Loriano Ballarin

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.

Genetic and cytological aspects of histocompatibility in ascidians

Abstract The three types of intraspecific histocompatibility in ascidians ‐fusibility, allograft rejection or retention, and cell contact reaction with mutual lysis ‐ are reviewed and compared under both genetic and histological profiles. The differences between solitary and colonial species are outlined. The genetics of fusibility in different populations of Botryllus schlossen is reconsidered with special reference to its bearing on sexual incompatibility, also in the light of new data. The different modes of allorejection are discussed with regard to the cellular and humoral factors involved. Some histological data on nonfusion reactions in B. schlossert are presented. The difficulties of finding a unifying model for the three types of ascidian alloreactivity are outlined.

Morula Cells as the Major Immunomodulatory Hemocytes in Ascidians: Evidences From the Colonial Species Botryllus schlosseri

Immunocytochemical methods were used to study the presence and distribution of IL-1-α- and TNF-α-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.

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.

Air exposure and functionality of Chamelea gallina haemocytes: effects on haematocrit, adhesion, phagocytosis and enzyme contents

The Venus clam Chamelea gallina is fairly common along the western coasts of the Adriatic and is subjected to intense fishing. Since over the last 20 years extensive hypoxic and anoxic conditions have repeatedly damaged this natural resource, we decided to study the effects of anoxic stress on the functionality of clam haemocytes and the consequences on immune responses. Clams, exposed to air, close their valves and tissues become anoxic and metabolism processes switch to anaerobiosis. In these conditions, a significant decrease in the haematocrit value and in the percentage of acid phosphatase-positive haemocytes was observed, while the number of cells with beta-glucuronidase significantly increased after day 1. The above indices generally returned to control values when clams were re-immersed in seawater after 1 day of treatment. Clams exposed to air for 2 days and then re-immersed, attempted to recover in the subsequent 3 days. Animals had fully recovered on day 4. Three-day-exposed clams did not recover. Phagocytic and adhesion indices decreased significantly after the first day of air exposure. The change in frequency of three types of circulating cells (spreading, round, apoptotic) was also monitored.

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.