Transmission of cyanobacterial symbionts during embryogenesis in the coral reef ascidians Trididemnum nubilum and T. clinides (Didemnidae, Ascidiacea, Chordata)

Vertical transmission of cyanobacterial symbionts occurs in didemnid ascidians harboring Prochloron as an obligate symbiont; the photosymbionts are transferred from the parental ascidian colony to the offspring in various ways depending on host species. Although several didemnids harbor non-Prochloron cyanobacteria in their tunics, few studies have reported the processes of vertical transmission in these didemnids. Here we describe the histological processes of the transmission of cyanobacteria in two didemnids, Trididemnum nubilum harboring Synechocystis and T. clinides harboring three cyanobacterial species. In both species, the photosymbionts in the tunic of the parent colony were apparently captured by the tunic cells of the host and transferred to the embryos brooded in the tunic. The symbiont cells were then incorporated into the inner tunic of the embryo. This mode of transmission is essentially the same as that of T. miniatum harboring Prochloron in the tunic, although there are some differences among species in the timing of the release of the symbionts from the tunic cells. We suggest that the similar modes of vertical transmission are an example of convergent evolution caused by constraints in the distribution patterns of symbiont cells in the host colony.     

An evaluation of chemical and physical defenses against fish predation in a suite of seagrass-associated ascidians

The palatability of two solitary and three colonial species of ascidians commonly found in sub-tropical seagrass meadows was evaluated using the abundant, sympatric, omnivorous pinfish Lagodon rhomboides as a model predator. Bite-sized pieces of fresh tissues of both solitary and one of the three colonial ascidian species were unpalatable to fish. Lipophilic and hydrophilic extracts of the three unpalatable species did not cause feeding deterrence indicating that secondary metabolites are not responsible for the lack of palatability. Distaplia bermudensis, the one colonial ascidian that was unpalatable to fish, had a highly acidified outer tunic (pH = 1.5). We tested the ability of acidified agar food pellets (pH = 1.5) to deter pinfish and found that the fish readily ingested acidified pellets. The toughness of the tunic of all five ascidian species was evaluated by measuring the Force (N) required to penetrate the tunic using a penetrometer. Tunic toughness is likely to explain the lack of palatability of the solitary ascidians Styela plicata and Molgula occidentalis as their tunics required a force of > 34 N to penetrate. Tunic toughness may be a particularly effective adaptation for ascidian defense in seagrass habitats where fish with strong crushing jaws, such as those that commonly occur in coral reef systems, are rare.     

Ultraviolet-light-absorbing tunic cells in didemnid ascidians hosting a symbiotic photo-oxygenic prokaryote,Prochloron

Coral reef invertebrates that host phototrophic symbionts are thought to protect themselves and their symbionts with mycosporine-like amino acids (MAAs)-UV-absorbing substances that act as sunscreens (Dunlap, W. C., and J. M. Shick, 1998. J. Phycol. 34: 418-430). However, the histological distribution of MAAs in the host tissues has not yet been visualized. We have localized the UV-absorbing substances in the tissues of two colonial didemnid ascidians-Lissoclinum patella and Diplosoma sp.-that contain the symbiotic photo-oxygenic prokaryote Prochloron sp. Cross-sections of unfixed tissue from these ascidians were examined by UV-light microscopy at 320 or 330 nm, wavelengths at which UV light is absorbed by MAAs. Within the tunic, the gelatinous integument of the colony, UV light was exclusively absorbed by a particular type of cell, the tunic bladder cell. Tunic bladder cells with strong UV absorption were denser in the upper tunic, which lies over a colony's zooids, than in the basal tunic underlying the zooid. In the upper tunic, those cells with strong UV absorption were most dense near the surface. The tunic bladder cell is highly vacuolated, and the vacuole contains strong acid, which destabilizes MAAs. Furthermore, the UV-absorbing portion of tunic bladder cells seemed to be cup-shaped, indicating that the MAAs are not localized in the vacuole, but in the cytoplasm. These results strongly suggest that didemnid ascidians accumulate MAAs in tunic bladder cells as a protection against UV radiation.     

Ascidian tunic cells: morphology and functional diversity of free cells outside the epidermis

Abstract. Tunic cells are free cells distributed in the tunic, the integumentary matrix of tunicates. In ascidians, various types of tunic cells have been described both in solitary and in colonial species. Many of them are functionally specialized and are related to the protection of the animal, such as phagocytosis to prevent infection, acid storage to avoid predation, and pigmentation to protect against solar radiation. While some tunic cells are known to play a role in colonial allorecognition, bioluminescence, and algal symbiosis, the functional roles of many cell types still remain to be determined. The composition of tunic-cell types varies among ascidian species, most likely reflecting the functional requirements of the tunic in each species. Although some cell types, e.g., tunic net cells and tunic bladder cells, are restricted to particular taxa of ascidians, tunic phagocytes are found in all known ascidians. Therefore, tunic phagocytes are hypothesized to be basal and shared with ancestral tunicates. In some ascidians, phagocytic cells are involved in other functions, such as pigmentation, intracellular photosymbiosis, and bioluminescence. These specialized phagocytic cells are hypothesized to be derived from tunic phagocytes, suggesting that tunic cells have a high potential to diversify and evolve a wide variety of cellular functions.     

Localization of symbiotic cyanobacteria in the colonial ascidian Trididemnum miniatum (Didemnidae, Ascidiacea)

Trididemnum miniatum is a colonial ascidian harboring the photosymbiotic prokaryote Prochloron sp. These bacterial cells are located in the tunic of the host animal. The present study revealed, by ultrastructural analysis, that the Prochloron cells were exclusively distributed and proliferated in the tunic. They were shown to be embedded in the tunic matrix and to have no direct contact with ascidian cells. Some tunic cells of the ascidians, however, did phagocytize and digest the symbiont. Round cell masses were sometimes found in the tunic and appeared to consist of disintegrating cyanobacterial cells. The thoracic epidermis of ascidian zooids was often digitated, and the epidermal cells extended microvilli into the tunic. Since there were no Prochloron cells in the alimentary tract of the ascidian zooids, the photosymbionts would not be considered part of the typical diet of the host ascidians. Thin layer chromatography showed that the symbionts possessed both chlorophyll a and b, while a 16S rRNA gene phylogeny supported the identification of the photosymbiont of T. miniatum as Prochloron sp.     

Comparative morphology of the stolonic vessel in a didemnid ascidian and some related tissues in colonial ascidians

The stolonic vessel is a tubular projection of the epidermis from the anterior part of the abdomen in the didemnid ascidians, and the vessel has been supposed to be closely related to the stolons, vascular appendages, and the posterior ends of the abdomen in other aplousobranch ascidians. We compared the morphology of the stolonic vessels of Diplosoma virens with similar or related tissue in other colonial ascidians, e.g. stolons of Clavelina, vascular appendages of Distaplia and Eudistoma, tunic vesicle of Aplidium, and vascular ampullae of Botrylloides. The epidermis of the stolonic vessel is composed of cuboidal cells in lateral wall and columnar cells at the distal tip of the vessel. The cuboidal cells have microvilli that probably anchor the stolonic vessel to the tunic. The columnar cells contain round granules that may concern with the secretion of some tunic components. The secretion of the granules, however, could not observed in this study. The stolonic vessel of D. virens is similar in morphology to the vascular ampullae of Botrylloides and the tunic vesicle of Aplidium rather than the other tissue examined here. Since the cell morphology is supposed to reflect its function but not the phylogenetic relationship, the present study could not provide conclusive evidences to prove the homology and the phylogenetic relationship among the tubular, epidermal projections in the colonial ascidians.     

Convergent evolution of the vertical transmission mode of the cyanobacterial obligate symbiont Prochloron distributed in the tunic of colonial ascidians

In chordates, obligate photosynthetic symbiosis has been reported exclusively in some colonial ascidians of the family Didemnidae. The vertical transmission of the symbionts is crucial in establishing the obligate symbiosis between the cyanobacteria and the host ascidians. The results of comparative surveys on the morphological processes of cyanobacterial transmission suggest the occurrence of convergent evolution of the vertical transmission in the host species harboring symbionts in the cloacal cavity. In Trididemnum species harboring cyanobacterial cells in the tunic, the symbiont cells are transported by the tunic cells to the tunic of embryos brooded in the tunic of the parent colony. The present study examined whether the mode of symbiont transmission is the same in host species harboring the symbionts in the tunic, regardless of host genera, or whether non-Trididemnum hosts have a different vertical transmission mode. Our results showed that the vertical transmission process in Lissoclinum midui was almost the same as in the Trididemnum species, supporting the occurrence of convergent evolution in the two distinct didemnid genera, that is, Trididemnum and Lissoclinum. High plasticity of the embryogenic process in didemnid ascidians may be important in developing the mechanism of vertical transmission; this assumption may also explain why the obligate cyanobacterial symbiosis has been exclusively established in didemnid ascidians among chordates.     

Grazing Characteristics and Growth Efficiencies at Two Different Temperatures for Three Nanoflagellates Fed with Vibrio Bacteria at Three Different Concentrations

Small inocula of one of the flagellates Paraphysomonas imperforata, Pteridomonas danica, and Cafeteria roenbergensis were added to suspensions of the bacterium Vibrio natriegens at each of three concentrations between 107 and 108 cells ml-1 and incubated at each of the temperatures 10 degrees C and 25 degrees C. Samples were taken at intervals for counting the flagellates and bacteria to determine the timing of the maximum of flagellate numbers and the concentrations at that time. Measurements of the protein concentration of the suspensions during incubation were used to determine the gross growth efficiency (GGE) or yield of flagellate grazing in each experiment. The most effective grazer was Pteridomonas, followed by Paraphysomonas, with Cafeteria being least effective, as judged by the threshold bacterial concentrations at which flagellate multiplication ceased, which were about 2 x 105, 2 x 106, and 2 x 107, respectively, and by the finding that Pteridomonas consumed 99%, Paraphysomonas about 95%, and Cafeteria only 60-70% of the available bacteria in the experiments. Peak concentrations of flagellates were reached later at the lower temperature, but the numbers of flagellates produced and of bacteria eaten were of a similar order at the two temperatures and the GGE was only slightly higher at the lower temperature. The time taken to reach peak flagellate numbers changed little with a threefold increase in bacterial concentrations, but the GGE increased and the numbers of bacteria eaten to produce one flagellate decreased when the bacterial concentration was increased. The three flagellates show clear evidence of niche specialization in differences in thresholds of bacterial prey concentration.     

Tunic phagocytes are involved in allorejection reaction in the colonial tunicate Aplidium yamazii (Polyclinidae, Ascidiacea)

The colonial ascidian Aplidium yamazii exhibited an allorejection reaction when two allogeneic colonies were brought into contact at their growing edges or at artificial cut surfaces. This species has no vascular network in the tunic, unlike the botryllid ascidians, which have a vascular network throughout the colony's common tunic. In the allorejection reaction induced by contact at the growing edges, some small, hard-packed tunic masses were formed at the contact points. Histological and electron microscopic investigation of these tunic masses revealed that they contained aggregates of tunic cells, with tunic phagocytes being the major cell type present. Some of the tunic phagocytes in these tunic masses appeared to be disintegrating. When allogeneic colonies were placed in contact at their artificial cut surfaces, the colonies partially fused, then separated. In this allorejection reaction, some loosely packed tunic masses remained in the gap between the two withdrawn colonies. These results strongly suggest that the tunic phagocytes are likely to be the major effector cells in the allorejection reaction. We also propose that the tunic phagocytes are not only the effector cells in the allorejection reaction but also bear the sites of allorecognition.     

Isolation and characterization of light-dependent hemolytic cytotoxin from harmful red tide phytoplankton Chattonella marina

Chattonella marina (C. marina), a raphidophycean flagellate, is a causative organism of red tide, and highly toxic to fish. In this study, we found that the cell-free methanol extract prepared from this flagellate exhibited potent hemolytic activity against rabbit erythrocytes. Interestingly, the hemolytic activity of the extract was absolutely light-dependent, and no hemolytic activity was detected in the dark even at very high concentration. Gel filtration chromatography of the methanol extract on a column of Sephadex LH-20 revealed that the extract contained hemagglutinin as well as hemolytic agents, and the substances responsible for these activities were separately eluted. These results suggest that the hemagglutinating and hemolytic activities were derived from distinct compounds. The hemolytic fraction obtained after gel filtration (F4) caused marked inhibition of the growth of C. marina itself and other species of phytoplanktons. Furthermore, F4 showed a potent cytotoxicity toward various mammalian cultured cell lines including human tumor cells (HeLa cells) in a dose-dependent manner. The cytotoxicity was also light-dependent, and no cytotoxic effect was exhibited in any cell lines tested in the dark. After further purification procedures via preparative thin-layer chromatography and subsequent HPLC, a major hemolytic agent was obtained as highly purified form. Since the methanol extracts prepared from other raphidophycean flagellates such as Heterosigma akashiwo, Olisthodiscus luteus, and Fibrocapsa japonica showed light-dependent hemolytic activity toward rabbit erythrocytes, it was suggested that the light-dependent hemolytic agents commonly exist at least in these raphidophycean flagellates.     

A common soil flagellate (Cercomonas sp.) grows slowly when feeding on the bacterium Rhodococcus fascians in isolation, but does not discriminate against it in a mixed culture with Sphingopyxis witflariensis

Flagellates are very important predators on bacteria in soil. Because of their high growth rates, flagellate populations respond rapidly to changes in bacterial numbers. Previous results indicate that actinobacteria are generally less suitable than proteobacteria as food for flagellates. In this study, we investigated the growth of the flagellate Cercomonas sp. (ATCC 50334) on each of the two bacteria Sphingopyxis witflariensis (Alphaproteobacteria) and Rhodococcus fascians (actinobacteria) separately and in combination. The growth rate of the flagellate was lower and the lag phase was longer when fed with R. fascians than when fed with S. witflariensis. This supports our initial hypothesis that the actinobacterium is less suitable as food than the alphaproteobacterium. However, after longer periods of growth the peak abundance of flagellates was higher on R. fascians, indicating that the food quality of bacterial prey depends on the time perspective of the flagellate-bacterial interaction. There was no evidence that the flagellates selected against the actinobacterium when feeding in mixed cultures of the two bacteria. Experiments where flagellates were fed with washed bacterial cells or with bacteria growing with different substrate concentrations suggested that the low food quality of R. fascians is related both to the intrinsic cell properties and to the extracellular metabolites.     

Cell types, microsymbionts, and pyridoacridine distribution in the tunic of three color morphs of the genus Cystodytes (Ascidiacea, Polycitoridae)

Abstract. The tunic of colonial ascidians of the genus Cystodytes is a dynamic and complex system where a variety of cell types and microsymbionts are found. The tunic is also the site where pyridoacridine alkaloids involved in chemical defense are found. We wanted to explore the composition of symbionts and tunic cell types and their relationship with localization of alkaloids in three color morphs (usually attributed to the species Cystodytes dellechiajei ). Tunic morphology was studied by means of transmission electron microscopy, and energy-dispersive X-ray microanalysis was performed for indirect localization of the bioactive alkaloids produced by these morphotypes. The main cell types identified are bladder cells, pigment cells, amebocytes, phagocytes, and morula cells. Amebocytes include several subtypes that may correspond to a sequence of ontogenetic stages; these cells also seem to give rise to other cell types. In the three morphotypes, the morphology of the tunic and tunic cells is basically the same. The alkaloids are localized in the pigment cells. At least three types of bacteria are present in the tunic, but they are scarce and do not store the targeted bioactive alkaloids. Our results indicate that, although pyridoacridine alkaloids are present in these ascidians, as in a variety of animal phyla, their wide taxonomic range is not necessarily the result of production by common microsymbionts, but rather of the convergent evolution of a successful biosynthetic pathway.     

Desmosomes and hemidesmosomes in the flagellate Crithidia fasciculata

Summary The flagellum of the trypanosomatid flagellate Crithidia fasciculata expands asymmetrically as it emerges from the reservoir. Where the flagellar memhrane approaches the membrane lining the reservoir, desmosomes are found. These structures are arranged in several slightly curved lines and have many features in common with vertebrate desmosomes.In cultures, the flagellates stick to each other by their flagella and form rosettes. In these bundles of cells, probable sites of adhesion between flagella, or between flagella and pieces of debris, are marked by a dense filamentous tract which passes posteriorly along the flagellum and by a thick band lying just below the flagellar membrane. It is suggested that similar adhesions are found in the insect host where the flagellate attaches itself to the gut wall.     

Tunic cells in pyrosomes (Thaliacea, Urochordata): cell morphology, distribution, and motility

Abstract. Cellular components of the tunic were histologically examined in 3 pyrosome species representing all 3 genera of the order: Pyrosoma atlanticum, Pyrosomella verticillata , and Pyrostremma spinosum . Three cell types are distributed in the tunic. Tunic amebocytes, irregularly shaped and motile, often contain granules and/or phagosomes. Spherical tunic cells contain many round vesicles with eosinophilic and acidic materials. Tunic net cells form a cellular network in which their long filopodia connect with one another. The net cells are densely distributed just beneath the tunic surface lining the common cloacal cavity and may produce tension to maintain the colony shape. The presence of net cells suggests a phylogenetic relationship between pyrosomes and some aplousobranch ascidians. Test fibers are multicellular cords that run in the tunic and connect the zooids. In P. atlanticum , they are attached to they are attached to the epidermal cells of the zooids, and transverse cloacal muscles are attached to the other (proximal) side of the epidermal cells. The test fibers may mediate coordination of the zooids and control muscle contraction.     

'Cup cell disease' in the colonial tunicate Botryllus schlosseri

A new progressive, fatal disease called 'cup cell disease' was characterized in ex situ cultures of Botryllus schlosseri, a colonial tunicate. The disease originated as a few dark spots growing within zooids. The infected colonies then started to deteriorate, morphologically diagnosed by ampullar retraction, lethargic blood circulation and by a swollen and soft tunic matrix. In later stages of the disease, developed buds were also affected. Many large black dots were scattered within the tunic matrix, and zooids were transformed to opaque, dilated, sac-like structures, signaling impending death. Colonies were infected periodically, even without direct tissue contact. The time course from first appearance to colony death ranged between 30 and 45 d. Histological studies, in vitro culturing of blood cells and blood smears revealed the existence of numerous cup-like cells (up to 4.8 microm diameter on average) with a yellowish cell wall and transparent cytoplasm that was not stained by various dyes (except azocarmine-G). Cells were refractive under bright-field illumination and revealed a flattened wall with flanges, characteristic of species of the phylum Haplosporidia. Cup cells aggregated in blood vessels and in internal parts of zooids and buds and were phagocytosed by blood cells. In a single case, plasmodia-like structures were found only in the tunic matrix of an infected colony. This is the first record in botryllid ascidians of an infectious lethal disease associated with haplosporidian protists.     

Dynamics of microbial communities on marine snow aggregates: colonization,growth, detachment,and grazing mortality of attached bacteria

We studied the dynamics of microbial communities attached to model aggregates (4-mm-diameter agar spheres) and the component processes of colonization, detachment, growth, and grazing mortality. Agar spheres incubated in raw seawater were rapidly colonized by bacteria, followed by flagellates and ciliates. Colonization can be described as a diffusion process, and encounter volume rates were estimated at about 0.01 and 0.1 cm(3) h(-1) for bacteria and flagellates, respectively. After initial colonization, the abundances of flagellates and ciliates remained approximately constant at 10(3) to 10(4) and approximately 10(2) cells sphere(-1), respectively, whereas bacterial populations increased at a declining rate to >10(7) cells sphere(-1). Attached microorganisms initially detached at high specific rates of approximately 10(-2) min(-1), but the bacteria gradually became irreversibly attached to the spheres. Bacterial growth (0 to 2 day(-1)) was density dependent and declined hyperbolically when cell density exceeded a threshold. Bacterivorous flagellates grazed on the sphere surface at an average saturated rate of 15 bacteria flagellate(-1) h(-1). At low bacterial densities, the flagellate surface clearance rate was approximately 5 x 10(-7) cm(2) min(-1), but it declined hyperbolically with increasing bacterial density. Using the experimentally estimated process rates and integrating the component processes in a simple model reproduces the main features of the observed microbial population dynamics. Differences between observed and predicted population dynamics suggest, however, that other factors, e.g., antagonistic interactions between bacteria, are of importance in shaping marine snow microbial communities.     

Status of the circulatory bed of the vascular membrane of the eyeball and retrobulbar structures during experimental venous stasis

In 43 test animals the state of the blood bed in the retrobulbar formations and the eyeball vasular tunic has been studied under venous congestion produced by ligation of the anterior vena cava (in dogs) and both external jugular veins (in rabbits). A complex of histological, histotopographic, morphometric and variation-statistical techniques has been used. The results obtained demonstrate that disturbances in the venous outflow in the anterior vena cava system produce certain responses in all parts of the retrotubular adipose tissue, of the eyeball muscles, of the optic nerve tunics, of the vascular tunic. Certain stageness is noted in the course of venous congestion. Places of the greatest morphological changes in the eyeball vascular tunic are determined. They are zones of vorticose veins formation and the area corresponding to the posterior pole of the eyeball. The analysis of the specific areas of the intermuscular arteries and veins cross sections demonstrates that in the reaction of these vessels to the different venous outflow in the anterior vena cava system these is certain unevenness in different ofthalmic muscles.     

Cytochemical investigations on tunic morphogenesis in the sea peach Halocynthia papillosa (Tunicata, Ascidiacea). 1: demonstration of polysaccharides

The distribution of carbohydrates was demonstrated in the embryonic, larval, and juvenile tunics of Halocynthia papillosa. An enzyme-gold marker (cellobiohydrolase-Au) was used to identify cellulose on ultrathin sections. This is the first time this biopolymer has been detected in the embryonic or larval tunic of an ascidian. Cellulose is present from the initial tail-bud stage onwards, as soon as the outer compartment of the tunic appears. Both compartments of the larval tunic also contain non-cellulosic polysaccharides, as demonstrated by the periodic acid-thiocarbohydrazide-silver proteinate (PA-TCH-SP) method. Our observations point to two types of cellulose synthesis. One occurs during the embryonic and larval stages, when glycogen-like material is stored in epidermal intracellular lacunae and discharged into the tunic where it is presumably used to synthesize cellulose throughout the depth of the tunic. The second occurs from the onset of metamorphosis onwards, just above the apical plasmalemma of epidermal cells, like cellulose biogenesis in plants.     

The role of actin, actomyosin and microtubules in defining cell shape during the differentiation of Naegleria amebae into flagellates

Differentiation of Naegleria amebae into flagellates was used to examine the interaction between actin, actomyosin and microtubules in defining cell shape. Amebae, which lack microtubules except during mitosis, differentiate into flagellates with a fixed shape and a complex microtubule cytoskeleton in 120 min. Based on earlier models of ameboid motility it has been suggested that actomyosin is quiescent in flagellates. This hypothesis was tested by following changes in the cytoskeleton using three-dimensional reconstructions prepared by confocal microscopy of individual cells stained with antibodies against actin and tubulin as well as with phalloidin and DNase I. F-actin as defined by phalloidin staining was concentrated in expanding pseudopods. Most phalloidin staining was lost as cells rounded up before the onset of flagellum formation. Actin staining with a Naegleria-specific antibody that recognizes both F- and G-actin was confined to the cell cortex of both amebae and flagellates. DNase I demonstrated G-actin throughout all stages. Most of the actin in the cortex was not bound by phalloidin yet was resistant to detergent extraction suggesting that it was polymerized. The microtubule cytoskeleton of flagellates was intimately associated with this actin cortex. Treatment of flagellates with cytochalasin D produced a rapid loss of flagellate shape and the appearance of phalloidin staining while latrunculin A stabilized the flagellate shape. These results suggest that tension produced by an actomyosin network is required to maintain the flagellate shape. The rapid loss of the flagellate shape induced by drugs, which specifically block myosin light chain kinase, supports this hypothesis.     

Lumichrome. A larval metamorphosis-inducing substance in the ascidian Hhalocynthia roretzi.

It has long been known that metamorphosis of ascidian larvae is induced by exposure to adult tunic extract or larval-conditioned seawater. However, such a natural 'inducer' has not been identified, probably due to its very low concentration in organisms. Here we have succeeded in isolating the same metamorphosis-inducing substance from the larvae, the larval-conditioned seawater, and the adult tunic of the ascidian Halocynthia roretzi. Structural analysis revealed that this substance was identical to lumichrome. Lumichrome was active toward H. roretzi larvae, but inactive toward another ascidian larvae, suggesting that lumichrome is species-specific. Riboflavin (vitamin B2), from which lumichrome might be derived from, was found to be inactive in induction of larval metamorphosis. In addition, it was demonstrated that lumichrome is localized predominantly in the basal region of the adhesive organ and the posterior part of the larval trunk. Thus, we propose that lumichrome functions as a natural inducer for larval metamorphosis in H. roretzi. This is the first natural metamorphosis-inducing substance to be identified in ascidians.