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

The amphibious teleost Giant mudskipper (

We report here on the histological and structural characteristics of the gas bladder, the vertebral morphology, and the bladder-vertebra relationships of the butterfly fish, Pantodon buchholzi. The bladder opens at the boundary between the pharynx and the esophagus by a middle slit. A pneumatic duct is absent. The bladder shows a dorsolateral wall that adapts to the anfractuosities of the coelomic cavity and a ventral wall in contact with the abdominal organs. The vertebral bodies are formed by an hourglass shaped autocentrum, and by an arcocentrum reduced to several longitudinal ridges. The transverse processes adopt the structure of a cage whose walls are formed by bone trabeculae of variable size and distribution pattern. The dorsolateral wall of the bladder is a membrane that covers the kidney, adapts to the irregular shape of the vertebrae, and invades the transverse processes at several points before extending laterally. However, invasion of the vertebral bodies, the presence of a labyrinth, or the formation of respiratory parenchyma were not observed. The luminal surface of this wall is a thin respiratory barrier containing a single epithelial cell type. In addition, the wall contains numerous eosinophils that may be implicated in immune defense. The bladder ventral wall is a membrane rich in collagen, vessels, smooth muscle, and nerves that lacks a respiratory barrier. Its luminal surface contains ciliated and nonciliated cells. The two cell types appear implicated in surfactant production.

In the last decade, zebrafish has been used as a model for the study of several human skin diseases. The epidermis of Danio rerio is composed of keratinocytes and two types of secretory cells: mucous cells and club cells. Club cells have multiple biological functions and among them may be important in the protection against ultraviolet damage through the proliferative response or through the increased production of protective substances. Calcium-binding proteins such as calbindin D28K and calretinin are used as markers of nervous and enteric nervous systems, but they are present in numerous other cells. These proteins are involved in a wide variety of cell activities, such as cytoskeletal organization, cell motility and differentiation, cell cycle regulation and neuroprotective function. In this study we demonstrated, for the first time, the presence of calretinin and calbindin D28K in skin club cells of Danio rerio exposed to different wavelengths by immunohistochemistry analysis. Exposure to white-blue light and blue light causes the expression and colocalization of calbindin-D28K and calretinin. These proteins were moderately expressed and no colocalization was observed in the club cells of the control fish. In zebrafish exposed to continuous darkness for 10 days, in the club cells the two antibodies did not detect any proteins specifically. These results demonstrate that calbindin and calretinin could be involved in the pathophysiology of skin injury due to exposure to short-wavelength visible light spectrums.

Acetylcholine (Ach) represents the old neurotransmitter in central and peripheral nervous system. Its muscarinic and nicotinic receptors (mAChRs and nAChRs) constitute an independent cholinergic system that is found in immune cells and play a key role in the regulation of the immune function and cytokine production. Gas exchanging surfaces of the gills and air-breathing organs (ABOs) of the sharptooth catfish Clarias gariepinus were investigated using ultrastructural and confocal immunofluorescence techniques. This study was predominantly focused on the structure of the immune cell types, the expression of their neurotransmitters, including the antimicrobial peptide piscidin 1, and the functional significance of respiratory gas exchange epithelia. A network of immune cells (monocytes, eosinophils, and mast cells) was observed in the gill and the ABO epithelia. Eosinophils containing 5-hydroxytryptamine (5-HT) immunoreactivity were seen in close association with mast cells expressing acetylcholine (Ach), 5-HT, neuronal nitric oxide synthase, and piscidin 1. A rich and dense cholinergic innervation dispersing across the islet capillaries of the gas exchange barrier and the localization of Ach in the squamous pavement cells covering the capillaries were evidenced byVAChT antibodies. We report for the first time that piscidin 1 (Pis 1)-positive mast cells interact with Pis 1-positive nerves found in the epithelia of the respiratory organs. Pis 1 immunoreactivity was also observed in the covering respiratory epithelium of the ABOs and associated with a role in local mucosal immune defense. The above results anticipate future studies on the neuro-immune interactions at mucosal barrier surfaces, like the gill and the skin of fish, areas densely populated by different immune cells and sensory nerves that constantly sense and adapt to tissue-specific environmental challenges.

Rodlet cells (RCs) have always been an enigma for scientists. RCs have been given a variety of activities over the years, including ion transport, osmoregulation, and sensory function. These cells, presumably as members of the granulocyte line, are present only in teleosts and play a role in the innate immune response. RCs are migratory cells found in a variety of organs, including skin, vascular, digestive, uropoietic, reproductive, and respiratory systems, and present distinct physical properties that make them easily recognizable in tissues and organs. The development of RCs can be divided into four stages: granular, transitional, mature, and ruptured, having different morphological characteristics. Our study aims to characterize the different stages of these cells by histomorphological and histochemical techniques. Furthermore, we characterized these cells at all stages with peroxidase and fluorescence immunohistochemical techniques using different antibodies: S100, tubulin, α-SMA, piscidin, and for the first time TLR-2. From our results, the immunoreactivity of these cells to the antibodies performed may confirm that RCs play a role in fish defense mechanisms, helping to expand the state of the art on immunology and immune cells of teleosts.

Pollution deriving from textile wastes, including industrial and household waste, is recently of great interest due to their environmental impacts. Anthropogenic and synthetic fibers are responsible for negative effects on the quality of water and soil, and, also, their presence damages plant and animal health. In this work, the authors revealed the occurrence of man-made cellulose fibers in specimens of Boops boops from the Northern Sicilian coasts. Bogue was chosen as target species as it has been used as an indicator within the European Marine Strategy Framework Directive (MSFD 2008/56/EC) in order to value the “microplastics status” in the stomach contents. Of the 30 specimens examined, 63.3% of these had ingested fibers items. The number of fibers ranged from 1 to 10 per specimens with an average of 2.7 items/specimen. Fibers length ranged from 0.5 to 30 mm, most of them were black (95%), and a small percentage was red (5%). The ingestion of man-made cellulose fibers, observed for the first time, in Boops boops in the Mediterranean Sea wake-up call and it should attract the attention of the EU for new guidelines where this new type of contaminant is classified harmful as well as plastics.

Vanadium accumulation levels in different tissues (muscle and organs) of the striped mullet Mugil cephalus (Linnaeus, 1758) and possible relationships with blood parameters were evaluated in a Natural Protected Area (Lake Faro, Sicily, Italy), during the winter of 2017. Hematological parameters (red blood cell, RBC; white blood cell, WBC; hemoglobin concentration, Hb; hematocrit, Hct; mean corpuscular volume, MCV; mean corpuscular hemoglobin, MCH; mean corpuscular hemoglobin concentration, MCHC; thrombocytes, TC), biometric indices (weight, total and fork length), and vanadium levels in muscles and organs (gills, liver, stomach and intestine) were determined. Statistical analyses showed significant differences in concentrations of vanadium of the analyzed tissue of M. cephalus and a positive relationship between vanadium concentration in the liver and some hematological parameters (RBC, Hb and Hct) and biometric indices. Our results underline the importance of fish blood parameters as sensitive indicators of toxic impact of environmental factors such as metals. This study, focusing on an ongoing topic, represents a valuable contribution to research concerning the monitoring and prevention of vanadium pollution in aquatic organisms and environments.

Cetacean mechanical senses, such as hearing, echolocation, active touch and the perception of water movements, are essential for their survival. Dolphins skin possesses dense packing of dermal papillae associated with the cutaneous ridges that suggests a sensory function, furthermore they are well innervated and very sensitive to touch. This is mediated by mechanoreceptors, abundant in the region of the head and in the dorsal part of the body. Most odontocetes possess vibrissae (i.e., sensory hair) that have been well described in literature and present a microanatomy similar to that of terrestrial mammals. The aim of this study was to characterize Merkel cell through use of specific antibodies: Substance P, Anti-calbindin DK28, Anti-5HT, Leu- enkephalin, Protein Gene Product 9.5 (PGP9.5) and Anti-Human Neuronal Protein, for the first time. Merkel cells (MCs) in the dolphin skin are specialized skin receptors, characterized by their particular location and close association with nerve terminals. The presence of neuroendocrine markers and different neuropeptides confirms that MCs play also neuroendocrine function and are considered as part of the diffuse neuroendocrine system. Furthermore, the presence of Leu-enkephalin in Merkel cells could involve these cells in inflammatory responses in the skin.

We have conducted a morphological and immunohistochemical study of the gills of juvenile specimens of the obligate air-breathing fish Heterotis niloticus. The study has been performed under normoxic and hypoxic conditions. The gills showed a reduced respiratory surface area by development of an interlamellar cellular mass (ILCM). The ILCM persisted without changes under both normoxia and hypoxia. Neuroepithelial cells (NECs), the major oxygen and hypoxia sensing cell type, were located in the distal end of the gill filaments and along the ILCM edges. These cells expressed 5HT, the neuronal isoform of the nitric oxide synthase (nNOS) and the vesicular acetylcholine transporter (VAChT). Furthermore, NECs appeared associated with nitrergic nerve fibres. The O2 levels did not modify the location, number or the immunohistochemical characteristics of NECs. Pavement cells covering the ILCM were also positive to nNOS and VAChT. The mechanisms of O

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are proteins that contain highly conserved functional domains and sequence motifs that are correlated with their unique biophysical activities, to regulate cardiac pacemaker activity and synaptic transmission. These pacemaker proteins have been studied in mammalian species, but little is known now about their heart distribution in lower vertebrates and c-AMP modulation. Here, we characterized the pacemaker system in the heart of the wild Atlantic cod (Gadus morhua), with respect to primary pacemaker molecular markers. Special focus is given to the structural, ultrastructural and molecular characterization of the pacemaker domain, through the expression of HCN channel genes and the immunohistochemistry of HCN isoforms, including the location of intracardiac neurons that are adjacent to the sinoatrial region of the heart. Similarly to zebrafish and mammals, these neurons are immunoreactive to ChAT, VAChT and nNOS. It has been shown that cardiac pacemaking can be modulated by sympathetic and parasympathetic pathways, and the existence of intracardiac neurons projecting back to the central nervous system provide a plausible link between them.

Acetylcholine (Ach) is the main neurotransmitter in the neuronal cholinergic system and also works as a signaling molecule in non-neuronal cells and tissues. The diversity of signaling pathways mediated by Ach provides a basis for understanding the biology of the cholinergic epithelial cells and immune cells in the gill of the species studied. NECs in the gill were not found surprisingly, but specialized cells showing the morphological, histochemical and ultrastructural characteristics of eosinophils were located in the gill filaments and respiratory lamellae. Much remains unknown about the interaction between the nerves and eosinophils that modulate both the release of acetylcholine and its nicotinic and muscarinic receptors including the role of acetylcholine in the mechanisms of O

Antimicrobial peptides (AMPs) are found widespread in nature and possess antimicrobial and immunomodulatory activities. Due to their multifunctional properties, these peptides are a focus of growing body of interest and have been characterized in several fish species. Due to their similarities in amino-acid composition and amphipathic design, it has been suggested that neuropeptides may be directly involved in the innate immune response against pathogen intruders. In this review, we report the molecular characterization of the fish-specific AMP piscidin1, the production of an antibody raised against this peptide and the immunohistochemical identification of this peptide and enkephalins in the neuroepithelial cells (NECs) in the gill of several teleost fish species living in different habitats. In spite of the abundant literature on Piscidin1, the biological role of this peptide in fish visceral organs remains poorly explored, as well as the role of the neuropeptides in neuroimmune interaction in fish. The NECs, by their role as sensors of hypoxia changes in the external environments, in combination with their endocrine nature and secretion of immunomodulatory substances would influence various types of immune cells that contain piscidin, such as mast cells and eosinophils, both showing interaction with the nervous system. The discovery of piscidins in the gill and skin, their diversity and their role in the regulation of immune response will lead to better selection of these immunomodulatory molecules as drug targets to retain antimicrobial barrier function and for aquaculture therapy in the future. Expression of the Antimicrobial Peptide Piscidin 1 and Neuropeptides in Fish Gill and Skin: A Potential Participation in Neuro-Immune Interaction

Serotonin [5-hydroxytryptamine (5-HT)] is an important neuromodulator involved in a wide range of physiological functions. The effects of serotonin are mediated by an extended family of receptors coupled to multiple heterotrimeric G-proteins, associated with cellular membrane. G proteins connect receptors to effectors and thus trigger intracellular signaling pathways. These cellular processes several regulate systemic functions such as embryonic development, gonadal development, learning and memory, and organismal homeostasis. Generally, elasmobranch fish dwell a hypersaline environment and utilize a specialized extrarenal salt secreting organ, the rectal gland, to face ionic homeostasis. In this study in addition to the morphological, histochemical and immunohistochemical description of the Scyliorhinus canicula rectal gland, for the first time, the presence of serotonin (5-HT), and distribution of different types of G protein alpha subunits (Gα o, Gα q/11, and Gα s/olf) has been investigated in the rectal gland epithelium by confocal immunofluorescence techniques. Colocalization G proteins and 5-HT in the secretory epithelium of the gland suggests serotonin acts as a hormone and involves G proteins in an autocrine-paracrine control of rectal gland homeostasis.

In the present work, the marine invertebrate Mytilus galloprovincialis was used as model organism to evaluate the toxic effects of the neonicotinoid Calypso 480 SC (CAL) following 20 days of exposure to sub-lethal concentrations of 7.77 mg L-1 (0.1 % 96h-LC50) and 77.70 mg L-1 (1 % 96h-LC50), and a recovery period of 10 days in uncontaminated seawater. Results revealed that exposure to both concentrations of CAL increased significantly mortality rate in the cells of haemolymph and digestive gland, while digestive gland cells were no longer able to regulate cell volume. Exposure significantly reduced haemolymph parameters (Cl-, Na+), affected the enzymatic activities of superoxide dismutase of digestive gland and catalase of gill, and caused also histopathological alterations in digestive gland and gills. Main histological damages detected in mussels were lipofuscin accumulation, focal points of necrosis, mucous overproduction and infiltrative inflammations. Interestingly, alterations persisted after the recovery period in CAL-free water, especially for haemocyte parameters (K+, Na+, Ca2+, lactate dehydrogenase, glucose). A slight recovery of histological conditions was detected. These findings suggested that sub-chronic exposure to the neonicotinoid insecticide caused important alterations in both cell and tissue parameters of M. galloprovincialis. Considering the ecologically and commercially important role of mussels in coastal waters, a potential risk posed by neonicotinoids to this essential aquatic resource can be highlighted.

Pesticides can enter aquatic environments potentially affecting non-target organisms. Unfortunately, the effects of such substances are still poorly understood. This study investigated the effects of the active neonicotinoid substance thiacloprid (TH) and the commercial product Calypso 480 SC (CA) (active compound 40.4% TH) on Mytilus galloprovincialis after short-term exposure to sublethal concentrations. Mussels were tested for seven days to 0, 1, 5 and 10 mg L−1 TH and 0, 10, 50 and 100 mg L−1 CA. For this purpose, several parameters, such as cell viability of haemocytes and digestive cells, biochemical haemolymph features, superoxide dismutase (SOD) and catalase (CAT) enzymatic activity of gills and digestive gland, as well as histology of such tissues were analysed. The sublethal concentrations of both substances lead to abatement or completely stopping the byssal fibres creation. Biochemical analysis of haemolymph showed significant changes (P

The airways and lungs of vertebrates are an entrance way for several microbial pathogens. Cetaceans present an upper and lower respiratory anatomy that allows the rapid flow of large air volumes, which may lead to high susceptibility to respiratory infections. Mortality and stranding rate of Cetaceans increased dramatically, so wide the knowledge about the immune system and specific antibodies identifying immune cells populations, is of fundamental importance to monitor and document cetacean health. The aim of this study was to identify the localization of dendritic cells marked by Langerin/CD207 in airways, lungs and associated lymph nodes, of the striped dolphin Stenella coeruleoalba. Samples of trachea, bronchi, lungs and lung-associated lymph nodes were obtained from a stranded adult male of Stenella coeruleoalba. Our results showed abundant lymphoid aggregates (LAs) in the lung of S. ceruleoalba. Langerhans-like dendritic cells were well distributed along the epithelium and interstitium of respiratory tract and in associated lymph nodes. The present study deepens the knowledge about the cetacean's immune system and report about the exploitability of a commercial antibody (Langerin/CD207) for cetacean species.

This study highlights plastics occurrence in five demersal fish species from the Southern Tyrrhenian Sea: the Red mullet Mullus barbatus barbatus, the Piper gurnard Trigla lyra, the Blackmouth catshark Galeus melastomus, the Lesser spotted dogfish Scyliorhinus canicula and the Brown ray Raja miraletus. Overall, 125 fish were examined: 21 Red mullets, 16 Piper gurnards, 75 Blackmouth catsharks, 72 Dogfish and 1 Brown ray. The percentage of fish with ingested plastics was 14.4% with 0.24 items per specimen. The majority of the debris were fibers and the application of infrared and Raman spectroscopy allowed the identification and discrimination of plastic and non-plastic fibers. The plastic debris isolated were mainly microplastics (94.1%), while macroplastics occurrence was very low (5.9%). The plastics were identified as polypropylene, Teflon, nylon, kraton G (triblock copolymer) and polyethylene. Also cellulose was detected. S. canicula was the species with the highest number of plastic pollutants.

The phylum Cnidaria is one of the most important contributors in providing abundance of bio- and chemodiversity. In this study, a comprehensive chemical investigation on the nutritional and nutraceutical properties of Mediterranean jellyfish Pelagia noctiluca was carried out. Also, compositional differences between male and female organisms, as well as between their main anatomical parts, namely bell and oral arms, were explored in an attempt to select the best potential sources of nutrients and/or nutraceuticals from jellyfish. With the exception of higher energy densities and total phenolic contents observed in females than males, no statistically significant differences related to the specimen&rsquo, s sex were highlighted for the other compound classes. Rather, the distribution of the investigated chemical classes varied depending on the jellyfish&rsquo, s body parts. In fact, crude proteins were more abundant in oral arms than bells, saturated fatty acids were more concentrated in bells than oral arms, whereas polyunsaturated fatty acids were distributed in the exact opposite way. On the other hand, major elements and trace elements demonstrated an opposite behavior, being the latter most accumulated in oral arms than bells. Additionally, important nutraceuticals, such as eicosapentaenoic and docosahexaenoic acids, and antioxidant minerals, were determined. Overall, obtained data suggest the potential employment of the Mediterranean P. noctiluca for the development of natural aquafeed and food supplements.

Toll-like receptors (TLRs) are a group of pattern recognition molecules that play a crucial role in innate immunity. The structural conservation of the archaic TLR system suggests that the regulation of the immune response might be similar in fish and mammals. Several TLRs (TLR-1, -2, and -4) are expressed by activated macrophages, “foam cells” in human atherosclerotic lesions. To date, 20 different TLRs were identified in more than a dozen different fish species. In this study we found that feeding goldfish, Carrassius auratus , a high-cholesterol diet (HCD) resulted macrophage foam cell formation in the intestinal tissues. The expression of TLR2 has been found in foam cells and in the cytoplasm of enterocytes, however the staining was more intense at the apical surface of polarized intestinal epithelial cells and in the lamina propria . In the intestinal epithelial cells and in the lamina propria cells of the control fish the TLR2 was expressed at low levels. The intestinal epithelium is directly involved in the mucosal immune response through its expression of proinflammatory genes, release of inflammatory cytokines, and recruitment of inflammatory cells.

The constant exposure of ocular surface to external environment and then to several microbial agents is often related to the pathogenesis of various inflammatory eye disorders. In the present study α-Smooth Muscle Actin (α-SMA) and Langerin CD/207 expression and function was investigated in a rabbit corneal keratitis. The inflammation was induced by the secreted form of glycoprotein B (gB1s) of HSV-1, in an ex vivo rabbit corneal model. α-SMA is often used as a marker for myofibroblasts. In this study, for the first time, we show α-SMA positive corneal epithelial cells, during HSV-1 cornea inflammation, demonstrating a crucial role in wound healing, especially during remodeling phase. Furthermore, we show the presence of Dendritic Cells Langerin CD/207 positive, located mainly in the basal epithelial layer and in corneal stroma during the inflammatory processes. Our result validating the ex vivo organotypic rabbit corneal model, for the study about pathogenesis of HSV-1 ocular infection.

We report the presence of microplastics on the external surface and in the gastrointestinal tract of white late-larval and juvenile stages (fry) of clupeid fishes caught in the Southern Tyrrhenian Sea. The average highest number of plastics debris was recorded on Sardina pilchardus (0.53 items/specimen); a lower average number of items was observed for Engraulis encrasicolus (0.26 items/specimen). The plastics were characterized by fibers that differed in shape, colour and composition. Polyester, polypropylene, polyacrylonitrile, polyethylene, polyamide, nylon, rayon and polyurethane segments were detected by Raman and FTIR spectroscopies. Traces of organic components and dyes, compounds that are generally included in the polymer matrix to modify its base properties, were also identified on microplastics. Our results raise concerns for the potential transfer of synthetic materials through the marine food web and into humans, given the prominent role of S. pilchardus and E. encrasicolus within the food web as main food source for many marine species.

The present study describes histochemical and immunohistochemical characteristics of the spiral valve and its associated lymphoid tissue (GALT) in the dogfish Scyliorhinus canicula. The mucosal surface of the spiral valve represents the first line of defense against pathogens coming from the external environment through food. Epithelial, mucus and immune cells play a key role in controlling the inflammatory response. Valve intestine of S. canicula had many folds lined by simple columnar cells and goblet cells, which later reacted positive to PAS, AB and AB-PAS, histochemical stains differentiated the different types of mucins; lectin histochemistry (PNA and WGA), detected neutral and acid mucins secreted that plays an important role in protection against invading pathogens. Integrin α5β1 was expressed in enterocytes that line the valve's folds with greater marking in the apical part of the cells. Laminin was found on the apical side of the epithelium, in fibrillar and cellular elements of the lamina propria and in the muscularis mucosa. In the spiral valve gut-associated lymphoid tissue (GALT) has been studied. For the first time, massive leucocytes aggregates were identified by confocal immunofluorescence techniques, using the following antibodies: TLR2, S100, Langerin/CD207. Our results expand knowledge about Dogfish valve intestine giving important news in understanding comparative immunology.

The present study investigates the occurrence of plastic pollution in two commercially important marine teleosts ( Zeus faber and Lepidopus caudatus ) from the northern coasts of Sicily (Tyrrhenian Sea). Plastics occurrence in the gastrointestinal tract was higher in Lepidopus caudatus (78.1%) than Zeus faber (51.4%). Debris characterization, carried out by micro-Raman spectroscopy, allowed identified the main types of found polymers as: polypropylene (PP), polyamide (PA), nylon and, to a lesser extent, polyethylene (PE). Of the two fish species studied, the silver scabbardfish appeared to be the more vulnerable to plastic ingestion. Our study represents a starting point that may pave the way for future investigation of the fate, accumulation and transfer of plastic debris to upper trophic levels, to verify their potential toxicity and to better understand strategies to mitigate this phenomenon.

The authors investigated the arsenic (As) accumulation in different tissues (muscle, gill, liver, stomach, and intestine) and the possible correlation between tissue concentration and hematological parameters in mullet (Mugil cephalus Linnaeus, 1758) caught in Faro Lake (Messina, Sicily, Italy). On all fish, hematological analyses of blood samples, measurement of biometric indices, and the removal of the muscles, gills, liver, stomach, and intestine for the determination of arsenic concentration were performed. A hemogram was performed to find effects of arsenic concentration in tissues on hematological variables. One-way analysis of variance showed significant differences of arsenic concentration in different tissues, with higher values in the gill. The correlation between hematological parameters and tissue arsenic concentration showed a statistical significance for red blood cell (RBC), hemoglobin concentration (Hb), and hematocrit (Hct) with the liver As concentration. Biometric indices (weight, length, and fork length) showed a significant correlation with As concentration of the muscle and liver also. Our results indicate the role of some hematological parameters as biomarkers useful to monitoring anthropogenic load of arsenic in water and sediment, because variations of these parameters represent one of the effects that arsenic exposure can have on fish.

The air-breathing specialization has evolved idependently in vertebrates, as many different organs can perfom gas exchange. The largest obligate air-breathing fish from South America Arapaima gigas breathe air using its gas bladder, and its dependence on air breathing increases during its growth. During its development, gill morphology shows a dramatic change, remodeling with a gradual reduction of gill lamellae during the transition from water breathing to air breathing . It has been suggested that in this species the gills remain the main site of O2 and CO2 sensing. Consistent with this, we demonstrate for the first time the occurrence of the neuroepithelial cells (NECs) in the glottis, and in the gill filament epithelia and their distal halves. These cells contain a broader spectrum of neurotransmitters (5-HT, acetylcholine, nNOS), G-protein subunits and the muscarininic receptors that are coupled to G proteins (G-protein coupled receptors). We report also for the first time the presence of G alpha proteins coupled with muscarinic receptors on the NECs, that are thought as receptors that initiate the cardiorespiratory reflexes in aquatic vertebrates. Based on the specific orientation in the epithelia and their closest vicinity to efferent vasculatures, the gill and glottal NECs of A. gigas could be regarded as potential O2 and CO2 sensing receptors. However, future studies are needed to ascertain the neurophysiological characterization of these cells.

In this work it is reported for the first time the characterization of microplastics from sea water samples and in two congener species of seabreams: Pagellus erythrinus and P. bogaraveo, Mediterranean fish species of great commercial importance. An experimental survey was conducted on May-June 2017 in the southernmost part of the Tyrrhenian Sea. Microplastics found in the sea water and in the grastrointestinal tract of two teleosts were characterized by Raman and IR spectroscopies. Microplastics found in sea water samples appeared in the form of fragments made of plastics of low and high density (PVC and LPDE). All the microplastics found in fish belonged to Nylon 66, typical fibers used in industry and in fisheries. Our findings highlighted the importance of further studies along the food web chain for a better understanding of the diffusion and possible consequences of this terrible threat.

Since a long time caffeine has been used in pharmaceutical and cosmetic preparations due to its favorable effects on the skin. The aim of this study was to evaluate the topical anti-inflammatory activity (carrageenan-induced paw oedema) of an ointment prepared using a methanolic extract from green beans of C. robusta via histology. Results showed that the treatment with the ointment reduced the paw oedema within 3 and 5 h post-carrageenan administration. The immunohistochemical evaluations of αSMA, Langerin and S100 gave further support to the morphological analysis. Finally, the methanolic extract from green beans of C. robusta proved to possess elevated free radical scavenger capability by DPPH assay, which may contribute to the observed anti-inflammatory activity.

This study reports the returning of the seahorse in the Faro Lake and the most recent finding in a brackish coastal Mediterranean lagoon. After about 50 years from the first documented occurrence of

In the present study zebrafish (Danio rerio) has been used as model organism to establish the effects of dietary supplementation of Gracilaria gracilis powder (GP) on mucosal and innate immune parameters, antioxidant enzymes, and growth. In order to establish these features, zebrafish were fed for eight weeks with experimental diets containing different levels of Red algae, 0.25, 0.5 and 1% of GP; also, a group was fed with control diet. At the end of the experimental period the antioxidant superoxide dismutase and catalase (SOD, CAT) genes expression, interleukin 1 beta (il-1β), lysozyme (LYZ), tumor necrosis factor alpha (TNF-α) for immune-related genes expression, total immunoglobulin (Ig), total protein, alkaline phosphatase (ALP) activity for innate immune parameters, and growth performance have been established. The GP dietary supplementation showed differences in SOD and CAT expression in zebrafish whole body respect to the control group. Non-signifcant differences were noticed among the different groups in case of TNF-α, LYZ and il-1expression (P 0.05). The skin mucus total Ig and total protein in the group fed on 1% of GP were significantly higher respect to control group (P 0.05). 0.25 and 0.5% of GP dietary supplementation significantly enhanced skin mucus ALP activity levels (P 0.05). No significant differences were recorded for growth performances among groups (P 0.05). The results obtained in the present study revealed that G. gracilis could be takes in account as fishes diet supplementation for its immune system stimulants effects.

The neuromast is the morphological unit of the lateral line of fishes and is composed of a cluster of central sensory cells (hair cells) surrounded by support and mantle cells. Heavy metals exposure leads to disruption of hair cells within the neuromast. It is well known that the zebrafish has the ability to regenerate the hair cells after damage caused by toxicants. The process of regeneration depends on proliferation, differentiation and cellular migration of sensory and non-sensory progenitor cells. Therefore, our study was made in order to identify which cellular types are involved in the complex process of regeneration during heavy metals exposure. For this purpose, adult zebrafish were exposed to various heavy metals (Arsenic, cadmium and zinc) for 72h. After acute (24h) exposure, immunohistochemical localization of S100 (a specific marker for hair cells) in the neuromasts highlighted the hair cells loss. The immunoreaction for Sox2 (a specific marker for stem cells), at the same time, was observed in the support and mantle cells, after exposure to arsenic and cadmium, while only in the support cells after exposure to zinc. After chronic (72h) exposure the hair cells were regenerated, showing an immunoreaction for S100 protein. At the same exposure time to the three metals, a Sox2 immunoreaction was expressed in support and mantle cells. Our results showed for the first time the regenerative capacity of hair cells, not only after, but also during exposure to heavy metals, demonstrated by the presence of different stem cells that can diversify in hair cells.

Little is known about the spinal sympathetic organization in the caecilian amphibians. We examined for the first time the location of sympathetic preganglionic neurons (SPNs) in the spinal cord using a panel of specific markers expressed in SPNs. The SPNs of anuran amphibians form two cell columns segregated mainly in the lateral and medial marginal areas of the central gray matter. In the caecilian Typhlonectes natans immunoreactivity for galanin and ChAT is found in most laterally arranged neurons lying in spinal segments 2–7. They are encircled by TH- and nNOS-immunoreactive nerve fibers. These neurons might project specifically to a population of adrenergic sympathetic postganglionic neurons in paravertebral ganglia and/or non-adrenergic sympathetic postganglionic neurons in the celiac ganglia. However the segmental restriction and target specificity of the neurons of the species studied are not known. As mucous and granular glands in the dermis may represent one of the peripheral targets of the adrenergic ganglion cells and reflect the prominent preganglionic cell columns, an immunohistochemical study was done also on these glands. Retrograde-tracing studies are, however, needed to study the segmental localization of the preganglionic neurons and their projections to the postganglionic neurons in sympathetic ganglia.

Mudskippers are amphibious fishes living in mudflats and mangroves. These fishes hold air in their large buccopharyngeal-opercular cavities where respiratory gas exchange takes place via the gills and higher vascularized epithelium lining the cavities and also the skin epidermis. Although aerial ventilation response to changes in ambient gas concentration has been studied in mudskippers, the localization and distribution of respiratory chemoreceptors, their neurochemical coding and function as well as physiological evidence for the gill or skin as site for O2 and CO2 sensing are currently not known. In the present study we assessed the distribution of serotonin, acetylcholine, catecholamines and nitric oxide in the neuroepithelial cells (NECs) of the mudskipper gill and skin epithelium using immunohistochemistry and confocal microscopy. Colocalization studies showed that 5-HT is coexpressed with nNOS, Na+/K+-ATPase, TH and VAChT; nNOS is coexpressed with Na+/K+-ATPase and TH in the skin. In the gill 5-HT is coexpressed with nNOS and VAhHT and nNOS is coexpressed with Na+/K+-ATPase and TH. Acetylcholine is also expressed in chain and proximal neurons projecting to the efferent filament artery and branchial smooth muscle. The serotonergic cells c labeled with VAChT, nNOS and TH, thus indicating the presence of NEC populations and the possibility that these neurotransmitters (other than serotonin) may act as primary transmitters in the hypoxic reflex in fish gills. Immunolabeling with TH antibodies revealed that NECs in the gill and the skin are innervated by catecholaminergic nerves, thus suggesting that these cells are involved in a central control of branchial functions through their relationships with the sympathetic branchial nervous system. The Na+/K+-ATPase in mitochondria-rich cells (MRCs), which are most concentrated in the gill lamellar epithelium, is colabeled with nNOS and associated with TH nerve terminals. TH-immunopositive fine varicosities were also associated with the numerous capillaries in the skin surface and the layers of the swollen cells. Based on the often hypercapnic and hypoxic habitat of the mudskippers, these fishes may represent an attractive model for pursuing studies on O2 and CO2 sensing due to the air-breathing that increases the importance of acid/base regulation and the O2-related drive including the function of gasotransmitters such as nitric oxide that has an inhibitory (regulatory) function in ionoregulation. This research was supported by project PAN LAB PONA3_00166. The authors would like to thank Michał Ignaszewski (TDT) for his kind help in statistical analysis.

Faro Lake is a coastal meromictic lagoon with singular characteristics in the Mediterranean (Messina, Sicily - Italy). It is part of the Natural Oriented Reserve of Capo Peloro (38° 15' 57″ N; 15° 37' 50″ E). In this area, traditional mollusc farming activity persists, producing 'autochthonous' mussels. This study reports of the Mytilus galloprovincialis haemolymph chemical profile and water variables determination of 1 year-lasted survey (April 2016 - March 2017). The determinations of electrolytes (Na+ , Cl- , K+ , Ca2+ , Mg2+ , P inorganic) and heavy metals in both Faro lake water and haemolymph have been carried out. Heavy metals are elements with high density and are quite toxic in low concentrations, such as Aluminum (Al), Arsenic (As), Cobalt (Co), Chrome (Cr), Copper (Cu), Iron (Fe), Magnesium (Mg), Manganese (Mn), Lead (Pb), Tin (Sn), Zinc (Zn). Heavy metals toxicity depends, principally, on bioaccumulation processes. M. galloprovincialis is a good bio-indicator, ideal for assess levels of environmental pollution thanks to its biological, ecological and physiological characteristics. The results of this study showed a typical fluctuation range in haemolymph and water parameters, related to the water ones; chemical-physical parameters affected the ions (electrolytes and metals) levels in some period of the year. The study reports the interactions between biotic (Mytilus galloprovincialis) and abiotic (water parameters) components of Faro Lake, and creates reference data for further future study on the same area or on similar ones.

Although the irritant effects of quaternium-15 have been established, little is known about the toxicological consequences induced by this xenobiotic on aquatic invertebrates. The present article reports toxicological, histological and physiological effects of quaternium-15 following the exposure of Mytilus galloprovincialis for 18 days at three different concentrations (0.1, 1.0 and 2.0 mg/L). The results demonstrate that at higher concentrations histological damages to M. galloprovincialis gills occur, like melanosis, light exfoliations, increase of mucus production and infiltrative inflammation. In addition digestive gland cells of M. galloprovincialis, were not able to perform the regulation volume decrease (RVD) owing to osmotic stress following the exposure to the preservative. Overall, this first study on quaternium-15 highlights that it can jeopardize both the morphology and vital physiological processes in marine invertebrates, depending on the duration of exposure and the concentration of the preservative, indicating that further studies are necessary to increase our knowledge about the effects of this substance, commonly added to our products of daily use.