Ovulation and egg segregation in the tunic of a colonial ascidian,Diplosoma listerianum (Tunicata,Ascidiacea)

Summary The process of egg segregation in the tunic of the ovoviviparous ascidian Diplosoma listerianum was studied by light and electron microscopy. One egg at a time was seen to mature in each zooid. The eggs had large yolk and grew on the ovary wall enveloped in four layers: (1) outer follicle cells (OFC), long and rich in RER (rough endoplasmic reticulum) and with dense granules in the Golgi region; (2) flat inner follicle cells (IFC); (3) a loosely fibrillar vitelline coat (VC); (4) test cells encased on the egg surface. The growing egg protrudes from the ovary wall and presses on the contiguous epidermis. Granulocytes enter the space between the epidermis and the egg and insinuate cytoplasmic protrusions, disrupting the continuity of the OFC layer. At ovulation, OFC and IFC are discharged and form a post-ovulatory follicle (corpus luteum). The epidermis shrinks and closes, possibly by activation of microfilaments, causing the egg to be completely surrounded by the tunic. In the zooid, the wound caused by the passage of the egg is repaired both by contraction of the epidermis and by phagocytic activity. Altered spermatozoans are found in phagocytosing cells in the lumen of the ovary. These are presumably remnants of those which entered to fertilize the egg before segregation.     

Unusual structures in the spermatozoa of the ascidians Lissoclinum perforatum and Diplosoma listerianum (Didemnidae)

Summary Sperm and spermiogenesis of L. perforatum and D. listerianum have been investigated by light- and electron microscopy. The study revealed basic ascidian organization of sperm cells with the nucleus flanked by a unique mitochondrion, but with specializations not previously reported: a dense groove on the head surface, extended nucleus-mitochondrion association, and well developed endoplasmic derivatives.The groove is a long furrow of the plasmalemma stretching the nucleus towards the cell periphery. At least two parallel, 20 nm high, ridges emerge from the bottom of the groove. At the opposite side of the groove the long mitochondrion embraces the nucleus by extending its outer membrane over the nuclear envelope; a restricted cytoplasmic space is always found between the two organelles. Filamentous inclusions organized in two strands are present in the mitochondrial matrix. Endoplasmic derivatives appear as a single, extended cistern in L. perforatum, and series of long tubules in D. listerianum. They appear to originate in the spermatid from the Golgi complex and endoplasmic reticulum. The spermatozoon lacks an evident acrosomal complex, but the possible homology of small apical densities with an acrosomal complex is considered. It is suggested that the functional role of these specialized structures is related to the special mode of internal fertilization of these species.     

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.     

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.     

Antileukemic effects of Didemnum psammatodes (Tunicata: Ascidiacea) constituents

Chemical investigation of the methanolic extract of the ascidian Didemnum psammatodes has led to the identification of fourteen known compounds: three methyl esters (methyl myristate, methyl palmitate and methyl stearate), four steroids (cholesterol, campesterol, stigmasterol and beta-sitosterol), two fatty acids (palmitic acid and stearic acid), three glyceryl ethers {(1,2-propanediol, 3-(heptadecyloxy), batyl alcohol and 1,2-propanediol, 3-[(methyloctadecyl)oxy]} and two nucleosides (thymidine and 2'-deoxyguanosine). Their structures were proposed by NMR and comparison with literature data and GC analysis in comparison with authentic sample. The cytotoxic activity of these compounds was evaluated against human leukemia cell line panel using the MTT assay. The mixture of the three methyl esters was the most active group of compounds, showing antiproliferative and cytotoxic effects. Further studies on their mode of action suggest that these activities are connected with inhibition of DNA synthesis and induction of both necrosis and apoptosis.     

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.     

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.     

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.     

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.     

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.     

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.     

Spread of Microcosmus squamiger (Ascidiacea: Pyuridae) in the Mediterranean Sea and adjacent waters

The Mediterranean Sea is subject to an ever-increasing arrival of non-indigenous marine organisms. Microcosmus squamiger is a solitary ascidian that inhabits shallow rocky littoral habitats. It probably originated in Australia and it has shown great invasive potential in other parts of the world. In the Mediterranean, M. squamiger has only been reported at a few sites in Spain and Italy. However, the closely related species Microcosmus exasperatus has been reported in several areas of the western Mediterranean. As these species can be easily confused, we re-examined most of the material from previous studies and our personal collections. In addition, sampling was done at several sites along the western Mediterranean and Atlantic coasts. The results showed that the majority of the M. exasperatus reports correspond to M. squamiger, and that M. squamiger is common on the Atlantic shores. This suggests that M. squamiger has entered the Mediterranean through the Gibraltar Strait, while the restricted distribution in the eastern Mediterranean of M. exasperatus suggests that this species is probably a Lessepsian migrant. In the Mediterranean Sea, M. squamiger has the ability to occupy extensive areas of hard substrata and to outcompete native species. Further studies are necessary to assess what impacts this invasive species have on native communities.     

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.     

Two new species of diplosoma (Ascidiacea: Didemnidae) bearing prokaryotic algae prochloron from Okinawajima (Ryukyu Archipelago, Japan)

Two new species of didemnid ascidians, Diplosoma ooru sp. nov. and Diplosoma simileguwa sp. nov., are described from coral reefs on Okinawajima (Ryukyu Archipelago, Japan). These two species form green colonies, having a symbiotic association with a prokaryotic alga Prochloron sp. The former species was found at the reef edges in the subtidal zone and the latter was found in a shallow reef lagoon. In these species, the colonies are thinner and the zooids are smaller than those of any other Prochloron-bearing Diplosoma species so far described. Moreover, each of the present new species has a unique combination of stigmatic numbers: 5 stigmata in the first and third rows, 6 in the second row, and 4 in the fourth in D. ooru; 4 stigmata in the first and third rows, 5 in the second row, and 3 in the fourth in D. simileguwa. In both of the new species, the retractor muscle emerges from the underside of the thorax. Larval morphology of D. ooru is also described.     

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.     

An ultrastructural study of the egg wall surrounding the miracidium of the digenean Brandesia turgida () (Plagiorchiida: Pleurogenidae), with the description of a unique cocoon-like envelope

The intrauterine eggs of the pleurogenid trematode Brandesia turgida (Brandes, 1888), exhibiting advanced stages of miracidial differentiation and fully formed, ciliated miracidia, were examined by means of transmission electron microscopy (TEM). Each embryonated egg is composed of a mature miracidium surrounded by a four-layered egg wall: (1) an outer, anucleate layer external to the eggshell, which forms a thick cocoon; (2) the operculate egg-shell; (3) a small remnant of the compact, granular cytoplasm of the outer embryonic envelope (sensu stricto); and (4) a relatively distinct cellular remnant of the inner embryonic envelope. Layers enveloping the egg apparently play an important role in the protection, metabolism and storage of nutritive reserves for the developing miracidium. The outer, anucleate layer, or cocoon, situated externally to the eggshell and composed of a transparent, electron-lucent substance with numerous dense, osmiophilic islands attached to its peripheral membrane, has never previously been seen in TEM studies of the eggs of parasitic platyhelminths. The origin, formation, functional ultrastructure and chemical composition of this peculiar layer remain enigmatic, although its function appears to be protective. The thick, electron-dense eggshell resembles that of other trematodes, exhibiting a characteristic fissure zone around the operculum. The very small, indistinct remnants of the outer embryonic envelope appear in the form of a very thin, compact, granular cytoplasm closely attached to the inner surface of the eggshell. Conversely, the inner embryonic envelope is frequently apparent at one or both poles of the developed egg as a syncytial envelope formed by the fusion of mesomeres. This envelope, even in eggs containing a fully formed miracidium, still has the features of a metabolically active layer with an energy storage capability. Lysosome-like structures observed in some eggs may be involved in the autolysis of the embryonic envelopes.     

Chemical and physical defenses against predators in Cystodytes (Ascidiacea)

Ascidians utilize both physical (spicules, tunic toughness) and chemical defenses (secondary metabolites, acidity) and suffer relatively little predation by generalist predators. The genus Cystodytes (Polycitoridae) is distributed widely in both tropical and temperate waters. Secondary metabolite composition, calcareous spicules and tunic acidity (pH < 1) may act as redundant defense mechanisms against predation in this genus. To assess the relative importance of chemical and physical defenses against predation in ascidians, we studied purple and blue morphs of Cystodytes from the western Mediterranean (formerly assigned to Cystodytes dellechiajei, but recently shown to belong to two different species), and a purple morph from Guam (USA), identified as Cystodytes violatinctus. Crude extracts, spicules, ascididemin (the major alkaloid of the blue morph) and acidity were used in feeding trials to evaluate chemical and physical defense mechanisms in Cystodytes spp. We performed feeding experiments in the field with a guild of generalist fish (mostly damselfish), and in the laboratory with a sea urchin and a puffer fish. Our results showed that all crude extracts and ascididemin significantly deterred fish predation, but not sea urchin predation. However, neither acidity alone nor spicules at natural concentrations deterred feeding. These results and other studies on sponges and gorgonians suggest that secondary metabolites are the primary means of defense against fish predators. Spicules and tunic acidity may perform other ecological roles and/or target certain specialist predators.