An experimental test of colonization ability in the potentially invasive Didemnum perlucidum (Tunicata, Ascidiacea)

Exotic species invasions are one of the greatest threats to marine systems and ascidians have many characteristics that favor transport, colonization and establishment into new regions. Didemnum perlucidum is a widespread species that has been introduced into tropical ports around the world. Here we examine the colonizing ability of D. perlucidum by experimental use of artificial plates in a shellfish culture. The experiment comprised paired plates for colonization (bare and occupied) in 16 monthly replicates. Recruitment and space occupation were compared between bare and occupied plates and an estimation of reproductive effort was based on the number of larvae produced in each of ten colonies collected on the culture structures. D. perlucidum reproduced continuously but greatest reproduction occurred between December 2006 and May 2007. While recruitment was somewhat greater (number of new colonies) on bare plates, this species can colonize already occupied substrates and, surprisingly, colony area was always similar between treatments. Thus, while fewer colonies formed on occupied plates, once formed, colonies grew at rates similar to those on clean plates. Thus, D. perlucidum colonizes substrates very efficiently, especially when unoccupied space is available.     

Genetic isolation among morphotypes in the photosymbiotic didemnid Didemnum molle (Ascidiacea, Tunicata) from the Ryukyus and Taiwan

Didemnum molle, a colonial ascidian that harbors the symbiotic cyanophyte Prochloron spp., is distributed throughout the coral reefs of the Indo-West Pacific Ocean. Several morphotypes of D. molle are characterized by the color and size of their colonies. Previous molecular phylogeny inferred from gene sequences for the cytochrome oxidase subunit I (COI) identified four morphotypes (i.e., gray, brown, white, and large) from several sites in the Ryukyu Islands, Japan. With the addition of 17 specimens, including another morphotype (small), from several collection sites (Taiwan and the Ryukyus), the present report demonstrates genetic separation among the five morphotypes based on COI sequences. A number of sexually mature specimens of the different morphotypes were collected at the same times and sites, indicating reproductive isolation among morphotypes.     

Cupular sense organs in Ciona (Tunicata: Ascidiacea)

The cupular organs of the atrial (exhalent) siphon of Ciona have been studied with scanning and transmission microscopy and are shown to resemble those of the vertebrate acoustico-lateralis system in several respects. The sensory cells are ciliated, and their cilia are apparently non-motile, having a modified inner tubular array. These cells lie amongst supporting cells that probably secrete the cupula, which is composed of polysaccharide and proteins as is the test. Ciona is sensitive to near-field vibrations, even after the brain has been removed; the significance of this observation and of the arrangement of the cupular organs is discussed. It is concluded that the tunicates show a suitable morphological starting point for the vertebrate acoustico-lateralis system.     

Cupular organs in two species of Corella (Tunicata: Ascidiacea)

Abstract. Simple cupular organs similar to those described in Ciona intestinalis were observed in Corella eumyota. They consist of a macula containing the cell bodies of 20–30 primary sensory neurons whose cilia project into a dome‐ or finger‐shaped structure, the cupula. Rather than being found in the mantle lining as in C. intestinalis, the organs were located on the atrial surface of the branchial sac. The sensory innervation was examined in whole‐mount preparations using anti‐tubulin immunohistochemistry. Sensory neurons in C. eumyota showed no immunoreactivity with antisera raised against gonadotropin‐releasing hormone (GnRH). A novel, elongated sense organ termed the cupular strand was found in Corella inflata. It has the same basic components as the simple type of cupular organ but consists of a single, long structure containing ～1500 sensory cells. Located on the atrial surface of the branchial sac, it extends along the midline of the dorsal fold, from the gonoduct openings almost as far as the brain. Preparations were examined using optical and electron microscopy. Nerves and cilia were visualized by anti‐tubulin immunofluorescence microscopy. It was possible to follow the sensory axons from the macula of the cupular strand to points where they joined branches of the visceral nerve, which enters a nerve root at the back of the brain. In C. inflata the sensory cell bodies and their axons were immunoreactive not only with anti‐tubulin but also with an antiserum raised against Tunicate I GnRH. There was no immunoreactivity, however, with Chicken II and catfish GnRH antisera. All three GnRH antisera labeled the dorsal strand plexus, a structure associated with production of GnRH in its role as a reproductive hormone. We concluded that the GnRH‐like molecule labeled in sensory neurons differs from the form of GnRH found in the dorsal strand plexus, and may have a different function, perhaps in the neural control of ciliary activity. The function of the cupular organs in species of Corella has not yet been investigated physiologically, but by analogy with such structures in other metazoans, cupular organs are probably hydrodynamic sensors registering local disturbances or changes in water flow through the atrial cavity.     

Morphological aspects of fertilization in Phallusia (Ascidia) nigra (Ascidiacea,Tunicata)

The spermatozoa of Phallusia (Ascidia) nigra have an elongated head (approximately 5 m in length) in which a nucleus and a single mitochondrion are located side by side. There is no midpiece. The apex of the head is wedge-shaped. Acrosomal vesicles (approximately 55–65 nm in diameter) and moderately electron-dense material (MEDM) are present between the plasmalemma and the nuclear membranes in the anterior tip of the head. The MEDM occupies a central position and three or four acrosomal vesicles are seen in a line alongside it. The acrosomal vesicles disappear as the sperm makes contact with the surface of the chorion. Gamete fusion most likely occurs between a small process extending from the peripheral margin of the sperm apex and the egg surface, resulting in incorporation of the sperm into the egg from the anterior region of its head.     

Ovulation in Diplosoma (Tunicata, Ascidiacea, Didemnidae): a light microscopical study

Serial sections of non-ovulated and ovulated eggs of Diplosoma migrans (Tunicata, Ascidiacea, Didemnidae) were compared in order to study the process of ovulation on the level of light microscopy. Fully grown eggs are surrounded by an outer follicular epithelium consisting of cubic cells, and an inner follicular epithelium consisting of flat cells. Egg release is initiated by a fusion of the epidermis with the outer follicular epithelium at the distal pole of the egg. A contraction of the outer follicular epithelium leads to an opening of both epidermis and outer follicular epithelium, and their removal from the egg. The envelope of ovulated eggs corresponds to the former inner follicular epithelium. Mainly due to a contraction of the epidermis, the outer follicular epithelium is shifted into the abdomen and separates from the epidermis. It transforms into a vestigial body (corpus luteum) and finally atrophies. The epidermis flattens and resumes its typical appearance. On the basis of these observations, the results of studies on the closely related Diplosoma listerianum are discussed and questioned. Electronic Publication     

Functional morphology of the branchial basket of ascidia paratropa (Tunicata,Ascidiacea)

Summary The filtering surface of the branchial basket of Ascidia paratropa (Huntsman, 1912) consists of two adjacent and connected sheets. The stigmatal surface is loosely folded. The secondary gill screen, composed of ciliated longitudinal vessels with primary and secondary papillae, is connected to the stigmatal surface on the inside of the branchial basket. Continuous mucus secretion results in a net with elastic and adhesive properties, and a pore size of less than 0.5 m in its longest dimension. Net production cannot be interrupted by external mechanical stimuli. The net is usually supported by and transported across the secondary screen, forming a filtering surface that parallels the undulations of the stigmatal surface; however, localized muscular activity of the dorsal laminar fold and longitudinal vessels are also involved in mucous net transport. Squirts occur more frequently than in many species. At these times the net is transported as a flat sheet, independent of the secondary screen. Squiring does not interrupt continuous feeding activity.     

The genus Herdmania Lahille, 1888 (Tunicata, Ascidiacea) in Australian waters

Revision of material from Australian waters previously assigned to Herdmania momus ( Savigny, 1816 ) has resulted in the recognition of five species (including two new indigenous species). A tropical Indo-West Pacific range is recorded for the type species ( H. momus ), although it is not recorded from Western Australia. A circum-continental range for the third and largest species, H. grandis ( Heller, 1878 ) is confirmed. Herdmania pallida (Heller, 1878), with a wide range in the western Pacific and Indian Ocean including the north-eastern and north-western coasts of Australia, is a different species from the Atlantic Ocean species (formerly thought to be conspecific). Herdmania fimbriae , a new species with a geographical range from southern to north-eastern Australia, is distinct from H. mentula , also a new species, from the north-western coast. The structure of the gonads and their ducts and accessory membranes and the arrangement and number of body wall muscles distinguish the species. Species differences are supported by the maximum size of individuals, nature of their test, number of branchial folds, number of internal longitudinal vessels on the folds and between the dorsal lamina and dorsal fold, the size and form of the dorsal tubercle and dorsal lamina, and the shape of the ciliated opening of the neural duct, although these characters are affected to some extent by growth. A variety of accessory membranes associated with both male and female gonoducal apertures are found in the majority of species in this genus. © 2002 The Linnean Society of London. Zoological Journal of the Linnean Society , 134 , 359–374.     

Cytotoxic activity of a dichloromethane extract and fractions obtained from Eudistoma vannamei (Tunicata: Ascidiacea)

This study consists of the bioassay-guided fractionation of the dichloromethane extract from Eudistoma vannamei and the pharmacological characterization of the active fractions. The dried hydromethanolic extract dissolved in aqueous methanol was partitioned with dichloromethane and chromatographed on a silica gel flash column. The anti-proliferative effect was monitored by the MTT assay. Four of the latest fractions, numbered 14 to 17, which held many chemical similarities amongst each other, were found to be the most active. The selected fractions were tested for viability, proliferation and death induction on cultures of HL-60 promyeloblastic leukemia cells. The results suggested that the observed cytotoxicity is related to apoptosis induction.     

Tunic cell populations during fusion events in the colonial ascidian Didemnum vexillum (Tunicata)

We documented changes in the abundance and distribution patterns of tunic cells involved in the allorecognition response of the colonial aplousobranch Didemnum vexillum, whose zooids do not share a common vascular system. A histological examination of the fusion zone of isogeneic (CIAs) and allogeneic (CAAs) fused colony assays revealed that tunic cuticles were rapidly regenerated. The underlying tunic matrix fused readily in all assays and controls. We identified four different types of tunic cells. Phagocytic cells represented the most abundant cell type in allogeneic fusions, followed by morula cells. These cells were more abundant at the immediate fusion junction than at 120 μm or 240 μm from the junction, most likely because they mediate the allorecognition reaction. Elongated filopodial cells also were present, although only at very low abundances, and a layer of bladder cells was located immediately below the cuticle. Our results provide quantitative evidence for the involvement of tunic cells in the allorecognition response of a highly invasive ascidian.     

Morpho-functional characterization of haemocytes of the compound ascidian Botrylloides leachi (Tunicata, Ascidiacea)

A morpho‐functional study of the colonial ascidian Botrylloides leachi haemocytes was carried out to propose their classification, relationships and specializations. This characterization was obtained by (i) investigations of both living and aldehyde‐fixed cells by light and electron microscopy; (ii) cytochemical and cytoenzymatic assays; (iii) lectin‐affinity assays; (iv) phagocytosis and haemagglutination assays; and (v) anti‐CD34 immunocytochemical assay for vertebrate haematopoietic stem cells. Results indicate that the haemoblast is a circulating stem cell and there are at least five haemocyte differentiation pathways, the last two of which have never been described in botryllids: (i) phagocytic line (hyaline amoebocytes and macrophage‐like cells) share ultrastructural features, the same hydrolytic enzymes and WGA lectin binding, and are involved in yeast phagocytosis and erythrocyte rosette formation; (ii) cytotoxic line (granular amoebocytes and morula cells) with vacuoles containing oxidative enzymes and polyphenolic substrates; (iii) vacuolated cell line (pigment cells and nephrocytes) involved in catabolite storage; (iv) compartment cell line (compartment amoebocytes and compartment cells) able to agglutinate erythrocytes and characterized by vacuoles with a moderately electron‐dense content, positive to arylsulphatase activity and binding DBA, UEA‐I, HPA lectins; and (v) granular cell line includes trophic cells, able to infiltrate the gut epithelium, showing a cytoplasm filled of PAS‐positive vacuoles with arylsulphatase, chloroacetylesterase and β‐glucuronidase activities.     

Glycogen and degeneration in the pyloric gland of Dendrodoa grossularia (Ascidiacea,Tunicata)

Summary Degeneration is observed in cells of the pyloric gland of Dendrodoa grossularia in which glycogen storage occurs. The ultrastructure of four phases of the degeneration cycle is described. Natural senescence seems to be the cause of the degeneration. Glycogen storage might be the result of metabolic disturbance, but its presence reveals the importance of glycogen in the function of the organ. The role of the pyloric gland is discussed.     

The alien ascidian Styela clava now invading the Sea of Marmara (Tunicata: Ascidiacea)

During the implementation of a large project aimed to investigate the benthic community structures of the Sea of Marmara, specimens of the invasive ascidian species Styela clava were collected on natural substrata (rocks) at 10 m depth at one locality (Karamürsel) in İzmit Bay. The specimens were mature, containing gametes, indicating that the species had become established in the area. The Sea of Marmara seems to provide suitable conditions for this species to survive and form proliferating populations.     

Biological activity in extracts of ascidians (Tunicata, Ascidiacea) from the northeastern Brazilian coast

Ascidians are marine animals with a great ability to synthesize bioactive substances. This study examined the cytotoxic potential of 10 ascidians found in the coastal waters of Northeast Brazil. Samples of the species Eudistoma vannamei Millar, 1977, Eudistoma sp., Didemnum ligulum Monniot F., 1983, Didemnum psammatodes (Sluiter, 1895), Didemnum sp., Polysyncraton sp., Trididemnum sp., Cystodytes dellechiajei (Della Valle, 1877), Euherdmania sp., and an unidentified species belonging to the Holozoidae family were extracted in methanol 5:1 (v/w). The extracts were tested for cytotoxicity using the brine shrimp lethality assay, sea urchin egg development assay, hemolysis assay, and MTT assay using tumor cell lines. The extract of E. vannamei showed the highest toxicity in brine shrimp (LD₅₀=34.7 μg/ml) and in all tumor cell lines tested, with an IC₅₀ of <2 μg/ml for CEM, 11.2 μg/ml for HL-60, 23.8 μg/ml for B16, and 14.3 μg/ml for HCT-8. In sea urchin eggs, it inhibited the cell cycle progression mainly at the blastula stage (IC₅₀=74.8 μg/ml). The extract of Euherdmania sp. also exhibited some toxicity in these assays, but at a lower potency than that of E. vannamei. The extracts of D. psammatodes and Polysyncraton sp. showed a strong inhibition of the sea urchin egg cell cycle during both phases examined, first cleavage and blastula, with a possible action on the cell microfilament apparatus. The extract of D. ligulum showed selective toxicity toward HCT-8 cells (IC₅₀=35.3 μg/ml). The extract from the Holozoidae was the only one that possessed a hemolytic effect, with an IC₅₀ of 175.2 μg/ml. Further studies are necessary for a better characterization of the active principles of these extracts and a possible elucidation of the mechanisms of action.     

Spatial competition induces the mobilization of morula cells in the colonial ascidian Didemnum perlucidum (Tunicata: Didemnidae)

Abstract. The effects of spatial competition among colonial marine organisms are often evident in the contact zones between colonies. These effects are especially pronounced when the interaction results in overgrowth or necrosis of one of the competitors. Ascidians, one of the dominant taxonomic groups in subtidal sessile communities, have specialized morula cells that provide a defense against microbial infections. Injuries resulting from interspecific competitive interactions might also act as a stimulus for this defensive mechanism. Therefore, we expected to see the recruitment of morula cells in tissues near competitor contact zones. To test the hypothesis that spatial competition elicits this immune response, we placed colonies of the ascidian Didemnum perlucidum from southeastern Brazil in four different types of competitive situations: (1) overgrowth of the competitor, (2) stand‐off interactions, (3) overgrowth by the competitor, and (4) free of competitors. Our results indicate that competitive interactions increase the population of morula cells in contact zones, as more cells were observed in interactions that resulted in the overgrowth of individuals of D. perlucidum, and fewer cells were observed in colonies that were free of competitors. We identified the defensive function of the morula cells by showing the presence of the enzyme phenoloxidase within its vacuoles. Phenoloxidase is a widespread enzyme among animals and plants, and is frequently used in defense by synthesizing toxic quinones from polyphenol substrates. This is the first study to document the presence of morula cells in didemnid ascidians and the mobilization of these cells by spatial competition by heterospecifics, and one of the first studies to identify phenoloxidase activity in morula cells.     

Spatial distribution patterns of ascidians (Ascidiacea: Tunicata) on the continental shelves off the northern Antarctic Peninsula

Ascidians (Ascidiacea: Tunicata) are sessile suspension feeders that represent dominant epifaunal components of the Southern Ocean shelf benthos and play a significant role in the pelagic–benthic coupling. Here, we report the results of a first study on the relationship between the distribution patterns of eight common and/or abundant (putative) ascidian species, and environmental drivers in the waters off the northern Antarctic Peninsula. During RV Polarstern cruise XXIX/3 (PS81) in January–March 2013, we used seabed imaging surveys along 28 photographic transects of 2 km length each at water depths from 70 to 770 m in three regions (northwestern Weddell Sea, southern Bransfield Strait and southern Drake Passage), differing in their general environmental setting, primarily oceanographic characteristics and sea-ice dynamics, to comparatively analyze the spatial patterns in the abundance of the selected ascidians, reliably to be identified in the photographs, at three nested spatial scales. At a regional (100-km) scale, the ascidian assemblages of the Weddell Sea differed significantly from those of the other two regions, whereas at an intermediate 10-km scale no such differences were detected among habitat types (bank, upper slope, slope, deep/canyon) on the shelf and at the shelf break within each region. These spatial patterns were superimposed by a marked small-scale (10-m) patchiness of ascidian distribution within the 2-km-long transects. Among the environmental variables considered in our study, a combination of water-mass characteristics, sea-ice dynamics (approximated by 5-year averages in sea-ice cover in the region of or surrounding the photographic stations), as well as the seabed ruggedness, was identified as explaining best the distribution patterns of the ascidians.     

A morphological and immunohistochemical study of programmed cell death in Botryllus schlosseri (Tunicata,Ascidiacea)

The blastogenic cycle of the colonial ascidian Botryllus schlosseri concludes in a phase of selective cell and zooid death called takeover. Every week, all asexually derived parental zooids synchronously regress over a 30-h period and are replaced by a new generation. Here we document the sequential ultrastructural changes which accompany cell death during zooid degeneration. The principal mode of visceral cell death during takeover occurred by apoptosis, the majority of cells condensing and fragmenting into multiple membrane-bounded apoptotic bodies. Cytoplasmic organelles (mitochondria, basal bodies, striated rootlets) within apoptotic bodies retained ultrastructural integrity. Dying cells and fragments were then swiftly ingested by specialized blood macrophages or intraepithelial phagocytes and subsequently underwent secondary necrotic lysis. Certain organs (stomach, intestine) displayed a combination of necrotic and apoptotic changes. Lastly, the stomach, which demonstrated some of the earliest regressive changes, exhibited intense cytoplasmic immunostaining with a monoclonal antibody to ubiquitin at the onset of takeover. Affinity-purified rabbit antiserum against sodium dodecyl sulfate-denatured ubiquitin detected a characteristic 8.6-kDa mono-ubiquitin band by Western blot analysis. Collectively, these findings raise the possibility that cell death during takeover is a dynamic process which requires active participation of cells in their own destruction.