Navigating spatio-temporal microbiome dynamics: Environmental factors and trace elements shape the symbiont community of an invasive marine species.

The proliferation of marine invasive species is a mounting concern. While the role of microbial communities in invasive ascidian species is recognized, the role of seasonal shifts in microbiome composition remains largely unexplored. We sampled five individuals of the invasive ascidian Styela plicata quarterly from January 2020 to October 2021 in two harbours, examining gills, tunics, and surrounding water. By analysing Amplicon Sequence Variants (ASVs) and seawater trace elements, we found that compartment (seawater, tunic, or gills) was the primary differentiating factor, followed by harbour. Clear seasonal patterns were evident in seawater bacteria, less so in gills, and absent in tunics. We identified compartment-specific bacteria, as well as seasonal indicator ASVs and ASVs correlated with trace element concentrations. Among these bacteria, we found that Endozoicomonas, Hepatoplasma and Rhodobacteraceae species had reported functions which might be necessary for overcoming seasonality and trace element shifts. This study contributes to understanding microbiome dynamics in invasive holobiont systems, and the patterns found indicate a potential role in adaptation and invasiveness.

Effects of Dermatan Sulfate from Marine Invertebrate Styela plicata in the Wound Healing Pathway: A Natural Resource Applied to Regenerative Therapy.

Acute and chronic dermatological injuries need rapid tissue repair due to the susceptibility to infections. To effectively promote cutaneous wound recovery, it is essential to develop safe, low-cost, and affordable regenerative tools. Therefore, we aimed to identify the biological mechanisms involved in the wound healing properties of the glycosaminoglycan dermatan sulfate (DS), obtained from ascidian Styela plicata, a marine invertebrate, which in preliminary work from our group showed no toxicity and promoted a remarkable fibroblast proliferation and migration. In this study, 2,4-DS (50 µg/mL)-treated and control groups had the relative gene expression of 84 genes participating in the healing pathway evaluated. The results showed that 57% of the genes were overexpressed during treatment, 16% were underexpressed, and 9.52% were not detected. In silico analysis of metabolic interactions exhibited overexpression of genes related to: extracellular matrix organization, hemostasis, secretion of inflammatory mediators, and regulation of insulin-like growth factor transport and uptake. Furthermore, in C57BL/6 mice subjected to experimental wounds treated with 0.25% 2,4-DS, the histological parameters demonstrated a great capacity for vascular recovery. Additionally, this study confirmed that DS is a potent inducer of wound-healing cellular pathways and a promoter of neovascularization, being a natural ally in the tissue regeneration strategy.

Localization of vasoactive intestinal peptide and toll-like receptor 2 immunoreactive cells in endostyle of urochordate Styela plicata (Lesueur, 1823).

The endostyle is the first component of the ascidian digestive tract, it is shaped like a through and is located in the pharynx's ventral wall. This organ is divided longitudinally into nine zones that are parallel to each other. Each zone's cells are physically and functionally distinct. Support elements are found in zones 1, 3, and 5, while mucoproteins secreting elements related to the filtering function are found in zones 2, 4, and 6. Zones 7, 8, and 9, which are located in the lateral dorsal section of the endostyle, include cells with high iodine and peroxidase concentrations. Immunohistochemical technique using the following antibodies, Toll-like receptor 2 (TLR-2) and vasoactive intestinal peptide (VIP), and lectin histochemistry (WGA-wheat-germagglutinin), were used in this investigation to define immune cells in the endostyle of Styela plicata (Lesueur, 1823). Our results demonstrate the presence of immune cells in the endostyle of S. plicata, highlighting that innate immune mechanisms are highly conserved in the phylogeny of the chordates. RESEARCH HIGHLIGHTS: Immune cells positive to TLR-2 and VIP in the endostyle of Styela plicata. Expression of WGA in several zones of endostyle. Use of comparative biology to improve the knowledge about immunology in ascidians.

Toll-like receptor 2 and α-Smooth Muscle Actin expressed in the tunica of a urochordate, Styela plicata.

The tunicate, Styela plicata (Lesueur, 1823) present an open circulator system with a tubular heart and blood flowing in lacunae among organs, bathing the tissues directly. Blood vascular lacunae are present in the tunica that is situated outside the epidermis and present a fibrous structure. The cells of the tunic are in straight contact with the blood vessels or are highly mobile. Ascidians are considered model organisms in comparative immunology of the chordate, and hold an important phylogenetic position as sister group of vertebrates. In recent years, numerous studies have reported the presence of Toll-like receptors (TLRs) in the genome of non-mammalian organisms including invertebrates. Two TLRs, designated Ci-TLR1 and Ci-TLR2 were expressed in the stomach, intestine and in numerous hemocytes of Ciona intestinalis, demonstrating that these key transmembrane proteins are evolutionarily conserved in ascidians. In this study for the first time, hemocytes aggregates were identified by confocal immunofluorescence techniques, using TLR2 antibody in the tunica of Styela plicata; furthermore, α-Smooth Muscle Actin (α-SMA) expression has been shown in the cells lining the vessels of the tunic. Our results support the view that the TLR-mediated innate immune functions are conserved in ascidian tissues.

Early detection of marine invasive species following the deployment of an artificial reef: Integrating tools to assist the decision-making process.

Early detection and rapid response plans are a set of principles to reduce the establishment, spread and impact of invasive species and it is a critical step in management in marine ecosystems. Two potentially invasive ascidians attached to the hull of a recently sunk fishing vessel were early detected in Patagonia. With the aim of assisting in the management decision-making process during the early steps of a rapid response, we conducted several analyses through different approaches. First, we identified the species through classic taxonomical and genetic analyses. Then, we evaluated the regional and international shipping connectivity to study potential donor regions and finally, we used species distribution models (SDMs) to predict the potential distribution of these species. The potentially invasive ascidians were identified as Styela clava and Styela plicata, and this is the first record for both species in the Nuevo gulf, Patagonia Argentina. Both species have a widespread distribution around the world with strong ecological and economic impacts documented. Shipping traffic analysis suggested that S. plicata could have arrived by secondary spread from regional ports, while the arrival of S. clava was likely to be associated with international shipping traffic. Furthermore, the SDM predicted that S. clava has suitable coastal areas along the entire Southwestern Atlantic shoreline, where it is currently absent. On the contrary, the SDM predicted that further southward spread of S. plicata is unlikely, being limited by the minimum annual temperature. We discussed the different approaches, tools, and expertise integrated in this work in the light of the decision-making process for the early detection of marine invasive species in the Southwestern Atlantic. Moreover, we call attention to the increased creation of artificial habitats through the intentional sinking of ships and the potential consequences of these actions in the conservation of marine ecosystems.

Attenuation of Rat Colon Carcinogenesis by Styela plicata Aqueous Extract. Modulation of NF-κB Pathway and Cytoplasmic Sod1 Gene Expression.

OBJECTIVE: In search for a unique natural combination of highly active biological components for treatment against colon cancer, we used aqueous extract of Ascidia, Styela plicata (ASCex), a marine invertebrate depending on its richness of high levels of biologically active components as indicated in our previous studies, against rat colon cancer, exploring its underlying mechanisms. METHODS: Rats chemically initiated for colon cancer were either non-treated or post-treated with highly saturated ASCex for 32 weeks after initiation, other groups of rats were administered ASCex without cancer initiation or served as normal controls. RESULTS: Rats treated with ASCex alone did not show any signs of non-favored health conditions. Treatment with ASCex after cancer initiation has significantly reduced the average incidences, multiplicities and volumes of colon tumors (adenomas and adenocarcinomas) as compared with the non-treated cancer group. ASCex has also significantly reduced the total numbers of aberrant crypt foci (ACF), surrogate biomarkers for colon cancer as compared with the non-treated cancer group. Moreover, anti-proliferative celluar nucular antigen (PCNA) immunohistochemical staining revealed that ASCex exerted significant antiproliferative characteristics in the carcinogen-treated colonic mucosa as compared with its corresponding control. Also, treatment with ASCex has markedly down-regulated the mRNA expression levels of Nuclear Factor-kappa B (NF-κB), a nuclear transcriptional activator as well as the mRNA expression of the cytoplasmic SOD1 gene which encodes Cu/Zn SOD, the first line defense against superoxide radicals. CONCLUSION: Collectively, ASCex could act as a potent chemotherapeutic drug against colon cancer, likely through the influence of its rich active metabolites which interfere with various biological pathways including inhibition of protein synthesis during cellular growth and marked induction of antioxidative capacity in the colonic mucosa. This role has been extensively discussed herein.

Dynamics of a biofouling community in finfish aquaculture: a case study from the South Adriatic Sea.

Biofouling is one of the challenges that can strongly affect the finfish farm economy. Although several studies on biofouling in aquaculture have been conducted in the Mediterranean Sea, they focused on specific taxa or were limited to a particular period of sampling. The present study investigated for the first time the development, composition and variation in a biofouling community in a finfish farm with immersion time, season and depth. The results indicate that all these factors influence biofouling succession and recruitment. Moreover, the species that had a crucial role in structuring the community and in the farm cleaning activities were the ascidian Styela plicata and the bivalve Mytilus galloprovincialis. Compared with the literature data, the results highlight the heterogeneity in the composition of the biofouling present in the Mediterranean Sea. Moreover, such knowledge of the biofouling community could provide important information about management efforts and the costs that farmers will face when siting new fish farms.

Core and Dynamic Microbial Communities of Two Invasive Ascidians: Can Host-Symbiont Dynamics Plasticity Affect Invasion Capacity?

Ascidians (Chordata, Ascidiacea) are considered to be prominent marine invaders, able to tolerate highly polluted environments and fluctuations in salinity and temperature. Here, we examined the seasonal and spatial dynamics of the microbial communities in the inner-tunic of two invasive ascidians, Styela plicata (Lesueur 1823) and Herdmania momus (Savigny 1816), in order to investigate the changes that occur in the microbiome of non-indigenous ascidians in different environments. Microbial communities were characterized using next-generation sequencing of partial (V4) 16S rRNA gene sequences. A clear differentiation between the ascidian-associated microbiome and bacterioplankton was observed, and two distinct sets of operational taxonomic units (OTUs), one core and the other dynamic, were recovered from both species. The relative abundance of the dynamic OTUs in H. momus was higher than in S. plicata, for which core OTU structure was maintained independently of location. Ten and seventeen core OTUs were identified in S. plicata and H. momus, respectively, including taxa with reported capabilities of carbon fixing, ammonia oxidization, denitrification, and heavy-metal processing. The ascidian-sourced dynamic OTUs clustered in response to site and season but significantly differed from the bacterioplankton community structure. These findings suggest that the associations between invasive ascidians and their symbionts may enhance host functionality while maintaining host adaptability to changing environmental conditions.

Acute exposure to water-soluble fractions of marine diesel oil: Evaluation of apoptosis and oxidative stress in an ascidian.

To understand the mechanisms involved in organisms' responses to toxicity from oil pollution, we studied the effect of acute exposure (24 h) to the marine water-soluble fraction of diesel oil (WFDO) on the ascidian Styela plicata. We evaluated the mortality and behavior by means of the siphon reflex, and the response of blood cells (hemocytes) contained in the pharynx, by means of the production of nitric oxide (NO) and reactive oxygen species (ROS), in addition to the activity of the antioxidant enzyme catalase (CAT). We also correlated oxidative stress with the activation of apoptotic pathways. No mortality occurred 24 h after the ascidians were exposed to 5% and 10% marine WFDO; however, the siphon reflex, a behavioral test based on the time that the animals took to close their siphons, increased. We also observed an inflammatory response, as estimated by the increase in the number of hemocytes in the pharynx. NO and ROS production and CAT activity were reduced, whereas caspase-3, a signaling molecule involved in apoptosis, was activated. This suggests that in ascidians acutely exposed to oil, another mechanism can occur in addition to oxidative stress. Another possibility is that WFDO may directly interact with cellular macromolecules and activate caspase-3, independently of generating oxidative stress. The results showed that components of diesel oil affected a marine organism, which showed reduced ROS production in the pharynx cells, including hemocytes, and activation of apoptotic pathways.

Styela plicata: a new promising bioindicator of heavy metal pollution for eastern Aegean Sea coastal waters.

As part of a research project, the concentrations of Cd, Cu, Pb, V, and Zn in the tissues of Styela plicata were investigated for the first time to determine if S. plicata is a suitable biological indicator for biomonitoring of heavy metals in eastern Aegean Sea coastal waters. To examine the relationships, heavy metal levels in suspended particulate matters (SPMs) and sediments were also determined. According to the results, the mean metal levels in SPM, sediments, and S. plicata samples could be arranged in the following order of abundance: Zn > Cu > Pb > V > Cd. As for heavy metal levels, significant positive correlations were noted between Cd-Pb, Cd-V, Cd-Zn, Cu-V, and Pb-V in SPM; Cd-Zn, Cu-Zn, Pb-Cd, Pb-Cu, and Pb-Zn in sediment; and Cu-Pb, Cu-Zn, and Pb-Zn in S. plicata samples. Positive relationships between these metals showed that they were originated from same sources and that they were associated with each other. Based on the findings, Zn, Cu, and Pb concentrations in suspended particulate matters, sediments, and S. plicata samples were generally represented with higher levels at stations that were used for boating, shipping, and related activities. As S. plicata is a strongest accumulator of V, the relatively low V levels observed in this study may indicate the lack of anthropogenic sources of this metal in the sampling stations. In conclusion, suspended particulate matter and sediment can be useful tool to detect the pollution status of the marine environment. Furthermore, the findings of this study highlighted that S. plicata is a promising alternative for the monitoring of heavy metal pollution for eastern Aegean Sea coasts.

Evaluation and comparison of trace metal accumulation in different tissues of potential bioindicator organisms: Macrobenthic filter feeders Styela plicata, Sabella spallanzanii, and Mytilus galloprovincialis.

Trace metal concentrations were measured in different tissues of Sabella spallanzanii, Styela plicata, and Mytilus galloprovincialis collected in the Termini Imerese Harbor (Sicily, Italy) to evaluate the potential use of these species as bioindicators. Higher bioaccumulation factors (BAFs) were calculated in the tube of S. spallanzanii, except for As, which had a higher BAF in the branchial crown of the same species. Regarding the other species analyzed, higher BAFs were found in the digestive gland of M. galloprovincialis. An exception was Pb, which was significantly more concentrated in the branchial basket and tunic of S. plicata. The BAFs calculated in the present study show that all the species analyzed accumulate a certain amount of metals as a consequence of filter feeding mechanisms, and thus it was possible to assess the suitability of S. plicata, S. spallanzanii, and M. galloprovincialis as indicators of water quality. In particular, the tube of S. spallanzanii is an important compartment in terms of metal retention and is more suitable for the evaluation of contamination from trace elements. Environ Toxicol Chem 2016;35:3062-3070. © 2016 SETAC.

Genetic Distances in Three Ascidian Species determined by PCR Technique.

Seven oligonucleotides primers were shown to generate the shared loci, specific loci, unique shared loci to each species and shared loci by the three species which could be obviously scored. In the present study, 7 oligonucleotides primers produced 401 total loci in the Styela clava (SC) species, 390 in the Halocynthia roretzi (HR) and 434 in the Styela plicata (SP), respectively. Seven oligonucleotides primers generated 275 specific loci in the SC, 341 in the HR and 364 in the SP species, respectively. The oligonucleotides primer BION-23 generated 28 unique loci to each species in the SP species. Especially, the oligonucleotides primer BION-25 produced 7 unique loci to each species, which were identifying each species in the SP species. BION-17 distinguished 21 shared loci by the three ascidian species, major and/or minor fragments of sizes, which were identical in almost all of the samples. Based on the average bandsharing values of all samples, the similarity matrix ranged from 0.519 to 0.774 in the SC species, from 0.261 to 0.683 in the HR species and from 0.346 to 0.730 in the SP species. As regards average bandsharing value (BS) results, individuals from SC species (0.661±0.081) exhibited higher bandsharing values than did individuals from HR species (0.555±0.074) (P<0.05). The dendrogram obtained by the seven oligonucleotides primers indicates three genetic groups. In three ascidian species, the shortest genetic distance (0.071) exhibiting significant molecular difference was also between individual no. 20 and no. 21 within the SP species.

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

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

Histamine regulates the inflammatory response of the tunicate Styela plicata.

Histamine is stored inside hemocytes of the tunicate Styela plicata (Chordata, Tunicata, Ascidiacea), but no evidence on its role in the regulation of the immune response of this species has been reported. We examined whether histamine participated in the regulation of inflammation and host defense in S. plicata. The presence of histamine inside S. plicata hemocytes was confirmed by flow cytometry, and histamine release was detected by ELISA, after in vitro hemocyte stimulation with different PAMPs. In vitro hemocyte treatment with histamine, or specific histamine-receptor agonists, reduced their phagocytic ability. Injection of histamine into the tunic recruited hemocytes to the site of injection. Systemic injection of histamine, or the histamine-releasing agent compound 48/80, decreased the phagocytic ability of hemocytes. Histamine promoted the constriction of tunic hemolymph vessels in vivo, having a direct effect on vasoconstriction in tunic explants. These results provide for the first time clear evidence for the involvement of histamine in the regulation of inflammation and host defense in tunicates.