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. Here, we review the main studies detailing rearing, staging methods, reproduction and colony growth of this species, emphasizing the asymmetry in sexual and asexual reproduction potential, sexual reproduction in the field and the laboratory, and self- and cross-fertilization. These data, opportunely matched with recent tanscriptomic and genomic outcomes, can give a valuable help to the elucidation of some important steps in chordate evolution.

Insight on signal transduction pathways involved in phagocytosis in the colonial ascidian Botryllus schlosseri.

Tunicates are chordate invertebrates, closely related to vertebrates, which represent valuable organisms for the study of a variety of biological processes from an evolutionary point of view. As invertebrates, they rely on innate immunity to cope with foreign, potentially pathogenic material. Among tunicates, the compound ascidian Botryllus schlosseri is emerging as a reliable model organism for the study of innate immune responses. However, there is a general lack of knowledge on the signalling pathways activated during immune responses and, in particular, in phagocytosis. In the present work, we carried out a preliminary investigation of the signalling pathways involved in phagocytosis, with particular reference to MAPK activation. We studied in vitro zymosan phagocytosis in the presence of manumycin A, which inhibit the activation of Ras, PD98059, SP600125 and SB202190, inhibitors of Erk, JNK and p38, respectively, parthenolide, N-acetyl-l-cysteine (NAC) and pyrrolidine dithiocarbamate (PDTC), inhibiting NF-kB activation. In addition we carried out immunoblot and immunocytochemistry analysis with the use of anti-pErk1/2, anti-pp38, anti-pJNK, anti-NF-kB (p50) and anti-pan Ras antibodies. Results demonstrate that the recognition of foreign cells triggers a phosphorylation cascade leading to the activation of Ras-like small GTPases, MAPKs and NF-kB and argue in favour of a conservation, also in ascidians, of the main signalling pathways.

Insight on cellular and humoral components of innate immunity in Squilla mantis (Crustacea, Stomatopoda).

For deeper insights into the function of crustacean haemocytes in immune responses, we studied the morphology and enzyme content of circulating cells of the mantis shrimp Squilla mantis from the North Adriatic Sea, together with their ability to phagocytose foreign cells. We also assayed the enzyme content and the agglutinating and haemolytic activities of cell-free haemolymph. Three haemocyte types, i.e., hyalinocytes, semigranulocytes and granulocytes, can be distinguished, according to cell and nuclear morphology and the presence of cytoplasmic granules. All of them share the same patterns of enzyme activities and are recognised by the same lectins. Spreading cells (hyalinocytes and semigranulocytes) can ingest foreign cells; granules of semigranular and granular cells have similar cytochemical properties. Injection of Micrococcus luteus into the heart sinus results in an increase in the frequency of hyaline cells and a decrease in the frequency of granulocytes. After 24 h from the injection, a decrease in the number of phagocytosing hyalinocytes, and a general decrease in the frequency of acid phosphatase-positive cells was reported. Our data match previous results and suggest the existence of a single differentiation pathway for Squilla haemocytes with the three haemocyte morphs as different stages of cell differentiation. Results also indicate that Squilla haemolymph performs immunosurveillance, through rapid changes in haemocyte distribution, increase of antimicrobial and antioxidant enzymes and secretion of lectins stimulating agglutination, phagocytosis and encapsulation.

Natural apoptosis during the blastogenetic cycle of the colonial ascidian Botryllus schlosseri: a morphological analysis.

Colonies of the compound ascidian Botryllus schiosseri undergo regular generation changes, during which adult zooids are progressively resorbed and replaced by growing buds. The generation change, or take-over, is characterized by massive cell death by apoptosis, as indicated by nuclear condensation, activation of caspases, overexpression of molecules recognized by antibodies against mammalian Bax, Fas, and FasL, changes in the expression of surface molecules by senescent cells of zooid tissues, and recruitment of circulating phagocytes in zooid tissues which ensure the complete clearing of dying cells. The entire process lasts 24-36 h at 20 degrees C and has been subdivided, on the basis of the degree of contraction of old zooids, into four substages. In the present work, we carried out a detailed morphological analysis of the events occurring in zooid tissues during the take-over substages. Results Indicate that traces of apoptosis can be found in the epidermis, peribranchial epithelium, and heart in the late substage but are easily found in the branchial basket 2-4 h after the beginning of the generation change, thus confirming the antero-posterior progression of cell death, at least in the alimentary system.

Recurrent phagocytosis-induced apoptosis in the cyclical generation change of the compound ascidian Botryllus schlosseri.

Colonies of the marine, filter-feeding ascidian Botryllus schlosseri undergo cyclical generation changes or takeovers. These events are characterised by the progressive resorption of adult zooids and their replacement by their buds that grow to adult size, open their siphons and start filtering. During the take-over, tissues of adult zooids undergo extensive apoptosis; circulating, spreading phagocytes enter the effete tissues, ingest dying cells acquiring a giant size and a round morphology. Then, phagocytes re-enter the circulation where they represent a considerable fraction (more than 20%) of circulating haemocytes. In this study, we evidence that most of these circulating phagocytes show morphological and biochemical signs of apoptosis. Accordingly, these phagocytes express transcripts of orthologues of the apoptosis-related genes Bax, AIF1 and PARP1. Electron microscopy shows that giant phagocytes contain apoptotic phagocytes inside their own phagocytic vacuole. The transcript of the orthologues of the anti-apoptotic gene IAP7 was detected only in spreading phagocytes, mostly abundant in phases far from the take-over. Therefore, the presented data suggest that, at take-over, phagocytes undergo phagocytosis-induced apoptosis (PIA). In mammals, PIA is assumed to be a process assuring the killing and the complete elimination of microbes, by promoting the disposal of terminally differentiated phagocytes and the resolution of infection. In B. schlosseri, PIA assumes a so far undescribed role, being required for the control of asexual development and colony homeostasis.

Data on four apoptosis-related genes in the colonial tunicate Botryllus schlosseri.

The data described are related to the article entitled "Recurrent phagocytosis-induced apoptosis in the cyclical generation change of the compound ascidian Botryllus schlosseri" (Franchi et al., 2016) [1]. Four apoptosis-related genes, showing high similarity with mammalian Bax (a member of the Bcl-2 protein family), AIF1 (apoptosis-inducing factor-1), PARP1 (poly ADP ribose polymerase-1) and IAP7 (inhibitor of apoptosis-7) were identified from the analysis of the trascriptome of B. schlosseri. They were named BsBax, BsAIF1, BsPARP1 and BsIAP7. Here, their deduced amino acid sequence were compared with known sequences of orthologous genes from other deuterostome species together with a study of their identity/similarity.

Looking for putative phenoloxidases of compound ascidians: haemocyanin-like proteins in Polyandrocarpa misakiensis and Botryllus schlosseri.

Phenoloxidases (POs) and haemocyanins constitute a family of copper-containing proteins widely distributed among invertebrates. Both of them are able, under appropriate conditions, to convert polyphenols to quinones and induce cytotoxicity through the production of reactive oxygen species, a fundamental event in many immune responses. In ascidians, PO activity has been described and studied in both solitary and colonial species and the enzyme is involved in inflammatory and cytotoxic reactions against foreign cells or molecules, and in the formation of the cytotoxic foci which characterise the nonfusion reaction of botryllids. Expressed genes for two putative POs (CiPO1 and CiPO2) have been recently identified in C. intestinalis. In the present study, we determined the cDNA sequences of two haemocyanin-like proteins from two colonial ascidians: Botryllus schlosseri from the Mediterranean Sea and Polyandrocarpa misakiensis from Japan. Multiple sequence alignments evidenced the similarity between the above sequences and crustacean proPOs whereas the analysis of the three-dimensional structure reveals high similarity with arthropod haemocyanins which share common precursors with arthropod proPOs. Botryllus HLP grouped in the same cluster with Ciona POs, whereas Polyandrocarpa HLP clustered with arthropod haemocyanins; all of them share the full conservation of the six histidines at the two copper-binding sites as well as of other motifs, also found in arthropod haemocyanin subunits, involved in the regulation of enzyme activity. In situ hybridisation indicated that the genes are transcribed inside morula cells, a characteristic haemocyte type in ascidians where PO activity is located, at the beginning of their differentiation. These results represent a first attempt to identify candidate molecules responsible of the PO activity in compound ascidians.

Immune roles of a rhamnose-binding lectin in the colonial ascidian Botryllus schlosseri.

The present paper describes the immune role played by a recently identified (Gasparini et al. 2008) member of the rhamnose-binding lectin (RBL) family from the colonial ascidian Botryllus schlosseri. B. schlosseri RBL (BsRBL) can activate phagocytes through: (i) induction of their directional movement towards the source of the molecule; (ii) modification of cytoskeleton, required for shape changes; (iii) stimulation of the respiratory burst, and consequent production of reactive oxygen species (ROS) with microbicidal activity, including superoxide anions and peroxides; and (iv) increase in the ability to phagocytose foreign particles. RBL also induces the synthesis and release, by cytotoxic morula cells (MCs), of cytokines recognised by anti-IL1α and anti-TNFα antibodies. At high concentrations, BsRBL induces degranulation of MCs and the consequent release of the cytotoxic enzyme phenoloxidase into the medium. Results are consistent with the existence of cross-talk between B. schlosseri immunocytes (phagocytes and MCs). In addition, a three-dimensional model for BsRBL is presented.

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. In addition, results suggest the conservation of apoptosis induction mechanisms throughout chordate evolution.