1,721,193 research outputs found
PHAGOCYTE BEHAVIOUR DURING THE COLONIAL BLASTOGENETIC CYCLE IN THE COMPOUND ASCIDIAN BOTRYLLUS SCHLOSSERI.
No full textColonies of the ascidian Botryllus schlosseri experience a cyclical generation change (takeover) during which adult zooids stop their filtering activity, their tissue undergo cell death by apoptosis and are progressively resorbed. In the meantime, a new blastogenic generation reaches its functional maturity, opens siphons and starts filtering. During the blastogenetic cycle, the phagocytic differentiation pathway of the circulating immunocytes plays a key role: the presence of both hydrolytic enzymes and mediators of local inflammation supports the hypothesis that the phagocytic cell line maintains characteristics of a primordial system with high functional versatility. Phagocytes cyclically change their morphology and frequency. During the mid-cycle, hyaline amoebocytes are numerous in blood circulation, inside the tunic and free of moving on the surface of the tunic that internally covers the oral siphon, where they play an immunosurveillance role of the pharynx by recognising and phagocytising foreign particles, and, after exposure of colonies to bacterial spores, forming a transitory plug in the siphonal lumen by exocytosis of floccular and colloidal material rich of heparin, histamine and proteases. In the takeover, the frequency of hyaline amoebocytes falls abruptly since, by engulfing apoptotic cells, they change their shape becoming large and spherical macrophage-like cells. These scavenger phagocytes are massively recruited from the circulation to the dying tissues of old zooids, where they assure the clearance of senescent cells. This is fundamental for the progression of the takeover, as during this phase lasting 24-36 hs, colonies do not feed and rely uniquely on the recycling of nutrients deriving from the digestion of senescent cells. The massive ingestion of effete cells causes, in turn, an increase in reactive oxygen metabolite production and nitric ion release leading to the death of phagocytes and subsequent clearance by other phagocytes, so that a “Russian doll effect” can be observed. Finally, large macrophage-like cells accumulate in the pharyngeal lacunae and are continuously eliminated through the peribranchial chamber and then the cloacal siphon with a discharging mechanism never previously described which continues in the first phases of the mid-cycle. The consistent disappearance of large phagocytes from the circulation is counterbalanced by a population of new, undifferentiated cells (haemoblasts) already beginning from the late takeover.
This work was supported by “Progetto di Ateneo” Grant CPDA082501 to F. Cim
THE HEPARIN-HISTAMINE SYSTEM IN THE PHAGOCYTIC LINE OF A TUNICATE: AN ANCIENT CELL SYSTEM EQUIVALENT TO VERTEBRATE MAST CELLS?
No full textIn the compound ascidian Botryllus schlosseri, sentinel-cells were observed in the oral siphon where they play an immunosurvellance role in the opening of the pharynx. Their morphology, histochemical characteristics and ability to engulf test-particles are typical of hyaline amebocytes of the blood phagocytic line.
Histochemical and immunohistochemical studies at light and electron microscope reveal that, like in vertebrate mast cells, heparin and histamine co-localize inside the granules of this cell type and exposure to compound 48/80, a specific degranulating agent of vertebrate mast cells, leads to cell degranulation, suggesting that polyfunctional cells separated functions and competences among various specialized cell types of the innate immunity throughout the chordate evolution. Heparin and histamine were found in the temporary “plug” of colloidal matter that closed the oral siphon after 15 min exposure to bacterial spores in seawater, resulting by degranulation of the sentinel-cells. The main physiological functions of these substances is discussed. Heparin might be involved in the releasing activity of proteases, antimicrobial peptides, histamine, cytokines and growth factors. Histamine might be involved in the modulation of the ciliary beat in the pharynx. In short-term branchial cultures, exogenous histamine significantly increases the ciliary beat frequency and the presence of H2 receptors was indirectly demonstrated by means of the specific antagonist ranitidine suggesting a model of clearance similar to the mucociliary transport in the vertebrate respiratory tract
First description of a histamine receptor of class 2 (HRH2) in a protochordate: expression during blastogenesis and role in regulation of ciliary beat frequency
Full text linkHistaminergic receptors belong to the family of seven-transmembrane α-helix domain receptors classified in mammals into four distinct classes. Despite being widely studied in vertebrates, few data are available on the invertebrate receptors, with only predicted H1 and H2 sequences for non-chordate deuterostomes. We report the first transcript evidence of an H2 receptor for histamine in the colonial ascidian Botryllus schlosseri showing a high degree of conservation with HRH2 mammalian and other vertebrate orthologous proteins. The transcript and protein localisation during blastogenic development through in situ hybridisation and immunohistochemistry has been described. The mRNA expression appears first in the ciliary tissues of the alimentary system in filter-feeding adults and the buds, with a particular intensity in the pharynx. Transcription is activated very early, beginning from the inner layer of the disc of the secondary bud. From one generation to the next, the transcript signals become more and more intense at the level of the emergence of primordia of the branchial and peribranchial chambers and, finally, in the cells bordering the stigmata, dorsal lamina, and non-glandular ciliated zones of the endostyle. The translated H2 receptor appears as soon as the primordia of branchial and peribranchial chambers form in the secondary bud, and, in the primary buds, is found mainly in the protostigmata before the two layers of branchial and peribranchial epithelial tissue perforate to form the stigmata. In the adult zooid, the H2 receptor is expressed by ciliated mucous cells involved in food progression throughout the whole length of the alimentary canal. The observation of the effects of histamine and histamine-receptor antagonist (ranitidine) and agonist (dimaprit) drugs on explanted branchial tissue has provided confirmation concerning the receptor class and its role in regulating the ciliary beat frequency. The involvement in the local regulation of ciliary activity is of particular concern for evolutionary considerations because HRH2 seems to have been conserved in the pharynx and its developmental derivatives (e.g. upper respiratory tract and middle ear of mammals) during the evolution of chordates
FURTHER INSIGHTS ON SIPHONAL GUARD CELLS OF ASCIDIANS
No full textIn the oral siphon of the colonial ascidian Botryllus schlosseri, hyaline amoebocytes directly exposed to the sea-water flow entering into the pharynx have been recently observed and described. These cells, named “siphonal guard cells” (SGC), are free of moving on the surface of the tunic that internally covers the siphons. Our previous observations by means of histochemical, histoenzymatic and immunohistochemical techniques showed that they share many morpho-functional characteristics with the phagocytic blood cell line, from which probably they originate, and are able to recognise and phagocytise various foreign particles.
After exposure of colonies to bacterial spores, the observations at both light and electronic microscope revealed that these cells are involved in a complex and unusual series of local and systemic immune events. Already after 5 min, the SGC showed bacteria inside their heterophagic vacuoles. After 10-15 min, as a transitory plug of floccular and colloidal material formed in the lumen of the siphon by exocytosis of some SGC, other ones with engulfed bacteria crossed the epidermis of the siphon reaching the siphonal sinus; cells of the cytotoxic blood cell line (morula cells) were drawn and crowded into the siphonal sinus, where most of them were positive to anti-TNF-α and anti-CD57 antibodies and degranulated stimulating, after this time and until 12 h, large scavenger phagocytes. The latter showed bacteria engulfed in their large phagosomes, increased in number in the blood circulation and were continuously eliminated through the peribranchial chamber with a mechanism which was never previously described.
As regards the ability to transfer an alert signal, the role of SGC appears important as regards the immunosurveillance of the opening of the alimentary canal, similarly to what occurs in the vertebrate oropharyngeal lymphatic tissues
CYCLIC APOPTOSIS IN THE DIGESTIVE TRACT OF A PROTOCHORDATE.
No full textTissue degeneration which occurs during development of organisms is often of morphogenetic importance as well as proliferation and differentiation. Cyclic apoptosis of organs was progressively lost in Chordates. Botryllus schlosseri is a colonial ascidian continuously forming new zooids by blastogenesis, through the recurrent formation of palleal buds, which grow and mature until an adult is formed. Three blastogenic generations are commonly co-present: adult, filtering zooids, their buds and budlets on buds. At a temperature of 19°C, adult zooids remain active for about one week (mid-cycle stages); then they contract, close their siphons and are gradually resorbed, being replaced by a new generation of adult zooids, represented by buds which reach functional maturity, open their siphons and begin their filtering activity (regression or take-over stage). This stage is characterised by the occurrence of diffuse programmed cell death by apoptosis in zooid tissues, as evidenced by TUNEL reaction for chromatin fragmentation and annexin V labelling for detection of exposed phosphatidylserine, whereas infiltration of circulating phagocytes, which appear engulfed with apoptotic cells, is observed. With these characteristics, colonial tunicates are suitable subjects for studies on cyclical involution and resorption of tissues. In residual zooids remaining for a long time in the centre of each colony, melanin and lipofuscins accumulate as detected with Masson-Fontana, Ziehl-Nielsen and H2O2 bleaching methods. Immunocytochemical assays to detect pro- and antiapoptotic factors reveal an opposite expression which progressively extends in tissues of adult zooids with an organ gradient starting from the branchial basket. Results support the idea that fundamental mechanisms for the induction of apoptosis are well conserved throughout Chordate evolution
Herbarium – un’inedita collezione di piante del XVIII secolo conservata presso l’Orto Botanico dell’Università di Padova
No full textEffects of geotextiles for coastal erosion control on marine fouling settlement.
No full textIn recent years, the prevention of coastal erosion has arisen as a top priority for reducing the damage to structures and loss of lands. Climatic change is making the erosion rates worse by increasing in storm frequency and intensity. The high coastline recession rates cause significant concern in heavily populated locations. New tools are represented by nonwoven geotextiles. They are made of polypropylene (PP) and polyester (PET) fibres, which are useful in artificial structures for coastal protection. On the other hand, they represent an alternative to natural hard substrates and could interfere with the settlement of fouling species in respect of natural substrates. To better understand the effects of geotextiles on the colonisation capability of macrofouling organisms, a 10-months study was carried out in the Lagoon of Venice (Italy).Three different needle-punched staple fibre geotextiles were chosen: 1) White-PP, 2) hot-calendered White PP&PET, and 3) multi-layered Coloured PP&PET. Fouling settlement was monitored and analysed on panels replaced monthly with the aim to investigate significant differences among the covering surfaces of each fouling species. The measures of the areas (cm2) per month were compared using PERMANOVA considering two fixed factors, i.e., geotextiles and month.
All geotextiles revealed a negative effect on the settlement of green and red algae, bivalve molluscs and barnacles. Conversely, they showed a positive selective effect towards both solitary and colonial ascidians. As regards the 18 organisms observed, PERMANOVA showed that for four of the organisms (Ulva rigida, Ceramium ciliatum, Janua heterostropha, Bugula neritina) the settlement takes place according to a significant negative selection on basis of both fixed parameters considered in the analysis (month and geotextile). Conversely, for two tunicates (Ciona robusta and
Diplosoma listerianum) the positive selection on settlement capacity appears to be significantly dependent only according to month and not to the type of geotextiles.
Therefore, it must be considered that a long-term and extensive use of geotextiles worldwide could negatively affect local biodiversity and community growth on coasts. The progressive loss of pivot species in favour of the selection of dominant and/or invasive species could trigger negative consequences in both trophic chains of coastal ecosystems and in economical relevant activities of fishing industry and aquaculture
Selective potential of geotextiles on marine fouling settlement
Full text linkIn recent years, the prevention of coastal erosion due to the action of waves, currents, tides and organisms has arisen as a top priority for reducing the damage to structures and loss of lands. Climatic change is making the erosion rates worse by increasing in storm frequency and intensity. The high coastline recession rates cause significant concern in heavily populated locations. New tools for application in hydraulic engineering are represented by nonwoven geotextiles. These materials are made of polypropylene (PP) and polyester (PET) fibres, have physical and hydraulic properties, which are useful in artificial structures for coastal protection as an alternative to natural hard substrates. On the other hand, they could interfere with the settlement of fouling species in respect of natural substrates. To better understand the potential effects of geotextiles on the colonization capability of fouling organisms, a 10-months study was carried out in the Lagoon of Venice (Italy), a particular environment of temperate transitional waters with high biodiversity. Three different needle punched staple fibre
geotextiles were chosen from Naue GmbH & Co. (Germany): 1) Secutex R601 (white PP), 2) Secutex 251 GRK 4C (hot-calendered white PP and PET), and 3) Terrafix B 609 (multi-layered coloured PP and white PET). Fouling settlement was monitored and analysed on panels replaced monthly and compared with the colonization on wood panels as a reference substrate. All geotextiles revealed a negative effect on the settlement of green and red algae, bivalve molluscs and barnacles. Secutex R601 and Terrafix B 609 also inhibited the settlement of serpulids. Conversely, they showed a positive selective effect towards various ascidian species. The loss of pivot species in favour of the selection of dominant and/or invasive species could trigger negative consequences in both trophic chains of coastal ecosystems and in economical relevant activities of fishing industry and aquaculture
Tributyltin induces cytoskeletal alterations in the colonial ascidian Botryllus schlosseri phagocytes via interaction with calmodulin
No full textIn 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 Ca2+-CaM hydrophobic region – by both interacting substances and, therefore, the formation of a TBT-CaM complex. Instead, isodynamic mixtures with thapsigargin, an inhibitor of Ca2+-ATPase of the endoplasmic reticulum, have an effect of potentiation, suggesting that TBT indirectly interacts with this Ca2+-ATPase activity. We hypothesise that the main mechanism of action of TBT in B. schlosseri phagocytes is alteration of Ca2+ homeostasis by means of direct interaction with endogenous CaM, which induces a conformational change preventing the regulative activity of CaM on Ca2+-ATPase. Consequently, an excess of cytosolic Ca2+ accumulates which, together with the inhibition of CaM-dependent kinases and Ca2+-regulated proteins, produces extensive cytoskeletal disorganisation
UNDIFFERENTIATING CELLS IN THE BLOOD OF THE COLONIAL ASCIDIAN BOTRYLLUS SCHLOSSERI: A MORPHO-FUNCTIONAL CHARACTERISATION.
No full textColonies of the ascidian Botryllus schlosseri undergo a periodic tissue renewal in the take-over stage of the colonial blastogenetic cycle, during which an extensive apoptosis occurs in the adult zooid tissues and the senescent cells are progressively removed by circulating phagocytes. The haemocytes which circulate in the common vascular system also die partly by apoptosis during this stage. These cells are replaced by new haemocytes, likely differentiating from stem cells. Up to now, haemopoiesis was observed only in solitary ascidians in which haematopoietic noduli were described in the branchial wall. Nothing is known on haemopoiesis in colonial species, in the blood circulation of which two cell types with the morphology of undifferentiated cells are recognizable: haemoblast and lymphocyte. We have studied the cytochemical and immunocytochemical properties of these haemocytes: results indicate the haemoblast as a pluripotent stem cell since it shows a basophilic nucleus labeled either with Hoechst 33342 for euchromatin or anti-Ki-67 and anti-PCNA antibodies specific markers of nuclear proteins involved in cell proliferation and its plasma membrane is labeled by anti-CD34 and anti-CD100 antibodies, specific for haemopoietic cells in vertebrates. Commercial antibodies for cytokine receptors, like interleukin 1 receptor I (IL-1RI) and stem cell factor receptor (SCF-R) label haemoblast plasma membrane, suggesting the presence of growth factor receptors. Both lymphocytes and haemoblasts during the colonial cycle show a significant increase in concentration during the blastogenetic replacement. However, mitosis figures were rarely observed in circulating haemocytes. In vitro assays of haemocyte exposure to colchicine showed the presence of mitosis figures, which significantly increase after exposure to bacteria indicating a proliferating capability in blood circulation mainly as an immune response as observed in other invertebrates like molluscs
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