Ascidian Phallusia Research Articles

Molecular control of cellulosic fin morphogenesis in ascidians

BackgroundThe tunicates form a group of filter-feeding marine animals closely related to vertebrates. They share with them a number of features such as a notochord and a dorsal neural tube in the tadpole larvae of ascidians, one of the three groups that make tunicates. However, a number of typical chordate characters have been lost in different branches of tunicates, a diverse and fast-evolving phylum. Consequently, the tunic, a sort of exoskeleton made of extracellular material including cellulose secreted by the epidermis, is the unifying character defining the tunicate phylum. In the larva of ascidians, the tunic differentiates in the tail into a median fin (with dorsal and ventral extended blades) and a caudal fin.ResultsHere we have performed experiments in the ascidian Phallusia mammillata to address the molecular control of tunic 3D morphogenesis. We have demonstrated that the tail epidermis medio-lateral patterning essential for peripheral nervous system specification also controls tunic elongation into fins. More specifically, when tail epidermis midline identity was abolished by BMP signaling inhibition, or CRISPR/Cas9 inactivation of the transcription factor coding genes Msx or Klf1/2/4/17, median fin did not form. We postulated that this genetic program should regulate effectors of tunic secretion. We thus analyzed the expression and regulation in different ascidian species of two genes acquired by horizontal gene transfer (HGT) from bacteria, CesA coding for a cellulose synthase and Gh6 coding for a cellulase. We have uncovered an unexpected dynamic history of these genes in tunicates and high levels of variability in gene expression and regulation among ascidians. Although, in Phallusia, Gh6 has a regionalized expression in the epidermis compatible with an involvement in fin elongation, our functional studies indicate a minor function during caudal fin formation only.ConclusionsOur study constitutes an important step in the study of the integration of HGT-acquired genes into developmental networks and a cellulose-based morphogenesis of extracellular material in animals.

The Ascidian–Amphipod Association between Phallusia mammillata (Cuvier, 1815) and Leucothoe richiardii (Lessona, 1865) in the Mar Grande of Taranto (Mediterranean Sea, Italy)

Amphipods are widespread in the marine environment, and some have adopted a symbiotic lifestyle, such as numerous species of the genus Leucothoe (Leach, 1814). Unfortunately, few examples of such a relationship are known for the Mediterranean Sea. In the present study, we report for the first time the symbiotic relationship between Leucothoe richiardii (Lessona, 1865) and the ascidian Phallusia mammillata (Cuvier, 1815) from the Mar Grande of Taranto. Two samplings were carried out in November 2021 and May 2022, and the population structure of L. richiardii was also analyzed in relation to host weight. L. richiardii was found to live its entire cycle in P. mammillata. The amphipod population was dominated by females, and in most cases, juveniles of various sizes were found together with adults, indicating prolonged parental care. The host selection by the amphipods proved to be random and was determined by the abundance/availability of ascidians rather than their weight. The ascidians showed no signs of disturbance by the inhabiting amphipods, although they were present in large numbers, and we assumed that the relationship between L. richiardii and P. mammillata was commensal.

Regulation of anterior neurectoderm specification and differentiation by BMP signaling in ascidians.

The most anterior structure of the ascidian larva is made of three palps with sensory and adhesive functions essential for metamorphosis. They derive from the anterior neural border and their formation is regulated by FGF and Wnt. Given that they also share gene expression profiles with vertebrate anterior neural tissue and cranial placodes, their study should shed light on the emergence of the unique vertebrate telencephalon. We show that BMP signaling regulates two phases of palp formation in Ciona intestinalis. During gastrulation, the anterior neural border is specified in a domain of inactive BMP signaling, and activating BMP prevented its formation. During neurulation, BMP defines ventral palp identity and indirectly specifies the inter-papilla territory separating the ventral and dorsal palps. Finally, we show that BMP has similar functions in the ascidian Phallusia mammillata, for which we identified novel palp markers. Collectively, we provide a better molecular description of palp formation in ascidians that will be instrumental for comparative studies.

Systematic analysis of cell morphodynamics in C. elegans early embryogenesis.

The invariant cell lineage of Caenorhabditis elegans allows unambiguous assignment of the identity for each cell, which offers a unique opportunity to study developmental dynamics such as the timing of cell division, dynamics of gene expression, and cell fate decisions at single-cell resolution. However, little is known about cell morphodynamics, including the extent to which they are variable between individuals, mainly due to the lack of sufficient amount and quality of quantified data. In this study, we systematically quantified the cell morphodynamics in 52C. elegans embryos from the two-cell stage to mid-gastrulation at the high spatiotemporal resolution, 0.5μm thickness of optical sections, and 30-second intervals of recordings. Our data allowed systematic analyses of the morphological features. We analyzed sphericity dynamics and found a significant increase at the end of metaphase in every cell, indicating the universality of the mitotic cell rounding. Concomitant with the rounding, the volume also increased in most but not all cells, suggesting less universality of the mitotic swelling. Combining all features showed that cell morphodynamics was unique for each cell type. The cells before the onset of gastrulation could be distinguished from all the other cell types. Quantification of reproducibility in cell-cell contact revealed that variability in division timings and cell arrangements produced variability in contacts between the embryos. However, the area of such contacts occupied less than 5% of the total area, suggesting the high reproducibility of spatial occupancies and adjacency relationships of the cells. By comparing the morphodynamics of identical cells between the embryos, we observed diversity in the variability between cells and found it was determined by multiple factors, including cell lineage, cell generation, and cell-cell contact. We compared the variabilities of cell morphodynamics and cell-cell contacts with those in ascidian Phallusia mammillata embryos. The variabilities were larger in C. elegans, despite smaller differences in embryo size and number of cells at each developmental stage.

Spawning-Induced pH Increase Activates Sperm Attraction and Fertilization Abilities in Eggs of the Ascidian, Phallusia philippinensis and Ciona intestinalis.

In Phlebobranchiata ascidians, oocytes and spermatozoa are stored in the oviduct and spermiduct, respectively, until spawning occurs. Gametes in the gonoducts are mature and fertilizable; however, it was found that the gametes of the ascidians Phallusia philippinensis and Ciona intestinalis could not undergo fertilization in the gonoductal fluids. The body fluids of the ascidians, especially in the gonoducts, were much more acidic (pH 5.5-6.8) than seawater (pH 8.2), and the fertilization rate was low under such acidic conditions. Hence, we examined the effect of pH on gametes. Pre-incubation of gonoductal eggs at pH 8.2 prior to insemination increased fertilization rates, even when insemination was performed under low pH conditions. Furthermore, an increase in ambient pH induced an increase in the intracellular pH of the eggs. It was also found that an increase in ambient pH triggered the release of sperm attractants from the egg and is therefore necessary for sperm chemotaxis. Hence, acidic conditions in the gonoductal fluids keep the gametes, especially eggs, infertile, and the release of eggs into seawater upon spawning induces an increase in ambient pH, which enables egg fertilization.

Determination of polycyclic aromatic sulfur heterocycles in ascidians (Phallusia nigra) using a green procedure

Ascidians as a pollution bioindicator are well reported based on metal analysis, but little is known about organic compounds accumulation in these marine organisms. In this study, a miniaturized solid–liquid extraction method, combined with dispersive micro-solid-phase extraction (MSLE-DµSPE) cleanup was applied to the determination of fourteen polycyclic aromatic sulfur heterocycles (PASH) from petrochemical sources in ascidian Phallusia nigra. The alumina amount in DµSPE step was optimized and the method was validated in terms of linearity, accuracy and precision using matrix-matched calibration. The ascidians were collected in Todos os Santos Bay, in a region close to a floating gas station. In the laboratory, samples were dissected in two parts (branchial basket and tunic), lyophilized, weighed and extracted by MSLE-DµSDE. Analytes were determined by gas chromatography coupled to mass spectrometry in selective ion monitoring (GC–MS/SIM). Under optimum conditions, the limit of detection ranged from 0.17 µg L−1 (4,6-dimethyldibenzothiophene) to 1.99 µg L−1 (2,3-dimethylbenzothiophene) and the limit of quantification the values were in the range from 0.57 µg L−1 to 6.65 µg L−1 for the same compounds. Determination coefficients (R2) ≥ 0.99 were obtained for all analytical curves. The recoveries of the PASH were between 76 and 119 % with intraday and interday precisions lower than 20 %. The method was successfully applied to ascidian samples and ten PASHs were quantified with highest concentrations in the branchial basket. This study represents the first application of a green miniaturized procedure to the determination of PASH from petrochemical source in marine organisms.

Functional characterization, antimicrobial and antioxidant properties from the biomass extraction of ascidian Phallusia nigra

Functional characterization, antimicrobial and antioxidant properties from the biomass extraction of ascidian Phallusia nigra

Highly distinct genetic programs for peripheral nervous system formation in chordates

BackgroundVertebrates develop their peripheral nervous system (PNS) from transient unique embryonic structures, the neural crest, and the ectodermal placodes that are located at the border of the forming central nervous system. By contrast, in the invertebrate chordates, amphioxus and ascidians, a large part of the PNS originates at the opposite of the embryo, in the ventral ectoderm. In both groups, a biphasic mechanism regulates ventral PNS formation: high BMP levels specify a neurogenic territory within which glutamatergic epidermal sensory neuron formation is controlled by the Notch pathway. Given these similarities and the phylogenetic relationships within chordates, it is likely that ventral PNS is an ancestral feature in chordates and that it has been lost in vertebrates.ResultsIn order to get insights into the molecular control of ventral PNS formation and to test the hypothesis of their homology and potential contribution to the emergence of vertebrate PNS, we undertook a close comparison of ventral PNS formation in the ascidian Phallusia mammillata and the amphioxus Branchiostoma lanceolatum. Using timed RNA-seq series, we identified novel markers of the ventral PNS during different phases of its development in both species. By extensively determining the expression of paralogous and orthologous genes, we observed that only a minority of genes have a shared expression in the ventral PNS. However, a large fraction of ventral PNS orthologous genes are expressed in the dorsally forming PNS of vertebrates.ConclusionsOur work has significantly increased the molecular characterization of ventral PNS formation in invertebrate chordates. The low observed conservation of gene expression in the ventral PNS suggests that the amphioxus and ascidian ventral PNS are either not homologous, or alternatively extensive drift has occurred in their regulatory mechanisms following a long period (600 My) of separate evolution and accelerated evolution in the ascidian lineage. The homology to genes expressed in the dorsally forming PNS of vertebrates suggests that ancestral sensory neurons gene networks have been redeployed in vertebrates.

Spatial Variability in a Symbiont-Diverse Marine Host and the Use of Observational Data to Assess Ecological Interactions

Despite a rich taxonomic literature on the symbionts of ascidians, the nature of these symbioses remains poorly understood. In the Egyptian Red Sea, the solitary ascidian Phallusia nigra hosted a symbiotic amphipod and four copepod species, with densities as high as 68 mixed symbionts per host. Correlation analyses suggested no competition or antagonism between symbionts. Ascidian mass, ash-free dry mass per wet mass (AFDM/WM), and both symbiont density and diversity per host, differed significantly among three reefs from El Gouna, Egypt. However, there was no correlation between amphipod, total copepod, or total symbiont densities and host mass or AFDM/WM. A host condition index based on body to tunic mass ratio was significantly related to symbiont density overall, but this positive pattern was only strong at a single site studied. Despite assumptions based on the habit of some of the symbiont groups, our analyses detected little effect of symbionts on host health, suggesting a commensal relationship.

Existence in cellulose shelters: industrial and pharmaceutical leads of symbiotic actinobacteria from ascidian Phallusia nigra, Andaman Islands.

The diversity of actinobacteria associated with marine ascidian Phallusia nigra from Andaman Islands was investigated. A total of 10 actinobacteria were isolated and based on the biochemical and molecular characterization, the isolates were assigned to 7 different actinobacterial genera. Eight putatively novel species belonging to genera Rhodococcus, Kineococcus, Kocuria, Janibacter, Salinispora and Arthrobacter were identified based on 16S rDNA sequence similarity with the NCBI database. The organic extracts of ten isolates displayed considerable bioactivity against test pathogens, which were Gram-positive and Gram-negative in nature. PCR-based screening for type I and type II polyketide synthases (PKS-I, PKS-II) and nonribosomal peptide synthetases (NRPS) revealed that, 10 actinobacterial isolates encoded at least one type of polyketide synthases biosynthesis gene. Majority of the isolates found to produce industrially important enzymes; amylase, protease, gelatinase, lipase, DNase, cellulase, urease, phosphatase and L-asparaginase. The present study emphasized that, ascidians are a prolific resource for novel bioactive actinobacteria with potential for novel drug discovery. This result expands the scope to functionally characterize the novel ascidian associated marine actinobacteria and their metabolites could be a source for the novel molecules of commercial interest.

Self-organized optimal packing of kinesin-5-driven microtubule asters scales with cell size.

Radial microtubule (MT) arrays or asters determine cell geometry in animal cells. Multiple asters interacting with motors, such as those in syncytia, form intracellular patterns, but the mechanical principles behind this are not clear. Here, we report that oocytes of the marine ascidian Phallusia mammillata treated with the drug BI-D1870 spontaneously form cytoplasmic MT asters, or cytasters. These asters form steady state segregation patterns in a shell just under the membrane. Cytaster centers tessellate the oocyte cytoplasm, that is divide it into polygonal structures, dominated by hexagons, in a kinesin-5-dependent manner, while inter-aster MTs form 'mini-spindles'. A computational model of multiple asters interacting with kinesin-5 can reproduce both tessellation patterns and mini-spindles in a manner specific to the number of MTs per aster, MT lengths and kinesin-5 density. Simulations predict that the hexagonal tessellation patterns scale with increasing cell size, when the packing fraction of asters in cells is ∼1.6. This self-organized in vivo tessellation by cytasters is comparable to the 'circle packing problem', suggesting that there is an intrinsic mechanical pattern-forming module that is potentially relevant to understanding the role of collective mechanics of cytoskeletal elements in embryogenesis. This article has an associated First Person interview with the first author of the paper.

Gene Editing in the Ascidian Phallusia mammillata and Tail Nerve Cord Formation.

Functional approaches for studying embryonic development have greatly advanced thanks to the CRISPR-Cas9 gene editing technique. Previously practiced in just a few organisms, these knockout techniques are now widely applied. Here we describe simple techniques for applying the CRISPR-Cas9 system to study the development of the nerve cord in the ascidian Phallusia mammillata.

Dermatan sulfate obtained from the Phallusia nigra marine organism is responsible for antioxidant activity and neuroprotection in the neuroblastoma-2A cell lineage

Neurodegenerative diseases are characterized by progressive loss of neurons in the central nervous system (CNS). Several molecules play a role in mammalian CNS regeneration, including glycosaminoglycans (GAGs). GAGs are found in abundance in many marine invertebrates, such as ascidians that belong to the phylum Chordata, which show a high CNS regeneration capacity even in adulthood. Here, we investigated the roles of dermatan sulfate, a type of GAG that was obtained from the ascidian Phallusia nigra. We investigated the neuroprotective and antioxidant properties of Phallusia nigra dermatan sulfate (PnDS) after neurotoxic damage induced by the pesticide rotenone using the Neuro-2A cell lineage. Neuroprotection was observed through a mitochondrial activity analysis. A morphometric analysis revealed long unbranched neurites after incubation with PnDS and co-incubation with PnDS and rotenone. Furthermore, PnDS showed antioxidant activity that reduced reactive oxygen species (ROS) even in co-incubation with rotenone. The reduced ROS probably occurred because PnDS increased the activity of the antioxidant enzymes superoxide dismutase and catalase and improved total antioxidant capacity, which protected cells from damage, as observed through decreased levels of lipid peroxidation. These data suggest a neuroprotective and antioxidant role of PnDS even under neurodegenerative conditions caused by rotenone.

Wettability and Substrate Selection in the Larval Settlement of the Solitary Ascidian Phallusia philippinensis (Phlebobranchia: Ascidiidae).

For marine benthic animals, the selection of substrate by larvae is important for their survival, with early post-settlement mortality often being affected by the microenvironment where they settle. We tested the substrate preference of the larvae of the ascidian Phallusia philippinensis toward nine commercially available substrates. In the present assay, the larvae settled on one of four substrates for seven substrate combinations with different wettabilities; we counted the number of settled larvae on each of the four substrates, and Manly's selection indices were compared to determine the preference rank of each substrate. Larvae significantly preferred more hydrophobic and oleophilic substrates to hydrophilic and oleophobic ones. While it is uncertain how the larvae detect the properties of the substrate surface, they might be able to sense the physical force, such as stickiness and repellent force. Although a hydrophobic surface is not common in a natural marine environment, the use of hydrophobic materials (as flypaper-like tools) for ascidian larvae might help to prevent the settlement of non-indigenous ascidians in aquaculture facilities.

Non-Anticoagulant Heparan Sulfate from the Ascidian Phallusia nigra Prevents Colon Carcinoma Metastasis in Mice by Disrupting Platelet-Tumor Cell Interaction

Although metastasis is the primary cause of death in patients with malignant solid tumors, efficient anti-metastatic therapies are not clinically available currently. Sulfated glycosaminoglycans from marine sources have shown promising pharmacological effects, acting on different steps of the metastatic process. Oversulfated dermatan sulfates from ascidians are effective in preventing metastasis by inhibition of P-selectin, a platelet surface protein involved in the platelet-tumor cell emboli formation. We report in this work that the heparan sulfate isolated from the viscera of the ascidian Phallusia nigra drastically attenuates metastases of colon carcinoma cells in mice. Our in vitro and in vivo assessments demonstrate that the P. nigra glycan has very low anticoagulant and antithrombotic activities and a reduced hypotension potential, although it efficiently prevented metastasis. Therefore, it may be a promising candidate for the development of a novel anti-metastatic drug.

Atlas of gastrulation

Developmental Biology We marvel at the progression of a single cell into a complex organism, and microscopy has long been used to visualize early developmental events. More recently, molecular networks and cellular lineages have been constructed by measuring cells' gene expression profile through single-cell RNA sequencing (scRNA-seq). Sladitschek et al. combined scRNA-seq and light-sheet imaging of the ascidian Phallusia mammillata to reveal gene expression in all cells of the embryo up to the formation of the multilayered gastrula. Every cell was followed for all cell divisions in 18 cell lineages. The result is a map of cell spatial position and lineage history that reveals embryonic bilateral symmetry and interembryonic variability, as well as the patterned expression of cell adhesion molecules. This resource can be used to examine any number of molecular mechanisms and developmental events occurring in the gastrulating embryo and can serve as model for the spatiotemporal map. Cell 10.1016/j.cell.2020.03.055 (2020).

MorphoSeq: Full Single-Cell Transcriptome Dynamics Up to Gastrulation in a Chordate

SummarySingle-cell RNA sequencing (scRNA-seq) provides a leap forward in resolving cellular diversity and developmental trajectories but fails to comprehensively delineate the spatial organization and precise cellular makeup of individual embryos. Here, we reconstruct from scRNA-seq and light sheet imaging data a canonical digital embryo that captures the genome-wide gene expression trajectory of every single cell at every cell division in the 18 lineages up to gastrulation in the ascidian Phallusia mammillata. By using high-coverage scRNA-seq, we devise a computational framework that stratifies single cells of individual embryos into cell types without prior knowledge. Unbiased transcriptome data analysis mapped each cell’s physical position and lineage history, yielding the complete history of gene expression at the genome-wide level for every single cell in a developing embryo. A comparison of individual embryos reveals both extensive reproducibility between symmetric embryo sides and a large inter-embryonic variability due to small differences in embryogenesis timing.

Oxidative stress: Noradrenaline as an integrator of responses in the neuroendocrine and immune systems of the ascidian Phallusia nigra

Neurotransmitters play key roles in regulating the homeostasis of organisms in stressful environments. Noradrenaline (NA) is the main neurotransmitter known to modulate immunological parameters, and is important in the crosstalk between the neuroendocrine and immune systems. In this study, using the ascidian Phallusia nigra, we analyzed the level of catecholamines (CA) in the plasma after mechanical stress, and the effect of NA on the oxidative stress (OS) displayed by immune cells. We measured the concentration of reactive oxygen species (ROS), and analyzed whether α- and/or β-adrenoreceptors (ARs) are involved in ROS modulation, lipid peroxidation (LPO), antioxidant capacity against peroxyl radicals (ACAP), and activity of the enzymes catalase (CAT) and glutathione S transferase (GST) in immune cells after incubation with different concentrations of NA, with or without zymosan (ZnA) challenge. The results showed that NA reduced ROS production, even in immune cells challenged with ZnA, and that this modulation occurred through α1-and β1-ARs. ACAP levels showed different responses, depending on whether immune cells were challenged or not with ZnA, and also depending on the NA concentration: 1.0 μM NA increased ACAP levels, but 10.0 μM reduced ACAP levels. NA enhanced the activity of CAT and GST in ZnA-challenged and non-challenged immune cells, while 1.0 and 10.0 μM NA effectively reduced LPO. Taken together, these results show that NA can protect cells from ROS damage, decreasing ROS production and LPO, and enhancing ACAP as well as the activity of CAT and GST. The approach used here with this model contributes to understanding the relationship between the neuroendocrine and immune systems, revealing new effects of NA on OS regulation in ascidians.

Recruitment patterns of the solitary ascidian Phallusia nigra Savigny, 1816 on artificial substrates submerged in the central Red Sea, Saudi Arabia

Abstract The solitary ascidian Phallusia nigra is commonly found on hard substrates along the Jeddah coastal waters of the central Red Sea. In this study, the recruitment pattern of P. nigra on artificial substrates was assessed in relation to their type, surface color and orientation. The results showed a higher recruitment rate of the ascidian species on concrete and dark panels. The abundance of the ascidian on test panels varied between the four seasons. The orientation (vertical or horizontal) of the panels did not show any major difference in the recruitment. Significant effects of light intensityon the recruitment of P. nigra were observed on test panels, with higher abundance on panels submerged in the shade. In conclusion, this study clearly indicates the importance of the type and color of substrates in the recruitment of ascidians on artificial materials.

Bisphenols disrupt differentiation of the pigmented cells during larval brain formation in the ascidian

The endocrine disruptor Bisphenol A (BPA), a widely employed molecule in plastics, has been shown to affect several biological processes in vertebrates, mostly via binding to nuclear receptors. Neurodevelopmental effects of BPA have been documented in vertebrates and linked to neurodevelopmental disorders, probably because some nuclear receptors are present in the vertebrate brain. Similarly, endocrine disruptors have been shown to affect neurodevelopment in marine invertebrates such as ascidians, mollusks or echinoderms, but whether invertebrate nuclear receptors are involved in the mode-of-action is largely unknown.In this study, we assessed the effect of BPA on larval brain development of the ascidian Phallusia mammillata. We found that BPA is toxic to P. mammillata embryos in a dose-dependent manner (EC50: 11.8μM; LC50: 21μM). Furthermore, micromolar doses of BPA impaired differentiation of the ascidian pigmented cells, by inhibiting otolith movement within the sensory vesicle. We further show that this phenotype is specific to other two bisphenols (BPE and BPF) over a bisphenyl (2,2 DPP). Because in vertebrates the estrogen-related receptor gamma (ERRγ) can bind bisphenols with high affinity but not bisphenyls, we tested whether the ascidian ERR participates in the neurodevelopmental phenotype induced by BPA. Interestingly, P. mammillata ERR is expressed in the larval brain, adjacent to the differentiating otolith. Furthermore, antagonists of vertebrate ERRs also inhibited the otolith movement but not pigmentation. Together our observations suggest that BPA may affect ascidian otolith differentiation by altering Pm-ERR activity whereas otolith pigmentation defects might be due to the known inhibitory effect of bisphenols on tyrosinase enzymatic activity.