Localizzazione di un recettore glucocorticoideo DLGR1 nei tessuti del teleosteo Dicentrarchus labrax

Bacillus thuringiensis treatment modulate the HSP70 expression in larva and adult brain of Rhynchophorus ferrugineus

To study the pathogen-host relationship, we used the model of the entomopathogenic bacterium Bacillus thuringiensis (Bt) and Rhynchophorus ferrugineus, a quarantine pest that attacks palm trees. In particular, we focused on the Bt stress-induced infections. We studied the effect of Bt on larval and adult growth, and on the expression of the heat shock proteins (HSPs), rapidly synthesized in the cell after exposure to stress including pathogens. Bt has negative effects on larval and adult growth, on total hemocytes counts and on the hemocyte type. HSP70 was evaluated in the supernatant of the brain lysate obtained from larvae and adults fed with sublethal doses of Bt. HSP70 expression was mo…

In vitro effect of cadmium and copper on separated blood leukocytes of Dicentrarchus labrax.

The immunotoxic effects of heavy metals on blood leukocytes of sea bass (Dicentrarchus labrax) were examined. The cells, separated by a discontinuous Percoll-gradients, were exposed in vitro to various sublethal concentrations of cadmium and copper (10(-7) M, 10(-5) M, and 10(-3) M) and their immunotoxic effect was then evaluated by measuring neutral red uptake, MU assay, DNA fragmentation and Hsp70 gene expression. First of all, we demonstrated that the cells treated in vitro could incorporate Cd and Cu. A relationship between heavy metal exposure and dose-time-dependent alterations in responses of leukocytes from blood was found for both metals, but copper was more immunotoxic than cadmiu…

Cytotoxic activity of Holothuria tubulosa (Echinodermata) coelomocytes.

Abstract The immune system of marine invertebrates, in particular that of holothurians, still requires further study. Our research showed that coelomocyte cells contained in the coelomic fluid of the sea cucumber, Holothuria tubulosa, are able to lyse, in vitro, red blood cells in rabbits and sheep. A plaque-forming assay showed spherule cells to be the effector cells, able to release cytotoxic molecules after xenogenic cell contact. The coelomocyte lysate supernatant, analysed by polyacrylamide gel electrophoresis overlay technique, using rabbit and sheep erythrocytes, showed two different haemolytic protein patterns: one calcium dependent and the other calcium independent. The fractions o…

Modulazione In vivo dell’Hsp70 negli emociti di Rhynchophorus ferrugineus dopo trattamento con Bacillus thuringiensis

Diversi tipi di stress ambientali possono indurre l’espressione delle Hsps vengono rapidamente sintetizzate all'interno delle cellule dopo esposizione ad uno stressor. Le HSPs sono raggruppate in diverse famiglie in base alle dimensioni delle proteine. Nella maggior parte degli organismi ci sono diversi geni che codificano i membri di questa famiglia HSP. In studi precedenti è stata valutata l'interazione tra Bacillus thuringiensis e gli emociti circolanti di Rhynchophorus ferrugineus il punteruolo rosso delle palme. Questo coleottero è grande, da due a cinque centimetri di lunghezza, ed è di colore rosso ruggine, le sue larve scavano buche fino a un metro lungo il tronco delle palme, e può…

Cytotoxic Potential of the Coelomic Fluid Extracted from the Sea Cucumber Holothuria tubulosa against Triple-Negative MDA-MB231 Breast Cancer Cells

Growing evidence has demonstrated that the extracts of different holothurian species exert beneficial effects on human health. Triple negative breast cancers (TNBC) are highly malignant tumors that present a poor prognosis due to the lack of effective targeted therapies. In the attempt to identify novel compounds that might counteract TNBC cell growth, we studied the effect of the exposure of the TNBC cell line MDA-MB231 to total and filtered aqueous extracts of the coelomic fluid obtained from the sea cucumber Holoturia tubulosa, a widespread species in the Mediterranean Sea. In particular, we examined cell viability and proliferative behaviour, cell cycle distribution, apoptosis, autophag…

Immune cells from sea urchins for the analysis of the effects of UV-B radiation

Immune cells from sea urchins for the analysis on the effects of UV-B radiation.

Components of hemocyte extracts from marine invertebrates exert antimicrobial activity

Effects of exogenous cortisol on the expression of glucocorticoid receptor (DIGR1) and hsp70 in head-kidney and peritonel cavity cells from Dicentrarchus labrax.

Effetti del cadmio sull'espressione delle HSP70 nelle cellule del sangue di Spigola (Dicentrachus labrax L. Osteichthyes, Moronidae)

Noise elicits hematological stress parameters in Mediterranean damselfish (Chromis chromis, perciformes): A mesocosm study

In the last few decades, technological developments and the widespread rise of anthropic activities have increased the exposure of organisms to noise pollution, thus evoking great interest in its biological effects, particularly on the immune system. The aim of the present work was to investigate some of the biochemical parameters in the blood of Chromis chromis (Linnaeus, 1758) following in vivo exposure to noise levels of 200 and 300 Hz. Our results revealed that, compared to the control specimens, the fish exposed to noise had significantly increased levels of stress biomarkers such as glucose, lactate and total proteins in plasma, as well as a rise in the expression of heat shock protei…

Behavioural and biochemical stress responses of Palinurus elephas after exposure to boat noise pollution in tank

This study examined the effects of boat noise on the behavioural and biochemical parameters of the Mediterranean spiny lobster (Palinurus elephas).The experiment was conducted in a tank equipped with a video and audio recording system. 18 experimental trials, assigned to boat noise and control conditions, were performed using lobsters in single and group of 4 specimens. After a 1. h habituation period, we audio- and video-recorded the lobsters for 1. h. During the experimental phase, the animals assigned to the boat groups were exposed to boat noise pollution (a random sequence of boat noises). Exposure to the noise produced significant variations in locomotor behaviours and haemolymphatic …

Effetti dell’esposizione a cadmio e rame sull’attività di cellule del sangue di spigola (Dicentrarchus labrax L. Osteichthyes, Moronidae).

Cadmium, Copper and Tributyltin effects on fertilization of Paracentrotus lividus (Echinodermata)

Marine environments are continuously being threatened by a large number of xenobi- otics from anthropogenic sources. The effect of chemical pollution on living organisms are numerous and may impair reproductive success of adults species of marine invertebrate and vertebrate through effects on gamete quality. Echinoderms are characterized by external fertilizzation and gametes, free of any type of protection, may be in contact with toxic substances so the reproductive success depends largely on the environment conditions. The purpose of this work is to assess the effects on the in vitro fertiliza- tion of exposure of sea urchin Paracentrotus lividus gametes to xenobiotic substances as CuSO4,…

Invertebrate lectins present cytokine properties.

Invertebrate lectins present cytockine properties.

Expression and distribution of the glucocorticoid receptor DlGR1 in the teleost Dicentrarchus labrax brain

Cortisol is the main corticosteroid secreted by the interrenal cells of the head kidney and it exerts a role in mantaining the omeostatic status in fish. In teleosts its effects are mediated through intracellular receptors expressed in several tissues, that are ligand-dependent transcription factors by binding to specific tissue DNA sequences. In Dicentrarchus labrax we previously cloned and sequenced a glucocorticoid receptor, DlGR1, isolated from leukocytes of peritoneal cavity. In this work we showed mRNA expression and tissue immunohistochemical localization of brain DlGR1 by in situ hybridization assays, with a riboprobe with DlGR1 cDNA trascriptional activation domain, and by immunohi…

Effects of waterg gun on echinoderms: biochemical changes on sea urchin (Arbacia lixula) and sea cucumber (Holothuria tubulosa)

Sources Airguns and Waterguns are used in seismic exploration the marine seafloor for different purposes such as oil and gas search, or geological-geodynamic reconstruction. In this technique a bubble of compressed air is produced with a consequently production of loud sound wave penetrating the ocean floor. The impact of seismic Airguns was assessed in some cetaceans (Kyhn et al. 2019), fish species and invertebrate (Carroll et al. 2017), but we need to know more. In this study we evaluate the biochemical changes in sea urchin (Arbacia lixula) and sea cucumber (Holothuria tubulosa) exposed to a 20 minutes of watergun noise (1 pulse each 10 s) at a distance of 50 m. For each species 40 indi…

Unità operativa "biotassonomia"

Vessel noise pollution as a human threat to fish: assessment of the stress response in gilthead sea bream (Sparus aurata, Linnaeus 1758)

This study examined the effects of boat noise pollution on the stress indices of gilthead sea bream (Sparus aurata, Linnaeus 1758). To assess the stress response in these fish, biometric values and plasma parameters such as ACTH, cortisol, glucose, lactate, haematocrit, Hsp70, total protein, cholesterol, triglycerides and osmolarity were analysed. After acclimatization of the animals, the experiment was carried out in a tank fitted with underwater speakers where the fish were exposed to sound treatments (in duplicate) consisting of: 10 days of no sound (control treatment; the animals were only exposed to the experimental tank’s background noise) and 10 days of noise derived from original re…

Monitoring chemical and physical stress using sea urchin immune cells.

Coelomocytes are the cells freely circulating in the body fluid contained in echinoderm coelom and constitute the defence system, which, in response to injuries, host invasion, and adverse conditions, is capable of chemotaxis, phagocytosis, and production of cytotoxic metabolites. Red and colourless amoebocytes, petaloid and philopodial phagocytes, and vibratile cells are the cell types that, in different proportions, constitute the mixed coelomocyte cell population found in sea urchins. Advances in cellular and molecular biology have made it possible to identify a number of specific proteins expressed in coelomocytes under resting conditions or when activated by experimentally induced stre…

Sea urchin cells and embryos as tools for bio-monitoring studies.

Effects of Sulfamethoxazole on Fertilization and Embryo Development in the Arbacia lixula Sea Urchin

To date, drugs released into the aquatic environment are a real problem, and among antibiotics, sulfamethoxazole is the one most widely found in wastewater; thus, the evaluation of its toxicity on marine organisms is very important. This study, for the first time, investigates the in vitro effects of 4 concentrations of sulfamethoxazole (0.05 mg/L, 0.5 mg/L, 5 mg/L, 50 mg/L) on the fertilization and development of the sea urchin Arbacia lixula. The gametes were exposed to drugs in three different stages: simultaneously with, prior to, and post-fertilization. The results show a significant reduction in the percentage of fertilized oocytes at the highest drug concentrations. Moreover, an incr…

Biotecnologie e monitoraggio ambientale: sviluppo e applicazione di biomarcatori molecolari.

Cellular responses and HSP70 expression during wound healing in Holothuria tubulosa (Gmelin, 1788).

Wound repair is a key event in the regeneration mechanisms of echinoderms. We studied, at the behavioural, cellular and molecular levels, the wound healing processes in Holothuria tubulosa after injuries to the body wall. The experiments were performed for periods of up to 72 h, and various coelomocyte counts, as well as the expression of heat shock proteins (HS27, HSP70 and HSP90), were recorded. Dermal wound healing was nearly complete within 72 h. In the early stages, we observed the injured animals twisting their bodies to keep their injuries on the surface of the water for the extrusion of the buccal pedicles. At the cellular level, we found time-dependent variations in the circulating…

Are mussels able to distinguish underwater sounds? Assessment of the reactions of Mytilus galloprovincialis after exposure to lab-generated acoustic signals

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 expos…

Profiling the physiological and molecular response to sulfonamidic drag in Procambarus clarkii

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 molecula…

Welfare status of cage farmed European sea bass (Dicentrarchus labrax): A comparison between submerged and surface cages

Abstract Submersible sea-cages represent an alternative method to standard surface-based cages, and could help solve several production problems that exist in surface-based farming, such as heavy storms, algal and jellyfish blooms and attacks by predators. In this study, we investigated growth, haematological, biochemical and immunological parameters of European sea bass ( Dicentrarchus labrax ) farmed in surface and submersible cages, to compare the physiological status under different rearing conditions. In addition, we evaluated the use of erythrocyte HSP70 levels as a biomarker of fish welfare. The study was conducted in two submerged and two surface cages filled with D. labrax . No sig…

Parametri sierologici e cellulari della specie alloctona Procambarus clarckii

Procambarus clarkii è un animale d’acqua dolce originario del Centro-Sud degli Stati Uniti e del Nord-Est del Messico. Questa specie rientra nella lista delle 100 specie invasive più pericolose al mondo (Delivering Alien Invasive Species Inventories for Europe) in quanto presenta una serie di caratteristiche biologiche, che la rendono una minaccia per la biodiversità. In Sicilia la sua presenza è stata registrata per la prima volta nel 2003 nella riserva naturale “Lago Preola e Gorghi Tondi”, Mazara del Vallo (TP). Allo scopo di conoscere meglio la biologia di questa specie, su esemplari di P. clarkii provenienti dalla riserva di Trapani, abbiamo esaminato i tipi emocitari ed alcune attivit…

Differential expression of two glucocorticoid receptors in seabass (teleost fish) head kidney after exogeneous cortisol inoculation

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 perit…

Stress and apoptosis in sea urchin coelomocytes short term cultures exposed to UV-B radiation

Effect of in vitro exposure to cadmiumand copper on sea bass blood cells

Blood cells freshly collected from sea bass (Dicentrarchus labrax) were exposed in vitro to different concentrations of cadmium (Cd) and copper (Cu) at 10-7 M, 10-5 M, 10-3 M, and exam- ined for neutral red retention capacity and for cell vitality with MTT assay. A relationship between heavy metal exposure and alteration in responses of blood cells in a dose-time-dependent was found. Our results showed that fish blood cells may constitute an interesting biological model for experimen- tal and applied toxicology, especially in the case of environmental pollution.

VALUTARE I SERVIZI: BUONE PRASSI NEL LAVORO DI RETE

Expression of a glucocorticoid receptor (D1GR1) in several tissues of the teleost fish Dicentrarchus labrax

Since glucocorticoids have a role in maintaining the homeostatic status in fish, in the present paper mRNA expression (in situ hybridization) and tissue immunohistochemical localization of a glucocorticoid receptor (DlGR1) in several Dicentrarchus labrax organs are reported. Riboprobe and specific antibodies were prepared by using the DlGR1 that has been previously cloned and sequenced from peritoneal cavity leukocytes. Both mRNA and receptor were identified in head kidney, spleen, gills, intestine, heart and liver tissues. The functional roles of DlGR1 localization are discussed.

Effetti modulanti del cadmio e del rame sulle attività cellulari del teleosteo Dicentrarchus labrax

Elevated cortisol modulates hsp70 and hsp90 gene expression and protein in sea bass head kidney and isolated leukocytes

In fish, interactions between Hsps and cortisol are involved in stress modulated physiological processes including innate immune responses. Cortisol exerts a role in the regulation of Hsps synthesis. Fish head kidney is a lymphomieloid and endocrine organ releasing cortisol, and it is the central organ for immune-endocrine interactions. In sea bass, cortisol intraperitoneal injection and in vitro treatment of head kidney cells show that inducible Hsp70 and Hsp90 are modulated by this hormone. However, an inverse relationship between mRNA expression (real-time PCR) and Hsp70 and Hsp90 protein levels (densitometric band analysis) was found. Time-course assays indicate a cortisol-mediated regu…

Does the immune answer to Bacillus thuringiensis infection is the same in larvae, females and males of Rhynchophorus ferrugineus?

Bacillus thuringiensis (Bt) è considerato un potenziale batterio entomopatogeno per la lotta al Rhynchophorus ferrugineus, coleottero da quarantena infestante le palme. In questo lavoro si considerano gli effetti dell’infezione di Bt in larve, maschi e femmine del punteruolo rosso. La patogenicità è stata valutata stimando la LD50 (Lethal Median Dose) e la LT50 (Lethal Median Time), il numero totale di emociti ed il tipo di emociti (Differential Haemocytes counts) ed inoltre l’espressione delle proteine Heat Shock Protein 70 (HSP70) negli emociti e nel cerebro. La mortalità di entrambi i sessi e delle larve aumentava all’aumentare della dose e del tempo di esposizione. Tuttavia le larve han…

Does Bacillus thuringiensis Affect the Stress and Immune Responses of Rhynchophorus ferrugineus Larvae, Females, and Males in the Same Way?

Simple Summary Rhynchophorus ferrugineus is a destructive quarantine pest of palm trees, now widely distributed. Although broad-spectrum insecticides are often used to protect palm against R. ferrugineus, there is increasing concern about their effects on the environment and human health, especially where palm trees are located in urban areas. As an environmentally friendly entomopathogen, Bacillus thuringiensis (Bt) has been widely used to prevent other pest infestations. Although Bt products are the most sold bio-insecticides, there are still many interesting features to be investigated in the relationship of Bt and its hosts. We investigated the effect of Bt on larvae, females, and males…

Bacillus thuringiensis treatment alters larval growth, hemocytes and modulation of Hsp70 in Rhynchophorus ferrugineus

To study the pathogen-host relationship, we used the model of the entomopathogenic bacterium Bacillus thuringiensis (Bt) and Rhynchophorus ferrugineus, a quarantine pest that attacks the palm trees. In particular, we focused on the Bt stress-induced infections. We studied the effect of Bt on larval growth, on hemocytes and on the expression of the heat shock proteins (Hsp70). HSPs are rapidly synthesized in the cells after a stress exposition including pathogens. The Hsp70 was evaluated in the supernatant of the hemocyte lysate (HLS) obtained from larvae fed with Bt. This is the first time that the presence of Hsp70 has been recorded in R. ferrugineus. Bt has negative effects on larval grow…

Shipping noise affecting immune responses of European spiny lobster (Palinuruselephas)

Marine crustaceans are influenced by numerous environmental factors. Environmental stress from pollutants seems to affect their metabolism, growth, moulting, survival, and immune defence. Recently, it has become clear that there is an impact of the global increase in sea noise levels due to shipping traffic on crustacean welfare. Considering the ecological and commercial importance of the European spiny lobster (Palinurus elephas (Fabricius, 1787)) in most parts of the Mediterranean coastal area, in the present study we investigated whether shipping noise pollution contribute to changing the immune parameters of stress in the European spiny lobster. The animals were exposed to a mix of nois…

Impacts of UV-B radiation on short-term cultures of sea urchin coelomocytes

Three specialized cell types constitute the heterogeneous population present in the coelomic cavity fluid of sea urchins. The list includes: phagocytes, which undergo a stress-induced petaloid-filopodial transition, white or red amoebocytes and vibratile cells. As a whole, they act as the immune defense system of the sea urchin and respond to environmental and experimental challenge triggering specific stress markers. Here we extended our studies on coelomocytes short-term cultures by describing the morphology and occurrence of each cell type and analyzing their response to UV-B radiation at the biochemical level and with respect to DNA damage. The effects of different doses, ranging from 5…

Physiological and agonistic behavioural response of Procambarus clarkii to an acoustic stimulus

Summary This study examined the effects of an acoustic stimulus on the haemolymph and agonistic behaviour of the red swamp crayfish Procambarus clarkii. The experiment was conducted in a tank equipped with a video recording system using 6 groups (3 control and 3 test groups) of five adult crayfish (30 specimens in total). After one hour of habituation, the behaviour of the crayfish was monitored for two hours. During the second hour, the animals in the test groups were exposed to a linear sweep (frequency range 0.1-25 kHz; peak amplitude 148 dBrms re 1 µPa at 12 kHz) acoustic stimulus for 30 minutes. Exposure to the noise produced significant variations in haemato-immunological parameters a…

Immune cells from sea urchins as biosensors for monitoring physical and chemical stress.

Expression of a glucocorticoid receptor (DlGR1) in several tissues of the teleost fish Dicentrarchus labrax

Since glucocorticoids have a role in maintaining the homeostatic status in fish, in the present paper mRNA expression (in situ hybridization) and tissue immunohistochemical localization of a glucocorticoid receptor (DlGR1) in several Dicentrarchus labrax organs are reported. Riboprobe and specific antibodies were prepared by using the DlGR1 that has been previously cloned and sequenced from peritoneal cavity leukocytes. Both mRNA and receptor were identified in head kidney, spleen, gills, intestine, heart and liver tissues. The functional roles of DlGR1 localization are discussed.

Underwater noise from boats: Measurement of its influence on the behaviour and biochemistry of the common prawn (Palaemon serratus, Pennant 1777)

Abstract This study examined the effects of recorded boat noises on the behaviour and biochemistry of the common prawn ( Palaemon serratus ) in laboratory settings. The experiment was carried out in a tank equipped with a video-recording system using six groups (three control and three tested) of eight common prawns (48 animals in total). After habituation for 1 h, the behaviour of the prawns was monitored for 1 h. During the last 30 min, the animals in the test groups were exposed to noise resembling a marine area with high anthropogenic acoustic pollution. The exposure to noise produced significant changes in locomotor patterns, presence inside or outside a shelter, total protein concentr…

Molecular and physiological characterization of in vivo Sulfamethoxazole response in Procambarus clark

Effects of cadmium on expression of the hsp70 in sea bass (Dicentrarchus labrax L., Osteichthyes. Moronidae) blood cells

Abstract To gain further insight into the role of cortisol in Wsh innate immune responses, we cloned and sequenced a 2592 bp cDNA from sea bass (Dicentrarchus labrax) peritoneal leukocytes (PCLs) encoding a glucocorticoid receptor (DlGR1). The deduced aminoacid sequence displayed that DlGR1 belong to a multigenic family of steroid hormone receptors, and exhibited high homology (80%) to the Burton’s mouth breeder (Haplochromis burtoni) HbGR1. The DlGR1 functional domains presented homologies with those of several vertebrate species. In situ hybridization assay revealed that DlGR1 was expressed in macrophages and neutrophils from the peritoneal cavity. Since in a previous paper, sea bass PCL …