Suppression of cell-spreading and phagocytic activity on nano-pillared surface: in vitro experiment using hemocytes of the colonial ascidian Botryllus schlosseri.

Nano-scale nipple array on the body surface has been described from various invertebrates including endoparasitic and mesoparasitic copepods, but the functions of the nipple array is not well understood. Using the hydrophilized nanopillar sheets made of polystyrene as a mimetic material of the nipple arrays on the parasites\u2019 body surface, we assayed the cell spreading and phagocytosis of the hemocytes of the colonial ascidian Botryllus schlosseri. On the pillared surface, the number of spreading amebocytes and the number of phagocytizing hemocytes per unit area were always smaller than those on the flat surface (Mann-Whitney test, p < 0.05 - 0.001), probably because the effective area for the cell attachment on the pillared surface is much smaller than the area on the flat sheet. The present results supports the idea that the nipple array on the parasites' body surface reduces the innate immune reaction from the host hemocytes

Complement-mediated cooperation between immunocytes in the compound ascidian Botryllus schlosseri

Two main kinds of innate immune responses are present in ascidians: phagocytosis and cytotoxicity. They are mediated by two different types of circulating immunocytes: phagocytes and cytotoxic morula cells (MCs). MCs, once activated by non-self-recognition, can stimulate phagocytosis by the release of soluble factors able to act as opsonins. BsC3, the complement C3 homologue, like mammalian C3, contains the thioester bond required to split the molecule into BsC3a and BsC3b. BsC3b likely represents the MC opsonin as it can enhances phagocytosis. The tenet is supported by the observed reduction in phagocytosing cells after exposure of hemocytes to compstatin, a drug preventing C3 activation, or after the bsc3 knockdown by iRNA injection. In addition, the transcript for BsCR1, homologous to mammalian CR1, is present in Botryllus phagocytes and the transcription is modulated during the blastogenetic cycle. MCs also release cytokines (chemokines) able to recruit immunocytes to the infection site. The activity is inhibited by antibodies raised against human TNFa. Since no genes for TNFa are present in the Botryllus genome, the observed activity is probably related to a TNF-domain containing protein, member of the Botryllus complement system. Conversely, activated phagocytes release a rhamnose-binding lectin able to interact with microbial surfaces and act as opsonin. It can also activate MCs by inducing the release of the reported cytokine and stimulate their degranulation. Overall, the results obtained so far indicate the presence of a well-defined cross-talk between the two types of immunocytes during the immune responses of B. schlosseri

BsTLR: a new member of the TLR family of recognition proteins from the colonial ascidian Botryllus schlosseri.

Toll-like receptors (TLRs) represent a well-known family of conserved pattern recognition receptors the importance of which, in non-self recognition, was demonstrated in both vertebrates and invertebrates. Tunicates represent the vertebrate sister group and, as invertebrates, they rely only on innate immunity for their defense. As regards TLRs, two transcripts have been described and characterized in the solitary species Ciona robusta, referred to as CiTLR1 and CiTLR2. Using the Ciona TLR nucleotide sequences, we examined the available transcriptomes of Botryllus schlosseri looking for similar sequences. We were able to identify a sequence, with similarity to CiTLR2 and, through in silico transduction and subsequent sequence analysis, we studied the domain content of the putative protein. The sequence, called BsTLR, has a TIR and a transmembrane domain, four LLR and two LRR-CT domains. In addition, we analised bstlr transcription in vivo and in vitro, under various experimental conditions and in different phases of the Botryllus blastogenetic cycle. Our data show that, in different phases, there is a change in gene transcription and mRNA location, according to the blastogenetic phase

Characterization of the complement system in a colonial tunicate: C3 complement receptors and opsonic role of C3

The compound ascidian Botryllus schlosseri is a reliable model organism for the study of immunobiology. As an invertebrate, it relies only on innate immunity for its defense. We already demonstrated the presence, in Botryllus, of homologues of mammalian C3, Bf, MBL and MASP1, referred to as BsC3, BsBf, BsMBL and BsMASP, respectively. All the complement components identified so far, are expressed by morula cells, the most abundant circulating hemocytes. In mammals, once the complement system is activated, a cascade of reactions occurs resulting in the cleavage of the third complement component (C3) to C3a and C3b, the former exerting a chemotactic activity, the latter acting as opsonin and, ultimately, activating the lytic pathway. The best-known receptor for C3a in mammals is C3aR, whereas CR1 is the receptor able to recognize and bind C3b on the microbial surfaces. Here, we describe, in B. schlosseri, new genes showing homology with vertebrate C3aR and CR1, respectively, and studied their transcription in the course of the colonial blastogenetic cycle. In addition, we continued our analysis of the role of C3 in Botryllus immunity by studying the modulation of BsC3 transcription during the colonial blastogenetic cycle and the effect of bsc3 knockdown on immune responses. Results indicate that only morula cells, and no other immunocytes type, are labelled by the antisense probe for BsC3aR, whereas phagocytes and young, undifferentiated cells, known as hemoblasts, are the cells stained by the probe for BsCR1. Both the bsc3ar and bscr1 genes are constitutively transcribed. However, a modulation in the extent of transcription occurs during the colonial blastogenetic cycle as the amount of BsC3aR mRNA abruptly decreased at TO, whereas no differences were observed when EC and MC were compared. This is probably related to the renewing of circulating cells at TO, that are replaced by new, differentiating cells entering the circulation in the same period

Characterization of the complement system in a colonial tunicate: C3 complement receptors and opsonic role of C3

The complement system is one of the most ancient immune modulator mechanism of bilaterian metazoans. In vertebrates, three complement-activation pathways are known: the classical, the alternative and the lectin pathways: all of them converge on the cleavage of C3. The compound ascidian Botryllus schlosseri is a reliable model organism for the study of immunobiology. As an invertebrate, B. schlosseri relies only on innate immunity for its defense and immunocytes. We already demonstrated the presence, in Botryllus, of homologues of mammalian C3, Bf, MBL and MASP1, referred to as BsC3, BsBf, BsMBL and BsMASP, respectively. All the complement components identified so far, are expressed by morula cells, the most abundant circulatingemocytes. In mammals, once the complement system isactivated, a cascade of reactions that involves proteolysis and polymerization occurs resulting in the cleavage of the third complement component (C3) to C3a and C3b, the former exerting a chemotactic activity, the latter acting as opsonin and, ultimately, activating the lytic pathway. The best-known receptor for C3a in mammals is C3aR, whereas CR1 is the receptor able to recognize and bind C3b on the microbial surfaces. Here, we describe, in B. schlosseri, two newgenes showing homology with vertebrate C3aR and CR1, respectively, and studied their transcription in the course of the colonial blastogeneticcycle. In addition, we continued our analysis ofthe role of C3 in Botryllus immunity by studying the modulation of BsC3 transcription during the colonial blastogenetic cycle and the effect of bsc3 knockdown on immune responses. Results indicate that only morula cells, and no other immunocytes type, are labelled by the antisense probe for BsC3aR, whereas phagocytes and young, undifferentiated cells, known as hemoblasts, are the cells stained by the probe for BsCR1. This suggests the presence of an important cross-talk between these two immunocytes types. Both the bsc3ar and bscr1 genes are constitutively transcribed as almost all morula cells and phagocytes, respectively, resulted labelled by the antisense probe in the ISH assay, independently of their previous challenge with zymosan, a known activator of B. schlosseri hemocytes. However, a modulation in the extent of transcriptionoccurs during the colonial blastogenetic cycle as the amount of BsC3aR mRNA abruptly decreased at TO, whereas no differences were observed when EC and MC were compared. This is probably related to the renewing of circulating cells at TO, when 20-30% of hemocytes undergo cell death by apoptosis and are replaced by new, differentiating cells entering the circulation in the same period

Expression study of molecular markers involved in staminality and differentiation in the colonial ascidians Botryllus schlosseri

Ascidians are invertebrate chordates, members of the subphylum Tunicata that represents the sister group of vertebrates. They offer the opportunity to investigate and compare the behaviour of both embryonic and adult stem cells. Morphological data suggest the presence of undifferentiated haemocytes (haemoblasts) able to proliferate and give rise to terminally differentiated cells. Relevant studies were also carried out in the neural lineage, in which neural progenitor cells regenerate the brain after extirpation. In B. schlosseri, during the cyclical generation change, bud primordial cells, probably deriving from a pool of long-living stem cells, are able to give rise to the neural complex. We screened the B. schlosseri genome and transcriptome, looking for transcripts/genes showing similarity to vertebrate molecular markers of haematopoietic and neural stem cells. Four sequences, orthologous to mammalian transcripts considered markers of haematopoietic progenitor cells, were identified in B. schlosseri. They are: bsabcg2, bscd133, bsgata1/2/3 and bsgata4/5/6. In situ hybridization on haemocyte monolayers and colony sections, resulted in labelling of cells in the sub-endostylar haemolymph lacunae. This results matches previously morphological data that identified the endostyle as a stem cell niche. Quantitative real time PCR (qRT-PCR) highlighted the over-expression of the considered genes in the mid-cycle phase of the blastogenetic cycle. During this phase, there is the formation of new secondary buds emerging from the primary buds. The high expression levels of bsabcg2, bscd133, bsgata1/2/3 and bsgata4/5/6 genes in the mid-cycle phase reflect the presence of undifferentiated cells involved in proliferative and differentiation events required for giving rise to the new blastogenetic generation. For the neural lineage, we identified and characterised two transcripts orthologues of vertebrate neural stem cell markers (BsSox2 and BsMsi2). We also studied the expression, during the blastogenetic cycle, of a panel of genes already known to be involved in ascidian larvae neurogenesis, i.e., orthologues of Pax2/5/8, Hox1 and Hox3. ISH with riboprobes for BsSox2, BsMsi2, BsPax2/5/8, BsHox1 and BsHox3 revealed a common labelling in the endostyle niche. The presence of bssox2, bsmsi2, bspax2/5/8, bshox1 and bshox3 transcripts in the cells of the region known to be a stem cell niche, led us to conclude, not only that our probes identified undifferentiated cells but even that in B. schlosseri are probably present a single population of pluripotent stem cells that could differentiate into haematopoietic or neural cells. The qRT-PCR, showed an high expression level in the mid-cycle phase of all the putative neural markers considered. In this phase new secondary buds are produced from primary buds. Each new bud needs its own neural complex and this requires the proliferation of undifferentiated cells to originate neural gland rudiment and cerebral ganglion. Bssox2, bsmsi2, bspax2/5/8, bshox1 and bshox3 increased their expression associated with these neurogenesis events and this support their involvement in neural stem cell differentiation

Semi-continuous Chlorella vulgaris Cultivation Using Anaerobic Digestate Liquid Fraction Pre-treated by Ultrasonic Cavitation to Improve Carbon Dioxide Solubilization

Nutrients-enriched effluents such as digestate can represent a suitable and economically appealing substrate for microalgae growth since they combine the effluent treatment with biomass production. Then, microalgae biomass can be exploited to produce several bio-based compounds. However, the use of digestate for microalgae cultivation can be challenging due to its high levels of ammonia nitrogen and its low C/N ratio. For this reason, an ultrasonic cavitation (UC) process combined with carbon dioxide (CO2) insufflation was tested on digestate, in order to obtain a faster solubilization of the CO2 in the medium and thus an increase in the C/N ratio. The test was carried out growing Chlorella vulgaris on both digestate (mixotrophic condition) and BG-11 medium (autotrophic condition) in 1 L photobioreactors. For the first 14 days of the experiment the reactors were maintained in batch conditions to acclimatize microalgae. Then, they were switched to semi-continuous for 32 days. The reactors were fed three times a week, with an HRT (Hydraulic Retention Time) of 10.5 d as weekly average. Regarding the test on digestate, both UC pre-treated and untreated conditions reached the highest biomass production at the end of the batch (4.8 and 4.1 g L-1 respectively) and a complete ammonium (NH4+) removal after 9 days. The switch to semi-continuous caused an increase in NH4+ concentration and a consistent decrease in biomass concentration. Biomass production reached the steady-state, with a concentration of 1.9 and 1.2 g L-1 for the UC pre-treated and untreated digestate, respectively (+55.6 % biomass production obtained with UC pre-treated digestate). Moreover, an NH4+ removal of 93.5 % and 92.3 % was reached for UC pre-treated and untreated conditions, respectively

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Amyloid and allorecognition in the colonial ascidian Botryllus schlosseri.

Allorecognition, i.e., the ability of intraspecific nonself recognition is widely distributed among colonial, sessile marine organisms in the form of colony specificity. In the cosmopolitan compound ascidian Botryllus schlosseri, colony specificity is controlled by a highly polymorphic Fu/HC locus: two colonies sharing at least one alleleat the Fu/HC locus can fuse into a chimeric colony; if no alleles are shared, a typical inflammatory reaction occurs, with the recruitment of a specific hemocyte type, the cytotoxic morula cells (MCs), inside the tips of the ampullae (the blind termini ofthe tunic vasculature) extending towards the alien colony, their extravasation in the tunic and their final degranulation. As a consequence of allorecognition, necrotic, melanic spots (points of rejection; PORs) form along the contact border, due to the release, by MCs, of their granular content, mainly represented by quinones, polyphenols and the enzyme phenoloxidase (PO), upon the perception of the allogeneic humoral factors diffusing from the alien colony through the partially fused tunics. It is remarkable that the deposition of melanin and the cell death is confined to the immediate outside of the ampullar tips, suggesting that the diffusion of PO and the products of its activity are, in some way, prevented in order to limit cytotoxicity to the immediate neighbourhood of the contact region. In this context, we looked for factors released by MCs that could limit the spreading of cytotoxicity and melanisation. We found that MCs share with vertebrate melanocytes similar packaging of melanin precursors, entrapped in a 3Dscaffold of amyloid fibrils. They contribute to form the electron dense content of MC granules that, after stimulation, flake off and is released in the surrounding medium. Released amyloid fibrils limit the diffusion of the produced melanin. The search for genes and factor controlling both melanogenesis and amyloidogenesis, revealed an evolutionary conserved machinery involved in the processes and an unexpected cross talk between the two Botryllus immunocyte types, i.e., phagocytes and MCs. Furthermore, this work confirms the physiological role of amyloid in tunicate immunity