Armando Sabbadin

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.

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.

Determination of polarity and bilateral asymmetry in palleal and vascular buds of the ascidian Botryllus schlosseri

Abstract Reversal of the bilateral asymmetry of the zooids was induced in a series of colonies of Botryllus schlosseri . Palleal buds from colonies with normal or reversed bilateral asymmetry were isolated in the early stages from the parental zooids and cultured in the vascularized tunic of the same colony or of another colony with opposite asymmetry. Vascular budding was induced in colonies with either type of asymmetry. The bud polarity was shown to depend on the vascularization; the test vessel entering the isolated palleal bud always causes the entrance point to become the posterior end of the developing zooid. On the contrary, the bilateral asymmetric type is predetermined in the bud primordium; the isolated palleal buds develop the type of asymmetry of their parents, even when grafted in the test of a colony with opposite asymmetry. Since the same was also true of the vascular buds, it is concluded that the information for the kind of bilateral asymmetry to be developed is conveyed by the epidermal envelope of the bud. The epidermis of the parental zooids influences the palleal buds, whereas the wall of the test vessels, epidermal extrusions of the zooids, influences the vascular buds.

Self- and cross-fertilization in the compound ascidianBotryllus schlosseri

Abstract In the viviparous colonies of the ascidianBotryllus schlosseri, which are protogynous hermaphrodite, selfing can be prevented by maintaining a difference of 2–3 days between the sexual cycles of the crossing colonies. The same expedient, applied to two pieces of single colonies, allows a successful selfing. Self- and cross-fertilization have been inferred from the phenotypes of the offspring yielded by colonies marked by different pigmentation genes. A comparative quantitative analysis of the sets of offspring, obtained by self- and cross-fertilization from colonies collected in the field and colonies raised in the laboratory, has given preliminary indications about the role played by environmental and genetic factors in the control of embryonic development, larval metamorphosis, survival and growth of newly founded colonies.

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.

Oxidative stress induces cytotoxicity during rejection reaction in the compound ascidian Botryllus schlosseri.

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.

Histoenzymatic staining and characterization of the colonial ascidian Botryllus schlosseri hemocytes

Abstract The distribution pattern of some hydrolytic and oxidizing enzymes among Botryllus schlosseri hemocytes has been investigated histochemically. Signet‐ring cells, macrophages, and some hyaline amoebocytes were found to stain positively for phosphatases, glycosidases, and esterases, and morula cells and some granular amoebocytes for arylsulfatase, peroxidase, and phenoloxidase. Our results suggest two differentiation pathways for these blood cell types: one involves hyaline amoebocytes, signet‐ring cells, and macrophages and leads to phagocytic cells; the other involves granular amoebocytes and morula cells and leads to cells responsible for inflammatory responses.

Chimeras and histocompatibility in the colonial ascidian Botryllus schlosseri

Chimeras of B. schlosseri were prepared by pairwise combination of colonies sharing one allele at the fusibility gene locus (AD = AC chimeras). A frequent resorption of one of the partners was observed and the resorption time was shown to be significantly correlated with the relative size, the resorbing partner being usually the larger. Both in the whole chimeras, AD = AC, and in the separated partners, (AD)AC, the fusibility of AC was frequently altered. In AD = AC the fusion did not prevent entirely AC fusion with BC. The fusion frequency with BC was significantly higher than with BD and significantly higher in (AD)AC than in AD = AC. Repeated rejections with BC or both BC and BD, repeated fusions with BD, simultaneous or successive fusions and rejections especially with BD, over a period of several months, indicated a long lasting competitive interaction of AD and AC in (AD)AC chimeras. The persistence in these chimeras of the AD cell population was also confirmed by the chimeric electrophoretic pattern of PGI in most of them.