Ecological Consequences of Altering the Timing Mechanism for Metamorphosis in Anural Ascidians

SYNOPSIS. In the present study the timing of metamorphosis inan anural ascidian, Molgula pacifica, was compared to metamorphosisin a urodele species Boltenia villosa. Metamorphosis in M. pacificawas triggered at a fixed time in development (32–36 hoursafter fertilization), just prior to hatching. In contrast, metamorphosiswas triggered in B. villosa after the hatched larvae respondedto substrate cues. The timing of metamorphosisin B. villosawas often delayed for up to four days, whereas delays in M.pacifica were not observed. An antibody, termed Epi-3, was foundto cross-react exclusively with epidermal cells in both species.The binding of FITC-labelled Epi-3 was very low prior to metamorphosisand then it increased dramatically after metamorphosis was triggered.The cytoplasm of ampulla tip cells and the tunic immediatelysurrounding each ampulla showed the highest levels of Epi-3fluorescence. The histological and ultrastructural featuresof the ampulla cells suggest that Epi-3 antibody recognizesgranules localized in the apical cytoplasm. How the evolution of an internal "clock" mechanism responsiblefor initiating metamorphosis may be beneficial to anural speciesis discussed. One possibility is that the anural type of timingmechanism reduces mortality rates during this critical phaseof its life cycle.     

Sperm Chemotaxis in Ascidians

SYNOPSIS. Sperm chemotaxis in ascidians, first demonstratedin the genera Ciona and Styela, has also been found in the generaAscidia, Halocynthia, Chelyosoma, Pyura, Corella and Boltenia.Species—specificity exists in many cases, but unlike theCnidaria, low level cross-specificity is more general. Thisis particularly true in the stolidobranchs. The sperm attractantof Ciona is a small molecule that is negatively charged andstable to heat and pronase; it binds avidly to glass surfaces.Calcium is required for sperm chemotaxis of both ascidian andcnidarian sperm. The mechanism by which turning occurs may involve both contractionof the pericentriolar process arms to shift the axis of thefiagellum relative to that of the head, and generation of asymmetricwaves in the fiagellum. Hydrozoan and ascidian sperm show aspectsof both behaviors, but the cnidarian sperm, known to possessa large asymmetric pericentriolar process, shows less shiftof the flagelluni axis and more dramatic fiagellar asymmetrythan the ascidian sperm, where an extensive pericentriolar complexhas yet to be demonstrated. These differences in flagellar behaviormay be adaptations to species' differences in the shape of thesperm head. They appear to prolong the turning maneuver, incontrast to the sudden turning seen in some unicellular flagellates.More data on flagellar behavior in ascidian sperm will be avaluable tool for the elucidation of the mechanism of chemotacticturning.     

Formal Genetics of Ascidians

SYNOPSIS. Our knowledge of ascidian genetics is reviewed. Thepaper is primarily concerned with the author's past and currentwork on the colonial species Bolryllus schlosseri. Five Mendelianloci account for most of its impressive polychromatism. Breedingexperiments have substantiated the hypothesis of a single multialleliclocus for each of three enzymes (MDH, SOD, PGI) suggested byelectrophoretic patterns. The nuclei of three linkage groupshave been revealed. Self—fertilization entails a severeinbreeding depression. A specific self, nonself recognition,expressed by fusion or repulsion of contacting colonies, occursin this species also. At variance with Botryllus primigenus,fusible colonies of B. schlosseri are completely interfertile.This has allowed a more direct genetic analysis of the phenomenon,confirming the alleged control by a single multiallelic locus.In order to fuse, the confronted colonies must share at leastone allele. Young buds grafted in the tunic after removal ofall the zooids develop a new colony at the host's expense onlyif donor and host are fusible. This means that fusibility andhistocompatibility are strictly correlated. Chimerical colonies,obtained either in this way or following the resorption of oneof two fused colonies, are now being investigated for theirrecognition specificity and electrophoretic pattern. Preliminarydata indicate that both can be durably altered, suggesting thatthe allogeneic cell populations are persistent and renewing.     

Coloniality has evolved once in Stolidobranch Ascidians

Ascidians exhibit a rich array of body plans and life history strategies. Colonial species typically consist of zooids embedded in a common test and brood large, fully developed larvae, while solitary species live singly and usually free-spawn eggs that develop into small, undifferentiated larvae. Ascidians in the order Stolidobranchia include both colonial and solitary species, as well as several species with intermediate morphologies. These include social species, which are colonial but do not live completely embedded in a common test, and a few solitary species that brood embryos and larvae until they are competent to metamorphose. We examined how many times coloniality has evolved within the Stolidobranchia, with phylogenetic analyses using full-length 18S rDNA and partial cytochrome oxidase B sequences for taxa in the families Molgulidae, Styelidae, and Pyuridae. Tunicata orders Phlebobranchia and Stolidobranchia are sister groups, and the family Molgulidae is a monophyletic group and should be raised to the subordinal level, as shown previously by analyses from this lab with partial 18S sequences. In contrast to previous studies, styelids and pyurids are separated into monophyletic groups by ML and Bayesian analyses. We show a single clade within the family Styelidae that contains two colonial (compound) botryllid species, a Symplegma (colonial compound), a colonial (social) species Metandrocarpa taylori, as well as four solitary species, thus confirming that the botryllids are a subfamily of the Styelidae. These results suggest that the ancestor of the Stolidobranchia was solitary and that coloniality has evolved only once within this clade of ascidians. Further phylogenetic analyses of aplousobranch and phlebobranch ascidians will be necessary to understand the number of times that coloniality has evolved within the class Ascidiacea.     

Self or Non--self Recognition in Compound Ascidians

SYNOPSIS. Certain species of compound ascidians have an abilityto distinguish self colonies from non—self colonies withinthe same species. This ability, called colony specificity, ismanifested by the fusibility between colonies. The fusibilityamong colonies of Japanese Botryllus is genetically controlledby a series of multiple alleles at a single locus. The fusibilityis determined by a factor(s) in blood, so that the fusibilitycan be altered by the exchange of blood. It is suggested thatrejection, called "nonfusion" reaction, may occur from the interactionbetween blood cells and blood humoral factor(s).     

The Renewing Cell Populations of Ascidians

SYNOPSIS. Renewing cell populations are tissues or groups ofcells which rapidly proliferate and whose cell division is balancedby cell loss. The rapid proliferation of cells can be determinedin autoradiograms by the uptake of tritiated thymidine intoDNA synthesizing cells, and the migration and loss of cellscan be followed by taking samples of tissues at increasing timeintervals after exposure to the radioisotope. In ascidians,renewing populations are the testis, ovary, blood cells, andepithelial lining of the digestive tract. They are made of subpopulationscalled compartments: 1) germinal compartments of relativelyundifferentiated, dividing stem cells (these cells are labeledat short time intervals), 2) mature compartments of fully differentiated,nondividing cells (these become labeled with time by the migrationof stem cells), and 3) transitional compartments of cells inintermediate stages. The stomach groove cell population is amodel system for cell differentiation and renewal in the digestivetract. Germinal compartments can be distinguished from maturecompartments by their morphology (pseudostratified vs. simplecolumnar epithelia), by the uptake of tritiated thymidine inautoradiograms, by histochemical staining, by their surfacefeatures in the scanning electron microscope, and by their intracellularorganelles when viewed in the transmission electron microscope.Stem cells differentiate into absorptive cells and zymogen cells.In differentiating, stem cells increase in size and lose theirabundant free ribosomes. Absorptive cells develop longer microvilli,a long cilium, smooth apical vesicles, and large supranuclearlysosomes. Zymogen cells produce abundant rough endoplasmicreticulum and numerous zymogen granules. The renewing cell populationsof blood (lymph nodule) and digestive tract are important modelsystems for studying cell differentiation, morphogenesis, andthe phylogeny of vertebrate hemopoietic, immune, and digestivesystems.     

Ascidians and the plasticity of the chordate developmental program

Little is known about the ancient chordates that gave rise to the first vertebrates, but the descendants of other invertebrate chordates extant at the time still flourish in the ocean. These invertebrates include the cephalochordates and tunicates, whose larvae share with vertebrate embryos a common body plan with a central notochord and a dorsal nerve cord. Tunicates are now thought to be the sister group of vertebrates. However, research based on several species of ascidians, a diverse and wide-spread class of tunicates, revealed that the molecular strategies underlying their development appear to diverge greatly from those found in vertebrates. Furthermore, the adult body plan of most tunicates, which arises following an extensive post-larval metamorphosis, shows little resemblance to the body plan of any other chordate. In this review, we compare the developmental strategies of ascidians and vertebrates and argue that the very divergence of these strategies reveals the surprising level of plasticity of the chordate developmental program and is a rich resource to identify core regulatory mechanisms that are evolutionarily conserved in chordates. Further, we propose that the comparative analysis of the architecture of ascidian and vertebrate gene regulatory networks may provide critical insight into the origin of the chordate body plan.     

Viviparous development in Botrylloides (Compound Ascidians)

The mode of sexual reproduction and embryogenesis was compared in 3 species of Botrylloides: B. simodensis, B. lenis, and B. violaceus. In all species, a testis and an egg (occasionally 2 eggs), the former being anterior to the latter, mature in the mantle on either side of a zooid. The egg is surrounded by 2 follicular layers and is attached by a vesicular follicle stalk (oviduct) to the atrial brood pouch. The egg is ovulated into the brood pouch, where it is fertilized and undergoes embryogenesis. The egg of B. simodensis is heavily yolked and measures about 180 μm in diameter. The course of embryogenesis in this species is that typical of ascidians. A mature tadpole larva is produced and shed in about 5 days; then, the mother zooid degenerates. The larva is smallest of the three species and has 8 ampullae. The metamorphosed oozooid bears a single bud on the right side only. Extraembryonic nutrition seems to be very limited. Both Botrylloides lenis and B. violaceus are species which display extreme examples of viviparity. Their eggs are devoid of yolk granules, measuring about 90 μm in diameter in the former species and 60 μm in the latter. The course of embryogenesis is similar in these 2 species. The neurula stage is characterized by a spherical vesicular shape owing to precocious differentiation of the embryonic pharynx, whose ectoderm becomes vacuolated. At the posterior end of the neurula, the mesodermal cells are located in a mass, from which the tail is extended later. In B. lenis, embryogenesis takes about 20 days. At the neurula stage of the embryo, the mother zooid becomes a mantle sac as a result of visceral disintegration. During further embryogenesis, the growth of buds of successive generations in the colony is characteristically arrested. A swimming larva of this species is somewhat larger than that of B. simodensis. It has 14–24 ampullae, and the oozooid carries a single bud on its right side. In B. violaceus, the gestation period lasts for more than a month. At the early gastrula stage of the embryo, the body of the mother zooid fully disintegrates. Only the brood pouch bearing the embryo survives and remains connected with the colonial vascular system. In this species, sexual reproduction does not affect the growth of buds in the colony. The swimming larva is gigantic, being furnished with 24–34 ampullae, and the oozooid always bears 3 buds, 2 on the right side and one on the left side.     

Morphogenesis in Marsilea

An account is given of investigations on the development of sporelings of Marsilea using aseptic culture techniques. Special attention was paid to the heteroblastic leaf development and to the conditions leading to the origin of land or water forms. A study was made of the effects of changes in the composition and concentration of the culture medium. The substances tested included sugars, nitrogen compounds, metabolic inhibitors, auxins, gibberellic acid and kinetin.
It was concluded that the changes in leaf segmentation of Marsilea , or heteroplastic development, are correlated with changes in the size of the apical meristematic regions of the shoot, which in turn are influenced by the nutritional status of the plant. On the other hand, the differences between land and water forms are probably due to differences in the available concentration of dissolved carbohydrates in the growing parts. The results are discussed in relation to similar morphogenetic problems in other vascular plants.     

Neural Complex of Ascidians and Features of Its Structure in Polyclinidae

The structure of the neural complex has been studied under a light microscope in the representatives of 4 genera belonging to the family Polyclinidae. Particular attention was given to the relationships between the ciliary organ, dorsal tube, and neural gland. No dorsal string was found in Polyclinidae. The neural gland is located in the proximal portion of the mediodorsal blood vessel of the branchial sac. This paper discusses probable reasons for the differences in the structure of the neural complex in different ascidians taking into account original data and the existing literature.     

Larval Tunic and the Function of the Test Cells in Ascidians

Abstract In normal ascidian development, cuticular fins begin to form at the late tailbud stage and are fully formed at hatching. When one or several neurulae were manually demembranated (follicle cells, vitelline coat and test cells removed) and cultured in seawater they failed to form caudal fins. Fins were normal when the follicle cells alone were removed. The shape of the fins was normal when demembranation was delayed to the late tailbud stage. Does demembranation cause the loss of an essential factor produced by the embryos themselves or do the test cells provide a factor for fin morphogenesis? Demembranated neurulae of Ascidia callosa were cultured in groups ranging in size from 2 to 80 in 1 ml volumes of seawater. The mean lengths of the caudal fins increased with group size. In larger groups, some embryos developed fins that were normal in shape and as long as undemembranated controls. Results were similar with Corella inflata. These experiments suggest that a diffusible substance from the embryos facilitates fin morphogenesis and that test cells are not required. Test cells deposit ‘ornaments’ on the tunic in some species. In other species no ornaments are produced. Ten families are compared. It is proposed that the test cells make the tunic hydrophilic.