The nuclear receptor coactivator 4 regulates ferritinophagy induced by vibrio splendidus in coelomocytes of Apostichopus japonicus.

Iron homeostasis is vital for the host's defense against pathogenic invasion and the ferritinophagy is a crucial mechanism in maintaining intracellular iron homeostasis by facilitating the degradation and recycling of stored iron. The nuclear receptor coactivator 4 (NCOA4) serves as a ferritinophagy receptor, facilitating the binding and delivery of ferritin to the autophagosome and lysosome. However, NCOA4 of the sea cucumber Apostichopus japonicus (AjNCOA4) has not been reported until now. In this study, we identified and characterized AjNCOA4 in A. japonicus. This gene encodes a polypeptide containing 597 amino acids with an open reading frame of 1794 bp. The inferred amino acid sequence of AjNCOA4 comprises an ARA70 domain. Furthermore, a multiple sequence alignment demonstrated varying degrees of sequence homology between AjNCOA4 from A. japonicus and other NCOA4 orthologs. The phylogenetic tree of NCOA4 correlates with the established timeline of metazoan evolution. Expression analysis revealed that AjNCOA4 is expressed in all tested tissues, including the body wall, muscle, intestine, respiratory tree, and coelomocytes. Following challenge with Vibrio splendidus, the coelomocytes exhibited a significant increase in AjNCOA4 mRNA levels, peaking at 24 h. We successfully obtained recombinant AjNCOA4 protein through prokaryotic expression and prepared a specific polyclonal antibody. Immunofluorescence and co-immunoprecipitation experiments demonstrated an interaction between AjNCOA4 and AjFerritin in coelomocytes. RNA interference-mediated knockdown of AjNCOA4 expression resulted in elevated iron ion levels in coelomocytes. Bacterial stimulation enhanced ferritinophagy in coelomocytes, while knockdown of AjNCOA4 reduced the occurrence of ferritinophagy. These findings suggest that AjNCOA4 modulates ferritinophagy induced by V. splendidus in coelomocytes of A. japonicus.

The multistressor effect of pH reduction, microplastic and lanthanum on sea urchin Arbacia lixula.

pH reduction (Low pH), microplastic (MP), and lanthanum (La) are substantial stressors due to their increasing trends in marine ecosystems and having adverse effects on marine species. This study investigates the single and combined effects of those stressors (Low pH: 7.45, polyethylene MP: 26 µg L-1, and La: 9 µg L-1) on the physiology and histology of sea urchin Arbacia lixula. Regarding physiological results, while the coelomocytes' quantity was slightly affected by stressors, their viability was significantly affected. The coelomocyte count and viability were suppressed most in Low pH-MP-La treatment. The stressors did not impact the respiration rate. According to the histological examination results, the crypt (villi-like structure) was shorter, and epithelial layers were thinner in single and dual stress treatments like MP, Low pH, Low pH-La, and MP-La. Overall, we suggest that the combination of variable types of those stressors causes negative effects on sea urchin's physiology and histology.

Comparison of Immune Indicators Related to Phagocytosis of Five Species of Sea Urchins under Artificial Infection with the Pathogenic Bacterium of Black Mouth Disease.

To screen for immune indicators closely related to disease resistance, two species of sea urchin susceptible to black mouth disease (Strongylocentrotus intermedius, S. intermedius ♀ × Heliocidaris crassispina ♂) and three species of sea urchin resistant to black mouth disease (H. crassispina, H. crassispina ♀ × S. intermedius ♂ and Mesocentrotus nudus) were artificially infected with the black mouth pathogen Vibrio echinoideorum. The phagocytosis-related immune indices of the five sea urchin species were compared at different time points post-infection. The results demonstrated that the parameters such as apoptotic rate of phagocytes, mean contribution value (MCV) of single effective phagocyte on Acid Phosphatase (ACP), Reactive Oxygen Species (ROS), and Total Antioxidant Capacity (T-AOC) of the five sea urchin species first increased and then decreased after infection. The key time points were 3 h to 6 h and 48 h post-infection when the black mouth disease-resistant and susceptible sea urchins demonstrated differences. At 3 h or 6 h post-infection, the up-regulation folds in MCV of ACP, ROS and T-AOC of black mouth disease-resistant sea urchins were considerably higher than that of the susceptible sea urchins. At 6 h post-infection, the apoptosis rate and the phagocytic index (PI) of the black mouth disease-resistant sea urchins were significantly higher than those of the susceptible sea urchins (p < 0.05). At 48 h post-infection, the necrosis rate of phagocytes, MCV of ACP and MCV of ROS of the black mouth disease-resistant sea urchins were significantly lower than those of the susceptible sea urchins (p < 0.05). The apoptosis and necrosis rate of phagocytes, PI, and MCV on ACP, ROS may be used as indicators of disease resistance in sea urchins. Disease resistance standards in immune indices can be summarized as phagocytosis increases greatly in the early infection stage and decreases timely to a normal level after killing the pathogen in a short period.

Physiological responses of Holothuria grisea during a wound healing event: An integrated approach combining tissue, cellular and humoral evidence.

Due to their tissue structure similar to mammalian skin and their close evolutionary relationship with chordates, holothurians (Echinodermata: Holothuroidea) are particularly interesting for studies on wound healing. However, previous studies dealing with holothuroid wound healing have had limited approaches, being restricted to tissue repair or perivisceral immune response. In this study, we combined tissue, cellular and humoral parameters to study the wound healing process of Holothuria grisea. The immune responses of the perivisceral coelom were assessed by analyzing the number, proportion and viability of coelomocytes and the volume and protein concentration of the coelomic fluid. Additionally, the morphology of the healing tissue and number of coelomocytes in the connective tissue of different body wall layers were examined over 30 days. Our results showed that perivisceral reactions started 3 h after injury and decreased to baseline levels within 24 h. In contrast, tissue responses were delayed, beginning after 12 h and returning to baseline levels only after day 10. The number of coelomocytes in the connective tissue suggests a potential cooperation between these cells during wound healing: phagocytes and acidophilic spherulocytes act together in tissue clearance/homeostasis, whereas fibroblast-like and morula cells cooperate in tissue remodeling. Finally, our results indicate that the major phases observed in mammalian wound healing are also observed in H. grisea, despite occurring at a different timing, which might provide insights for future studies. Based on these data, we propose a model that explains the entire healing process in H. grisea.

Novel mechanistic insights into Cr(VI) and Cr(III) induced discrepancies of cellular toxicity and oxidative injury events in Eisenia fetida.

Chromium (Cr) poses a high ecological risk, however the toxic mechanisms of Cr in different valence states to soil organisms at cellular and molecular levels are not exactly. In this study, the Eisenia fetida coelomocytes and Cu/Zn-superoxide dismutase (Cu/Zn-SOD) were chosen as the target subjects to investigate the effects and mechanisms of cellular toxicity induced by Cr(VI) and Cr(III). Results indicated that Cr(VI) and Cr(III) significantly reduced the coelomocytes viability. The level of reactive oxygen species (ROS) was markedly increased after Cr(VI) exposure, which finally reduced antioxidant defense abilities, and induced lipid peroxidation and cellular membrane damage in earthworm coelomocytes. However, Cr(III) induced lower levels of oxidative stress and cellular damage with respect to Cr(VI). From a molecular perspective, the binding of both Cr(VI) and Cr(III) with Cu/Zn-SOD resulted in protein backbone loosening and reduced ß-Sheet content. The Cu/Zn-SOD showed fluorescence enhancement with Cr(III), whereas Cr(VI) had no obvious effect. The activity of Cu/Zn-SOD continued to decrease with the exposure of Cr. Molecular docking indicated that Cr(III) interacted more readily with the active center of Cu/Zn-SOD. Our results illustrate that oxidative stress induced by Cr(VI) and Cr(III) plays an important role in the cytotoxic differences of Eisenia fetida coelomocytes and the binding of Cr with Cu/Zn-SOD can also affect the normal structures and functions of antioxidant defense-associated protein.

Biomarker response index in earthworms following chronic exposure to leachate from a closed dumpsite: Behavioral, cytotoxicity and antioxidant system alterations.

Leachate, an effluent produced during solid waste decomposition, interacts directly with soil, mainly in dumpsite areas. Studies on terrestrial animal exposure to leachate are, however, lacking. Plants are the most frequently studied organisms, while animal studies, especially earthworms, are limited. Nevertheless, ecotoxicological assessments involving earthworms are crucial due to their role in soil health and ecosystem maintenance, which are paramount in understanding potential terrestrial ecosystem leachate effects. In this context, this study aimed to evaluate behavioral effects, sublethal cytotoxicity and antioxidant system alterations in Eisenia andrei earthworms chronically exposed to leachate from a closed dumpsite. Cytotoxicity was determined by coelomocyte density, viability and cell typing, while antioxidant system alterations were assessed through superoxide dismutase (SOD), catalase (CAT), glutathione-S-transferase (GST), reduced glutathione (GSH) and metallothionein (MT) determinations. Malondialdehyde (MDA) and protein carbonylation (PTC) levels were also determined as oxidative effect markers. Finally, the Biomarker Response Index (BRI) was assessed, aiming to quantitatively integrate the results of the investigated endpoints and establish a biological health state (BHS) for each leachate concentration. Leachate exposure led to leak responses at concentrations of up to 50%, but attraction at higher concentrations. Decreased cell density (28%) was observed after 48 days and reduced viability (50%), after 14 days of leachate exposure. The observed cell typing changes indicate anti-inflammatory immune system effects. Leachate exposure led to several antioxidant system alterations, increasing SOD (2-6 %), CAT (5-35 %) and GST (5-70 %) activities and GSH (7-37%) and MT (3-67%) levels. Earthworm antioxidant defenses were, however, able to prevent lipid peroxidation, which decreased (11-37%) following leachate exposure to concentrations above 12.5%, and PTC, which increased at 42 days (26%) and reduced at 56 days (12 %). This is the first PTC assessment in leachate-exposed earthworms. The increased carbonylation levels observed after 42 days alongside MDA decreases highlight the need for further research employing oxidative effect biomarkers other than MDA. Finally, an integrated approach employing the BRI was carried out, revealing mild initial changes evolving to moderate to major effects at the highest leachate exposure concentration, with an effect attenuation detected at the end of the experiment. In this sense, this study brings forth a significant novelty, employing a biomarker previously not assessed in earthworms, demonstrating an oxidative effect, alongside the use of the BRI as an integrative tool for the endpoints applied in this assessment.

Copper toxicity on Eisenia fetida in a vineyard soil: a combined study with standard tests, genotoxicity assessment and gut metagenomic analysis.

Copper (Cu) toxicity is a pressing concern for several soils, especially in organic viticulture. The objective of this work was to assess Cu toxicity on the non-target organism Eisenia fetida, employing both traditional and novel tools for early identification of Cu-induced damages. In addition to traditional tests like avoidance and reproductive toxicity experiments, other tests such as the single cell gel electrophoresis (SCGE) and gut microbiome analysis were evaluated to identify early and more sensitive pollution biomarkers. Four sub-lethal Cu concentrations were studied, and the results showed strong dose-dependent responses by the earthworm avoidance test and the exceeding of habitat threshold limit at the higher Cu doses. An inverse proportionality was observed between reproductive output and soil Cu concentration. Bioaccumulation was not detected in earthworms; soil concentrations of potentially bioavailable Cu were not affected by E. fetida presence or by time. On the contrary, the SCGE test revealed dose-dependent genotoxicity for the 'tail length' parameter already at the second day of Cu exposition. Gut microbiome analysis a modulation of microbial composition, with the most aboundant families being Pectobateriaceae, Comamonadaceae and Microscillaceae. Bacillaceae increased over time and showed adaptability to copper up to 165 mg/kg, while at the highest dose even the sensitive Acetobacteriaceae family was affected. The research provided new insights into the ecotoxicity of Cu sub-lethal doses highlighting both alterations at earthworms' cellular level and changes in their gut microbiota.

Molecular cloning, biological description, and functional analysis of Ajfos transcription factor in pathogen-induced Apostichopus japonicus.

Activator protein-1 subfamily member c-Fos wields significant influence over cellular activities, such as regulation of cell growth and division, cell death, and immune responses under various extracellular situations. In this study, the full-length c-Fos of sea cucumber, Apostichopus japonicus (Ajfos) was successfully cloned and analyzed. The anticipated 306 amino acid sequences of Ajfos displayed a basic-leucine zipper (bZIP) domain, similar to invertebrate counterparts. In addition, the qPCR results suggested Ajfos expressed in all tissues, with the highest level in coelomocytes from polian vesicle (vesicle lumen cells), followed by coelomocytes from coelom (coelomocytes). Moreover, the expression levels of Ajfos in the coelomocytes and vesicle lumen cells of sea cucumber showed significant changes after the Vibrio splendidus challenge, especially reaching a peak at 6 h. Compared with the silencing negative control RNA interference (siNC) group, silencing Ajfos (siAjfos) in vivo decreased the downstream proliferation-related gene expression of vesicle lumen cells after infection with V. splendidus while no significant influence was observed on coelomocytes. Furthermore, the proliferation proportion of vesicle lumen cells in the siAjfos group was significantly reduced under pathogen stimulation conditions. Finally, based on the fluctuation trend of total coelomocyte density (TCD) from coelom and polian vesicle previously discovered, it is evident that Ajfos played a critical role in facilitating the swift proliferation of vesicle lumen cells in response to V. splendidus stimulation. Altogether, this research provided an initial reference of the function of Ajfos in echinoderms, unveiling its participation in host coelomocyte proliferation of sea cucumbers during bacterial challenges.

Earthworm Coelomocyte Internalization of MoS2 Nanosheets: Multiplexed Imaging, Molecular Profiling, and Computational Modeling.

Fully understanding the cellular uptake and intracellular localization of MoS2 nanosheets (NSMoS2) is a prerequisite for their safe applications. Here, we characterized the uptake profile of NSMoS2 by functional coelomocytes of the earthworm Eisenia fetida. Considering that vacancy engineering is widely applied to enhance the NSMoS2 performance, we assessed the potential role of such atomic vacancies in regulating cellular uptake processes. Coelomocyte internalization and lysosomal accumulation of NSMoS2 were tracked by fluorescent labeling imaging. Cellular uptake inhibitors, proteomics, and transcriptomics helped to mechanistically distinguish vacancy-mediated endocytosis pathways. Specifically, Mo ions activated transmembrane transporter and ion-binding pathways, entering the coelomocyte through assisted diffusion. Unlike molybdate, pristine NSMoS2 (P-NSMoS2) induced protein polymerization and upregulated gene expression related to actin filament binding, which phenotypically initiated actin-mediated endocytosis. Conversely, vacancy-rich NSMoS2 (V-NSMoS2) were internalized by coelomocytes through a vesicle-mediated and energy-dependent pathway. Mechanistically, atomic vacancies inhibited mitochondrial transport gene expression and likely induced membrane stress, significantly enhancing endocytosis (20.3%, p < 0.001). Molecular dynamics modeling revealed structural and conformational damage of cytoskeletal protein caused by P-NSMoS2, as well as the rapid response of transport protein to V-NSMoS2. These findings demonstrate that earthworm functional coelomocytes can accumulate NSMoS2 and directly mediate cytotoxicity and that atomic vacancies can alter the endocytic pathway and enhance cellular uptake by reprogramming protein response and gene expression patterns. This study provides an important mechanistic understanding of the ecological risks of NSMoS2.

Morphological and physiological impairments in algae-infested sea urchins of the species Echinometra lucunter (Echinodermata).

Epibiotic species, which may be characterized as those living attached to the outer surface of a host (the basibiont), have hardly been described as living on echinoderms, probably because the outer surface of these latter is covered by the epidermis. Studies analyzing epibiotic associations usually focus on taxonomical and ecological aspects, while a physiological approach - even being able to reveal the costs and/or benefits of such interactions - has been neglected. Here, we not only report an unprecedented algal epibiotic association for the sea urchin Echinometra lucunter, but we mainly investigate how such kind of interaction could affect spine morphology and basibiont physiology, and consequently its health. To achieve this, we compared the spine morphology of Echinometra lucunter with and without algal infestation using histology, microcomputed tomography, and SEM. Immunological parameters, such as the number, proportion, and viability of the coelomocytes were evaluated. Algal-infested individuals showed a higher number and lower viability of coelomocytes, as well as an altered proportion of phagocytes and red spherulocytes. Additionally, spine stroma and stereom were severely degraded in comparison to non-infested ones. Thus, our findings suggest that algal-infested E. lucunter may be less efficient in carrying out routine activities than non-infested individuals, such as physical protection, anchorage, or coping with immune challenges.

Environmental relevant concentrations of polystyrene nanoplastics and lead co-exposure triggered cellular cytotoxicity responses and underlying mechanisms in Eisenia fetida.

Heavy metal pollution of soils and the widespread use of plastics have caused environmental problems worldwide. Nanoplastics (NPs) contaminants in water and soil environments can adsorb heavy metals, thereby affecting the bioavailability and toxicity of heavy metals. In this paper, the effect of co-exposure of polystyrene microspheres with 100 nm particle size and lead acetate (Pb) on the Eisenia fetida coelomocytes was investigated. The environmental concentration of NPs used was 0.01 mg/L and the concentration of Pb ranged from 0.01 to 1 mg/L, and the exposed cells were incubated at 298 k for 24 h. Our study demonstrated that exposure of cells to environmental relevant concentrations of NPs did not significantly affect the cytotoxicity of Pb exposure. It was shown that co-exposure induced cellular production of reactive oxygen species (ROS, increased to 134.4 %) disrupted the antioxidant system of earthworm body cavity cells, activated superoxide dismutase and catalase (CAT), produced reduced glutathione, and inhibited glutathione-dependent enzyme (GST) activity (Reduced to 64 %). Total antioxidant capacity (T-AOC) is first enhanced against ROS due to the stress of NPs and Pb. When the antioxidant reserves of cells are exhausted, the antioxidant capacity will decrease. The level of malondialdehyde, a biomarker of eventual lipid peroxidation, increased to 231.7 %. At the molecular level, due to co-exposure to NPs and Pb, CAT was loosely structured and the secondary structure is misfolded, which was responsible for exacerbating oxidative damage in E. fetida coelomocytes. The findings of this study have significant implications for the toxicological interaction and future risk assessment of co-contamination of NPs and Pb in the environment.

Immune and Reproductive Biomarkers in Female Sea Urchins Paracentrotus lividus under Heat Stress.

The functioning of the immune and reproductive systems is crucial for the fitness and survival of species and is strongly influenced by the environment. To evaluate the effects of short-term heat stress (HS) on these systems, confirming and deepening previous studies, female sea urchin Paracentrotus lividus were exposed for 7 days to 17 °C, 23 and 28 °C. Several biomarkers were detected such as the ferric reducing power (FRAP), ABTS-based total antioxidant capacity (TAC-ABTS), nitric oxide metabolites (NOx), total thiol levels (TTL), myeloperoxidase (MPO) and protease (PA) activities in the coelomic fluid (CF) and mitochondrial membrane potential (MMP), H2O2 content and intracellular pH (pHi) in eggs and coelomocytes, in which TAC-ABTS and reactive nitrogen species (RNS) were also analyzed. In the sea urchins exposed to HS, CF analysis showed a decrease in FRAP levels and an increase in TAC-ABTS, TTL, MPO and PA levels; in coelomocytes, RNS, MMP and H2O2 content increased, whereas pHi decreased; in eggs, increases in MMP, H2O2 content and pHi were found. In conclusion, short-term HS leads to changes in five out of the six CF biomarkers analyzed and functional alterations in the cells involved in either reproductive or immune activities.

Regeneration of starfish radial nerve cord restores animal mobility and unveils a new coelomocyte population.

The potential to regenerate a damaged body part is expressed to a different extent in animals. Echinoderms, in particular starfish, are known for their outstanding regenerating potential. Differently, humans have restricted abilities to restore organ systems being dependent on limited sources of stem cells. In particular, the potential to regenerate the central nervous system is extremely limited, explaining the lack of natural mechanisms that could overcome the development of neurodegenerative diseases and the occurrence of trauma. Therefore, understanding the molecular and cellular mechanisms of regeneration in starfish could help the development of new therapeutic approaches in humans. In this study, we tackle the problem of starfish central nervous system regeneration by examining the external and internal anatomical and behavioral traits, the dynamics of coelomocyte populations, and neuronal tissue architecture after radial nerve cord (RNC) partial ablation. We noticed that the removal of part of RNC generated several anatomic anomalies and induced behavioral modifications (injured arm could not be used anymore to lead the starfish movement). Those alterations seem to be related to defense mechanisms and protection of the wound. In particular, histology showed that tissue patterns during regeneration resemble those described in holothurians and in starfish arm tip regeneration. Flow cytometry coupled with imaging flow cytometry unveiled a new coelomocyte population during the late phase of the regeneration process. Morphotypes of these and previously characterized coelomocyte populations were described based on IFC data. Further studies of this new coelomocyte population might provide insights on their involvement in radial nerve cord regeneration.

Short-Term Thermal Stress Affects Immune Cell Features in the Sea Urchin Paracentrotus lividus.

Due to global warming, animals are experiencing heat stress (HS), affecting many organic functions and species' survival. In this line, some characteristics of immune cells in sea urchins subjected to short-term HS were evaluated. Paracentrotus lividus adult females were randomly divided into three groups and housed in tanks at 17 °C. In two of these tanks, the temperatures were gradually increased up to 23 and 28 °C. Celomatic fluid was collected after 3 and 7 days. The coelomocytes were morphologically typed and evaluated for their mitochondrial membrane potential (MMP), lipoperoxidation extent (LPO), and hydrogen peroxide content (H2O2). Respiratory burst was induced by treatment with phorbol 12-myristate 13-acetate (PMA). HS caused a significant change in the coelomocytes' type distribution. MMP increased in the 23 °C-group and decreased in the 28 °C-group at both 3 and 7 days. LPO only increased in the 28 °C-group at 7 days. H2O2 progressively decreased together with the temperature increase. Respiratory burst was detected in all groups, but it was higher in the 17 °C group. In conclusion, the increase in temperature above the comfort zone for this animal species affects their immune cells with possible impairment of their functions.

Coelomocytes of the Oligochaeta earthworm Lumbricus terrestris (Linnaeus, 1758) as evolutionary key of defense: a morphological study.

Metazoans have several mechanisms of internal defense for their survival. The internal defense system evolved alongside the organisms. Annelidae have circulating coelomocytes that perform functions comparable to the phagocytic immune cells of vertebrates. Several studies have shown that these cells are involved in phagocytosis, opsonization, and pathogen recognition processes. Like vertebrate macrophages, these circulating cells that permeate organs from the coelomic cavity capture or encapsulate pathogens, reactive oxygen species (ROS), and nitric oxide (NO). Furthermore, they produce a range of bioactive proteins involved in immune response and perform detoxification functions through their lysosomal system. Coelomocytes can also participate in lithic reactions against target cells and the release of antimicrobial peptides. Our study immunohistochemically identify coelomocytes of Lumbricus terrestris scattered in the epidermal and the connective layer below, both in the longitudinal and in the smooth muscle layer, immunoreactive for TLR2, CD14 and α-Tubulin for the first time. TLR2 and CD14 are not fully colocalized with each other, suggesting that these coelomocytes may belong to two distinct families. The expression of these immune molecules on Annelidae coelomocytes confirms their crucial role in the internal defense system of these Oligochaeta protostomes, suggesting a phylogenetic conservation of these receptors. These data could provide further insights into the understanding of the internal defense system of the Annelida and of the complex mechanisms of the immune system in vertebrates.

Cytotoxicity and Oxidative Stress Effects of Indene on Coelomocytes of Earthworm (Eisenia foetida): Combined Analysis at Cellular and Molecular Levels.

Indene (IND) is a kind of important aromatic hydrocarbon that is extracted from coal tar and has important applications in industry and biology. In the process of production and utilization, it is easy to enter the soil and produce toxic effects on the soil or organisms. The earthworm is an important organism in the soil. The toxicity of indene on earthworm coelomocytes is rarely studied, and the oxidative stress effects of IND on earthworm coelomocytes remain unclear. In this study, coelomocytes from earthworms and antioxidant enzymes were selected as the research targets. In addition, IND caused oxidative stress, and its related toxic effects and mechanisms were systematically studied and evaluated at the cellular and molecular levels. The results showed that IND destroyed the redox balance in earthworm coelomocytes, and the large accumulation of reactive oxygen species (ROS) significantly inhibited the activities of the antioxidant system, including superoxide dismutase (SOD), catalase (CAT), and glutathione (GSH), and caused lipid peroxidation and membrane permeability changes, resulting in a decrease in cell viability to 74.5% of the control group. At the molecular level, IND was bound to SOD by the arene-H bond, and the binding constant was 4.95 × 103. IND changed the secondary structure of the SOD and led to a loosening of the structure of the SOD peptide chain. Meanwhile, IND caused SOD fluorescence sensitization, and molecular simulation showed that IND was mainly bound to the junction of SOD subunits. We hypothesized that the changes in SOD structure led to the increase in SOD activity. This research can provide a scientific basis for IND toxicity evaluation.

Response to microplastic exposure: An exploration into the sea urchin immune cell proteome.

It is now known that the Mediterranean Sea currently is one of the major hotspot for microplastics (MPs; < 5 mm) pollution and that the risks will be even more pronounced in the coming years. Thus, the in-depth study of the mechanisms underlying the MPs toxicity in key Mediterranean organisms, subjected to high anthropic pressures, has become a categorical imperative to pursue. Here, we explore for the first time the sea urchins immune cells profile combined to their proteome upon in vivo exposure (72 h) to different concentrations of polystyrene-microbeads (micro-PS) starting from relevant environmental concentrations (10, 50, 103, 104 MP/L). Every 24 h, immunological parameters were monitored. After 72 h, the abundance of MPs was examined in various organs and coelomocytes were collected for proteomic analysis based on a shotgun label free proteomic approach. While sea urchins treated with the lowest concentration tested (10 and 50 micro-PS/L) did not show the presence of micro-PS in any tissue, in the specimens exposed to the highest concentration (103 and 104 micro-PS) there was an internalisation of 9.75 ± 2.75 and 113.75 ± 34.5 MP/g, respectively. Proteomic analyses revealed that MPs exposure altered coelomocytes protein profile not only compared to the control group but also among the different micro-PS concentrations and these variations are micro-PS concentration dependent. The proteins exclusively expressed in the coelomocytes of specimens exposed to MPs are mainly metabolite interconversion enzymes, involved in cellular processes, indicating a severe alteration of the cellular metabolic pathways. Overall, these findings provide new insights on the mode of action of MPs in the sea urchin immune cells both at the molecular and cellular level.

Transcriptomic profiling of immune-associated molecules in the coelomocytes of lugworm Arenicola marina (Linnaeus, 1758).

Organization and functioning of immune system remain unevenly studied in different taxa of lophotrochozoan animals. We analyzed transcriptomic data on coelomocytes of the lugworm Arenicola marina (Linnaeus, 1758; Annelida, Polychaeta) to gain insights into the molecular mechanisms involved in polychaete immunity. Coelomocytes are specialized motile cells populating coelomic fluid of annelids, responsible for cellular defense reactions and providing humoral immune factors. The transcriptome was enriched with immune-related transcripts by challenging the cells in vitro with lipopolysaccharides of Escherichia coli and Zymosan from Saccharomyces cerevisiae. Our analysis revealed a multifaceted and complex internal defense system of the lugworm. A. marina possesses orthologs of proto-complement-like factors: six thioester-containing proteins, a complement-like receptor, and a MASP-related serine protease (MReM2). A. marina coelomocytes employ pattern-recognition receptors to detect pathogens and regulate immune responses. Among them, there are 18 Toll-like receptors and various putative lectin-like proteins with evolutionary conserved and taxa-specific domains. C-type lectins and a novel family of Gal-binding and CUB domains containing receptors were the most abundant in the transcriptome. The array of pore-forming proteins in the coelomocytes was surprisingly reduced compared to that of other invertebrate species. We characterized a set of conserved proteins metabolizing reactive oxygen species and nitric oxide and expanded the arsenal of potential antimicrobial peptides. Phenoloxidase activity in immune cells of lugworm is mediated only by laccase enzyme. The described repertoire of immune-associated molecules provides valuable candidates for further functional and comparative research on the immunity of annelids.

Response pathways of superoxide dismutase and catalase under the regulation of triclocarban-triggered oxidative stress in Eisenia foetida: Comprehensive mechanism analysis based on cytotoxicity and binding model.

Triclocarban (TCC) is an emerging environmental contaminant, posing potential ecological risks. Displaying a high accumulation effect and 120-day half-life in the soil environment, the toxic effects of TCC to soil organisms have been widely reported. Previous studies have confirmed that TCC can induce the oxidative stress and changes in superoxide dismutase (SOD) and catalase (CAT) activities in earthworms, but the underlying mechanisms of oxidative stress and disorder in antioxidant enzyme activities induced by TCC have not yet been elucidated. Here, we explored the multiple response mechanisms of SOD and CAT under the regulation of oxidative stress induced by TCC. Results indicated that higher-dose (0-2.0 mg/L) TCC exposure triggered the overproduction of ROS in Eisenia foetida coelomocytes, causing oxidative damage and a decrease in cell viability that was response to ROS accumulation. The TCC-induced inhibition of intracellular SOD/CAT activity was found under the regulation of oxidative stress (SOD: 29.2 %; CAT: 18.5 %), and this effect was blunted by antioxidant melatonin. At the same time, the interaction between antioxidative enzymes and TCC driven by various forces (SOD: electrostatic interactions; CAT: van der Waals forces and hydrogen bonding) led to inhibited SOD activity (9.84 %) and enhanced CAT activity (17.5 %). Then, to elucidate the binding mode of TCC, we explored the changes in SOD and CAT structure (protein backbone and secondary structure), the microenvironment of aromatic amino acids, and aggregation behavior through multispectral techniques. Molecular docking results showed that TCC inhibited SOD activity in a substrate competitive manner and enhanced CAT activity by the stabilizing effects of TCC on the heme groups. Collectively, this study reveals the response mechanisms of SOD/CAT under the regulation of TCC-triggered oxidative stress and shed a new light on revealing the toxic pathways of exogenous pollutants on antioxidant-related proteins function.

Innovative in vivo and in vitro bioassays for the establishment of toxicity thresholds of pollutants in sediment quality assessment using polychaetes and their immune cells.

Sediment toxicity testing has become a crucial component for assessing the risks posed by contaminated sediments and for the development of sediment quality assessment strategies. Commonly used organisms for bioassays with estuarine sediments include amphipods, Arenicola marina polychaetes and echinoids. Among the latter, the Sea Urchin Embryo test (SET) is the most widely used. However, one relevant limitation of this bioassay is the unavailability of gametes all year-round, particularly outside the natural spawning seasons. Consequently, the establishment of an appropriate and complementary model organism for a continuous assessment of sediment quality is recommended. A reliable assessment of the hazards resulting from pollutants in sediments or pore water, can be achieved with ecologically relevant species of sediment such as the polychaete Hediste diversicolor, which is widespread in estuaries and has the capacity to accumulate pollutants. The aim of this work was to develop reliable in vivo and in vitro bioassays with H. diversicolor and its coelomocytes (immune cells) to determine the toxicity thresholds of different contaminants bounded to sediments or resuspended into water. Polychaetes were exposed to sublethal concentrations of CuCl2 (in vivo) and a non-invasive method for collection of polychaetes coelomocytes was applied for the in vitro bioassay, exposing cells to a series of CuCl2 and AgNPs concentrations. Same reference toxicants were used to expose Paracentrotus lividus following the SET (ICES Nº 51; Beiras et al., 2012) and obtained toxicity thresholds were compared between the two species. In vivo exposure of polychaetes to high concentrations of Cu produced weight loss and histopathological alterations. After in vitro approaches, a significant decrease in coelomocytes viability was recorded for both toxicants, in a monotonic dose-response curve, at very short-exposure times (2 h). The toxicity thresholds obtained with polychaetes were in line with the ones obtained with the SET, concluding that their sensitivity is similar. In conclusion, in vivo and in vitro bioassays developed with H. diversicolor are accurate toxicity screenings of pollutants that could be bounded to sediments or dissolved in the pore water, and may complement the SET outside the spawning period of the echinoderms. The bioassays herein developed could be applied not only to establish the toxicity thresholds of individual compounds or mixtures, but also to assess the toxicity of field collected sediments.