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.

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.

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.

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.

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.

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.

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.

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.

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.

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.