Branchial Sac Research Articles

Distinct microbial communities in an ascidian-crustacean symbiosis.

Ascidians are marine invertebrates known to occasionally host symbiotic crustaceans. Although the microbiomes of both ascidians and free-living crustaceans have been characterized, there is no documentation of microbial communities in an ascidian-crustacean symbiosis. Samples of the solitary ascidian Ascidia sydneiensis and ambient seawater were collected in Belize. Four symbiotic amphipod crustaceans were retrieved from the branchial sac of the animal, and their microbiomes were compared with those from their ascidian host (tunic and branchial sac compartments) and seawater. Microbiome richness and diversity differed significantly between sample types, with amphipod microbiomes exhibiting significantly lower diversity than tunic and ambient seawater samples. Microbiome composition also differed significantly between sample types and among all pairwise comparisons, except for branchial sac and amphipod microbiomes. Differential operational taxonomic unit (OTU) analyses revealed that only 3 out of 2553 OTUs had significantly different relative abundances in amphipods compared with ascidian branchial sacs, whereas 72 OTUs differed between amphipod and tunic and 315 between amphipod and seawater samples. Thus, different body compartments of A. sydneiensis hosted distinct microbiomes, and symbiotic amphipods contained microbiomes resembling the region they inhabit (i.e., the branchial sac), suggesting that environmental filtering and co-evolutionary processes are determinants of microbiome composition within ascidian-crustacean symbioses.

Comparison of Evolutionary Relationships between Branchiostoma floridae, Ciona intestinalis, and Homo sapiens Globins Provide Evidence of Gene Co-Option and Convergent Evolution.

Globins have been studied as model proteins to elucidate the principles of protein evolution. This was achieved by understanding the relationship between amino acid sequence, three-dimensional structure, physicochemical properties, and physiological function. Previous molecular phylogenies of chordate globin genes revealed the monophyletic evolution of urochordate globins and suggested convergent evolution. However, to provide evidence of convergent evolution, it is necessary to determine the physicochemical and functional similarities between vertebrates and urochordate globins. In this study, we determined the expression patterns of Ciona globin genes using real-time RT-PCR. Two genes (Gb-1 and Gb-2) were predominantly expressed in the branchial sac, heart, and hemocytes and were induced under hypoxia. Combined with the sequence analysis, our findings suggest that Gb-1/-2 correspond to vertebrate hemoglobin-α/-β. However, we did not find a robust similarity between Gb-3, Gb-4, and vertebrate globins. These results suggested that, even though Ciona globins obtained their unique functions differently from vertebrate globins, the two of them shared some physicochemical features and physiological functions. Our findings offer a good example for understanding the molecular mechanisms underlying gene co-option and convergence, which could lead to evolutionary innovations.

Differentially expressed chaperone genes reveal a stress response required for unidirectional regeneration in the basal chordate Ciona

BackgroundUnidirectional regeneration in the basal chordate Ciona intestinalis involves the proliferation of adult stem cells residing in the branchial sac vasculature and the migration of progenitor cells to the site of distal injury. However, after the Ciona body is bisected, regeneration occurs in the proximal but not in the distal fragments, even if the latter include a part of the branchial sac with stem cells. A transcriptome was sequenced and assembled from the isolated branchial sacs of regenerating animals, and the information was used to provide insights into the absence of regeneration in distal body fragments.ResultsWe identified 1149 differentially expressed genes, which were separated into two major modules by weighted gene correlation network analysis, one consisting of mostly upregulated genes correlated with regeneration and the other consisting of only downregulated genes associated with metabolism and homeostatic processes. The hsp70, dnaJb4, and bag3 genes were among the highest upregulated genes and were predicted to interact in an HSP70 chaperone system. The upregulation of HSP70 chaperone genes was verified and their expression confirmed in BS vasculature cells previously identified as stem and progenitor cells. siRNA-mediated gene knockdown showed that hsp70 and dnaJb4, but not bag3, are required for progenitor cell targeting and distal regeneration. However, neither hsp70 nor dnaJb4 were strongly expressed in the branchial sac vasculature of distal fragments, implying the absence of a stress response. Heat shock treatment of distal body fragments activated hsp70 and dnaJb4 expression indicative of a stress response, induced cell proliferation in branchial sac vasculature cells, and promoted distal regeneration.ConclusionsThe chaperone system genes hsp70, dnaJb4, and bag3 are significantly upregulated in the branchial sac vasculature following distal injury, defining a stress response that is essential for regeneration. The stress response is absent from distal fragments, but can be induced by a heat shock, which activates cell division in the branchial sac vasculature and promotes distal regeneration. This study demonstrates the importance of a stress response for stem cell activation and regeneration in a basal chordate, which may have implications for understanding the limited regenerative activities in other animals, including vertebrates.

Apoptosis is a generator of Wnt-dependent regeneration and homeostatic cell renewal in the ascidian Ciona.

ABSTRACTIn the ascidian Ciona intestinalis, basal body parts regenerate distal structures but distal body parts do not replace basal structures. Regeneration involves the activity of adult stem cells in the branchial sac, which proliferate and produce migratory progenitor cells for tissue and organ replacement. Branchial sac-derived stem cells also replenish recycling cells lining the pharyngeal fissures during homeostatic growth. Apoptosis at injury sites occurs early during regeneration and continuously in the pharyngeal fissures during homeostatic growth. Caspase 1 inhibitor, caspase 3 inhibitor, or pan-caspase inhibitor Z-VAD-FMK treatment blocked apoptosis, prevented regeneration, and suppressed branchial sac growth and function. A pharmacological screen and siRNA-mediated gene knockdown indicated that regeneration requires canonical Wnt signaling. Wnt3a protein rescued both caspase-blocked regeneration and branchial sac growth. Inhibition of apoptosis did not affect branchial sac stem cell proliferation but prevented the survival of progenitor cells. After bisection across the mid-body, apoptosis occurred only in the regenerating basal fragments, although both fragments contained a part of the branchial sac, suggesting that apoptosis is unilateral at the wound site and the presence of branchial sac stem cells is insufficient for regeneration. The results suggest that apoptosis-dependent Wnt signaling mediates regeneration and homeostatic growth in Ciona.

High mucous-mesh production by the ascidian Herdmania momus

Suspension feeders, including ascidians (Phylum Chordata, Class Ascidiacea), experience a dilute prey field composed of extremely small particles. The filtration apparatus of ascidians is based on a mucous-mesh that is continuously secreted and ingested. The rate and metabolic cost of this mesh secretion has not been quantified to date. We used video boroscopy to quantify the mucous-mesh production rate of the solitary ascidian Herdmania momus under different food and temperature treatments. H. momus individuals with an average (±95% CI) biomass of 30.7 ± 1.1 mg and a branchial sac area of 10.3 ± 1.2 cm2 produced an average of 276 ± 33.5 cm2 of mucous-mesh h-1, corresponding to a median turnover rate of 625 ± 82 mesh d-1. Since the mean mesh mass was 2.44 ± 0.58 mg, this production rate corresponds to roughly 50 ± 8 times the individual’s biomass per day. Food concentration had no detectable effect on mesh production rate, whereas a temperature difference of ~9°C (20 vs. 29°C) moderately increased mesh production by 30-50%. The current study reveals that the feeding process of H. momus involves a high expenditure on mucous-mesh synthesis that, combined with low food availability, may limit its growth in oligotrophic waters and under changing climate regimes.

Effect of Polycyclic Aromatic Hydrocarbons on Development of the Ascidian Ciona intestinalis Type A.

Polycyclic aromatic hydrocarbons (PAHs) are pollutants that exert harmful effects on marine invertebrates; however, the molecular mechanism underlying PAH action remains unclear. We investigated the effect of PAHs on the ascidian Ciona intestinalis type A (Ciona robusta). First, the influence of PAHs on early Ciona development was evaluated. PAHs such as dibenzothiophene, fluorene, and phenanthrene resulted in formation of abnormal larvae. PAH treatment of swimming larva induced malformation in the form of tail regression. Additionally, we observed the Ciona aryl hydrocarbon receptor (Ci-AhR) mRNA expression in swimming larva, mid body axis rotation, and early juvenile stages. The time correlation between PAH action and AhR mRNA expression suggested that Ci-AhR could be associated with PAH metabolism. Lastly, we analyzed Ci-AhR mRNA localization in Ciona juveniles. Ci-AhR mRNA was localized in the digestive tract, dorsal tubercle, ganglion, and papillae of the branchial sac, suggesting that Ci-AhR is a candidate for an environmental pollutant sensor and performs a neural function. Our results provide basic knowledge on the biological function of Ci-AhR and PAH activity in marine invertebrates.

<i>Pyura gangelion</i> (Savigny, 1816) (Tunicata: Pyuridae) from the Persian Gulf

We report the occurrence of Pyura gangelion (Savigny, 1816) from two sites in the Persian Gulf and its morphological description is also provided. This species has a wide distribution in the Indian and Pacific oceans as well as the Red Sea region. However, no record is available on its distribution in the Persian Gulf. The species is distinguished from other species by characteristics such as individuals are solitary with leathery, brownish to reddish tunic, siphons not easily visible; both siphons are in upright position with four lobes, mantle thin and covers entire body, muscle bands are denser adjacent to the siphons, oral tentacles branched with different sizes, the branchial sac is folded with six folds on each side of the body. Both internal longitudinal and transverse vessels are present, there is only one hermaphroditic gonad on the right side, endocarps, conspicuous projections into the atrium are visible in both sides, a hepatic cecum is positioned on the left side with a close connection to the branchial basket.

Symbiotic bacteria associated with ascidian vanadium accumulation identified by 16S rRNA amplicon sequencing

Ascidians belonging to Phlebobranchia accumulate vanadium to an extraordinary degree (≤ 350 mM). Vanadium levels are strictly regulated and vary among ascidian species; thus, they represent well-suited models for studies on vanadium accumulation. No comprehensive study on metal accumulation and reduction in marine organisms in relation to their symbiotic bacterial communities has been published. Therefore, we performed comparative 16S rRNA amplicon sequence analyses on samples from three tissues (branchial sac, intestine, and intestinal lumen) involved in vanadium absorption, isolated from two vanadium-rich (Ascidia ahodori and Ascidia sydneiensis samea) and one vanadium-poor species (Styela plicata). For each sample, the abundance of every bacteria and an abundance value normalized to their abundance in seawater were calculated and compared. Two bacterial genera, Pseudomonas and Ralstonia, were extremely abundant in the branchial sacs of vanadium-rich ascidians. Two bacterial genera, Treponema and Borrelia, were abundant and enriched in the intestinal content of vanadium-rich ascidians. The results suggest that specific selective forces maintain the bacterial population in the three ascidian tissues examined, which contribute to successful vanadium accumulation. This study furthers the understanding of the relationship between bacterial communities and metal accumulation in marine life.

Progenitor targeting by adult stem cells in Ciona homeostasis, injury, and regeneration

In the ascidian Ciona intestinalis, oral siphon amputation activates adult stem cell niches in the branchial sac to divide and dispatch migratory progenitor cells to a regeneration blastema at the site of injury. This study shows that progenitor cells derived from branchial sac stem cell niches have roles in homeostasis, wound repair, and regeneration of the siphons and neural complex (NC). During homeostasis, progenitor cells targeted the pharyngeal stigmata to replace ciliated cells involved in filter feeding. After individual or double siphon amputations, progenitor cells specifically targeted the oral or atrial siphons or both siphons, and were involved in the replacement of siphon circular muscle fibers. After oral siphon wounding, progenitor cells targeted the wound sites, and in some cases a supernumerary siphon was formed, although progenitor cell targeting did not predict the induction of supernumerary siphons. Following NC ablation, progenitor cells specifically targeted the regenerating NC, and supplied the precursors of new brain and neural gland cells. The tissues and organs targeted by branchial sac stem cells exhibited apoptosis during homeostasis and injury. It is concluded that branchial sac progenitor cells are multipotent and show targeting specificity that is correlated with apoptosis during homeostatic growth, tissue repair, and regeneration.

Not-so-simple sieving by ascidians: re-examining particle capture at the mesh and organismal scales

The particle capture mechanisms of biological filters determine the particle spectrum that is ingested by filter-feeding animals. Although ascidian feeding has been extensively investigated, the organismal-scale fluid dynamics and mesh-scale particle-filter interactions are not fully characterized. Fluorescein dye visualization of flow through the branchial sac of the ascidian Ciona intestinalis showed organismal-scale flow was laminar and moved both parallel and perpendicular to the mucous mesh. Endoscopic investigations of Herdmania momus revealed the mesh-scale filtration process, including the pre-capture velocities, particle approach angles, and mesh behavior. The mesh speed was variable (range 0–0.4 mm s−1). To determine how particle shape affects hydrosol capture, Styela plicata was fed differently shaped polystyrene particles (ellipsoids and spheres); sampling the inhaled and exhaled water revealed that microellipsoids (0.3 × 0.7 µm) were captured at significantly lower efficiency (32%) than 1 µm microspheres (86%). The capture efficiency of microellipsoids resembled that of microspheres with a diameter similar to the microellipsoids’ minor axis (0.3 µm, 31%) suggesting that the minimum diameter of ellipsoidal particles determines the capture efficiency. Flow near the filter was parallel to the mesh even ~ 10 s of micrometers away, implicating a “crossflow” component to ascidian filtration, where the fluid being filtered is directed along the surface of the filter rather than exclusively perpendicular to it. Collectively, these results suggest that ascidian filtration acts as a hybrid-flow filtration system rather than a classical direct sieve.

First Record of Synoicum clavatum (Ascidiacea: Aplousobranchia: Polyclinidae) from Korea

A colonial ascidian, Synoicum clavatum has previously been reported from Japan. Here, we provide detailed descriptions and illustrations of S. clavatum as the first record in Korea. Synoicum clavatum is characterized by club­ shaped colonies with oval head and elongate stalk, circular cloacal systems, a branchial sac with 14-16 stigmata rows of 10-17 stigmata per row and a stomach with smooth surface. The specimens of S. clavatum were collected in the subtidal zone (15-25 m depth) at Beomseom and Mumseom in Jeju­do while SCUBA diving from 2000 to 2014.

Feeding cessation alters host morphology and bacterial communities in the ascidian Pseudodistoma crucigaster.

BackgroundAscidians can associate with abundant and diverse consortia of microbial symbionts, yet these communities remain unexamined for the majority of host ascidians and little is known about host-symbiont interactions.MethodsWe coupled electron microscopy and 16S rRNA gene tag pyrosequencing to investigate the bacterial communities associated with the colonial ascidian Pseudodistoma crucigaster, a species endemic to the Mediterranean Sea that has a life cycle with two phases: actively-filtering (active) and non-filtering (resting) forms.ResultsResting colonies exhibited a reduced branchial sac (feeding apparatus) and a thickened cuticle. Electron microscope images also suggested higher abundance of colonizing microorganisms on surfaces of resting colonies. Accordingly, bacterial sequences associated with environmental sources (sediment and biofilms, >99 % similarity) were detected exclusively in resting colonies. Bacterial communities of P. crucigaster colonies (active and resting) were dominated by 3 core taxa affiliated (>94 % similarity) with previously described symbiotic Alphaproteobacteria in marine invertebrates. Shifts in rare bacteria were detected when ascidians entered the resting phase, including the appearance of strictly anaerobic lineages and nitrifying bacterial guilds.ConclusionsThese findings suggest that physical (thickened cuticle) and metabolic (feeding cessation) changes in host ascidians have cascading effects on associated bacteria, where modified oxygen concentrations and chemical substrates for microbial metabolism may create anaerobic microhabitats and promote colonization by environmental microorganisms.Electronic supplementary materialThe online version of this article (doi:10.1186/s12983-016-0134-4) contains supplementary material, which is available to authorized users.

The nephridial hypothesis of the gill slit origin.

Metameric gill slits are mysterious structures, unique for Chordata and Hemichordata, and also, perhaps, for the extinct Cambrian Calcichordata. There is a discussed hypothesis of the gill slits origin from the metameric nephridia. According to the hypothesis, the hypothetical metameric deuterostome ancestor had in each segment a pair of coelomoducts and a pair of intestinal pockets. In the anterior segments, the coelomoducts have fused with the intestinal pockets. As a result, each nephridium opened both into the gut and into the environment. Then the dissepiments and funnels reduced in all segments except the collar one. Thus, in recent enteropneusts, only the first pair of gill slits keeps the ancestral arrangement communicating at the same time with the gut, with the environment, and with the coelom of the preceding (collar) segment. In the anterior part of the branchio-genital trunk region of enteropneusts, the metameric intestinal pockets remained, as well as the metameric coelomoducts functioning as the ducts of the metameric gonads, i.e., as the gonoducts. The consequence of the hypothesis is that the metameric gill pores originate from the metameric excreting pores, and the metameric branchial sacs originate from the metameric endodermal pockets of the gut fused with the coelomoducts. The metameric gill slits by themselves correspond with metameric openings connecting the gut with metameric intestinal pockets. J. Exp. Zool. (Mol. Dev. Evol.) 324B: 647-652, 2015. © 2015 Wiley Periodicals, Inc.

Evolution of the chordate regeneration blastema: Differential gene expression and conserved role of notch signaling during siphon regeneration in the ascidian Ciona

The regeneration of the oral siphon (OS) and other distal structures in the ascidian Ciona intestinalis occurs by epimorphosis involving the formation of a blastema of proliferating cells. Despite the longstanding use of Ciona as a model in molecular developmental biology, regeneration in this system has not been previously explored by molecular analysis. Here we have employed microarray analysis and quantitative real time RT-PCR to identify genes with differential expression profiles during OS regeneration. The majority of differentially expressed genes were downregulated during OS regeneration, suggesting roles in normal growth and homeostasis. However, a subset of differentially expressed genes was upregulated in the regenerating OS, suggesting functional roles during regeneration. Among the upregulated genes were key members of the Notch signaling pathway, including those encoding the delta and jagged ligands, two fringe modulators, and to a lesser extent the notch receptor. In situ hybridization showed a complementary pattern of delta1 and notch gene expression in the blastema of the regenerating OS. Chemical inhibition of the Notch signaling pathway reduced the levels of cell proliferation in the branchial sac, a stem cell niche that contributes progenitor cells to the regenerating OS, and in the OS regeneration blastema, where siphon muscle fibers eventually re-differentiate. Chemical inhibition also prevented the replacement of oral siphon pigment organs, sensory receptors rimming the entrance of the OS, and siphon muscle fibers, but had no effects on the formation of the wound epidermis. Since Notch signaling is involved in the maintenance of proliferative activity in both the Ciona and vertebrate regeneration blastema, the results suggest a conserved evolutionary role of this signaling pathway in chordate regeneration. The genes identified in this investigation provide the foundation for future molecular analysis of OS regeneration.

Gut-spilling in chordates: evisceration in the tropical ascidian Polycarpa mytiligera.

The ejection of internal organs, i.e., evisceration, is a well-known phenomenon in sea-cucumbers. We report the ability of a member of the Chordate phyla, the tropical ascidian Polycarpa mytiligera, to eviscerate and regenerate its gut within 12 days, and to rebuild its branchial sac within 19 days. Evisceration occurred within 4–43 seconds of gentle mechanical pressure exerted on the tunic in 47% of the tested P. mytiligera. Individuals were able to discard up to 3/4 of their digestive tract via the incurrent siphon by rupture of the branchial sac in this area. Although chemical analysis revealed no significant levels of toxic compounds, the eviscerated guts were unpalatable to the triggerfish and pufferfish on which they were tested, suggesting evisceration as a defense mechanism. Given the close affinity of ascidians to vertebrates, the regeneration pathway of the viscera and branchial sac of ascidians suggests its potential beneficial application in soft tissue regeneration research.

Regeneration, Stem Cells, and Aging in the Tunicate Ciona: Insights from the Oral Siphon.

Regeneration studies in the tunicate Ciona intestinalis have recently been focused on the potential of adult stem cells to replace injured tissues and organs during the adult life cycle using the oral siphon (OS) as a model. The OS has oral siphon pigment organs (OPOs) along its rim and an underlying network of muscle fibers in its tube. Different regeneration processes are triggered by OS amputation at the tip, along the tube, or at the base. One process involves the replacement of OPOs without new cell division by direct differentiation of locally deployed stem cells or stem cells that migrate from the branchial sac. Another process involves blastema formation by the migration of progenitor cells produced from branchial sac stem cells. The capacity for complete and accurate OS regeneration declines continuously during the adult life cycle. Finally, after an age threshold is reached, OS regeneration ceases in old animals. The loss of regeneration capacity in old animals involves the depletion of stem cells in the branchial sac, the inability of branchial sac progenitor cells to migrate to the sites of regeneration, and defective oral pigment organ replacement. The significance of the OS model for studying regeneration, stem cells, and aging will be enhanced by the application of molecular methods.

Distal Regeneration Involves the Age Dependent Activity of Branchial Sac Stem Cells in the Ascidian Ciona intestinalis.

Tunicates have high capacities for regeneration but the underlying mechanisms and their relationship to life cycle progression are not well understood. Here we investigate the regeneration of distal structures in the ascidian tunicate Ciona intestinalis. Analysis of regenerative potential along the proximal−distal body axis indicated that distal organs, such as the siphons, their pigmented sensory organs, and the neural complex, could only be replaced from body fragments containing the branchial sac. Distal regeneration involves the formation of a blastema composed of cells that undergo cell proliferation prior to differentiation and cells that differentiate without cell proliferation. Both cell types originate in the branchial sac and appear in the blastema at different times after distal injury. Whereas the branchial sac stem cells are present in young animals, they are depleted in old animals that have lost their regeneration capacity. Thus Ciona adults contain a population of age‐related stem cells located in the branchial sac that are a source of precursors for distal body regeneration.

A First Record of the Genus Eusynstyela (Ascidiacea: Stolidobranchia: Styelidae) from Korea

Colonial ascidian, Eusynstyela monotestis (Tokioka, 1953), is newly reported from Korean waters. The specimens of E. monotestis examined in this study were collected at subtidal zone of Beomseom, Munseom, Seopseom and Chagwido in Jeju-do by SCUBA diving. The genus Eusynstyela Michaelsen, 1904 is also new to Korean waters and it is distinct from other genera by having branchial sac with folds, longitudinal stigmata, hermaphrodite gonads on both sides, 1-2 male follicles in each gonad and body wall with endocarps. Eusynstyela monotestis is distinct from other species by having gonad with only single male follicle. In this paper, detailed descriptions and photographs of Eusynstyela monotestis (Tokioka, 1953) are provided.

Botryllophilus kozloffi,new species (Copepoda: Cyclopoida: Ascidicolidae), living in the compound ascidianClavelina lepadiformis(Müller) from Roscoff, France

Botryllophilus kozloffi is described, based on females living in the branchial sac of the zooid of the compound ascidian Clavelina lepadiformis (Müller, 1776) collected at Roscoff, France. The new copepod belongs to subgroup 2 (leg armature formula pattern II) of female morphotype A (urosome 5-segmented) of the genus, and is primarily characterized by the following morphological features: 1) metasome (segmentation indistinct) large and inflated dorsally, anterior prosome strongly curved from left to right, and urosome long cylindrical; 2) on medial margin of mandibular coxa, 2 conical spines each associated with 1 small subconical spine anteroproximally; 3) right leg 1 exopod approximately trapezoidal, bearing 7 long spines (I–VII) on wide distal margin, its central distal portion bearing 4 spines (II–V) distinctly protruded; and 4) leg 5 short stout, directed posterolaterally.

The ascidian-associated mysid Corellamysis eltanina gen.nov., sp.nov. (Mysida, Mysidae, Heteromysinae): a new symbiotic relationship from the Southern Ocean.

A new mysid species representing a new genus is described based on specimens collected in the 1968 cruise of the U.S. Navy Ship Eltanin from the Macquarie Island region (Southern Ocean). The new mysid, Corellamysis eltanina, is characterized by the globular eyes lacking definite eyestalks, the seven and eighth thoracic endopods specialized as gnathopods forming a strong subchela, and by the armature and shape of the uropod endopod and telson. Corellamysis eltanina lives only in the branchial sacs of the ascidian Corella brewinae suggesting an obligate endocommensal symbiotic association. Therefore, this is the first known report of a mysid living symbiotically with a benthic tunicate, as well as the first report of a mysid symbiosis from the Southern Ocean. The distribution and habitats of known symbiont mysids are reviewed. An update of identification key to world genera and subgenera of Heteromysinae is suggested.