Francesca Cima

Phagocytosis in the colonial ascidian Botryllus schlosseri

Phagocytosis by Botryllus schlosseri hemocytes is influenced by temperature, pH, concentration, and physicochemical properties of the test particles and requires Ca2+ or Mg2+ ions to occur. Phagocytes recognize glucosyl or mannosyl residues on the surface of yeast cells, and a respiratory burst is associated with phagocytosis, as indicated by increased superoxide production. Factors that enhance phagocytosis of yeast, sheep red blood cells, and latex beads and reduce the uptake of yeast and sheep erythrocytes are present in the plasma.

Botryllus schlosseri: a model ascidian for the study of asexual reproduction.

Botryllus schlosseri, a cosmopolitan colonial ascidian reared in the laboratory for more than 50 years, reproduces both sexually and asexually and is used as a model organism for studying a variety of biological problems. Colonies are formed of numerous, genetically identical individuals (zooids) and undergo cyclical generation changes in which the adult zooids die and are replaced by their maturing buds. Because the progression of the colonial life cycle is intimately correlated with blastogenesis, a shared staging method of bud development is required to compare data coming from different laboratories. With the present review, we aim (1) to introduce B. schlosseri as a valuable chordate model to study various biological problems and, especially, sexual and asexual development; (2) to offer a detailed description of bud development up to adulthood and the attainment of sexual maturity; (3) to re‐examine Sabbadins ( 1955 ) staging method and re‐propose it as a simple tool for in vivo recognition of the main morphogenetic events and recurrent changes in the blastogenetic cycle, as it refers to the developmental stages of buds and adults. Developmental Dynamics 236:335–352, 2007.

PHENOLOXIDASE AND CYTOTOXICITY IN THE COMPOUND ASCIDIAN BOTRYLLUS SCHLOSSERI

The vacuoles of morula cells (MC) of the colonial ascidian Botryllus schlosseri contain phenoloxidase (PO). As the release of their vacuolar content at the border of incompatible contacting colonies is associated with the formation of necrotic masses which characterize the rejection reaction, the role of PO in Botryllus cytotoxicity was investigated. When hemocytes are incubated with blood plasma from incompatible (heterologous) colonies, MC degranulate and, after 60 min, the cytotoxicity index becomes significantly greater than that observed in controls incubated with autologous plasma. The rise in cell mortality is completely inhibited by the addition of PO inhibitors sodium benzoate, tropolone and phenylthiourea, and serine protease inhibitors phenylmethylsulfonyl fluoride, benzamidine, N-tosyl-L-phenylalanine chloromethyl ketone and N-tosyl-L-lysine chloromethyl ketone. The addition of either reducing agents L-cysteine and ascorbic acid or reactive oxygen species scavenger enzymes superoxide dismutase and catalase has a similar effect. Significant inhibition of cytotoxicity is also observed with the quinone scavenger, 3-methyl-2-benzothiazolinone hydrazone. In the presence of sodium benzoate and phenylthiourea, there is a significant reduction in the number, size and color intensity of necrotic masses along the contact border of incompatible colonies. A significant increase in superoxide anion production, completely inhibited by sodium benzoate, is observed when hemocytes are incubated with heterologous blood plasma. These results indicate that: (i) PO is the enzyme responsible for the cytotoxicity observed in both hemocyte cultures and rejection reactions; (ii) PO is present inside MC vacuoles as a proenzyme which is activated, upon release, by humoral proteases; (iii) cytotoxicity appears to be mainly due to oxidative stress generated by PO during oxidation of polyphenols to quinones without the involvement of other oxidases such as NADPH oxidase and peroxidase.

Morula Cells and Histocompatibility in the Colonial Ascidian Botryllus schlosseri

Abstract The role of morula cells (MC) in the formation of necrotic regions characterizing the rejection reaction between incompatible (i.e. nonfusible) colonies was investigated in the colonial ascidian Botryllus schlosseri. These blood cells share several chemical and histochemical properties with the pigment of the necrotic masses: both of them show strong reducing activities and contain high quantities of sulphur and iron. Sulphur is present mainly as sulphates and thiols in MC and as sulphates and disulphides in necrotic regions; iron is in the form of ferrous iron in MC and as ferric iron in the necrotic regions. Inside MC vacuoles phenoloxidase (PO) activity and polyphenol substrata are present: the latter are oxidized by PO to quinones, which then polymerize to form the melanin-like substances of dark-brown colour of the necrotic regions. When hemocytes are incubated with heterologous incompatible blood plasma (BP), MC change their morphology, and a significant increase in PO activity is found in the recovered medium as compared with the activity of untreated plasma. No increase in PO activity is observed after incubation with autologous or heterologous compatible blood plasma. These results are consistent with the hypothesis of a humoral factor diffusing from incompatible colonies which is recognized by MC and leads to their degranulation, with the consequent release of the content of their vacuoles, mainly oxidative enzymes, responsible for the localized cell death in necrotic regions.

Embryotoxicity of butyltin compounds to the sea urchin Paracentrotus lividus

Tributyltin (TBT) has been widely employed in marine anti-fouling paints as a biocide, although it represents a serious risk, particularly in estuarine and coastal water/sediment ecosystems. In this study, the embryotoxic effects of TBT and its degradation products, dibutyltin (DBT) and monobutyltin (MBT), were analyzed during the development of the sea urchin Paracentrotus lividus from post-fertilization to the pluteus stage, to better clarify ecotoxicological impact. The embryotoxicity of butyltins is concentration-dependent and increases proportionally with number of butyl groups. Significant growth reduction was observed at TBT concentrations as low as 0.01 microgram l-1; 1 microgram l-1 was the maximum concentration allowing embryos to reach the pluteus stage at 48 h post-fertilization. Development was blocked at the morula or blastula stage with higher TBT concentrations. DBT and MBT are less toxic: slowed development and a decrease in pluteus size occurred at 10 micrograms l-1 DBT and 0.5 mg l-1 MBT. Effects on both skeletal deposition and blocked embryonic development are suggested to be due to the interference of organotin compounds with intracellular calcium homeostasis.

Immunotoxic effects of organotin compounds in Tapes philippinarum.

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 TBT > or = DBT > MBT for butyltins and TPTC > TPTA > or = TPTH for triphenyltins.

Biomarkers for TBT immunotoxicity studies on the cultivated clam Tapes philippinarum (Adams and Reeve, 1850).

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.

Tributyltin induces cytoskeletal alterations in the colonial ascidian Botryllus schlosseri phagocytes via interaction with calmodulin

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.

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 α‐tubulin. The mean number of circulating haemocytes was 5 (×106) cells/ml haemolymph. Two main haemocyte populations were found in the haemolymph: small cells, 2–3 μm in diameter and 10–100 fl in volume; and large cells, 6–10 μm in diameter and 150–400 fl 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.

Apoptosis and phosphatidylserine-mediated recognition during the take-over phase of the colonial life-cycle in the ascidian Botryllus schlosseri

Colonies of the ascidian Botryllus schlosseri undergo recurrent generation changes in which adult zooids are gradually resorbed and replaced by new blastogenic generations. During these periods, known as take-over phases, programmed cell death, which, on the basis of morphological analysis is ascribed to apoptosis, occurs widely in zooid tissues. In the present report, we re-investigate cell death during the take-over process. Results confirm the occurrence of diffuse apoptosis, as evidenced by chromatin condensation, positivity to the TUNEL reaction and expression of phosphatidylserine on the outer leaflet of the plasma membrane. Apoptosis also occurs among haemocytes, and senescent blood cells are actively recognised and ingested by circulating professional phagocytes. Both phosphatidylserine and CD36, a component of the thrombospondin receptor, are involved in the recognition of apoptotic haemocytes, which fosters the idea that fundamental recognition mechanisms are well conserved throughout chordate evolution.