Ascidian larval tunic: Extraembryonic structures influence morphogenesis

Summary The larval tunic of Corella inflata is composed of two cuticular layers, extracellular filaments and ground substance. It lies outside the epidermis and most of it is known to be produced by the epidermis. The dorsal, ventral and caudal fins are specialized parts of the tunic that are essential for larval locomotion. The following hypothesis was tested: Morphogenesis of the larval fins is dependent upon the presence of extraembryonic structures (test cells, chorion or follicle cells) before completion of the late tail bud stage of development. We tested this by dechorionating embryos of Corella inflata and Ascidia paratropa. The operation removes all extraembryonic structures. It was performed mainly on neurula, early tail-bud and late tail-bud stages.Fin formation is inhibited when neurulae are dechorionated but not when late tail-bud or older embryonic stages are dechorionated. Dechorionated neurulae produce all of the major components of the tunic (cuticular layers, filaments and ground substance) but they are unable to form functional fins. At the time of dechorionation, in all experiments, the embryos had no fins.Removal of the follicle cells does not inhibit fin formation. The test cells are known to secrete granular ornaments that attach to the surface of the tunic. The fibrous, acellular chorion may serve to contain the test cells and their products or products of the embryo that are not firmly attached. The test cells may induce or control the morphogenesis of the larval fins in ascidians before the late tail-bud stage of development. We suggest ways of testing this hypothesis and an alternative hypothesis.     

Bacterial community in the tunic matrix of a colonial ascidian<Emphasis Type="Italic"> Diplosoma migrans</Emphasis>

This paper provides the first information on the morphology of different morphotypes of bacteria in the tunic matrix of the colonial ascidian Diplosoma migrans. Ascidians were collected from waters near Helgoland (German Bight, North Sea). The dominant type is represented by extremely high numbers of long, needle-like rods (length 10–30 µm, width 0.5 µm). The bacteria are motile by means of bipolar monotrichous flagella, generating swift sigmoidal movement. Bacteria are already present during different embryonic stages. It is assumed that they are transferred during sexual propagation from the parental colony to its offspring. As a second morphotype, the tunic harbors screw-like bacteria in low numbers (length 4–10 µm, width 0.5 µm). Besides these conspicuous morphotypes, occasionally motile rods with spore-like globules at one end and additional coccoid forms in large quantities of unknown meaning (possibly spores) were found. The taxonomic status and ecological functions of these differently shaped bacterial groups are unclear.Communicated by H.-D. Franke     

A new cellulosic structure, the tunic cord in the ascidianPolyandrocarpa misakiensis

Summary A specialized structure of tunic cord inPolyandrocarpa misakiensis is investigated by electron microscopy. The tunic cord is a cord-like coiled structure of 5–30 m in diameter and 0.1–9.0 mm in length. The tunic cords originate and elongate from the dorsal tunic, and their termini have a swollen and ornamented structure. Scanning and transmission electron micrographs and the electron diffractogram show that the tunic cords are composed of bundled microfibrils of cellulose I with high crystallinity. The tunic cord is completely surrounded by single-layered epidermal cells, which have been found as the site of cellulose biosynthesis. A number of tunic cords are connected to the internal tunic of the siphon by forming eyelet structures at their termini. These observations suggest that the tunic cords act as a connector between dorsal and internal tunic of the siphon.     

Oxidative transformation of tunichromes – Model studies with 1,2-dehydro-N-acetyldopamine and N-acetylcysteine

Tunichromes are 1,2-dehydrodopa containing bioactive peptidyl derivatives found in blood cells of several tunicates. They have been implicated in metal sequestering, tunic formation, wound healing and defense reaction. Earlier studies conducted on these compounds indicate their extreme liability, high reactivity and easy oxidative polymerization. Their reactions are also complicated by the presence of multiple dehydrodopyl units. Since they have been invoked in crosslinking and covalent binding, to understand the reactivities of these novel compounds, we have taken a simple model compound that possess the tunichrome reactive group viz., 1,2-dehydro-N-acetyldopamine (Dehydro NADA) and examined its reaction with N-acetylcysteine in presence of oxygen under both enzymatic and nonenzymatic conditions. Ultraviolet and visible spectral studies of reaction mixtures containing dehydro NADA and N-acetylcysteine in different molar ratios indicated the production of side chain and ring adducts of N-acetylcysteine to dehydro NADA. Liquid chromatography and mass spectral studies supported this contention and confirmed the production of several different products. Mass spectral analysis of these products show the potentials of dehydro NADA to form side chain adducts that can lead to polymeric products. This is the first report demonstrating the ability of dehydro dopyl units to form adducts and crosslinks with amino acid side chains.     

Blue-green algalike cells associated with the tunic of Ciona intestinalis L.

Summary Certain organisms resembling blue-green algae embedded in the tunic of the solitary ascidian Ciona intestinalis L. are described. Their probable symbiotic role as related to the peculiar habitat of this ascidian is suggested.     

The identification and localization of two intermediate filament proteins in the tunic of Styela plicata (Tunicata, Styelidae)

The intermediate filament (IF) proteins Styela C and Styela D from the tunicate Styela (Urochordata) are co-expressed in all epidermal cells and they are thought to behave as type I and type II keratins. These two IF proteins, Styela C and Styela D, were identified in immunoblots of proteins isolated from the tunic of Styela plicata. The occurrence and distribution of these proteins within the tunic of this ascidian was examined by means of immunofluorescence and immunoperoxidase techniques, using anti-Styela C and anti-Styela D antibodies. In addition, immuno-electron microscopy of the tunic showed that the two proteins are located in the cuticle layer and in the tunic matrix. These results represent the first data about the presence of IF proteins in the tunic of adult ascidian S. plicata. The possible involvement of these IF proteins in reinforcing the integrity of the tunic, that represents the interface between the animal body and the external environment, is discussed.     

Evidence for the role of the glomerulocyte in cellulose synthesis in the tunicate,Metandrocarpa uedai

Summary The tunicate,Metandrocarpa uedai, contains a large quantity of cellulose; however, it is not known how and where the cellulose is synthesized. Based on evidence from electron diffraction and conventional thin-sectioning for electron microscopy, this study shows that the glomerulocyte is involved in the synthesis of cellulose. The bundles of microfibrils in the glomerulocyte as well as the tunic were identified as cellulose I using selected area electron diffraction analysis. The diffraction pattern of cellulose in the glomerulocyte was similar to that from the tunic, suggesting that the crystallization of cellulose already is initiated in the glomerulocyte. The diameter of cellulose microfibrils, both in the glomerulocyte and the tunic was the same, about 16 nm. These results suggest that the glomerulocyte is the most probable site for the synthesis of cellulose in the tunic ofM. uedai. Using thin-sectioning techniques, a series of observations showed that individual microfibrils are primarily assembled in structures tentatively identified as vacuole-like structures, then they are bundled by a tapering region within the vacuole-like structures. These bundles of microfibrils are deposited in a continuously circular arrangement. The microtubules are oriented parallel to the bundles of microfibrils at the tapering vacuole-like structure, and they may be involved in the tapering of these structures (perhaps controlling the shape). This study also provides the first account for the involvement of a vacuole-like structure in the synthesis of cellulose microfibrils among living organisms.     

The prophenoloxidase system is activated during the tunic inflammatory reaction of Ciona intestinalis

Phenoloxidase (PO) activity was examined in the tunic tissue of Ciona intestinalis following lipopolysaccharide (LPS) intratunic injection. Tunic homogenate supernatant (THS), assayed with the Dopa-MBTH reaction, displayed Ca(2+)-independent PO activity that was raised by LPS and further enhanced by proteases. Specific inhibitors (tropolone, phenylthiourea, diethylthiocarbamate) supported the specificity of the reaction. Assay with soybean trypsin inhibitor showed that, in the tunic, PO activation with trypsin was not significantly inhibited suggesting that proteases diverse from serine proteases were involved. In vivo experiments were carried out by injecting isosmotic medium or LPS, and THS was assayed for its PO activity. Analysis of variance of the time-course profiles showed that LPS was more effective in activating proPO. To disclose the PO response at the injured site, an assay with Dopa-MBTH was performed in vitro. Quinones were mainly contained in the tunic matrix enriched with inflammatory cells around the injection site. Microscopic observations and immunohistochemistry with anti-CinPO-2 antibodies showed granulocytes and unilocular refractile granulocytes containing PO, whereas few morula cells were stained. In THS zymograms (SDS-polyacrylamide gel electrophoresis), PO activity linked to 90-kDa and 120-kDa bands was observed as an effect of LPS injection, whereas the density of 170-kDa PO was weak. A third presumptive PO enzyme (CinPO-3) containing the CinPO-2 peptide was identified in the recent Ciona genome version. Presumably, LPS stimulated the production and dimerization (120 kDa) of CinPO-3 (66 kDa). Thus, the activated proPO system includes several POs that are distinguishable by size and that are contained and presumably released by tunic inflammatory cells and hemocytes of the pharynx bars.     

Diversity of intratunical bacteria in the tunic matrix of the colonial ascidian Diplosoma migrans

This paper provides the first information on diversity based on sequence data of the 16S rDNA of intratunical bacteria in the colonial ascidian Diplosoma migrans and its embryonic offspring. Ascidians were collected from waters near Helgoland (German Bight, North Sea). Sample material comprised tunic tissue, bacteria collected from tunic tissue, eggs with single embryos at different developmental stages, and free-swimming larvae. Bacterial 16S rDNA from D. migrans was directly amplified using PCR. DNA species were separated using denaturing gradient gel electrophoresis (DGGE). DGGE profiles generated ca. ten different distinguishable operational taxonomic units. Eleven bands from different sample materials were successfully re-amplified and sequenced. Sequence data generated five different subgroups of intratunical proteobacteria. The dominant band, detected in all of the samples tested, showed a low degree of relationship (84–86%) to Ruminococcus flavefaciens (-subgroup). A weaker band, located above, which was not detected in all of the samples, was also similarly related to R. flavefaciens. Other bands derived from tunic material and embryonic stages showed closer relationship (ca. 97–99%) to Pseudomonas saccherophilia, a knallgas bacterium, and Ralstonia pickettii, a pathogen bacterium (both members of the -subgroup). A solitary band generated from tunic material was assigned to a typical marine Flavobacterium symbiont (95%). Finally, a band from isolated bacteria was related (96%) to pathogen Arcobacter butzleri (-subgroup). At this state of the investigation, a reliable interpretation of the ecological functions of intratunical bacteria cannot yet be given. This is due to the low degree of relationship of some of the bacteria and the fact that not all of the characteristic bands were successfully sequenced. However, the intratunical bacteria represent a unique bacterial community. Their DGGE profiles do not correspond to the profiles of the planktonic bacteria generated from surface seawater close to the ascidian habitat. The allocation of DNA sequences to the different morphotypes, their isolation and culturing, and the elucidation of the physiological functions of intratunical bacteria are in progress.     

Oxidative transformation of a tunichrome model compound provides new insight into the crosslinking and defense reaction of tunichromes

Tunichromes, small oligopeptides with dehydrodopa units isolated from the blood cells of ascidians, have been implicated in the defense reactions, metal binding, wound repair, or tunic formation. Their instability and high reactivity has severely hampered the assessment of their biological role. Experiments conducted with the model compound, 1,2-dehydro-N-acetyldopamine, indicated that the instability of tunichromes is due to this basic structure. Exposure of this catecholamine derivative to even mild alkaline condition such as pH 7.5 causes rapid nonenzymatic oxidation. High performance liquid chromatography and mass spectrometry studies confirmed the production of dimeric and other oligomeric products in the reaction mixture. The nonenzymatic reaction seemed to proceed through the intermediary formation of semiquinone free radical and superoxide anion. Ultraviolet and visible spectral studies associated with the oxidation of tunichromes isolated from Ascidia nigra by tyrosinase indicated the probable formation of oligomeric tunichrome products. Attempts to monitor the polymerization reaction by mass spectrometry ended in vain. Tunichrome also exhibited instability in mild alkaline conditions generating superoxide anions. Based on these studies, a possible role for oxidative transformation of tunichrome in defense reaction, tunic formation and wound healing is proposed.