Insights on cytotoxic cells of the colonial ascidian Botryllus schlosseri

Morula cells (MCs) represent the most abundant circulating hemocyte of the compound ascidian Botryllus schlosseri. They are cytotoxic cells involved in the rejection reaction between contacting, genetically incompatible colonies. Upon the recognition of foreign substances, they degranulate and release their content, which contribute to the cell death along the contact borders. A major role in MC-related cytotoxicity is exerted by the enzyme phenoloxidase (PO) that converts polyphenol substrata to quinones which, then, polymerize to form melanins. During this reaction, reactive oxygen species are formed which are the cause of MC-related cytotoxicity. Here, we carried out new analyses to investigate further the nature of MC content and its role in cytotoxicity. Results confirm that PO is located inside MC vacuoles together with arylsulfatase, iron and polyphenols/quinones, the latter probably representing ready-to-use cytotoxic molecules, deriving from the oxidation of DOPA-containing proteins. In addition, small DOPA-containing peptides, called tunichromes, are also present inside MCs. MC degranulation and PO-mediated cytotoxicity are prevented by secretion inhibitors and by H89 and calphostin C. The observation that PO activity is always detectable in MCs in the absence of protease treatment, and its inhibition by sulfites and sulfates, suggest a non-classical pathway of PO modulation in botryllid ascidians

Tunic Cell Morphology and Classification in Botryllid Ascidians(Morphology)

The morphology of tunic cells was investigated in ten botryllid ascidians, six Botryllus and four Botrylloides species, by means of light and electron microscopy. Three types of tunic cells were described, I.e. amoeboid, vacuo-granular and large granule tunic cells. Amoeboid tunic cells were irregularly shaped, and had many pseudopodia. They were found in the all species studied here. Vacuo-granular tunic cell had many vacuoles which contained round granules, and was found in four Botryllus and four Botrylloides species. The diameters of the granules varied amond species (ca. 1.4-0.8 ^m). Large granule tunic cells were peculiar to Botryllus scalaris, and each of them contained a single large granule with lamellate substructure. Some tunic cells showed intermediate characteristics of amoeboid and vacuo-granular tunic cells. It seems that amoeboid tunic cells probably differentiate into vacuo-granular tunic cells in the tunic. There are thread-like bacteria in the tunic, and they often dangle around vacuo-granular tunic cells. There may be some interactions between tunic cells and the bacteria

A QUNANITATIVE EVALUATION OF MORPHOLOGICAL CHANGES DURING FUSION EVENTS IN A COLONY OF DIDEMNUM VEXILLUM AND OF POLYCUNUM CONSTELLATUM

We documented changes in the abundance and distribution patterns of cells involved in the allorecognition response in two species of colonial aplousobranch tunicates lacking a common vascular system. A histological examination of the fusion junction of isogeneic (CIAs) and allogeneic (CAAs) colony assays revealed that tunic cuticles were rapidly regenerated. The underlying tunic matrix fused readily in Didemnum vexillum in all assay and control types, however it did not fuse in CIAs in Polyclinum constellatum. We identified four and five different cell types in P. constellatum and D. vexillum, respectively. Phagocytic cells represented the most abundant cell type in both species and were most abundant in CAAs, followed by morula (or morula-like) cells. Additionally, the two cell types were more abundant at the fusion junction than at 120 mum or 240 mum from the junction (p\u3c0.05 for P. constellatum; p=0.000 for D. vexillum). Hence, we concluded that phagocytes and morula cells are likely involved in the allorecognition reaction. Filopodial and granular cells, were present, although only at very low abundances. A layer of bladder cells was located immediately below the cuticle in D. vexillum. No bladder cells were found in P. constellatum.

An elongated COI fragment to discriminate botryllid species and as an improved ascidian DNA barcode

Botryllids are colonial ascidians widely studied for their potential invasiveness and as model organisms, however the morphological description and discrimination of these species is very problematic, leading to frequent specimen misidentifications. To facilitate species discrimination and detection of cryptic/new species, we developed new barcoding primers for the amplification of a COI fragment of about 860 bp (860-COI), which is an extension of the common Folmer's barcode region. Our 860-COI was successfully amplified in 177 worldwide-sampled botryllid colonies. Combined with morphological analyses, 860-COI allowed not only discriminating known species, but also identifying undescribed and cryptic species, resurrecting old species currently in synonymy, and proposing the assignment of clade D of the model organism Botryllus schlosseri to Botryllus renierii. Importantly, within clade A of B. schlosseri, 860-COI recognized at least two candidate species against only one recognized by the Folmer's fragment, underlining the need of further genetic investigations on this clade. This result also suggests that the 860-COI could have a greater ability to diagnose cryptic/new species than the Folmer's fragment at very short evolutionary distances, such as those observed within clade A. Finally, our new primers simplify the amplification of 860-COI even in non-botryllid ascidians, suggesting their wider usefulness in ascidians

Assessing the ecotoxicity of copper and polycyclic aromatic hydrocarbons: Comparison of effects on Paracentrotus lividus and Botryllus schlosseri, as alternative bioassay methods

Adult sea urchins and their embryos are ideal targets to investigate the medium- and long-term effects of various toxic agents, such as organic and inorganic pollutants, to forecast and mitigate their environmental effects. Similarly, small colonial tunicates such as Botryllid ascidians may reveal acute toxicity processes and permit quick responses for the management of contaminants impacting coastal waters, to preserve the functional integrity of marine ecosystems. This investigation compares the functional responses of two model invertebrates, the sea urchin Paracentrotus lividus and the sea squirt Botryllus schlosseri, to chronic and acute exposures to organic and inorganic toxic agents. Such heavy metals as copper produce both acute and chronic effects on marine biota, while polycyclic aromatic hydrocarbons (PAHs) mainly produce chronic effects at the concentrations ordinarily measured in marine coastal waters. Both models were tested over a range of concentrations of copper and PAHs. Copper triggered a clear effect in both species, producing a delay in the embryo development of P. lividus and a rapid death of sea squirts. B. schlosseri was less sensitive to PAHs than P. lividus. The results on both species may synergistically contribute to assess the toxicity of organic and inorganic compounds at various concentrations and different physiologic levels

Behavioural Responses of the Colonial Sea Squirt Botrylloides violaceus Oka to Suspended Food Micro-Particles in Laboratory Cultures

none4openChiara Gregorin; Luigi Musco; Emanuele Somma; Valerio ZupoGregorin, Chiara; Musco, Luigi; Somma, Emanuele; Zupo, Valeri

Life history and ecological genetics of the colonial ascidian Botryllus schlosseri

The colonial ascidian Botryllus schlosseri is a cosmopolitan, marine filter feeder, introduced as a laboratory research organism in the 1950s. Currently, it is widely used in many laboratories to investigate a variety of biological questions. Recently, it has become a species of concern, as it is an invasive species in many coastal environments. Here, we review studies on the geographical distribution of the species, sexual and asexual reproduction in the field, tolerance to temperature, salinity and anthropogenic activity, polychromatism, enzymatic polymorphism, and the genetic basis of pigmentation. Studying the relationship between genetic polymorphism and the adaptation of B. schlosseri to environmental stress is a challenge of future research and will improve our understanding of its evolutionary success and invasive potential

Urochordate Histoincompatible Interactions Activate Vertebrate-Like Coagulation System Components

The colonial ascidian Botryllus schlosseri expresses a unique allorecognition system. When two histoincompatible Botryllus colonies come into direct contact, they develop an inflammatory-like rejection response. A surprising high number of vertebrates' coagulation genes and coagulation-related domains were disclosed in a cDNA library of differentially expressed sequence tags (ESTs), prepared for this allorejection process. Serine proteases, especially from the trypsin family, were highly represented among Botryllus library ortholgues and its “molecular function” gene ontology analysis. These, together with the built-up clot-like lesions in the interaction area, led us to further test whether a vertebrate-like clotting system participates in Botryllus innate immunity. Three morphologically distinct clot types (points of rejection; POR) were followed. We demonstrated the specific expression of nine coagulation orthologue transcripts in Botryllus rejection processes and effects of the anti-coagulant heparin on POR formation and heartbeats. In situ hybridization of fibrinogen and von Willebrand factor orthologues elucidated enhanced expression patterns specific to histoincompatible reactions as well as common expressions not augmented by innate immunity. Immunohistochemistry for fibrinogen revealed, in naïve and immune challenged colonies alike, specific antibody binding to a small population of Botryllus compartment cells. Altogether, molecular, physiological and morphological outcomes suggest the involvement of vertebrates-like coagulation elements in urochordate immunity, not assigned with vasculature injury

Behavioural responses of the colonial sea squirt botrylloides violaceus oka to suspended food micro‐ particles in laboratory cultures

Violet sea squirts are noteworthy model organisms, because they provide insights into various physiologic processes, including cell senescence, ageing, apoptosis and allorecognition. Consequently, their culture is critical to permit experimental studies. Most papers refer to short periods of rearing using various feeds, both living and conserved, missing a formal justification for their use or indications of their actual nutritional value. Here, we use two behavioural responses— the percentage of open siphons and the frequency of zooid contractions—as compared to the abundance of suspended microparticles during feeding tests, to identify feeds able to promote filter-feeding. The results will enable to formulate compound diets that maximise positive physiological responses. Our tests demonstrated that plant items, such as dry microalgae and cyanobacteria (Arthrospira platensis, commercially known as Spirulina), along with living planktonic Haptophyta (Isochrysis galbana), trigger clear positive reactions, represented by a higher frequency of zooid contractions and larger proportions of open siphons. These responses correspond to decreases in the concentrations of suspended microparticles during the experiment and indicate higher filter-feeding activity. In contrast, feeds commonly administered to colonies, such as milk powder, dried eggs and artificial plankton, triggered negative behavioural responses, and their intake was lower during the feeding trials

Cryptic diversity and database errors challenge non-indigenous species surveys: An illustration with Botrylloides spp. in the English Channel and Mediterranean Sea

Molecular tools have been extensively used in recent decades to examine biological invasion processes, and are increasingly being adopted as efficient tools to support non-indigenous species surveys, notably through barcoding approaches, i.e., the use of a reference sequence specific to a given species to validate its identification. The technique is easy to use but requires reliable reference sequences to be available in public databases. In addition, the increasing discovery of cryptic species in marine taxa may complicate taxonomic assignment. We illustrate these two issues in the ascidian genus Botrylloides, in which at least three global marine invaders have been recognized, including B. violaceus and B. diegensis. We obtained COI sequences from >750 colonies of Botrylloides spp. sampled in W Europe or provided by expert colleagues from other regions. Phylogenetic trees clearly distinguished our targeted taxa [i.e., B. violaceus, B. diegensis and B. leachii (native)]. They also revealed another discrete lineage apparently related to a recently described eastern Mediterranean species. By examining public databases, we found sequences of B. diegensis erroneously assigned to B. leachii. This observation has major implications as the introduced B. diegensis can be misidentified as a putatively native species. We also checked published sequences of the genus Botrylloides in the Mediterranean Sea, complemented with new samples. Based on our custom reference database, all published sequences of B. leachii corresponded to B. diegensis, although this NIS has hardly been reported at all in the Mediterranean region. Such database errors are unfortunate, as the barcoding approach is a powerful tool to identify the recognized Botrylloides species currently present in European seas. This is of particular importance because a trait often used during field assessment, i.e., single-color vs. two-color colonies, is misleading to distinguish B. violaceus and B. diegensis respectively: a substantial proportion of the single-color morph are actually B. diegensis in both the Mediterranean Sea and the English Channel. Altogether, this study exemplifies the advantages and disadvantages of molecular barcoding in NIS surveys and studies. The limitations that were identified are all easy to resolve once proper vouchers and collections are set up