Adrenoreceptor phylogeny and novel functions of nitric oxide in ascidian immune cells.

Nitric oxide (NO) is a simple molecule involved in many biological processes and functions in the cardiovascular, neural, and immune systems. In recent years, NO has also been recognized as a crucial messenger in communication between the nervous and immune systems. Together with NO, catecholamines are the main group of neurotransmitters involved in cross-talk between the nervous and immune systems. Catecholamines such as noradrenaline, can act on immune cells through adrenoreceptors (ARs) present on the cell surface, and NO can cross the cell membrane and interact with secondary messengers, modulating catecholamine production. Here, we analyzed the mutual modulation by noradrenaline and NO in Phallusia nigra immune cells for specific subtypes of ARs. We also investigated the involvement of protein kinases A and C as secondary messengers to these specific subtypes of ARs in the adrenergic signaling pathway that culminates in NO modulation, and the phylogenetic distribution of ARs in deuterostome genomes. This analysis provided evidence for single-copy orthologs of α1, α2 and ß-AR in ascidian genomes, suggesting that NO and NA act on a less diverse set of ARs in urochordates. Pharmacological assays showed that high levels of NO can induce ascidian immune cells to produce catecholamines. We also observed that protein kinases A and C are the secondary messengers involved in downstream modulation of NO production through an ancestral ß-AR. Taken together, these results provide new information on NO as a modulator of immune cells, and reveal the molecules involved in the signaling pathway of ARs. The results also indicate that ARs may participate in NO modulation. Finally, our results suggest that the common ancestor of urochordates possessed a less complex system of ARs required for immune action and diverse pharmacological responses, since the α-ARs are phylogenetically more related to D1-receptors than are the ß-ARs.

Mechanical and genetic control of ascidian endoderm invagination during gastrulation.

Gastrulation is a near universal developmental process of animal embryogenesis, during which dramatic morphogenetic events take place: the mesodermal and endodermal tissues are internalized, the ectoderm spreads to cover the embryo surface, and the animal body plan and germ layers are established. Morphogenesis during gastrulation has long been considered the result of spatio-temporally localised forces driven by the transcriptional programme of the embryo. Recent work has shown that tissue rheological properties, which define the mechanical response of tissues to internally-generated or external forces, are also important dynamic regulators of gastrulation. Here, we first introduce how embryonic mechanics can be represented, before outlining current knowledge of the mechanical and genetic control of gastrulation in ascidians, invertebrate marine chordates which develop with invariant cell lineages and a solid-like rheological behaviour until the neurula stages. We discuss the potential of these organisms for the experimental and computational whole-embryo characterisation of the mechanisms shaping gastrulation, and how they may inform the more complex tissue internalization strategies used by other model organisms.

Aurasperone A Inhibits SARS CoV-2 In Vitro: An Integrated In Vitro and In Silico Study.

Several natural products recovered from a marine-derived Aspergillus niger were tested for their inhibitory activity against SARS CoV-2 in vitro. Aurasperone A (3) was found to inhibit SARS CoV-2 efficiently (IC50 = 12.25 µM) with comparable activity with the positive control remdesivir (IC50 = 10.11 µM). Aurasperone A exerted minimal cytotoxicity on Vero E6 cells (CC50 = 32.36 mM, SI = 2641.5) and it was found to be much safer than remdesivir (CC50 = 415.22 µM, SI = 41.07). To putatively highlight its molecular target, aurasperone A was subjected to molecular docking against several key-viral protein targets followed by a series of molecular dynamics-based in silico experiments that suggested Mpro to be its primary viral protein target. More potent anti-SARS CoV-2 Mpro inhibitors can be developed according to our findings presented in the present investigation.

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.

Practical Guide for Ascidian Microinjection: Phallusia mammillata.

Phallusia mammillata has recently emerged as a new ascidian model. Its unique characteristics, including the optical transparency of eggs and embryos and efficient translation of exogenously introduced mRNA in eggs, make the Phallusia system suitable for fluorescent protein (FP)-based imaging approaches. In addition, genomic and transcriptomic resources are readily available for this ascidian species, facilitating functional gene studies. Microinjection is probably the most versatile technique for introducing exogenous molecules such as plasmids, mRNAs, and proteins into ascidian eggs/embryos. However, it is not practiced widely within the community; presumably, because the system is rather laborious to set up and it requires practice. Here, we describe in as much detail as possible two microinjection methods that we use daily in the laboratory: one based on an inverted microscope and the other on a stereomicroscope. Along the stepwise description of system setup and injection procedure, we provide practical tips in the hope that this chapter might be a useful guide for introducing or improving a microinjection setup.

Electroporation in Ascidians: History, Theory and Protocols.

Embryonic development depends on the orchestration of hundreds of regulatory and structural genes to initiate expression at the proper time, in the correct spatial domain(s), and in the amounts required for cells and tissues to become specified, determined, and ultimately to differentiate into a multicellular embryo. One of the key approaches to studying embryonic development is the generation of transgenic animals in which recombinant DNA molecules are transiently or stably introduced into embryos to alter gene expression, to manipulate gene function or to serve as reporters for specific cell types or subcellular compartments. In some model systems, such as the mouse, well-defined approaches for generating transgenic animals have been developed. In other systems, particularly non-model systems, a key challenge is to find a way of introducing molecules or other reagents into cells that produces large numbers of embryos with a minimal effect on normal development. A variety of methods have been developed, including the use of viral vectors, microinjection, and electroporation. Here, I describe how electroporation was adapted to generate transgenic embryos in the ascidian, a nontraditional invertebrate chordate model that is particularly well-suited for studying gene regulatory activity during development. I present a review of the electroporation process, describe how electroporation was first implemented in the ascidian, and provide a series of protocols describing the electroporation process, as implemented in our laboratory.

Transgenic Techniques for Investigating Cell Biology During Development.

Ascidians are increasingly being used as a system for investigating cell biology during development. The extreme genetic and cellular simplicity of ascidian embryos in combination with superior experimental tractability make this an ideal system for in vivo analysis of dynamic cellular processes. Transgenic approaches to cellular and sub-cellular analysis of ascidian development have begun to yield new insights into the mechanisms regulating developmental signaling and morphogenesis. This chapter focuses on the targeted expression of fusion proteins in ascidian embryos and how this technique is being deployed to garner new insights into the cell biology of development.

Catecholamines are produced by ascidian immune cells: The involvement of PKA and PKC in the adrenergic signaling pathway.

The stress response is a complex mechanism, which includes changes in the immune system to enable organisms to maintain homeostasis. The neurohormones dopamine, noradrenaline (NA) and adrenalin are responsible for the physiological modulations that occur during acute stress. In the present study, we analyzed the effects of NA on the immune system specific to nitric-oxide (NO) production by subpopulations of immune cells (hemocytes) of the ascidian Phallusia nigra. We also investigated the capability of immune cells to produce catecholamine (CA). Finally, we tested the involvement of protein kinase A (PKA) and C (PKC) in the NA downstream signaling pathway. The results revealed that NA can reduce NO production by P. nigra hemocytes threefold, and that signet-ring cells, univacuolar refractile granulocytes and morula cells are the cell types most involved in this event. A challenge effected with Zymosan A induced CA production, and co-incubation with both inhibitors of the second messengers PKA and PKC revealed the involvement of these molecules in the adrenergic pathway of P. nigra hemocytes. Taken together, these results suggest that NO production can be down-regulated by NA through α- and ß-adrenoceptors via the second messengers PKA and PKC.

Phallusiasterol C, A New Disulfated Steroid from the Mediterranean Tunicate Phallusia fumigata.

A new sulfated sterol, phallusiasterol C (1), has been isolated from the Mediterranean ascidian Phallusia fumigata and its structure has been determined on the basis of extensive spectroscopic (mainly 2D NMR) analysis. The possible role in regulating the pregnane X receptor (PXR) activity of phallusiasterol C has been investigated; although the new sterol resulted inactive, this study adds more items to the knowledge of the structure-PXR regulating activity relationships in the case of sulfated steroids.

Taxonomic Composition and Biological Activity of Bacterial Communities Associated with Marine Ascidians from Andaman Islands, India.

Marine invertebrates, particularly ascidians, constitute an important source of potential active and biofunctional natural products. The microbial diversity associated with ascidians is little recognized, although these microorganisms play a vital role in marine ecosystems. The objective of this study was to investigate bacterial population diversity in four ascidian samples: Phallusia nigra, Phallusia fumigata, Eudistoma viride, and Rhopalaea macrothorax, collected from the North Bay, Andaman and Nicobar Islands. Microbial strains identified up to the species level revealed 236 distinct species/ribotypes out of 298 bacterial strains. Of 298 ascidian-associated bacteria, 72 isolates belong to the class Gammaproteobacteria and the genus Endozoicomonas. The results from this investigation will contribute a broaden knowledge of microbial diversity associated to marine ascidians, and as a promising source for the discovery of new natural products.

En masse DNA Electroporation for in vivo Transcriptional Assay in Ascidian Embryos.

Ascidian embryos are powerful models for functional genomics, in particular, due to the ease of generating a large number of transgenic embryos by electroporation. In addition, the small size of their genome makes them an attractive model for studying cis-regulatory elements that control gene expression during embryonic development. Here, I describe the adaptation of the seminal method developed 25 years ago in Ciona robusta for en masse DNA electroporation for in vivo transcription to additional species belonging to three genera. It is likely that similar optimizations would make electroporation successful in other ascidian species, where in vitro fertilization can be performed on a large number of eggs.

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.

Proceraea exoryxae sp. nov. (Annelida, Syllidae, Autolytinae), the first known polychaete miner tunneling into the tunic of an ascidian.

While studying organisms living in association with the solitary tunicate Phallusia nigra (Ascidiacea, Ascidiidae) from a shallow fringing reef at Zeytouna Beach (Egyptian Red Sea), one of the collected ascidians showed peculiar perforations on its tunic. Once dissected, the perforations revealed to be the openings of a network of galleries excavated in the inner tunic (atrium) by at least six individuals of a polychaetous annelid. The worms belonged to the Autolytinae (Syllidae), a subfamily that is well known to include specialized predators and/or symbionts, mostly associated with cnidarians. The Red Sea worms are here described as Proceraea exoryxae sp. nov., which are anatomically distinguished by the combination of simple chaetae only in anterior chaetigers, and a unique trepan with 33 teeth in one outer ring where one large tooth alternates with one medium-sized tricuspid tooth, and one inner ring with small teeth located just behind the large teeth. Male and female epitokes were found together with atokous individuals within galleries. Proceraea exoryxae sp. nov. constitutes the first known miner in the Autolytinae and the second species in this taxon known to live symbiotically with ascidians. The implications of finding this specialized parasite are discussed considering that Phallusia nigra has been introduced worldwide, in tropical and sub-tropical ecosystems, where it has the potential of becoming invasive.

The invariant cleavage pattern displayed by ascidian embryos depends on spindle positioning along the cell's longest axis in the apical plane and relies on asynchronous cell divisions.

The ascidian embryo is an ideal system to investigate how cell position is determined during embryogenesis. Using 3D timelapse imaging and computational methods we analyzed the planar cell divisions in ascidian early embryos and found that spindles in every cell tend to align at metaphase in the long length of the apical surface except in cells undergoing unequal cleavage. Furthermore, the invariant and conserved cleavage pattern of ascidian embryos was found to consist in alternate planar cell divisions between ectoderm and endomesoderm. In order to test the importance of alternate cell divisions we manipulated zygotic transcription induced by ß-catenin or downregulated wee1 activity, both of which abolish this cell cycle asynchrony. Crucially, abolishing cell cycle asynchrony consistently disrupted the spindle orienting mechanism underpinning the invariant cleavage pattern. Our results demonstrate how an evolutionary conserved cell cycle asynchrony maintains the invariant cleavage pattern driving morphogenesis of the ascidian blastula.

Morphological aspects of fertilization in Phallusia (Ascidia) nigra (Ascidiacea, Tunicata).

The spermatozoa of Phallusia (Ascidia) nigra have an elongated head (approximately 5 µm in length) in which a nucleus and a single mitochondrion are located side by side. There is no midpiece. The apex of the head is wedge-shaped. Acrosomal vesicles (approximately 55-65 nm in diameter) and moderately electron-dense material (MEDM) are present between the plasmalemma and the nuclear membranes in the anterior tip of the head. The MEDM occupies a central position and three or four acrosomal vesicles are seen in a line alongside it. The acrosomal vesicles disappear as the sperm makes contact with the surface of the chorion. Gamete fusion most likely occurs between a small process extending from the peripheral margin of the sperm apex and the egg surface, resulting in incorporation of the sperm into the egg from the anterior region of its head.