Colocalization of heparin and histamine in the intracellular granules of test cells from the invertebrate Styela plicata (Chordata-Tunicata)

In most ascidian species the oocytes are surrounded by two types of accessory cells called follicle cells and test cells. Test cells are located on the periphery of oocytes and remain in the perivitelline space during egg development until hatching. Heparin and histamine were previously described in the test cells of the ascidian Styela plicata. In the present study, electron microscopy techniques were used to characterize the ultrastructure of the S. plicata test cells and to localize heparin and histamine in these cells. Test cells contain several intracellular granules with unique ultrastructural features. They are formed by elongated filaments composed of serial globules with an electron-lucent circle, containing a central electron-dense spot. Immunocytochemistry showed that heparin and histamine colocalize at the border of granule filaments in the test cell. Compound 48/80, a potent secretagogue of heparin-containing mast cells, also induced degranulation of test cells. According to these results, we suggest that test cells represent ancient effector cells of the innate immunity in primitive chordates.     

The identification and localization of two intermediate filament proteins in the tunic of Styela plicata (Tunicata, Styelidae)

The intermediate filament (IF) proteins Styela C and Styela D from the tunicate Styela (Urochordata) are co-expressed in all epidermal cells and they are thought to behave as type I and type II keratins. These two IF proteins, Styela C and Styela D, were identified in immunoblots of proteins isolated from the tunic of Styela plicata. The occurrence and distribution of these proteins within the tunic of this ascidian was examined by means of immunofluorescence and immunoperoxidase techniques, using anti-Styela C and anti-Styela D antibodies. In addition, immuno-electron microscopy of the tunic showed that the two proteins are located in the cuticle layer and in the tunic matrix. These results represent the first data about the presence of IF proteins in the tunic of adult ascidian S. plicata. The possible involvement of these IF proteins in reinforcing the integrity of the tunic, that represents the interface between the animal body and the external environment, is discussed.     

Occurrence of different secretin-like cells in the digestive tract of the ascidian Styela plicata (Urochordata,Ascidiacea)

Summary Secretin-like cells have been detected in the digestive tract of the ascidian Styela plicata by means of immunofluorescent and immunocytochemical methods.Especially, in the esophageal epithelium there are immunoreactive cells (S₂) in which a biogenic amine (5-HT) and a regulatory peptide (secretin) occur together. In the gastric epithelium only secretin-like cells (S₁) are present.Tests of cross-reactivity performed with glucagon, GIF and VIP, have confirmed the presence of a secretin-like molecule only in the S₁ and S₂ cells.     

Metabolite profiling of ascidian Styela plicata using LC–MS with multivariate statistical analysis and their antitumor activity

To identify the metabolite distribution in ascidian, we have applied an integrated liquid chromatography– tandem mass spectrometry (LC–MS) metabolomics approach to explore and identify patterns in chemical diversity of invasive ascidian Styela plicata. A total of 71 metabolites were reported among these alkaloids, fatty acids and lipids are the most dominant chemical group. Multivariate statistical analysis, principal component analysis (PCA) showed a clear separation according to chemical diversity and taxonomic groups. PCA and partial least square discriminant analysis were applied to discriminate the chemical group of S. plicata crude compounds and classify the compounds with unknown biological activities. In this study, we reported for the first time that a partially purified methanol extract prepared from the ascidian S. plicata and Ascidia mentula possess antitumor activity against four tumor cell lines with different tumor histotype, such as HeLa (cervical carcinoma), HT29 (colon carcinoma), MCF-7 (breast carcinoma) and M14 (melanoma). S. plicata fraction SP-50 showed strong inhibition of cell proliferation and induced apoptosis in HeLa and HT29 cells, thus indicating S. plicata fraction SP-50 a potential lead compound for anticancer therapy. The molecular mechanism of action and chemotherapeutic potential of these ascidian unknown biomolecules need further research.     

Regulation of the fertilization current in ascidian oocytes by intracellular second messengers

Neomycin, injected into ascidian oocytes to a final concentration of 10–50 mM, inhibits both the fertilization current and the surface contraction, showing that phosphoinositide hydrolysis is required for these early activation events. Sperm-activated fertilization currents are not inhibited in the presence of 100 μg/ml intracellular heparin, suggesting that these currents are not directly gated by InsP₃. The sulfhydryl reagent thimerosal at 100 μM, in contrast, significantly increases the fertilization current presumably by sensitizing the channel receptor. Since heparin inhibits the surface contraction, InsP₃ receptors are shown to play a role in the propagation of the activation response in ascidian oocyte. Depleting intracellular calcium stores by microinjecting 50 mM EGTA into oocytes does not activate fertilization channels; however, subsequent fertilization of these EGTA loaded oocytes leads to a significantly larger and faster fertilization current. Thus in contrast to somatic cells studied to date, second messenger operated plasma membrane channels in ascidian oocytes are not gated by calcium released from intracellular stores. © 1994 Wiley-Liss, Inc.     

The morphology of allograft rejection in Styela plicata (Urochordata: Ascidiacea)

Summary This study identifies three discrete processes responsible for the rejection of tunic tissue transplanted between individuals of the solitary ascidian Styela plicata. The first stage of rejection is characterized by the destruction of blood vascular components within incompatible allografts. In the second phase, dense boundaries of extracellular material are deposited between grafts and the surrounding host tunic, effectively amputating the transplanted tissues. Finally, detached transplants undergo a gradual necrosis which results in the total degeneration of extracellular graft matrices. Of these three phases, the initial cellular depletion of allografts is responsible for the immunological specificity that is characteristic of histocompatibility in S. plicata. The subsequent amputation and necrosis of extracellular graft matrices are taken to be non-specific consequences of the initial cellular reactivity.     

A comparative study of the organization of the sarcotubular system in ascidian muscle

The caudal musculature of ascidian tadpole larvae consists of mononucleated muscle cells joined end to end in long rows flanking the notochord. A comparative study of the fine structure of these cells in larvae from different families has revealed wide variations in the pattern of organization of the sarcotubular system. The species examined can be distinguished in two groups according to the presence or absence of a system of plasma membrane invaginations equivalent to the T system of vertebrate and invertebrate striated muscle. Muscle cells from the first group of species, Clavelina lepadiformis, Ciona intestinalis and Molgula socialis, are characterized by absence of T system and show peripheral couplings of sarcoplasmic reticulum cisternae directly with the plasma membrane. In contrast, a T system is present in muscle cells of Diplosoma listerianum, Styela plicata and Botrylloides leachi. The presence of T system in ascidian muscle is not related to the taxonomic position of the various species, but rather to the intracellular disposition of the myofibrils, which are peripheral in the species of the first group whereas they occupy a more internal position in the species of the second group. The T system displays unique structural features in ascidian muscle. It consists of wide laminae invaginating from the plasma membrane and associated in longitudinally oriented dyads with sarcoplasmic reticulum cisternae in register with the I band of the myofibrils. It is apparent from these observations that, in contrast with the uniformity of myofibrillar structure in all chordates, there are basic differences between ascidians and vertebrates as regards the organization of the sarcotubular system. On the other hand, there are significant similarities in this respect between ascidian and invertebrate muscle.     

Localization of α-MSH-like immunoreactive cells in the neural gland of the ascidian Styela plicata

Summary An -MSH-like immunoreactivity has been localized in the neural gland of the ascidian Styela plicata. In particular, immunoreactive cells occur in some lobules and are weakly lead-haematoxylin positive.On the basis of the results, the homology of the ascidian neural gland with the vertebrate adenohyphysis is suggested and discussed. Furthermore, some hypotheses are presented about the possible functions of -MSH-like material in ascidians.This work was supported by a grant from the Ministero della Pubblica Istruzione (60%)     

Small core communities and high variability in bacteria associated with the introduced ascidian Styela plicata

The solitary ascidian Styela plicata is an introduced species in harbors of temperate and tropical oceans around the world. The invasive potential of this species has been studied through reproductive biology and population genetics but no study has yet examined the microbial diversity associated with this ascidian and its potential role in host ecology and invasiveness. Here, we used 16S rRNA gene tag pyrosequencing and transmission electron microscopy to characterize the abundance, diversity and host-specificity of bacteria associated with 3 Mediterranean individuals of S. plicata. Microscopy revealed low bacterial abundance in the inner tunic and their absence from gonad tissues, while pyrosequencing revealed a high diversity of S. plicata-associated bacteria (284 OTUs from 16 microbial phyla) in the inner tunic. The core symbiont community was small and consisted of 16 OTUs present in all S. plicata hosts. This core community included a recently described ascidian symbiont (Hasllibacter halocynthiae) and several known sponge and coral symbionts, including a strictly anaerobic Chloroflexi lineage. Most recovered bacterial OTUs (79.6 %) were present in single S. plicata individuals and statistical analyses of genetic diversity and community structure confirmed high variability of bacterial communities among host individuals. These results suggest that diverse and variable bacterial communities inhabit the tunic of S. plicata, including environmental and host-associated bacterial lineages that appear to be re-established each host generation. We hypothesize that bacterial communities in S. plicata are dynamic and have the potential to aid host acclimation to new habitats by establishing relationships with beneficial, locally sourced bacteria.     

Renewal of the gonads in Styela clava (Urochordata: Ascidiacea) as revealed by autoradiography with tritiated thymidine

DNA-synthesizing cells in the gonads of the ascidian Styela clava were labeled with tritiated thymidine and detected with autoradiography. In the testis, spermatogonia and primary spermatocytes are labeled after 1 hr. Labeled spermatozoa occur in the lumen of the testis follicles after 10 days and in the sperm ducts after 20 days. In the ovary, only germ cells (oogonia and pre-leptotene primary oocytes) and follicle cells are labeled after 1 hr. By 60 days, oocytes with basophilic cytoplasm (15–65 μ in diameter) are labeled; test cells embedded in larger eosinophilic oocytes (150 μ in diameter) are also labeled. Germ cells give rise to both oocytes and follicle cells. Through continued cell division, follicle cells give rise to test cells.     

Anatomy of the ovaries of the starfish Asterias rubens (Echinodermata)

Summary The ovaries of the starfish Asterias rubens were studied histologically and ultrastructurally. The reproductive system in female specimens consists of ten separate ovaries, two in each ray. Each ovary is made up of a rachis with lateral primary and secondary folds: the acini maiores and acini minores. The ovarian wall is composed of an outer and an inner part, separated by the genital coelomic sinus. The ovarian lumen contains oocytes in various phases of oogenesis, follicle cells, nurse cells, phagocytosing cells and steroid-synthesizing cells.Oogenesis is divided into four phases: (i) multiplication phase of oogonia, (ii) initial growth phase of oocytes I, (iii) growth phase proper of oocytes I, and (iv) post-growth phase of oocytes I. The granular endoplasmic reticulum and the Golgi complex of the oocytes appear to be involved in yolk formation, while the haemal system, haemal fluid and nurse cells may also be important for vitellogenesis. The haemal system is discussed as most likely being involved in synchronizing the development of the ovaries during the annual reproductive cycle and in inducing, stimulating and regulating the function of the ovaries.Steroid-synthesizing cells are present during vitellogenesis; a correlation between the presence of these cells and vitellogenesis is discussed.     

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.     

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.     

Evolution of the chordate muscle actin gene

Summary The ascidians Styela plicata, S. clava, and Mogula citrina are urochordates. The larvae of urochordates are considered to morphologically resemble the ancestral vertebrate. We asked whether larval and adult ascidian muscle actin sequences are nonmusclelike as in lower invertebrates, musclelike as in vertebrates, or possess characteristics of both. Nonmuscle and muscle actin cDNA clones from S. plicata were sequenced. Based on 27 diagnostic amino acids, which distinguish vertebrate muscle actin from other actins, we found that the deduced protein sequences of ascidian muscle actins exhibit similarities to both invertebrate and vertebrate muscle actins. A comparison to muscle actins from different vertebrate and invertebrate phylogenetic groups suggested that the urochordate muscle actins represent a transition from a nonmusclelike sequence to a vertebrate musclelike sequence. The ascidian adult muscle actin is more similar to skeletal actin and the larval muscle actin is more similar to cardiac actin, which indicates that the divergence of the skeletal and cardiac isoforms occurred before the emergence of urochordates. The muscle actin gene may be a powerful probe for investigating the chordate lineage. Offprint requests to: C.R. Tomlinson     

Ultrastructural and histochemical study of anural development in the ascidian Molgula pacifica (Huntsman)

Summary Tadpole development is eliminated in the life cycle of the ascidian Molgula pacifica. The elimination of a tailed larva is termed anural development, in contrast to urodele development which is exhibited by most ascidian species. In the present study, transmission electron microscopy and histochemistry were used to gain a better understanding of anural development in M. pacifica. The fine structure of M. pacifica oocytes and fertilized eggs was similar to urodele oocytes and eggs, except that a perivitelline space and test cells were absent. M. pacifica embryos exhibited the typical cleavage pattern of urodele embryos. Gastrulation was initiated at the vegetal pole, as in urodeles, and occurred at the same time as in two urodele species (Molgula manhattensis and Pyura haustor). However, changes in cell shapes and cell movements of the vegetal pole cells that participate in gastrulation were highly modified compared to commonly studied ascidians. The changes in shapes and movements of the vegetal pole cells were minimal and resulted in embryos having a very small archenteron and blastopore. The presence of large, yolky cells in the interior of the embryo likely restricted vegetal cell movements. Two ultrastructurally distinct types of epidermal cells were evident at the gastrula stage. When gastrulae were manually dechorionated from their surrounding mucous-follicular envelope layers, the embryos were already surrounded by a thin tunic. When day 1 juveniles in the process of hatching were sectioned along the anterior-posterior axis, regional differences in cell types were evident. Differentiated muscle cells in the posterior region were not evident. Day 1 M. pacifica juveniles, anural-developing M. provisionalis juveniles and tadpoles from three urodele species were tested for their abilities to express AchE activity. The highest levels of AchE activity were detected in the larval tail muscle cells of urodeles, low levels of activity were detected in the posterior region of M. provisionalis juveniles, whereas M. pacifica juveniles did not exhibit AchE activity. The results are discussed in terms of evolutionary mechanisms responsible for anural development in ascidians. Offprint requests to: W.R. Bates     

3‐acetylpyridine‐induced degeneration in the adult ascidian neural complex: Reactive and regenerative changes in glia and blood cells

Ascidians are interesting neurobiological models because of their evolutionary position as a sister‐group of vertebrates and the high regenerative capacity of their central nervous system (CNS). We investigated the degeneration and regeneration of the cerebral ganglion complex of the ascidian Styela plicata following injection of the niacinamide antagonist 3‐acetylpyridine (3AP), described as targeting the CNS of several vertebrates. For the analysis and establishment of a new model in ascidians, the ganglion complex was dissected and prepared for transmission electron microscopy (TEM), routine light microscopy (LM), immunohistochemistry and Western blotting, 1 or 10 days after injection of 3AP. The siphon stimulation test (SST) was used to quantify the functional response. One day after the injection of 3AP, CNS degeneration and recruitment of a non‐neural cell type to the site of injury was observed by both TEM and LM. Furthermore, weaker immunohistochemical reactions for astrocytic glial fibrillary acidic protein (GFAP) and neuronal βIII‐tubulin were observed. In contrast, the expression of caspase‐3, a protein involved in the apoptotic pathway, and the glycoprotein CD34, a marker for hematopoietic stem cells, increased. Ten days after the injection of 3AP, the expression of markers tended toward the original condition. The SST revealed attenuation and subsequent recovery of the reflexes from 1 to 10 days after 3AP. Therefore, we have developed a new method to study ascidian neural degeneration and regeneration, and identified the decreased expression of GFAP and recruitment of blood stem cells to the damaged ganglion as reasons for the success of neuroregeneration in ascidians. © 2014 Wiley Periodicals, Inc. Develop Neurobiol 75: 877–893, 2015     

MORPHOLOGY AND DEVELOPMENT OF AHNFELTIA PLICATA (RHODOPHYTA): PROPOSAL OF AHNFELTIALES ORD. NOV.1

Ahnfeltia plicata (Hudson) Fries, the type species of Ahnfeltia Fries, is currently assigned to the Phyllophoraceae (Gigartinales). Several morphological and biochemical characters distance A. plicata from the Phyllophoraceae but, because sexual reproduction has never been demonstrated, an alternative placement has not been possible. A. plicata now is shown to have a heteromorphic sexual life history. Erect branched gametophytes are dioecious. In male sori, spermatangia are cut off transversely from spermatangial mother cells. Female sori form numerous terminal sessile carpogonia. Following fertilization, several zygotes in each sorus fuse facultatively with undifferentiated intercalary cells of the female sorus and cut off gonimoblast initials obliquely outwards. These initials give rise to branching gonimoblast filaments that fuse with apical and intercalary female sorus cells and with each other, then grow radially outward in the compound external carposporophyte and terminate in carposporangia. Carpospores develop in culture into crustose tetrasporophytes identical to Porphyrodiscus simulans Batters. Field-collected P. simulans tetraspores grew into erect A. plicata axes. Tetrasporangia are formed by division and enlargement of crust apical cells followed by sequential enlargement and maturation of tetrasporocytes in an erosive process. Monosporangia are formed in sori on male gametophytes. Pit plugs of both gametophyte and tetrasporophyte phases consist of naked plug cores without cap layers of membranes. Gametophytes exhibit both cell fusions and secondary pit connections whereas tetrasporophytes form cell fusions but lack secondary pit connections. On the basis of the unique female and postfertilization reproductive development and in conjunction with the pit plug structure which is unique among florideophytes, the order Ahnfeltiales, containing the family Ahnfeltiaceae, is proposed.     

Host cell binding of the flagellar tip protein of Campylobacter jejuni

Flagella are nanofibers that drive bacterial movement. The filaments are generally composed of thousands of tightly packed flagellin subunits with a terminal cap protein, named FliD. Here, we report that the FliD protein of the bacterial pathogen Campylobacter jejuni binds to host cells. Live‐cell imaging and confocal microscopy showed initial contact of the bacteria with epithelial cells via the flagella tip. Recombinant FliD protein bound to the surface of intestinal epithelial cells in a dose‐dependent fashion. Search for the FliD binding site on the host cell using cells with defined glycosylation defects indicated glycosaminoglycans as a putative target. Heparinase treatment of wild type cells and an excess of soluble heparin abolished FliD binding. Binding assays showed direct and specific binding of FliD to heparin. Addition of an excess of purified FliD or heparin reduced the attachment of viable C. jejuni to the host cells. The host cell binding domain of FliD was mapped to the central region of the protein. Overall, our results indicate that the C. jejuni flagellar tip protein FliD acts as an attachment factor that interacts with cell surface heparan sulfate glycosaminoglycan receptors.