The oral sensory organs in Bathochordaeus stygius (Tunicata Appendicularia) are unique in structure and homologous to the coronal organ.

BACKGROUND: Appendicularia consists of approximately 70 purely marine species that belong to Tunicata the probable sister taxon to Craniota. Therefore, Appendicularia plays a pivotal role for our understanding of chordate evolution. In addition, appendicularians are an important part of the epipelagic marine plankton. Nevertheless, little is known about appendicularian species, especially from deeper water. RESULTS: Using µCT, scanning electron microscopy, and digital 3D-reconstruction techniques we describe three pairs of complex oral sensory organs in the mesopelagic appendicularian Bathochordaeus stygius. The oral sensory organs are situated at the anterior and lateral margin of the mouth and inside the mouth cavity. A single organ consists of 22-90 secondary receptor cells that project apical cilia through a narrow hole in the epidermis. The receptor cells are innervated by branches of the second brain nerve. CONCLUSIONS: Based on position, morphology, and innervation we suggest that the oral sensory organs are homologues of the coronal organs in other tunicates. We discuss the hypothesized homology of coronal organs and the lateral line system of primary aquatic vertebrates. The complex oral sensory organs of B. stygius are unique in tunicates and could be adaptations to the more muffled environment of the mesopelagic.

In vitro induction of Entamoeba gingivalis cyst-like structures from trophozoites in response to antibiotic treatment.

Background: Entamoeba gingivalis (E. gingivalis) is an anaerobic protozoan that is strongly associated with inflamed periodontal pockets. It is able to invade the mucosal epithelium of the human host, where it can feed on epithelial cells and elicit a severe innate immune response. Unlike other Entamoeba species, it is considered that E. gingivalis cannot form cysts, because it is a non-infectious protozoan. The lack of encystation capability would make it susceptible to periodontal treatment. However, it is not clear how the human host becomes infected with E. gingivalis trophozoites. We investigated the ability of E. gingivalis to encapsulate in response to an unfavorable environment in vitro. Methods: Different strains of E. gingivalis, isolated from inflamed periodontal pocket samples, were cultured for 8 days in the presence or absence of the antimicrobials amoxycillin and metronidazole. To reveal cyst formation, we investigated the morphology and ultrastructure of the amoeba by light, fluorescence, transmission and scanning electron microscopy. We also used the fluorescent dye calcofluor white M2R to demonstrate chitin present in the cyst wall. Results: We observed exocysts and an intra-cystic space separating the encapsulated trophozoite from the environment. Remarkably, cysts showed a smooth surface, polygonal edges and smaller size compared to free-living trophozoites. In addition, encapsulated trophozoites that detached from the cyst wall had a dense cytoplasma without phagocytic vesicles. The cyst walls consisted of chitin as in other Entamoba species. The encapsulated trophozoids were mononuclear after antibioticinduced encapsulation. Discussion: We conclude that E. gingivalis cyst formation has significant implications for dissemination and infection and may explain why established treatment approaches often fail to halt periodontal tissue destruction during periodontitis and peri-implantitis.

Evolutionary traces of miniaturization in a giant-Comparative anatomy of brain and brain nerves in Bathochordaeus stygius (Tunicata, Appendicularia).

Appendicularia comprises 70 marine, invertebrate, chordate species. Appendicularians play important ecological and evolutionary roles, yet their morphological disparity remains understudied. Most appendicularians are small, develop rapidly, and with a stereotyped cell lineage, leading to the hypothesis that Appendicularia derived progenetically from an ascidian-like ancestor. Here, we describe the detailed anatomy of the central nervous system of Bathochordaeus stygius, a giant appendicularian from the mesopelagic. We show that the brain consists of a forebrain with on average smaller and more uniform cells and a hindbrain, in which cell shapes and sizes vary to a greater extent. Cell count for the brain was 102. We demonstrate the presence of three paired brain nerves. Brain nerve 1 traces into the epidermis of the upper lip region and consists of several fibers with some supportive bulb cells in its course. Brain nerve 2 innervates oral sensory organs and brain nerve 3 innervates the ciliary ring of the gill slits and lateral epidermis. Brain nerve 3 is asymmetric, with the right nerve consisting of two neurites originating posterior to the left one that contains three neurites. Similarities and differences to the anatomy of the brain of the model species Oikopleura dioica are discussed. We interpret the small number of cells in the brain of B. stygius as an evolutionary trace of miniaturization and conclude that giant appendicularians evolved from a small, progenetic ancestor that secondarily increased in size within Appendicularia.

The filter-house of the larvacean Oikopleura dioica. A complex extracellular architecture: From fiber production to rudimentary state to inflated house.

While cellulose is the most abundant macromolecule in the biosphere, most animals are unable to produce cellulose with the exception of tunicates. Some tunicates have evolved the ability to secrete a complex house containing cellulosic fibers, yet little is known about the early stages of the house building process. Here, we investigate the rudimentary house of Oikopleura dioica for the first time using complementary light and electron microscopic techniques. In addition, we digitally modeled the arrangement of chambers, nets, and filters of the functional, expanded house in three dimensions based on life-video-imaging. Combining 3D-reconstructions based on serial histological semithin-sections, confocal laser scanning microscopy, transmission electron microscopy, scanning electron microscopy (SEM), and focused ion beam (FIB)-SEM, we were able to elucidate the arrangement of structural components, including cellulosic fibers, of the rudimentary house with a focus on the food concentration filter. We developed a model for the arrangement of folded structures in the house rudiment and show it is a precisely preformed structure with identifiable components intricately correlated with specific cells. Moreover, we demonstrate that structural details of the apical surfaces of Nasse cells provide the exact locations and shapes to produce the fibers of the house and interact among each other, with Giant Fol cells, and with the fibers to arrange them in the precise positions necessary for expansion of the house rudiment into the functional state. The presented data and hypotheses advance our knowledge about the interrelation of structure and function on different biological levels and prompt investigations into this astonishing biological object.

The Pericardial Body of Ciona intestinalis Contains Hemocytes and Degenerating Muscle Cells, But No Parasites.

PURPOSE: A ventral heart positioned posterior to the branchial basket and equipped with a pericardium is homologous in tunicates and their sister group, the craniates, yet the tunicate model organism Ciona intestinalis features a pericardial body, a structure peculiar to few ascidian species. Here, we set out to distinguish between two competing hypotheses regarding the function of the pericardial body found in the literature: (H1) The pericardial body performs a role in the removal of dysfunctional myocardial cells, and (H2) it is a specialized niche of the immune system involved in defense against parasites. METHODS: We used histological techniques, transmission electron microscopy, and PCR-based gene sequencing to investigate whether individual ascidians parasitized with apicomplexan protists show signs of infections within the pericardial body. RESULTS: In individuals of C. intestinalis from the German North Sea infested with apicomplexan protists, the pericardial body contains numerous myocardial cells in various stages of degeneration while no remnants of parasitic cells could be identified. CONCLUSION: Thus, we conclude that H2-the pericardial body is a specialized niche of the immune system involved in defense against parasites-can be refuted. Rather, our observations support H1, the hypothesis that the pericardial body performs a role in the removal of dysfunctional myocardial cells.

An apicoplast-resident folate transporter is essential for sporogony of malaria parasites.

Malaria parasites are fast replicating unicellular organisms and require substantial amounts of folate for DNA synthesis. Despite the central role of this critical co-factor for parasite survival, only little is known about intraparasitic folate trafficking in Plasmodium. Here, we report on the expression, subcellular localisation and function of the parasite's folate transporter 2 (FT2) during life cycle progression in the murine malaria parasite Plasmodium berghei. Using live fluorescence microscopy of genetically engineered parasites, we demonstrate that FT2 localises to the apicoplast. In invasive P. berghei stages, a fraction of FT2 is also observed at the apical end. Upon genetic disruption of FT2, blood and liver infection, gametocyte production and mosquito colonisation remain unaltered. But in the Anopheles vector, FT2-deficient parasites develop inflated oocysts with unusual pulp formation consisting of numerous single-membrane vesicles, which ultimately fuse to form large cavities. Ultrastructural analysis suggests that this defect reflects aberrant sporoblast formation caused by abnormal vesicular traffic. Complete sporogony in FT2-deficient oocysts is very rare, and mutant sporozoites fail to establish hepatocyte infection, resulting in a complete block of parasite transmission. Our findings reveal a previously unrecognised organellar folate transporter that exerts critical roles for pathogen maturation in the arthropod vector.

Common plant flavonoids prevent the assembly of amyloid curli fibres and can interfere with bacterial biofilm formation.

Like all macroorganisms, plants have to control bacterial biofilm formation on their surfaces. On the other hand, biofilms are highly tolerant against antimicrobial agents and other stresses. Consequently, biofilms are also involved in human chronic infectious diseases, which generates a strong demand for anti-biofilm agents. Therefore, we systematically explored major plant flavonoids as putative anti-biofilm agents using different types of biofilms produced by Gram-negative and Gram-positive bacteria. In Escherichia coli macrocolony biofilms, the flavone luteolin and the flavonols myricetin, morin and quercetin were found to strongly reduce the extracellular matrix. These agents directly inhibit the assembly of amyloid curli fibres by driving CsgA subunits into an off-pathway leading to SDS-insoluble oligomers. In addition, they can interfere with cellulose production by still unknown mechanisms. Submerged biofilm formation, however, is hardly affected. Moreover, the same flavonoids tend to stimulate macrocolony and submerged biofilm formation by Pseudomonas aeruginosa. For Bacillus subtilis, the flavonone naringenin and the chalcone phloretin were found to inhibit growth. Thus, plant flavonoids are not general anti-biofilm compounds but show species-specific effects. However, based on their strong and direct anti-amyloidogenic activities, distinct plant flavonoids may provide an attractive strategy to specifically combat amyloid-based biofilms of some relevant pathogens.

A lipocalin mediates unidirectional heme biomineralization in malaria parasites.

During blood-stage development, malaria parasites are challenged with the detoxification of enormous amounts of heme released during the proteolytic catabolism of erythrocytic hemoglobin. They tackle this problem by sequestering heme into bioinert crystals known as hemozoin. The mechanisms underlying this biomineralization process remain enigmatic. Here, we demonstrate that both rodent and human malaria parasite species secrete and internalize a lipocalin-like protein, PV5, to control heme crystallization. Transcriptional deregulation of PV5 in the rodent parasite Plasmodium berghei results in inordinate elongation of hemozoin crystals, while conditional PV5 inactivation in the human malaria agent Plasmodium falciparum causes excessive multidirectional crystal branching. Although hemoglobin processing remains unaffected, PV5-deficient parasites generate less hemozoin. Electron diffraction analysis indicates that despite the distinct changes in crystal morphology, neither the crystalline order nor unit cell of hemozoin are affected by impaired PV5 function. Deregulation of PV5 expression renders P. berghei hypersensitive to the antimalarial drugs artesunate, chloroquine, and atovaquone, resulting in accelerated parasite clearance following drug treatment in vivo. Together, our findings demonstrate the Plasmodium-tailored role of a lipocalin family member in hemozoin formation and underscore the heme biomineralization pathway as an attractive target for therapeutic exploitation.

Phylogenetic analysis of phenotypic characters of Tunicata supports basal Appendicularia and monophyletic Ascidiacea.

With approximately 3000 marine species, Tunicata represents the most disparate subtaxon of Chordata. Molecular phylogenetic studies support Tunicata as sister taxon to Craniota, rendering it pivotal to understanding craniate evolution. Although successively more molecular data have become available to resolve internal tunicate phylogenetic relationships, phenotypic data have not been utilized consistently. Herein these shortcomings are addressed by cladistically analyzing 117 phenotypic characters for 49 tunicate species comprising all higher tunicate taxa, and five craniate and cephalochordate outgroup species. In addition, a combined analysis of the phenotypic characters with 18S rDNA-sequence data is performed in 32 OTUs. The analysis of the combined data is congruent with published molecular analyses. Successively up-weighting phenotypic characters indicates that phenotypic data contribute disproportionally more to the resulting phylogenetic hypothesis. The strict consensus tree from the analysis of the phenotypic characters as well as the single most parsimonious tree found in the analysis of the combined dataset recover monophyletic Appendicularia as sister taxon to the remaining tunicate taxa. Thus, both datasets support the hypothesis that the last common ancestor of Tunicata was free-living and that ascidian sessility is a derived trait within Tunicata. "Thaliacea" is found to be paraphyletic with Pyrosomatida as sister taxon to monophyletic Ascidiacea and the relationship between Doliolida and Salpida is unresolved in the analysis of morphological characters; however, the analysis of the combined data reconstructs Thaliacea as monophyletic nested within paraphyletic "Ascidiacea". Therefore, both datasets differ in the interpretation of the evolution of the complex holoplanktonic life history of thaliacean taxa. According to the phenotypic data, this evolution occurred in the plankton, whereas from the combined dataset a secondary transition into the plankton from a sessile ascidian is inferred. Besides these major differences, both analyses are in accord on many phylogenetic groupings, although both phylogenetic reconstructions invoke a high degree of homoplasy. In conclusion, this study represents the first serious attempt to utilize the potential phylogenetic information present in phenotypic characters to elucidate the inter-relationships of this diverse marine taxon in a consistent cladistic framework.

Structure and ultrastructure of eyes and brains of Thalia democratica (Thaliacea, Tunicata, Chordata).

Salps are marine planktonic chordates that possess an obligatory alternation of reproductive modes in subsequent generations. Within tunicates, salps represent a derived life cycle and are of interest in considerations of the evolutionary origin of complex anatomical structures and life history strategies. In the present study, the eyes and brains of both the sexual, aggregate blastozooid and the asexual, solitary oozooid stage of Thalia democratica (Forskål, ) were digitally reconstructed in detail based on serial sectioning for light and transmission electron microscopy. The blastozooid stage of T. democratica possesses three pigment cup eyes, situated in the anterior ventral part of the brain. The eyes are arranged in a way that the optical axes of each eye point toward different directions. Each eye is an inverse eye that consists of two different cell types: pigment cells (pigc) and rhabdomeric photoreceptor cells (prcs). The oozooid stage of T. democratica is equipped with a single horseshoe-shaped eye, positioned in the anterior dorsal part of the brain. The opening of the horseshoe-shaped eye points anteriorly. Similar to the eyes of the blastozooid, the eye of the oozooid consists of pigment cells and rhabdomeric photoreceptor cells. The rhabdomeric photoreceptor cells possess apical microvilli that form a densely packed presumably photosensitive receptor part adjacent to the concave side of the pigc. We suggest correspondences of the individual eyes in the blastozooid stage to respective parts of the single horseshoe-shaped eye in the oozooid stage and hypothesize that the differences in visual structures and brain anatomies evolved as a result of the aggregate life style of the blastozooid as opposed to the solitary life style of the oozooid.

High-precision morphology: bifocal 4D-microscopy enables the comparison of detailed cell lineages of two chordate species separated for more than 525 million years.

BACKGROUND: Understanding the evolution of divergent developmental trajectories requires detailed comparisons of embryologies at appropriate levels. Cell lineages, the accurate visualization of cleavage patterns, tissue fate restrictions, and morphogenetic movements that occur during the development of individual embryos are currently available for few disparate animal taxa, encumbering evolutionarily meaningful comparisons. Tunicates, considered to be close relatives of vertebrates, are marine invertebrates whose fossil record dates back to 525 million years ago. Life-history strategies across this subphylum are radically different, and include biphasic ascidians with free swimming larvae and a sessile adult stage, and the holoplanktonic larvaceans. Despite considerable progress, notably on the molecular level, the exact extent of evolutionary conservation and innovation during embryology remain obscure. RESULTS: Here, using the innovative technique of bifocal 4D-microscopy, we demonstrate exactly which characteristics in the cell lineages of the ascidian Phallusia mammillata and the larvacean Oikopleura dioica were conserved and which were altered during evolution. Our accurate cell lineage trees in combination with detailed three-dimensional representations clearly identify conserved correspondence in relative cell position, cell identity, and fate restriction in several lines from all prospective larval tissues. At the same time, we precisely pinpoint differences observable at all levels of development. These differences comprise fate restrictions, tissue types, complex morphogenetic movement patterns, numerous cases of heterochronous acceleration in the larvacean embryo, and differences in bilateral symmetry. CONCLUSIONS: Our results demonstrate in extraordinary detail the multitude of developmental levels amenable to evolutionary innovation, including subtle changes in the timing of fate restrictions as well as dramatic alterations in complex morphogenetic movements. We anticipate that the precise spatial and temporal cell lineage data will moreover serve as a high-precision guide to devise experimental investigations of other levels, such as molecular interactions between cells or changes in gene expression underlying the documented structural evolutionary changes. Finally, the quantitative amount of digital high-precision morphological data will enable and necessitate software-based similarity assessments as the basis of homology hypotheses.

Neurogenesis in directly and indirectly developing enteropneusts: of nets and cords.

Concerning the evolution of deuterostomes, enteropneusts (acorn worms) occupy a pivotal role as they share some characteristics with chordates (e.g., tunicates and vertebrates) but are also closely related to echinoderms (e.g., sea urchin). The nervous system in particular can be a highly informative organ system for evolutionary inferences, and advances in fluorescent microscopy have revealed overwhelming data sets on neurogenesis in various clades. However, immunocytochemical descriptions of neurogenesis of juvenile enteropneusts are particularly scarce, impeding the reconstruction of nervous system evolution in this group. We followed morphogenesis of the nervous system in two enteropneust species, one with direct (Saccoglossus kowalevskii) and the other with indirect development (Balanoglossus misakiensis), using an antibody against serotonin and electron microscopy. We found that all serotonin-like immunoreactive (LIR) neurons in both species are bipolar ciliary neurons that are intercalated between other epidermal cells. Unlike the tornaria larva of B. misakiensis, the embryonic nervous system of S. kowalevskii lacks serotonin-LIR neurons in the apical region as well as an opisthotroch neurite ring. Comparative analysis of both species shows that the projections of the serotonin-LIR somata initially form a basiepidermal plexus throughout the body that disappears within the trunk region soon after settlement before the concentrated dorsal and ventral neurite bundles emerge. Our data reveal a highly conserved mode of neurogenesis in enteropneusts that is independent of the developing mode and is inferred to be a common feature for Enteropneusta. Moreover, all detected serotonin-LIR neurons are presumably receptor cells, and the absence of serotonin-LIR interneurons from the enteropneust nervous system, which are otherwise common in various bilaterian central nervous systems, is interpreted as a loss that might have occurred already in the last common ancestor of Ambulacraria.

Morphological Differences between Larvae of the Ciona intestinalis Species Complex: Hints for a Valid Taxonomic Definition of Distinct Species.

The cosmopolitan ascidian Ciona intestinalis is the most common model species of Tunicata, the sister-group of Vertebrata, and widely used in developmental biology, genomics and evolutionary studies. Recently, molecular studies suggested the presence of cryptic species hidden within the C. intestinalis species, namely C. intestinalis type A and type B. So far, no substantial morphological differences have been identified between individuals belonging to the two types. Here we present morphometric, immunohistochemical, and histological analyses, as well as 3-D reconstructions, of late larvae obtained by cross-fertilization experiments of molecularly determined type A and type B adults, sampled in different seasons and in four different localities. Our data point to quantitative and qualitative differences in the trunk shape of larvae belonging to the two types. In particular, type B larvae exhibit a longer pre-oral lobe, longer and relatively narrower total body length, and a shorter ocellus-tail distance than type A larvae. All these differences were found to be statistically significant in a Discriminant Analysis. Depending on the number of analyzed parameters, the obtained discriminant function was able to correctly classify > 93% of the larvae, with the remaining misclassified larvae attributable to the existence of intra-type seasonal variability. No larval differences were observed at the level of histology and immunohistochemical localization of peripheral sensory neurons. We conclude that type A and type B are two distinct species that can be distinguished on the basis of larval morphology and molecular data. Since the identified larval differences appear to be valid diagnostic characters, we suggest to raise both types to the rank of species and to assign them distinct names.

Cell lineages and fate maps in tunicates: conservation and modification.

Comparison of features of the cell lineages and fate maps of early embryos between related species is useful in inferring developmental mechanisms and amenable to evolutionary considerations. We present cleavage patterns, cell lineage trees, and fate maps of ascidian and appendicularian embryos side by side to facilitate comparison. This revealed a number of significant differences in cleavage patterns and cell lineage trees, whereas the fate maps were found to be conserved. This fate map similarity can be extended to vertebrates, thus representing the fate map characteristics of chordates. Cleavage patterns and cell lineages may have been modified during evolution without any drastic changes in fate maps. Selective pressures that constrain developmental mechanisms at early embryonic stages might not be so strong as long as embryos are still able to generate a chordate-type fate map. Aquatic chordates share similar fate maps and morphogenetic movements during gastrulation and neurulation, eventually developing into tadpole-shaped larvae. As swimming by tail beats, and not by cilia, is advantageous, selective pressure may maintain the basic elements of the tadpole shape. We also discuss the evolutionary origin of the vertebrate neural crest and the embryonic origin of the appendicularian heart to illustrate the usefulness of cell lineage data. From an evolutionary standpoint, cell lineages behave like other characteristics such as morphology or protein sequences. Both novel and primitive features are present in extant organisms, and it is of interest to identify the relative degree of evolutionary conservation as well as the level at which homology is inferred.

Larval anatomy of the pterobranch Cephalodiscus gracilis supports secondarily derived sessility concordant with molecular phylogenies.

Pterobranchs have been interpreted as "missing links" combining primitive invertebrate features with advanced vertebrate-like characteristics. The first detailed morphological description of an ontogenetic stage of a pterobranch, based on digital 3D-reconstruction at electron microscopic resolution, reveals a triploblastic animal with monociliated epithelia, an extensive coelomic cavity, a through gut with an asymmetrically developed gill slit but no signs of planktonic specializations, such as ciliated bands. Therefore, this crawling larva supports the hypothesis proposed in previous molecular phylogenetic studies that pterobranchs could be derived within enteropneusts rather than being "missing links".

A detailed description of the development of the hemichordate Saccoglossus kowalevskii using SEM, TEM, Histology and 3D-reconstructions.

INTRODUCTION: Traditionally, the origin of the third germ layer and its special formation of coelomic cavities by enterocoely is regarded to be an informative character in phylogenetic analyses. In early deuterostomes such as sea urchins, the mesoderm forms through a single evagination pinching off from the apical end of the archenteron which then gives off mesocoela and metacoela on each side. This echinoid-type coelom formation has conventionally been assumed to be ancestral for Deuterostomia. However, recent phylogenetic analyses show that Echinodermata hold a more derived position within Deuterostomia. In this regard a subgroup of Hemichordata, namely enteropneusts, seem to host promising candidates, because they are supposed to have retained many ancestral deuterostome features on the one hand, and furthermore share some characteristics with chordates on the other hand. In enteropneusts a wide range of different modes of coelom formation has been reported and in many cases authors of the original observations carefully detailed the limitations of their descriptions, while these doubts disappeared in subsequent reviews. In the present study, we investigated the development of all tissues in an enteropneust, Saccoglossus kowalevskii by using modern morphological techniques such as complete serial sectioning for LM and TEM, and 3D-reconstructions, in order to contribute new data to the elucidation of deuterostome evolution. RESULTS: Our data show that in the enteropneust S. kowalevskii all main coelomic cavities (single protocoel, paired mesocoela and metacoela) derive from the endoderm via enterocoely as separate evaginations, in contrast to the aforementioned echinoid-type. The anlagen of the first pair of gill slits emerge at the late kink stage (~96 h pf). From that time onwards, we documented a temporal left-first development of the gill slits and skeletal gill rods in S. kowalevskii until the 2 gill slit juvenile stage. CONCLUSIONS: The condition of coelom formation from separate evaginations is recapitulated in the larva of amphioxus and can be observed in crinoid echinoderms in a similar way. Therefore, coelom formation from separated pouches, rather than from a single apical pouch with eventual subdivision is suggested as the ancestral type of coelom formation for Deuterostomia. Left-right asymmetries are also present in echinoderms (rudiment formation), cephalochordates (larval development), tunicates (gut coiling) and vertebrates (visceral organs), and it is known from other studies applying molecular genetic analyses that genes such as nodal, lefty and pitx are involved during development. We discuss our findings in S. kowalevskii in the light of morphological as well as molecular genetic data.

Evolutionary diversification of secondary mechanoreceptor cells in tunicata.

BACKGROUND: Hair cells are vertebrate secondary sensory cells located in the ear and in the lateral line organ. Until recently, these cells were considered to be mechanoreceptors exclusively found in vertebrates that evolved within this group. Evidence of secondary mechanoreceptors in some tunicates, the proposed sister group of vertebrates, has recently led to the hypothesis that vertebrate and tunicate secondary sensory cells share a common origin. Secondary sensory cells were described in detail in two tunicate groups, ascidians and thaliaceans, in which they constitute an oral sensory structure called the coronal organ. Among thaliaceans, the organ is absent in salps and it has been hypothesised that this condition is due to a different feeding system adopted by this group of animals. No information is available as to whether a comparable structure exists in the third group of tunicates, the appendicularians, although different sensory structures are known to be present in these animals. RESULTS: We studied the detailed morphology of appendicularian oral mechanoreceptors. Using light and electron microscopy we could demonstrate that the mechanosensory organ called the circumoral ring is composed of secondary sensory cells. We described the ultrastructure of the circumoral organ in two appendicularian species, Oikopleura dioica and Oikopleura albicans, and thus taxonomically completed the data collection of tunicate secondary sensory cells. To understand the evolution of secondary sensory cells in tunicates, we performed a cladistic analysis using morphological data. We constructed a matrix consisting of 19 characters derived from detailed ultrastructural studies in 16 tunicate species and used a cephalochordate and three vertebrate species as outgroups. CONCLUSIONS: Our study clearly shows that the circumoral ring is the appendicularian homologue of the coronal organ of other tunicate taxa. The cladistic analysis enabled us to reconstruct the features of the putative ancestral hair cell in tunicates, represented by a simple monociliated cell. This cell successively differentiated into the current variety of oral mechanoreceptors in the various tunicate lineages. Finally, we demonstrated that the inferred evolutionary changes coincide with major transitions in the feeding strategies in each respective lineage.

Ascidia subterranea sp. nov. (Phlebobranchia: Ascidiidae), a new tunicate belonging to the A. sydneiensis Stimpson, 1855 group, found as burrow associate of Axiopsis serratifrons A. Milne-Edwards, 1873 (Decapoda: Axiidae) on Derawan Island, Indonesia.

A new tunicate, Ascidia subterranea sp. nov., was found in burrows of the axiid crustacean Axiopsis serratifrons on Derawan Island, Indonesia. It differs from other ascidians in its habitat as well as numerous morphological peculiarities which are described in detail. The shrimp Rostronia stylirostris Holthuis, 1952 was found inside A. subterranea sp. nov., and 4 species of bivalves, 3 species of polychaetes, 1 gastropod, 1 polyplacophoran and 1 sponge species were found as burrow associates besides the ascidian.

The calcineurin dependent transcription factor TacA is involved in development and the stress response of Dictyostelium discoideum.

Calcineurin is an important signalling protein in a plethora of Ca(2+)-regulated cellular processes. In contrast to what is known about the function of calcineurin in various organisms, information on calcineurin substrates is still limited. Here we describe the identification and characterisation of the transcription factor activated by calcineurin (TacA) in the model organism Dictyostelium discoideum. TacA is a putative zinc-finger transcription factor orthologue of yeast Crz1. In resting unstimulated cells the protein is located in the cytosol and translocates to the nucleus in a calcineurin-dependent manner after Ca(2+)-stimulation. Nuclear export of TacA is partially dependent on GskA, the Dictyostelium orthologue of mammalian GSK3. The expression of tacA is developmentally regulated with its kinetics roughly paralleling calcineurin regulation. Silencing of tacA via RNAi leads to developmental defects and dysregulation of developmentally regulated and Ca(2+)-regulated marker genes. Additionally, TacA is involved in the stress response of D. discoideum during development in a separate pathway to the well-known stress response in Dictyostelium via STATc. Finally we provide evidence that TacA is not only an orthologue of yeast Crz1 but also functionally related to mammalian NFAT.