physical, mechanical and hydraulic properties useful in coastal protection as an alternative to natural stone, slag, and concrete. In a 10-month experiment, the colonisation of macrofouling organisms on different substrata based on polypropylene (PP), polyester (PET) or high density polyethylene (HDPE) fibres was investigated in the Lagoon of Venice, Italy - an environment with temperate transitional waters with high biodiversity - and compared with the colonisation on wood as a reference substratum, because of its occurrence in artificial structures at the study location, until a stable stage was reached in the development of the macrofouling community. Geotextile fabrics showed implications for community development.

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.

Immunological response to bacterial infection in a pelagic tunicate: Inflammation in the salp Thalia democratica.

Thaliaceans are pelagic tunicates that play a key role in trophic chains of the oceans. In the field of tunicate immunity, a notable gap is the lack of data on their inflammatory response. The common salp, Thalia democratica, possesses scant immunocytes, represented by a phagocytic line (hyaline amebocytes) and a mast cell-like line (granular cells).

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.

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.

Spermatogenesis as a tool for staging gonad development in the gonochoric appendicularian Oikopleura dioica Fol 1872.

Oikopleura dioica, the only gonochoric species among appendicularians, has a spematozoon with a mid-piece and a conspicuous acrosome that, during fertilisation, undergoes a reaction forming an acrosomal process. To provide more insight into the spermatogenesis of a holoplanktonic tunicate species that completes its life cycle in three to five days, changes in the testis during individual growth have been examined. Spermatogenesis has been subdivided into seven stages based on ultrastructural features during the formation and organisation of the male gonad and the relationships between its macroscopic anatomy and the events of sperm differentiation.

Proliferation and differentiation of circulating haemocytes of Ruditapes philippinarum as a response to bacterial challenge.

Ultrastructural investigation confirmed the presence of four cell types (granulocytes, hyalinocytes, serous cells, and haemoblasts) in the haemolymph of the Manila clam, Ruditapes philippinarum. Granulocytes were characterised by numerous electron-dense granules, whereas hyalinocytes had a considerable number of small clear vesicles. Serous cells exhibited large vacuoles, which filled the cytoplasm, and haemoblasts (the undifferentiated cells) were small roundish cells characterised by a high nucleus/cytoplasm ratio.

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.

Enzyme Histochemistry for Functional Histology in Invertebrates.

In invertebrates, enzyme histochemistry has recently found a renaissance regarding its applications in morphology and ecology. Many enzyme activities are useful for the morphofunctional characterization of cells, as biomarkers of biological and pathologic processes, and as markers of the response to environmental stressors. Here, the adjustments to classic techniques, including the most common enzymes used for digestion, absorption, transport, and oxidation, as well as techniques for azo-coupling, metal salt substitution and oxidative coupling polymerization, are presented in detail for various terrestrial and aquatic invertebrates.

An intracellular adrenomedullin system reduces IL-6 release via a NF-kB-mediated, cAMP-independent transcriptional mechanism in rat thymic epithelial cells.

Thymic epithelial cells (TECs) play a key role in the regulation of central immune tolerance by expressing autoantigens and eliminating self-reactive T cells. In a previous paper we reported that adrenomedullin (ADM) and its co-receptor protein RAMP2 are located intracellularly in newborn human thymic epithelial cells (TECs). This work has two main aims: (1) to examine the cellular localization of ADM and its receptor in TECs of adult Wistar rats to validate this animal model for the study of the ADM system and its function(s) in thymus; (2) to investigate the potential modulating effect of ADM on the NF-kB pathway, which is involved through the production of cytokines such as IL-6, in the maturation of T-lymphocytes and immunological tolerance.

Histamine Stimulates Ciliary Beat Frequency via the H2 Receptor in the Protochordate Botryllus schlosseri.

Histamine is a biogenic molecule that plays a role in many physiological pathways via binding to a specific receptor. Histaminergic receptors belong to the large family of seven-transmembrane α-helix domain receptors classified in mammals into four distinct classes: H1, H2, H3, and H4. Despite being widely studied in vertebrates, few data are available on the invertebrate receptors, with only predicted H1 and H2 sequences for nonchordate deuterostomes.

Immunotoxicity in ascidians: antifouling compounds alternative to organotins-IV. The case of zinc pyrithione.

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.

Sexual and asexual reproduction in the colonial ascidian Botryllus schlosseri.

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.

Preliminary evaluation of the toxic effects of the antifouling biocide Sea-Nine 211™ in the soft coral Sarcophyton cf. glaucum (Octocorallia, Alcyonacea) based on PAM fluorometry and biomarkers.

Sea-Nine 211™ is a new biocide specifically formulated for antifouling paints and being considered to have a low environmental impact. Even with a short environmental half-life, this compound can cause toxic effects on marine organisms. This study used PAM fluorometry and biomarkers of oxidative stress (GST, CAT and LPO) to monitor potential toxic effects of Sea-Nine 211™ on fragments of the soft coral Sarcophyton cf.

Immunotoxicity in ascidians: antifouling compounds alternative to organotins: III--the case of copper(I) and Irgarol 1051.

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.

Hovering between death and life: natural apoptosis and phagocytes in the blastogenetic cycle of the colonial ascidian Botryllus schlosseri.

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.

Immunotoxicity in ascidians: antifouling compounds alternative to organotins - II. The case of Diuron and TCMS pyridine.

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.

First evidence of cell division in circulating haemocytes from the Manila clam Tapes philippinarum.

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.

A tale of death and life: natural apoptosis in the colonial ascidian Botryllus schlosseri (Urochordata, Ascidiacea).

The colonial ascidian Botryllus schlosseri forms new zooids by blastogenesis, through the formation of palleal buds which progressively grow and mature until adults are formed. At a temperature of 19 degrees C, adult zooids remain active for about one week; then they contract, close their siphons and are gradually resorbed, being replaced by buds which reach functional maturity, open their siphons and begin their filtering activity as adult zooids. This recurrent generation change, known as take-over, is characterised by the occurrence of diffuse programmed cell death by apoptosis.

Toxic effects of new antifouling compounds on tunicate haemocytes I. Sea-nine 211 and chlorothalonil.

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.