Anti-Trichomonas vaginalis Activity of Marine Ascidians (Tunicates; Ascidiacea) from the Bushehr Province, Iran

Objective: The aim of the current research is to evaluate the antiparasite effects of compounds isolated from marine ascidian tunicates on Trichomonas vaginalis. Methods: Ascidian tunicates after collection were cut into small pieces, freeze-dried, and powdered. The resulting material was subjected to extraction in double-distilled water, ethanol, n-hexane, and dichloromethane. To fractionate the extracts and identify the most bioactive compound, silica gel column chromatography and GC-M/S analysis were used. Results: Fraction 18 of silica gel column chromatography of ethanol extract was the most effective against T. vaginalis. The respective IC50, CC50, and SI values for fraction 18 were 28.62 µg/mL, ˃800 µg/mL, and ˃27.95. GC-M/S analysis of this fraction identified a major phenolic compound (2, 4-bis (1, 1-dimethyl ethyl), whose toxicity against vero cells was only 10.15%. Conclusion: The ethanolic fraction containing phenol-2,4-bis (1,1-dimethylethyl), which has a potent lethality effect on T. vaginalis, may be considered as an antiparasite drug candidate.

An organic extract from ascidian Ciona robusta induces cytotoxic autophagy in human malignant cell lines.

The last decades have seen an increase in the isolation and characterization of anticancer compounds derived from marine organisms, especially invertebrates, and their use in clinical trials. In this regard, ascidians, which are included in the subphylum Tunicata, represent successful examples with two drugs, Aplidine© and Yondelis© that reached the market as orphan drugs against several malignancies. Here, we report that an organic extract prepared from homogenized tissues of the Mediterranean ascidian Ciona robusta inhibited cell proliferation in HT-29, HepG2, and U2OS human cells with the former being the most sensitive to the extract (EC50 = 250 µg/mL). We demonstrated that the ascidian organic extract was not cytotoxic on HT-29 cells that were induced to differentiate with sodium butyrate, suggesting a preference for the mixture for the malignant phenotype. Finally, we report that cell death induced by the organic extract was mediated by the activation of a process of cytotoxic autophagy as a result of the increased expression of the LC3-II marker and number of autophagic vacuoles, which almost doubled in the treated HT-29 cells. In summary, although the detailed chemical composition of the Ciona robusta extract is still undetermined, our data suggest the presence of bioactive compounds possessing anticancer activity.

Henri de Lacaze-Duthiers and the ascidian hypothesis.

In 1830, Cuvier and Geoffroy Saint-Hilaire confronted each other in a famous debate on the unity of the animal kingdom, which permeated the zoology of the 19th century. From that time, a growing number of naturalists attempted to understand the large-scale relationships among animals. And among all the questions, that of the origin of vertebrates was one of the most controversial. Analytical methods based on comparative anatomy, embryology and paleontology were developed to identify convincing homologies that would reveal a logical sequence of events for the evolution of an invertebrate into the first vertebrate. Within this context, several theories have clashed on the question of the identity of the ancestor of vertebrates. Among the proposals, a group of rather discrete organisms, the ascidians, played a central role. Because he had discovered an ascidian with a particularly atypical larval development, the Molgula, Henri de Lacaze-Duthiers, a rigorous and meticulous naturalist, became involved in the ascidian hypothesis. While the visionary mind of Lacaze-Duthiers led him to establish a particularly innovative methodology and the first marine biology station in Europe, at Roscoff, the tailless tadpole of the Molgula prevented him from recognizing the ancestor of vertebrates. This old 19th century story echoes the ever-present questions driving the field of Eco-Evo-Devo.

Women in tunicate research: Pioneers of the past and their present legacy.

The search for female scientists who pioneered the research on tunicates is hindered by the tradition of reporting only the first initials of authors' names on scientific publications using only the initials of their first names. While this practice has the theoretical merit of broadening the readership by preventing the possible bias that could be caused by the gender of the author(s) in some of the readers, it rendered the identification of female researchers active in, or before, the first half of the 20th century quite challenging. Sifting through several dozen electronic records, and with the help of references and/or quotes found online, we have stitched together the information that we were able to retrieve on the life of female scientists who authored some of the earliest publications on tunicates, and we have organized them in (approximate) chronological order. We have also compiled brief synopses of the findings of scientists active in the field of tunicate biology in more recent times, and organized them by subdiscipline.

Extraction, Isolation, Characterization, and Biological Activity of Sulfated Polysaccharides Present in Ascidian Viscera Microcosmus exasperatus.

Ascidians are marine invertebrates that synthesize sulfated glycosaminoglycans (GAGs) within their viscera. Ascidian GAGs are considered analogues of mammalian GAGs and possess great potential as bioactive compounds, presenting antitumoral and anticoagulant activity. Due to its worldwide occurrence and, therefore, being a suitable organism for large-scale mariculture in many marine environments, our main objectives are to study Microcosmus exasperatus GAGs regarding composition, structure, and biological activity. We also aim to develop efficient protocols for sulfated polysaccharides extraction and purification for large-scale production and clinical applications. GAGs derived from M. exasperatus viscera were extracted by proteolytic digestion, purified by ion-exchange liquid chromatography, and characterized by agarose gel electrophoresis and enzymatic treatments. Anticoagulant activity was evaluated by APTT assays. Antitumoral activity was assessed in an in vitro model of tumor cell culture using MTT, clonogenic, and wound healing assays, respectively. Our results show that M. exasperatus presents three distinct polysaccharides; among them, two were identified: a dermatan sulfate and a fucosylated dermatan sulfate. Antitumoral activity was confirmed for the total polysaccharides (TP). While short-term incubation does not affect tumor cell viability at low concentrations, long-term TP incubation decreases LLC tumor cell growth/proliferation at different concentrations. In addition, TP decreased tumor cell migration at different concentrations. In conclusion, we state that M. exasperatus presents great potential as an alternative GAG source, producing compounds with antitumoral properties at low concentrations that do not possess anticoagulant activity and do not enhance other aspects of malignancy, such as tumor cell migration. Our perspectives are to apply these molecules in future preclinical studies for cancer treatment as antitumoral agents to be combined with current treatments to potentiate therapeutic efficacy.

Ciona spp. and ascidians as bioindicator organisms for evaluating effects of endocrine disrupting chemicals: A discussion paper.

In context of testing, screening and monitoring of endocrine-disrupting (ED) type of environmental pollutants, tunicates could possibly represent a particularly interesting group of bioindicator organisms. These primitive chordates are already important model organisms within developmental and genomics research due to their central position in evolution and close relationship to vertebrates. The solitary ascidians, such as the genus Ciona spp. (vase tunicates), could possibly be extra feasible as ED bioindicators. They have a free-swimming, tadpole-like larval stage that develops extremely quickly (<20 h under favorable conditions), has a short life cycle (typically 2-3 months), are relatively easy to maintain in laboratory culture, have fully sequenced genomes, and transgenic embryos with 3D course data of the embryo ontogeny are available. In this article, we discuss possible roles of Ciona spp. (and other solitary ascidians) as ecotoxicological bioindicator organisms in general but perhaps especially for effect studies of contaminants with presumed endocrine disrupting modes of action.

Physiological changes during torpor favor association with Endozoicomonas endosymbionts in the urochordate Botrylloides leachii.

Environmental perturbations evoke down-regulation of metabolism in some multicellular organisms, leading to dormancy, or torpor. Colonies of the urochordate Botrylloides leachii enter torpor in response to changes in seawater temperature and may survive for months as small vasculature remnants that lack feeding and reproductive organs but possess torpor-specific microbiota. Upon returning to milder conditions, the colonies rapidly restore their original morphology, cytology and functionality while harboring re-occurring microbiota, a phenomenon that has not been described in detail to date. Here we investigated the stability of B. leachii microbiome and its functionality in active and dormant colonies, using microscopy, qPCR, in situ hybridization, genomics and transcriptomics. A novel lineage of Endozoicomonas, proposed here as Candidatus Endozoicomonas endoleachii, was dominant in torpor animals (53-79% read abundance), and potentially occupied specific hemocytes found only in torpid animals. Functional analysis of the metagenome-assembled genome and genome-targeted transcriptomics revealed that Endozoicomonas can use various cellular substrates, like amino acids and sugars, potentially producing biotin and thiamine, but also expressing various features involved in autocatalytic symbiosis. Our study suggests that the microbiome can be linked to the metabolic and physiological states of the host, B. leachii, introducing a model organism for the study of symbioses during drastic physiological changes, such as torpor.

The impact of ascidian biofouling on the farmed Mediterranean mussel Mytilus galloprovincialis physiology and welfare, revealed by stress biomarkers.

In biofouling communities, ascidians are among the most damaging species, presenting severe threats, such as depressed growth rates and decreased chances of lower survival, to shellfish aquaculture. However, little is known concerning the fouled shellfish physiology. In an effort to obtain information for the magnitude of stress caused by ascidians to farmed Mytilus galloprovincialis, five seasonal samplings took place in a mussel aquaculture farm suffering from ascidian biofoulants, in Vistonicos Bay, Greece. The dominant ascidian species were recorded and several stress biomarkers, including Hsp gene expression at both mRNA and protein levels, as well as MAPKs levels, and enzymatic activities of intermediate metabolism were examined. Almost all investigated biomarkers revealed elevated stress levels in fouled mussels compared to non-fouled. This enhanced physiological stress seems to be season-independent and can be attributed to the oxidative stress and/or feed deprivation caused by ascidian biofouling, thus illuminating the biological impact of this phenomenon.

Azaspiracid accumulation in Japanese coastal bivalves and ascidians fed with Azadinium poporum producing azaspiracid-2 as the dominant toxin component.

The filter-feeding bivalves often accumulate marine toxins by feeding on toxic dinoflagellates that produce marine toxins. Azaspiracids (AZAs) are a group of lipophilic polyether toxins which have been detected in a variety of organisms in many countries. In our present study, accumulation kinetics and toxin distributions in the tissues of seven bivalve species and ascidians relevant to Japanese coastal waters were investigated by experimentally feeding a toxic dinoflagellate Azadinium poporum, which produces azaspiracid-2 (AZA2) as the dominant toxin component. All bivalve species and ascidians investigated in this study had the capability to accumulate AZA2 and no metabolites of AZA2 were detected in the bivalves and the ascidians. Japanese short-neck clams, Japanese oysters, Pacific oysters and ascidians accumulated AZA2 with the highest concentrations on the hepatopancreas, whereas the highest concentrations of AZA2 were found on the gills in surf clams and horse clams. Hard clams and cockles accumulated high levels of AZA2 in both the hepatopancreas and the gills. As far as we know, this is the first report describing detailed tissue distribution of AZAs in several bivalve species other than mussels (M. edulis) and scallops (P. maximus). Variation of accumulation rates of AZA2 in Japanese short-neck clams on different cell densities or temperatures were observed.

Prioritizing management of high-risk routes and ports by vessel type to improve marine biosecurity efforts.

The shipping industry constitutes the main vector of marine bioinvasions. Over 90,000 vessels world-wide create a highly complex shipping network that requires appropriate management tools. Here we characterized a novel vessel category, Ultra Large Container Vessels (ULCV), in terms of potential contribution to the dispersal of Non-Indigenous Species (NIS) in comparison to smaller vessels traveling similar routes. Such approach is essential for providing precise information-based risk analysis necessary to enforce biosecurity regulations and reduce the adverse global effects of marine NIS. We used Automatic Identification System (AIS) based websites to extract shipping data that will enable us to test for differences in two vessel behaviors linked to NIS dispersal: port visit durations and voyage sailing times. We then examined the geographic spread of ULCVs and small vessels, quantifying the accumulation of new port visits, countries, and ecoregions for each vessel category. Finally, Higher Order Network (HON) analysis revealed emergent patterns within shipping traffic, species flow, and invasion risk networks of these two categories. Compared to the smaller vessels, ULCVs spent significantly longer time in 20% of the ports and were more geographically constrained, with fewer port visits, countries, and regions. HON analysis revealed that the ULCV shipping species flow and invasion risk networks were more similar to each other than to those of the smaller vessels. However, HON port importance shifts were discernible for both vessel categories, with major shipping hubs not necessarily being major invasion hubs. Overall, compared to smaller vessels, ULCVs behave differently in ways that potentially increase biofouling risk, albeit in a smaller set of ports. Future studies using HON analysis of other dispersal vectors appears critical for prioritizing management of high-risk routes and ports.

New Data on the Rhamnose-Binding Lectin from the Colonial Ascidian Botryllus schlosseri: Subcellular Distribution, Secretion Mode and Effects on the Cyclical Generation Change.

Botryllus schlosseri in a cosmopolitan ascidian, considered a reliable model organism for studies on the evolution of the immune system. B. schlosseri rhamnose-binding lectin (BsRBL) is synthesised by circulating phagocytes and behaves as an opsonin by interacting with foreign cells or particles and acting as a molecular bridge between them and the phagocyte surface. Although described in previous works, many aspects and roles of this lectin in Botryllus biology remain unknown. Here, we studied the subcellular distribution of BsRBL during immune responses using light and electron microscopy. In addition, following the hints from extant data, suggesting a possible role of BsRBL in the process of cyclical generation change or takeover, we investigated the effects of interfering with this protein, by injecting a specific antibody in the colonial circulation, starting one day before the generation change. Results confirm the requirement of the lectin for a correct generation change and open new queries on the roles of this lectin in Botryllus biology.

Botryllin, a Novel Antimicrobial Peptide from the Colonial Ascidian Botryllus schlosseri.

By mining the transcriptome of the colonial ascidian Botryllus schlosseri, we identified a transcript for a novel styelin-like antimicrobial peptide, which we named botryllin. The gene is constitutively transcribed by circulating cytotoxic morula cells (MCs) as a pre-propeptide that is then cleaved to mature peptide. The synthetic peptide, obtained from in silico translation of the transcript, shows robust killing activity of bacterial and unicellular yeast cells, causing breakages of both the plasma membrane and the cell wall. Specific monoclonal antibodies were raised against the epitopes of the putative amino acid sequence of the propeptide and the mature peptide; in both cases, they label the MC granular content. Upon MC degranulation induced by the presence of nonself, the antibodies recognise the extracellular nets with entrapped bacteria nearby MC remains. The obtained results suggest that the botryllin gene carries the information for the synthesis of an AMP involved in the protection of B. schlosseri from invading foreign cells.

Marine life as a source for breast cancer treatment: A comprehensive review.

Breast cancer, one of the most significant tumors among all cancer cells, still has deficiencies for effective treatment. Moreover, substitute treatments employing natural products as bioactive metabolites has been seriously considered. The source of bioactive metabolites are not only the most numerous but also represent the richest source. A unique source is from the oceans or marine species which demonstrated intriguing chemical and biological diversity which represents an astonishing reserve for discovering novel anticancer drugs. Notably, marine sponges produce the largest amount of diverse bioactive peptides, alkaloids, terpenoids, polyketides along with many secondary metabolites whose potential is mostly therapeutic. In this review, our main focus is on the marine derived secondary metabolites which demonstrated cytotoxic effects towards numerous breast cancer cells and have been isolated from the marine sources such as marine sponges, cyanobacteria, fungi, algae, tunicates, actinomycetes, ascidians, and other sources of marine organisms.

Ascidian gastrulation and blebbing activity of isolated endoderm blastomeres.

Gastrulation is the first dynamic cell movement during embryogenesis. Endoderm and mesoderm cells are internalized into embryos during this process. Ascidian embryos provide a simple system for studying gastrulation in chordates. Gastrulation starts in spherical late 64-cell embryos with 10 endoderm blastomeres. The mechanisms of gastrulation in ascidians have been investigated, and a two-step model has been proposed. The first step involves apical constriction of endoderm cells, followed by apicobasal shortening in the second step. In this study, isolated ascidian endoderm progenitor cells displayed dynamic blebbing activity at the gastrula stage, although such a dynamic cell-shape change was not recognized in toto. Blebbing is often observed in migrating animal cells. In ascidians, endoderm cells displayed blebbing activity, while mesoderm and ectoderm cells did not. The timing of blebbing of isolated endoderm cells coincided with that of cell invagination. The constriction rate of apical surfaces correlated with the intensity of blebbing activity in each endoderm-lineage cell. Fibroblast growth factor (FGF) signaling was both necessary and sufficient for inducing blebbing activity, independent of cell fate specification. In contrast, the timing of initiation of blebbing and intensity of blebbing response to FGF signaling were controlled by intrinsic cellular factors. It is likely that the difference in intensity of blebbing activity between the anterior A-line and posterior B-line cells could account for the anteroposterior difference in the steepness of the archenteron wall. Inhibition of zygotic transcription, FGF signaling, and Rho kinase, all of which suppressed blebbing activity, resulted in incomplete apical constriction and failure of the eventual formation of cup-shaped gastrulae. Blebbing activity was involved in the progression and maintenance of apical constriction, but not in apicobasal shortening in whole embryos. Apical constriction is mediated by distinct blebbing-dependent and blebbing-independent mechanisms. Surface tension and consequent membrane contraction may not be the sole mechanical force for apical constriction and formation of cup-shaped gastrulae. The present study reveals the hidden cellular potential of endodermal cells during gastrulation and discusses the possible roles of blebbing in the invagination process.

The forgotten ones of ports: The filter feeders at the heart of siltation processes.

Siltation is a major concern in dynamic and complex ecosystems, such as ports. The mud must be regularly dredged to avoid disturbing the navigation channels. Sediments are carried by the waters entering the port and are partially trapped by harbour structures. Numerous studies have been conducted on the physical factors influencing siltation in port areas, whereas, few have focused on the role of biotic factors in mud formation. However, research in other contexts has shown that organisms that are abundant in pontoons, such as bivalves and tunicates, play an important role in this siltation process. All of these organisms belong to the filter feeder group. The sediments sucked in by the filter feeders are excreted in the form of faeces or mucus-bound pseudo-faeces. These waste materials, called bioproducts, settle efficiently and are involved in the composition of the mud. This study aimed to highlight the role of filter feeders in the siltation process in port areas and to determine the factors that influence the production of bioproducts by filter feeders. To investigate the role of filter feeders in the siltation processes, an experimental analysis was conducted in the largest marina in Europe (La Rochelle, France). It is divided into four basins with distinct filter feeder communities and environmental conditions, allowing for a detailed study of the environmental factors that influence the production of bioproducts. This analysis consisted of recovering and studying the bioproducts generated by the filter feeders using sediment traps fixed under pontoons. To explore the evolution of this biological production, 16 campaigns were conducted from January to March 2020 and May to July 2020. The total amount of dry matter produced was constant between seasons at approximately 130 g/m2/d; marina-wide, this amount represents a total daily production of 3.2 tons. However, the production amount varies spatially and temporally in relation to marine hydrodynamics and the organisms involved. Bioproduction was taxon-dependent: areas abundant in oysters and mussels were the areas with the most pronounced bioproduction, whereas there was no significant relationship between bioproduction and the presence of tunicates or scallops. If we consider bioproduction on a seasonal scale, we can see that the campaigns with the greatest production correspond to the periods when the sediment supply was the highest, i.e. when the tidal range was the highest. The quality of the bioproducts (organic matter content) differed between seasons, which can be explained by both environmental and metabolic changes. Understanding the role of filter feeders in siltation processes appears to be essential in port environments that need to be regularly dredged to ensure safe navigation.

Shipping voyage simulation reveals abiotic barriers to marine bioinvasions.

The shipping industry is considered the main vector of introduction of marine non-indigenous species (NIS). NIS distributions are often a consequence of frequent trade activities that are affected by economic trends. A dominant trend in the shipping industry is the operation of Ultra Large Container Vessels (ULCV), which are over 395 m long and sail mostly on the East-Asia - northern-Europe route. Understanding the risk of NIS introduction by this emerging shipping category is needed for devising strategies for sustainable shipping. Here, we conducted a controlled simulation of key abiotic factors that determine marine bioinvasion success: temperature, salinity, and food availability along selected routes, under two treatments: ULCV and intermediate-size vessels. We tested the effect of each treatment and the varying environmental conditions on the survival of two invasive ascidians (Chordata, Ascidiacea). We used survival analysis methods to locate predictors of ascidian mortality; Environmental conditions at ports with high mortality were used to identify similar major ports on a global scale as potential abiotic barriers. The key factors in ascidian mortality varied between the two species, but for both species, the treatment and salinity were dominant predictors for survival. We identified Port Klang, Rotterdam, and Dammam as ports with high mortality and located several globally distributed major ports that present similar environmental conditions. Our results highlight the potential role of selected major ports as abiotic barriers to fouling organisms during ocean voyages. The tolerance of the tropical-origin Microcosmus exasperatus to the northern-Europe conditions, and of the temperate/sub-tropical origin Styela plicata, to high temperature conditions, point out the urgent need to modify international fouling regulations in view of global change. Further studies on the survival of fouling organisms during a cascade of changing environmental conditions will contribute to the advancement of science-based regulations to reduce the adverse effects of NIS.

Co-expression network analysis of environmental canalization in the ascidian Ciona.

BACKGROUND: Canalization, or buffering, is defined as developmental stability in the face of genetic and/or environmental perturbations. Understanding how canalization works is important in predicting how species survive environmental change, as well as deciphering how development can be altered in the evolutionary process. However, how developmental gene expression is linked to buffering remains unclear. We addressed this by co-expression network analysis, comparing gene expression changes caused by heat stress during development at a whole-embryonic scale in reciprocal hybrid crosses of sibling species of the ascidian Ciona that are adapted to different thermal environments. RESULTS: Since our previous work showed that developmental buffering in this group is maternally inherited, we first identified maternal developmental buffering genes (MDBGs) in which the expression level in embryos is both correlated to the level of environmental canalization and also differentially expressed depending on the species' gender roles in hybrid crosses. We found only 15 MDBGs, all of which showed high correlation coefficient values for expression with a large number of other genes, and 14 of these belonged to a single co-expression module. We then calculated correlation coefficients of expression between MDBGs and transcription factors in the central nervous system (CNS) developmental gene network that had previously been identified experimentally. We found that, compared to the correlation coefficients between MDBGs, which had an average of 0.96, the MDBGs are loosely linked to the CNS developmental genes (average correlation coefficient 0.45). Further, we investigated the correlation of each developmental to MDBGs, showing that only four out of 62 CNS developmental genes showed correlation coefficient > 0.9, comparable to the values between MDBGs, and three of these four genes were signaling molecules: BMP2/4, Wnt7, and Delta-like. CONCLUSIONS: We show that the developmental pathway is not centrally located within the buffering network. We found that out of 62 genes in the developmental gene network, only four genes showed correlation coefficients as high as between MDBGs. We propose that loose links to MDBGs stabilize spatiotemporally dynamic development.