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Sea urchins are the best-known models in echinoderm immunology and only four coelomocytes – i.e. phagocytes, vibratile cells, and red and colorless spherulocytes – have traditionally been observed in these animals. However, recent studies have demonstrated that this number could be underestimated, which consequently may hinder a better understanding of sea urchin physiology. Here, we investigated the cells of one of the most known sea urchin genera in the world: the genus Paracentrotus. This genus comprises two species with distinct geographic distributions, thus providing an excellent scenario for comparative analyses. In this context, through an integrative approach consisting of living cells, cytological preparations, and scanning electron microscopy (SEM), we analyzed morphological, morphometric, and cytochemical characteristics of the coelomocytes of Paracentrotus lividus (Lamarck, 1816) and Paracentrotus gaimardi (Blainville, 1825). Seven different coelomic subpopulations were found in these species, including the traditional types and the granular spherulocyte, crystal cell, and progenitor cell. For all spherulocyte subpopulations, a set of morphologically similar cells were observed in both cytological and SEM preparations. Based on morphology, morphometry, and cytochemistry, we observed that these morphotypes could be organized in a sequence, which we interpreted as a maturation process. Then, we raise one hypothesis that explains how spherulocytes mature. Lastly, taking into account the all results achieved by this study, we discuss (1) how the morphological, morphometric, and cytochemical characteristics observed in Paracentrotus coelomocytes may be used to identify the coelomocytes of other sea urchins; (2) the physiological implications of our findings. Considering that P. lividus is one of the most studied sea urchins in the world, the new findings obtained here may shed new light on traditional aspects of sea urchin immunology (e.g. coelomocytes function), as well as stimulate research on new aspects of Echinoidea and even in Echinodermata immunology. 1 – The coelomocytes of the only two species belonging to the genus Paracentrotus were studied. 2 – The integrative approach used here allowed the identification of three new cell types. 3 – Morphological, morphometric, and cytochemical data revealed a putative maturation process for spherulocytes. 4 – The data obtained for Paracentrotus sea urchins will be useful to identify coelomocytes in other echinoids. [ABSTRACT FROM AUTHOR] more...

This study examined the effects of lab-generated acoustic signals on the behaviour and biochemistry of Mediterranean mussels ( Mytilus galloprovincialis ). The experiment was carried out in a tank equipped with a video-recording system using six groups of five mussels exposed to five acoustic treatments (each treatment was replicated three times) for 30 min. The acoustic signals, with a maximum sound pressure level of 150 dB rms re 1 μPa, differed in frequency range as follows: low (0.1–5 kHz), mid-low (5–10 kHz), mid (10–20 kHz), mid-high (20–40 kHz) and high (40–60 kHz). The exposure to sweeps did not produce any significant changes in the mussels' behaviour. Conversely, the specimens exposed to the low frequency band treatment showed significantly higher values of the following biochemical stress parameters measured in their plasma and tissues: glucose, total proteins, total haemocyte number (THC), heat shock protein 70 (Hsp70) expression, and Acetylcholinesterase (AChE) activity. The responses observed in the mussels exposed to low frequency sweeps enable us to suppose a biological and ecological role for this sound, which contains the main frequencies produced by both shipping traffic and the acoustic emissions of fish. [ABSTRACT FROM AUTHOR] more...

Highlights: [•] This study evaluated the impact of boat noise on lobsters (Palinurus elephas). [•] Effects on lobsters were assessed using behavioural and biochemical parameters. [•] High levels of locomotor activity were observed in lobsters exposed to boat noise. [•] Exposure to noise produced significant high levels of haemolymphatic stress indexes. [ABSTRACT FROM AUTHOR] more...

Abstract: In the immune system of vertebrates, gender-specific differences in individual immune competence are well known. In general, females possess more powerful immune response than males. In invertebrates, the situation is much less clear. For this purpose we have chosen to study the immune response of the two sexes of the echinoderm Paracentrotus lividus in pre- and post-spawning phases. The coelomic fluid from the echinoderms contains several coelomocyte types and molecules involved in innate immune defenses. In this article we report that the degree of immune responses in the P. lividus differs according to sex in both pre- and post-spawning phases. We found in all tests that females were more active than males. The results indicate that females possess a significant higher number of immunocytes consisting of phagocytes and uncolored spherulocytes. Since the immunological activity is mainly based on immunocytes, it was not surprising that females possessed the highest values of cytotoxicity and hemolysis activity and showed a greater ability to uptake neutral red and phagocyte yeasts cells, while the average number of ingested particles per active phagocyte was not significantly different. Furthermore, agglutinating activity was more evident in the coelomocyte lysate and coelomic fluid of females than in those of males. Finally we found that the acidic extract of female gonads possessed greater antimicrobial activity than that of male gonads. These results make it very likely that gender differences in the immune response are not restricted to vertebrates; rather, they are a general evolutionary phenomenon. [Copyright &y& Elsevier] more...

Abstract: Stressful conditions include a prompt release of corticosteroid hormones which can mediate gene expression through glucocorticoid receptors (GR). Since two seabass (Dicentrarchus labrax) GRs have been cloned and sequenced from peritoneal cavity cells (DlGR1) and liver (DlGR2), a comparative amino acid sequence analysis that included Haplochromis burtoni HbGRs, was carried out and homologies disclosed. The DlGR1 and DlGR2 deduced aminoacid sequences showed 61% identity (I) and 70% similarity (S). Moreover, DlGR2 was similar to HbGR2b (69% I, 73% S), and the DlGR1 to HbGR1 (72% I, 78% S). In addition, we examined the expression of the DlGRs after exogeneous cortisol inoculation into the peritoneal cavity, mimicking stress effects. At various times after the administration (3h, 24h, 1week), gene expressions was evaluated in head kidney by real-time PCR. In addition, immunoblotting and densitometry analyses were performed with anti-DlGR1 antibodies. Although sea bass head kidney expressed both DlGR1 and DlGR2 they were differentially modulated by intraperitoneal implant of exogeneous cortisol. [Copyright &y& Elsevier] more...

Marine life is extremely sensitive to the effects of environmental noise due to its reliance on underwater sounds for basic life functions, such as searching for food and mating. However, the effects on invertebrate species are not yet fully understood. The aim of this study was to determine the biochemical responses of Arbacia lixula exposed to high-frequency noise. Protein concentration, enzyme activity (esterase, phosphatase and peroxidase) and cytotoxicity in coelomic fluid were compared in individuals exposed for three hours to consecutive linear sweeps of 100 to 200 kHz lasting 1 s, and control specimens. Sound pressure levels ranged between 145 and 160 dB re 1μPa. Coelomic fluid was extracted and the gene and protein expression of HSP70 with RT-PCR was evaluated on coelomocytes. A significant change was found in enzyme activity and in the expression of the HSP70 gene and protein compared to the control. These results suggested that high-frequency stimuli elicit a noise-induced physiological stress response in A. lixula , confirming the vulnerability of this species to acoustic exposure. Furthermore, these findings provide the first evidence that cell-free coelomic fluid can be used as a signal to evaluate noise exposure in marine invertebrates. Unlabelled Image • A. lixula was used as a model to investigate the impact of anthropogenic noise pollution. • The high frequency noise, from 100 to 200 kHz, interferences on homeostasis of sea urchin. • Cell-free coelomic fluid can be used as a matrix to evaluate noise exposure in marine invertebrates. [ABSTRACT FROM AUTHOR] more...

Sulfamethoxazole (SMZ) is one of the most widely employed sulfonamides. Because of the widespread use of SMZ, a considerable amount is indeed expected to be introduced into the environment. The cytotoxicity of SMZ relies mainly on arylhydroxylamine metabolites (S-NOH) of SMZ and it is associated with the production of reactive oxygen species (ROS). There is limited information about the toxic potential of SMZ at the cellular and molecular levels, especially in aquatic and/or non-target organisms. In the present study, the red swamp crayfish ( Procambarus clarkii ), being tolerant to extreme environmental conditions and resistant to disease, was used as a model organism to profile the molecular and physiological response to SMZ. Haemolymphatic–immunological parameters such as glucose serum levels and total haemocyte counts were altered; moreover, a significant increase in Hsp70 plasma levels was detected for the first time. Variations at the transcriptional level of proinflammatory genes (cyclooxygenase-1, COX 1, and cyclooxygenase-2, COX 2 ), antioxidant enzymes (glutathione- S -transferase, GST and manganese superoxide dismutase MnSOD ), stress response and Fenton reaction inhibitor genes (heat-shock protein 70 HSP70, metallothionein, MT and ferritin, FT ) were evaluated, and alterations in the canonical gene expression patterns emerged. Considering these results, specific mechanisms involved in maintaining physiological homeostasis and adaptation in response to perturbations are suggested. [ABSTRACT FROM AUTHOR] more...

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. [Display omitted] • Physiological responses of the perivisceral coelom and tissue are used to study wound healing in Holothuria grisea. • Coelomic fluid responses begin at 3 h and returns to basal levels at 24 h. • Tissue responses begin at 12 h and does not return to basal level until day 10. • Cell cooperation for homeostasis and tissue remodeling may be observed. • Perivisceral coelom and tissue cooperate to keep homeostasis during wound healing in H. grisea. [ABSTRACT FROM AUTHOR] more...

Summary: Based on previous cloning and sequencing study, real-time PCR and in situ hybridization assays of the inflamed body wall of LPS-injected Ciona intestinalis showed the enhanced gene expression of a collagen with FACIT structural features (Ci-type IX-Col 1α-chain). By using specific antibodies raised against an opportunely chosen Ci-type IX-Col synthetic peptide, the fibroblast property of hemocytes challenged in vitro with LPS (at 4h) was displayed by flow cytometry, while immunocytochemistry identified hemocytes with large granules (morula cells) as collagen-producing cells. Hemocyte lysate supernatant analyzed in immunoblotting contained a 60kDa band identifiable as 1α-chain-Ci-type IX-Col. Observations of body wall sections (immunohistochemistry method) supported the role of hemocytes and showed that epidermis expressed Ci-type IX-Col 1α-chain in the time course of the inflammatory reaction (within 24h). Transcript and protein were mainly found in the epidermis that outlined the proximal side of the tunic matrix (at 24h after LPS injection), in cells associated with the epidermis at 4 and 192h. In conclusion, the C. intestinalis inflammatory response to LPS challenge appeared to be composed of a complex reaction set, and for the first time we showed in ascidians a granulation tissue with FACIT-collagen production that could participate in inflammation and wound healing. Like in vertebrates, C. intestinalis acute inflammatory reactions result in a regulated pattern of tissue repair with collagen expression during remodelling. Ci-type IX-Col could be involved in a network of non-fibril-forming collagens that participates in the organization of extracellular matrix and defense responses. [Copyright &y& Elsevier] more...

Different bioactive molecules extracted from macroalgae, including oxylipins, showed interesting potentials in different applications, from healthcare to biomaterial manufacturing and environmental remediation. Thus far, no studies reported the effects of oxylipins-containing macroalgae extracts on embryo development of marine invertebrates and on neuroblastoma cancer cells. Here, the effects of an oxylipins-containing extract from Ericaria brachycarpa, a canopy-forming brown algae, were investigated on the development of Arbacia lixula sea urchin embryos and on SH-SY5Y neuroblastoma cells viability. Embryos and cells were exposed to concentrations covering a full 0–100% dose-response curve, with doses ranging from 0 to 40 μg mL−1 for embryos and from 0 to 200 μg mL−1 for cells. These natural marine toxins caused a dose-dependent decrease of normal embryos development and of neuroblastoma cells viability. Toxicity was higher for exposures starting from the gastrula embryonal stage if compared to the zygote and pluteus stages, with an EC 50 significantly lower by 33 and 68%, respectively. Embryos exposed to low doses showed a general delay in development with a decrease in the ability to calcify, while higher doses caused 100% block of embryo growth. Exposure of SH-SY5Y neuroblastoma cells to 40 μg mL−1 for 72 h caused 78% mortality, while no effect was observed on their neuronal-like cells derivatives, suggesting a selective targeting of proliferating cells. Western Blot experiments on both model systems displayed the modulation of different molecular markers (HSP60, HSP90, LC3, p62, CHOP and cleaved caspase-7), showing altered stress response and enhanced autophagy and apoptosis, confirmed by increased fragmented DNA in apoptotic nuclei. Our study gives new insights into the molecular strategies that marine invertebrates use when responding to their environmental natural toxins and suggests the E. brachycarpa 's extract as a potential source for the development of innovative, environmentally friendly products with larvicide and antineoplastic activity. [Display omitted] • Seven oxylipins were identified in extract of seaweed Ericaria brachycarpa (EBE). • Stage-specific EBE effect was found in developing Arbacia lixula sea urchin embryos. • EBE was toxic for neuroblastoma cells and not for the differentiated counterpart. • Autophagy and apoptosis were induced by EBE in embryos and neuroblastoma cells. • Expression levels of heat shock proteins were modulated by EBE in embryos and cells. [ABSTRACT FROM AUTHOR] more...

In this study, 1029 fish and cephalopod samples came from Central-Western Mediterranean (FAO 37.1.1 and FAO 37.1.3) were analysed for Anisakidae larvae research with the aim to identify possible hybridisations between Anisakis pegreffii and Anisakis simplex s.s. species. A total of 1765 larvae were detected, with prevalence values between 8.1% and 100%. The morphologic analysis revealed characters attributable to morphotype I of Anisakis in 98.5% of the examined larvae, while 1.5% belonged to the morphotype II. PCR-based Restriction Fragment Length Polymorphism (PCR–RFLP) analysis of the entire ITS region (ITS1, 5.8S and ITS2) of nuclear ribosomal DNA (rDNA) was performed with Hinf I and Hha I restriction enzymes. The majority of the larvae examined by PCR-RFLP were identified as A. pegreffii (71%), with a prevalence on horse mackerel from FAO 37.1.3, while 10% were identified as A. simplex s.s., 2% as A. physeteris and 17% as A. pegreffii × A. simplex s.s. hybrid genotype. The sequence analysis confirmed the hybridisation in the 85% of the larvae recognised as hybrid forms by PCR- RFLP, suggesting this form as the product of natural interspecific recombination due to the presence of sympatry areas. The presence of hybrid forms were mostly found in fish samples from FAO subzone 37.1.1. This is the first report of A. pegreffii x A. simplex s.s. hybrid genotype in fishes caught off the coasts of Sicily (Southern Italy). Finally, this study provided substantial information about the geographical distribution of Anisakidae family in Central-Western Mediterranean Sea. [ABSTRACT FROM AUTHOR] more...

The much-publicised increased resistance of pathogenic bacteria to conventional antibiotics has focused research effort on the characterization of new antimicrobial drugs. In this context, antimicrobial peptides (AMPs) extracted from animals are considered a promising alternative to conventional antibiotics. In recent years, freshwater crayfish species have emerged as an important source of bioactive compounds. In fact, these invertebrates rely on an innate immune system based on cellular responses and on the production of important effectors in the haemolymph, such as AMPs, which are produced and stored in granules in haemocytes and released after stimulation. These effectors are active against both Gram-positive and Gram-negative bacteria. In this review, we summarise the recent progress on AMPs isolated from the several species of freshwater crayfish and their prospects for future pharmaceutical applications to combat infectious agents. [Display omitted] • Antimicrobial peptides (AMPs), a potential natural alternative to conventional antibiotics. • Freshwater crayfish as an important source of AMPs. • Potential applications of AMPs isolated from freshwater crayfish. [ABSTRACT FROM AUTHOR] more...

Wounds in living organisms trigger tissue-repair mechanisms. The sea cucumber (Holoturia tubulosa) is an excellent model species for achieving a better understanding of the humoral and cellular aspects involved in such healing processes. Consequently, this study assesses data on its morphometric, physiological and humoral responses 1, 2, 6, 24 and 48h after wound induction. In particular, morphometric data on the weight, width, length and coelomic-fluid volume of the species were estimated at different times during our experiments. In addition, the humoral aspects related to the enzymatic activity of esterase, alkaline phosphatase and peroxidase, as well as the cytotoxic activity of cell lysates (CL) and cell-free coelomic fluids (CfCf) are evaluated for the first time. Our results reveal a significant decrease in body length and weight, along with time-dependent, significant changes in the esterase, alkaline phosphatase, peroxidase and cytotoxic activity in both the CL and CfCf. The data obtained lead to the pioneering finding that there is an important time-dependent involvement of morphometric (changes in weight and length) and humoral (enzymatic and cytotoxic) responses in wound healing. Unlabelled Image • Holothuria tubulosa as a model organism to study wound healing. • Humoral responses after cut at different experimental times. • Time-dependent significant change in enzyme activity. [ABSTRACT FROM AUTHOR] more...

• Complete Allograft inflammatory factor 1 protein sequence and structure • AIF-1 and early immune response • AIF-1 immunomodulation response to Gram- LPS treatment Echinoderms are a phylum of deuterostomic invertebrates that play a key role in maintaining the biodiversity of marine ecosystems. They represent a good study model for immunity because their coelomic fluid contains different types of cells involved in the inflammatory response: the coelomocytes. In the case of the sea urchin Paracentrotus lividus , the coelomocyte population is mainly represented by amoebocytes and uncoloured spherulocytes that implement a defence program through phagocytosis, encapsulation, cytotoxicity, and production of antimicrobial agents. The purpose of this study was to find evidence of a possible modulating effect of lipopolysaccharide LPS on the expression of the AIF-1 gene and therefore to determine whether or not there is a correlation between the P. lividus immune response and the expression of this gene when the homeostasis of the animals is disturbed by a bacterial infection which, in this case, was simulated with treatment with LPS. AIF-1 (Allograft Inflammatory Factor 1) is a 17 kDa calcium-binding protein that, in vertebrates, is involved in the activation of macrophages. Similarly, in Echinoids and in general in invertebrates, the expression of this gene increases considerably after a bacterial attack and this suggests that it plays a key role during the immune responses. [ABSTRACT FROM AUTHOR] more...

Among the diseases that afflict the human population, cancer is one for which many drug treatments are not yet known or effective. Moreover, the pharmacological treatments used often create serious side effects in sick patients and for this reason, it is essential to find effective and less harmful treatments. To date, marine biodiversity is a real source of metabolites with antitumoral activity and among invertebrates' ascidians have been the main source to obtain them. Mediterranean area is the richest in biodiversity and contains several ascidian species used in drugs development during the years. However, many more Mediterranean ascidian species have not been studied and could be a source of useful bioactive compounds. This review aims to summarize the scientific studies that analyzed the antitumor compounds obtained from different Mediterranean ascidians species, encouraging them to search further compounds in other new species to improve pharmacological treatments and human population life. • Antitumoral compounds from Mediterranean ascidian extracted to date. • Baseline to search further compounds in other species to improve pharmacological treatments. • Mediterranean ascidian species as a useful source of bioactive compounds for incurable cancers. [ABSTRACT FROM AUTHOR] more...