Cellulose in the house of the appendicularianOikopleura rufescens

Summary By electron diffraction analysis, highly crystalline cellulose Iβ was found in the house (a special structure in which the tunicate lives) of the appendicularianOikopleura rufescens. Cellulose microfibrils 20 nm in width were observed in a random array or highly organized with rectangular spacing of 2 to 10 (im in the house. The bundled cellulose microfibrils formed in the inlet filters, which are highly ordered meshwork structures. This paper provides the first account of the existence of cellulose in the house of an appendicularian. Our findings showed that the house and tunic are homologous tissues among the tunicates, and that the common ancestor of the tunicates (ascidians, thaliaceans, and appendicularians) already possessed cellulose-biosynthetic ability.     

Anatomy of the trunk mesoderm in tunicates: homology considerations and phylogenetic interpretation

The tadpole stage of tunicates has played a pivotal role in understanding chordate evolution. While the organization of the mesoderm has been given high importance in comparative anatomical studies of Bilateria, this morphological character remains largely unexplored in tunicate tadpoles. For larvae of the phlebobranch ascidian Ciona intestinalis, the presence of two mesodermal pockets had been claimed, raising the possibility that paired coelomes are present in the larval ascidian. Using computer assisted 3D-reconstructions based on complete series of 1 μm-sections analyzed by light microscopy complemented by TEM-investigation of selected regions a comparative anatomical study of tadpole stages from four major tunicate clades, Aplousobranchiata, Phlebobranchiata, Stolidobranchiata, and Appendicularia is presented. In the aplousobranch Clavelina lepadiformis numerous mesodermal cells are found throughout the entire trunk plus the unpaired ventral rudiment of the pericardium. In the phlebobranch Ascidia interrupta, massive mesodermal components occur in the posterior trunk, whereas more anteriorly situated mesoderm consists of loose streaks of cells or isolated cells. This is also the case in the stolidobranch ascidians Herdmania momus and Styela plicata. In the stolidobranch Molgula occidentalis and the appendicularian Oikopleura dioica the anterior trunk is entirely devoid of mesodermal cells. TEM-investigation revealed that all mesodermal structures in the trunk of tunicate tadpoles were mesenchymal with the exception of a ventral portion of the mesoderm in C. lepadiformis, which probably corresponds to the developing pericardium, and the differentiated pericardium of the juvenile O. dioica. Thus no evidence for paired coelomic cavities in Tunicata was found. Outgroup comparison suggests that the reduction of paired coelomic cavities is an apomorphic trait of Tunicata. Within Tunicata a stepwise evolutionary reduction of the anterior larval mesenchyme is documented.     

Cleavage pattern,gastrulation, and neurulation in the appendicularian, <Emphasis Type="Italic">Oikopleura dioica</Emphasis>

The appendicularian, Oikopleura dioica is a chordate. Its life cycle is extremely short—approximately 5 days—and its tadpole shape with a beating tail is retained throughout entire life. The tadpole hatches after 3 h of development at 20°C. Here, we describe the cleavage pattern and morphogenetic cell movements during gastrulation and neurulation. Cleavage showed an invariant pattern. It is basically bilateral but also shows various minor left–right asymmetries starting from the four-cell stage. We observed two rounds of unequal cleavage of the posterior-vegetal B-line cells at the posterior pole. The nature of the unequal cleavages is reminiscent of those in ascidian embryos and suggests the presence of a centrosome-attracting body, a special subcellular structure at the posterior pole. The representation of the cell division pattern in this report will aid the identification of each cell, a prerequisite for clarifying the gene expression patterns in early embryos. Gastrulation started as early as the 32-cell stage and progressed in three phases. By the end of the second phase at the 64-cell stage, every vegetal cell had ingressed into the embryo, and animal cells had covered the entire embryo by epiboly. There was no archenteron formation. In the anterior region, eight A-line cells were aligned as a 2 × 4 array along the anterior–posterior axis and become internalized during the 64-cell stage. This process was considered to correspond to neurulation. The simple and accelerated development of Oikopleura, nevertheless giving rise to a conserved chordate body plan, is advantageous for studying developmental mechanisms using molecular and genetic approaches and makes this animal the simplest model organism in the phylum Chordata. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Fertilization in Oikopleura dioica (Tunicata,Appendicularia): Acrosome reaction,cortical reaction and sperm-egg fusion

Summary Fine structural changes in the egg and sperm are described during gamete interaction in Oikopleura dioica, an appendicularian tunicate. The unfertilized egg has a vitelline layer 80 nm thick and a perivitelline space about 5 m wide. In the peripheral cytoplasm are a few cortical granules 0.6×0.7 m in diameter and areas rich in parallel cisternae of rough endoplasmic reticulum alternating with areas rich in long mitochondria. In the deeper cytoplasm the predominant organelles are multivesicular bodies. From 25 s to 60 s after insemination, the egg transiently elongates, although with no obvious cytoplasmic rearrangement, and the egg surface becomes bumpy. During this interval sperm enter the egg, and the cortical granules undergo exocytosis. After expulsion into the perivitelline space, the cortical granule contents do not appear to change their shape or blend with the vitelline layer, which neither elevates further nor loses its ability to bind sperm. On encountering the egg, the sperm undergoes an acrosome reaction involving exocytosis of the acrosome and production of an acrosomal tubule. The acrosomal contents bind the sperm to the vitelline layer, and the posterior portion of the acrosomal membrane and the anterior portion of the nuclear envelope evaginate together to form an acrosomal tubule, which fuses with the egg plasma membrane to form a fertilization cone. By 45 s after insemination, the sperm nucleus, centriole, mitochondrion and at least the anterior portion of the axoneme are within the fertilization cone. By 60 s sperm entry is complete. In having eggs with a cortical reaction and sperm with an acrosome reaction, O. dioica resembles echinoderms and enteropneusts and differs markedly from ascidian tunicates, which lack both these features. The relatively unmodified pattern of gamete interaction in O. dioica in comparison with the highly modified pattern in ascidians is difficult to reconcile with the neoteny theory that appendicularians have evolved via ascidian ancestors. The present results are more consistent with the idea that an appendicularian-like ancestor gave rise to ascidians.     

Comparison of the serotonergic nervous system among Tunicata: implications for its evolution within Chordata

Using immunohistochemistry in combination with confocal laser scanning microscopy, the serotonergic nervous systems of major tunicate taxa were studied in three-dimensional detail. Organisms analyzed included aplousobranchiate, phlebobranchiate, and stolidobranchiate ascidian larvae, appendicularian juveniles and adults, and doliolid oozooids. Outgroup comparisons to notochordates showed that the serotonergic nervous system of the last common ancestor of Chordata consisted of two elements: (i) an anterior concentration of serotonergic cell bodies, and (ii) a fiber network that followed posteriorly and gave rise to fiber tracts that descended towards the effective somatic lateral musculature. Within Tunicata, the nervous systems of Appendicularia and Aplousobranchiata appear serotonin-reduced or negative. This could be interpreted as a heterochronic reduction and a synapomorphy between Appendicularia and Aplousobranchiata. In this hypothesis, free-living Appendicularia are derived within Tunicata, and a biphasic life cycle with a free-swimming larva and a sessile, ascidian-like adult is most parsimoniously reconstructed for the last common ancestor of Tunicata. The close spatial relation of the serotonergic cell cluster with the statocyte complex suggests a function as an integrative control center for the coordination of locomotion. A similar anterior concentration of serotonergic nerve cells is known from tornaria larvae.See also Electronic Supplement at: http://www.senckenberg.de/odes/05-02.htm     

Primitive Coronet Cells in the Brain of Oikopleura (Appendicularia,Tunicata)

Abstract A peculiar cell type is described from the sensory vesicle of the brain of the appendicularian tunicate Oikopleura dioica. The cells carry globular, modified cilia and resemble in several other respects the coronet cells of Saccus vasculosus in many fishes. This finding, together with earlier reports of similar cells in the sensory vesicle of ascidian tadpoles, makes it probable that a primitive form of coronet cells is a regular constituent of the tunicate brain cavity. The existence of coronet cells in the tunicate line of evolution is of interest from a phylogenetical as well as functional point of view. The author proposes that the tunicate sensory vesicle corresponds to part of the craniate third ventricle and that the tunicate coronet cells are involved in the regulation of the ventricle fluid composition.     

Continued growth and cell proliferation into adulthood in the notochord of the appendicularian Oikopleura dioica

The appendicularian urochordate Oikopleura dioica retains a free-swimming chordate body plan throughout life, in contrast to ascidian urochordates, whose metamorphosis to a sessile adult form involves the loss of chordate structures such as the notochord and dorsal nerve cord. Development to adult stages in Oikopleura involves a lengthening of the tail and notochord and an elaboration of the repertoire of tail movements. To investigate the cellular basis for this lengthening, we have used confocal microscopy and BrdU labeling to examine the development of the Oikopleura notochord from hatching through adult stages. We show that as the notochord undergoes the typical urochordate transition from a stacked row of cells to a tubular structure, cell number begins to increase. Addition of new notochord cells continues into adulthood, multiplying the larval complement of 20 cells by about 8-fold by the third day of life. In parallel, the notochord lengthens by about 4-fold. BrdU incorporation and a cell-cycle marker confirm that notochord cells continue to proliferate well into adulthood. The extensive postlarval proliferation of notochord cells, together with their arrangement in four circumferentially distributed longitudinal rows, presumably provides the Oikopleura tail with the necessary mechanical support for the complex movements exhibited at adult stages.     

Larval convergence in a colonial tunicate: the organization of the sarcotubular complex in <Emphasis Type="Italic">Ecteinascidia turbinata</Emphasis> (Perophoridae,Phlebobranchiata, Tunicata,Chordata)

Ecteinascidia turbinata is a colonial ascidian that as an adult shares characters with phlebobranch ascidians, whereas the larvae are similar to aplousobranch ascidian larvae. The sarcotubular complex consists of invaginations of the sarcolemma that contact the sarcoplasmatic reticulum via dyads or triads. If present, the invaginations of the sarcolemma in tunicates have been characterized as laminar or tubular. We comparatively investigated the sarcotubular complex of E. turbinata and seven other tunicate species using 3D-reconstruction techniques based on electron micrographs of serial sections. The mononucleate muscle cells in E. turbinata possess intermediate and close junctions and contain several layers of peripheral myofibrillae. The myofibrillae are surrounded by continuous cisternae of the sarcoplasmic reticulum that forms interconnected rings around the z-bands. The invaginations of the sarcolemma are laminar, contacting the sarcoplasamatic reticulum at the height of the z-bands via dyads and triads. We present a clear definition of character states encountered in Tunicata: laminar invaginations are characterized by a width to length ratio of smaller than 1:20, tubular invagination by a width to length ratio of larger than 1:10. Laminar invaginations are found in stolidobranch ascidians and E. turbinata. Tubular invaginations are present in aplousobranch ascidians and appendicularians. This character state distribution across taxa supports the hypothesis that E. turbinata should be included in Phlebobranchiata as suggested by adult characters and that the larval similarities with Aplousobranchiata arose by convergent evolution. An erratum to this article can be found at     

The haemocytes of the salp Thalia democratica (Tunicata,Thaliacea): an ultrastructural and histochemical study in the oozoid

In comparative immunology and evolution of the chordate immune system, tunicates hold an important phylogenetic position as sister group of vertebrates. However, knowledge of the tunicate immune system is limited to the class Ascidiacea, in which some species are now considered model organisms. In the class Thaliacea, represented by fragile pelagic species, the few studies on their haemocytes go back to several decades ago and do not consider comparative aspects with ascidian haemocytes. In this study, we identified various haemocyte types and their distribution in the common salp Thalia democratica by comparative observations under light and electron microscopy and by histochemical, histoenzymatic and immunohistochemical techniques. By comparing specialisations with those of ascidian haemocytes, we detected an undifferentiated cell type (lymphocyte‐like cell) and three categories with four cell types, that is, (i) phagocytic line (hyaline amoebocyte and amoebocyte with large vacuoles), (ii) mast cell‐like line (granular cell) and (iii) storage cells (nephrocyte). Both phagocytes and granular cells appear to migrate in the tunic. Phagocytes adhere to the tunic which internally covers the oral siphon, where they probably function as sentinel cells of the pharynx. Results show the variety of haemolymph cells in the salp similar to phlebobranch ascidians.     

Long-distance cell migration during larval development in the appendicularian, Oikopleura dioica

The appendicularian, Oikopleura dioica, is a planktonic chordate. Its simple and transparent body, invariant cell lineages and short life cycle of 5 days make it a promising model organism for studies of chordate development. Here we describe the cell migration that occurs during development of the O. dioica larva. Using time-lapse imaging facilitated by florescent labeling of cells, three cell populations exhibiting long-distance migration were identified and characterized. These included (i) a multinucleated oral gland precursor that migrates anteriorly within the trunk region and eventually separates into the left and right sides, (ii) endodermal strand cells that are collectively retracted from the tail into the trunk in a tractor movement, and (iii) two subchordal cell precursors that individually migrate out from the trunk to the tip of the tail. The migration of subchordal cell precursors starts when all of the endodermal strand cells enter the trunk, and follows the same path but in a direction opposite to that of the latter. Labeling of these cells with a photoconvertible fluorescent protein, Kaede, demonstrated that the endodermal strand cells and subchordal cell precursors have distinct origins and eventual fates. Surgical removal of the trunk from the tail demonstrated that the endodermal strand cells do not require the trunk for migration, and that the subchordal cell precursors would be attracted by the distal part of the tail. This well-defined, invariant and traceable long-distance cell migration provides a unique experimental system for exploring the mechanisms of versatile cell migration in this simple organism with a chordate body plan.     

Appendicularians: an important food supply for the Argentine anchovy Engraulis anchoita in coastal waters

The stomach contents of 532 adult specimens of the Argentine anchovy, Engraulis anchoita, caught in coastal waters of the Argentine Sea from 1994 to 1996, were analyzed. Larger amounts of food were found in the stomachs of anchovies collected in the northern surveys (35–40°S), with the highest values attained in 1994. Main diet items were copepods, appendicularians, cladocerans, fish eggs, and pteropods. Anchovies caught in the southern surveys (40–45°S) ingested less food, whereby the main items were copepods and appendicularians. Plankton samples collected simultaneously with the fishing trawls were also analyzed. Except for Fritillaria borealis, which occurred only in plankton samples, the same appendicularian species (Oikopleura dioica and O. fusiformis) were found in both stomach contents and in plankton samples. The Ivlev selectivity index calculated for O. dioica and O. fusiformis and for the different maturity stages of both species were in all cases ～0, supporting the hypothesis of a non‐selective feeding.     

The evolving proteome of a complex extracellular matrix, the Oikopleura house

Extracellular matrices regulate biological processes at the level of cells, tissues, and in some cases, entire multicellular organisms. The subphylum Urochordata exemplifies the latter case, where animals are partially or completely enclosed in "houses" or "tunics". Despite this common strategy, we show that the house proteome of the appendicularian, Oikopleura, has very little in common with the proteome of the sister class, ascidian, Ciona. Of 80 identified house proteins (oikosins), ～half lack domain modules or similarity to known proteins, suggesting de novo appearance in appendicularians. Gene duplication has been important in generating almost 1/3 of the current oikosin complement, with serial duplications up to 8 paralogs in one family. Expression pattern analyses revealed that individual oikosins are produced from specific fields of cells within the secretory epithelium, but in some cases, migrate up to at least 20 cell diameters in extracellular space to combine in defined house structures. Interestingly, peroxidasin and secretory phospholipase A(2) domains, implicated in innate immune defence are secreted from the anlage associated with the food-concentrating filter, suggesting that this extra-organismal structure may play, in part, such a role in Oikopleura. We also show that sulfation of proteoglycans is required for the hydration and inflation of pre-house rudiments into functional houses. Though correct proportioning in the production of oikosins would seem important in repetitive assembly of the complex house structure, the genomic organization of oikosin loci appears incompatible with common enhancers or locus control regions exerting such a coordinate regulatory role. Thus, though all tunicates employ extracellular matrices based on a cellulose scaffold as a defining feature of the subphylum, they have evolved radically different protein compositions associated with this common underlying structural theme.     

The house of Oikopleura dioica (Tunicata,Appendicularia): Structure and functions

Summary Using appropriate techniques, we have studied in detail the structure and functions of the house of the appendicularian Oikopleura dioica Fol, 1872. In particular, we describe the structure of the inlet funnel, the reinforcing chamber on each side of the tail chamber and its function, the role and the structure of the valves located between the dorsal and water exit chambers and the structure and the role of the exit sphincter. In conclusion, we show that in addition to filtration of particles, movement of the tail also helps location of a favorable particulate environment in the sea.     

Behavior and cellular morphology of the test cells during embryogenesis of the ascidian Halocynthia roretzi

During the early stages of embryogenesis of the ascidian Halocynthia roretzi the test cells creep exclusively on the inner surface of the chorion. Concomitant with elongation of the embryonic tail, however, the test cells begin to gather around the embryo and finally cover the whole embryo. The time at which the test cells surround the embryo almost coincides with that of initiation of larval tunic formation. Scanning electron microscope observations revealed that the test cells extend numerous cytoplasmic processes or pseudopodia. During larval tunic formation, the test cells compose a net by intertwining their filopodia, and the cell net covers the whole embryo.