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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 O2 chemosensing. In this study we report for the first time the expression of Ach in the pavement cells of the gill lamellae in fish, the mast cells associated with eosinophils and nerve interaction for both immune cell types, in the gill of the extant butterfly fish Pantodon buchholzi. Multiple roles have been hypothesized for Ach and alpha nAChR in the gills. Among these there are the possible involvement of the pavement cells of the gill lamellae as O2 chemosensitive cells, the interaction of Ach positive mast cells with eosinophils and interaction of eosinophils with nerve terminals. This could be related to the use of the vesicular acetylcholine transporter (VAChT) and the alpha 2 subunit of the acetylcholine nicotinic receptor (alpha 2 nAChR). These data demonstrate the presence of Ach multiple sites of neuronal and non-neuronal release and reception within the gill and its ancestral signaling that arose during the evolutionary history of this conservative fish species.

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.

Otolith features are useful tools for studying taxonomy, ecology, paleontology, and fish biology since they represent a permanent record of life history. Nevertheless, the functional morphology of otoliths remains an open research question that is useful to completely understand their eco-morphology. This study aims to deepen the knowledge of intra- and interspecific variation in sagitta morphology in three congeneric seabreams, to understand how such variability could be related to the lifestyles of each species. Therefore, the sagittae (n = 161) of 24 Pagellus bogaraveo, 24 Pagellus acarne, and 37 Pagellus erythrinus specimens, collected from the south Tyrrhenian Sea, were analyzed using scanning electron microscopy and a stereomicroscope to assess morphometric features, variability between otolith pairs and the external crystalline structure the of sulcus acusticus. Statistical analysis demonstrated that, between the species, variability in sagittal otolith rostral length growth and sulcus acusticus features, correlated with increased fish total length and body weight. Moreover, slight differences between otolith pairs were detected in P. acarne and P. erythrinus (P

Environmental pollution by plastic particles is of major global concern, as a potential threat to aquatic organisms and ecosystems. The accumulation of microplastics in freshwater and marine environments has strong ecological implications due to their long persistence, potential toxicity, and ability to adsorb other pollutants, acting as vectors of pathogens. Nevertheless, while the number of studies on the presence of microplastics in the wild fish increased, less attention has been paid to the farmed fish species. Here, we investigated the occurrence of microparticles in the digestive tracts of Sparus aurata and Cyprinus carpio at different life stages and reared by an intensive and semi-intensive production system, respectively. Our results showed the presence of natural microfibers and microplastics, with microfibers (~ 90%) being the dominant type. In both fish species, the presence of microparticles was not revealed at larval stage. Fry and adult S. aurata specimens showed microfiber abundances of 0.21 and 1.3 items/individual, respectively. A lower load of microparticles (p < 0.05) occurred in fry (0.06 items/individual) and adult C. carpio specimens (0.25 items/individual). As to the chemical composition of the micro-items, natural (20%), semi-synthetic (28%), and single or blended synthetic fibers (52%) were identified in S. aurata. Linen, rayon, lyocell, cotton: polyester and polyester (12.5% concentration for each polymer) fibers were identified in C. carpio, while PTFE (37.5%) was present as fragments. Rayon was the most frequent chemical type (21.2%). The polymer composition of the extracted microparticles showed significant differences between the fish species analysed (p < 0.05). Notably, a considerably lower contamination level of synthetic polymers (average 0.11 items/individual) was detected in the farmed fishes compared with the data obtained in other studies on feral ones. To the best of our knowledge, this is the first study reporting the plastic and the non-plastic microfiber contamination in the farmed S. aurata and C. carpio at different life stages.

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.

This study aims to evaluate some biochemical parameters and serum electrolytes in cultured rainbow trout Oncorhynchus mykiss (Walbaum, 1792) to evaluate a potential correlation with biometric parameters (weight and length). For this purpose, 100 cultured trout (300–700 g weight range, 25–38 cm length range) bred on a fish-farm were used for the study. Physico-chemical characteristics of water were measured on the farm. Blood samples were collected from each fish to analyze the following parameters: glucose, triglycerides, cholesterol, aspartate aminotransferase (AST); alanine aminotransferase (ALT), calcium (Ca2+), chlorine (Cl−), iron (Fe2+), phosphorus (P), magnesium (Mg2+), potassium (K+), sodium (Na+) and urea. Statistical data analysis showed a significant correlation between size and glucose and cholesterol. No correlation was found between size and other parameters studied. These results represent a contribution to the study of fish size leading to better understanding some biochemical parameters and serum electrolyte profiles in cultured rainbow trout. This research contributes to understanding the intra-individual variability of some blood parameters in cultured rainbow trout O. mykiss offering reliable information on chronic stress status, metabolic disorders and deficiencies in relation to different sizes. These could help in improve the health monitoring in trout fish farms., Biochemical parameters; Intra-variability blood parameters; Serum electrolytes; Size; Rainbow trout.

It is well known that microplastics are ingested by a wide range of marine organisms. Little is known about their occurrence in cnidarians. The paper reports microplastic contamination in Pelagia n...

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.

We reviewed literature on the diet of the Galeus melastomus Rafinesque, 1810, from the Mediterranean Sea. Specific keywords (“Galeus melastomus diet”, “feeding habits”, “trophic position”, “biology”, “deep environment adaptation”) in the principal data sources, such as Web of Science, PubMed, and Google Scholar were used. Seventeen studies conducted on the diet and trophic position of G. melastomus have been considered for Mediterranean Sea regions. The feeding habits have been analyzed in many areas of the western basin; instead, for the Tyrrhenian, Adriatic, and central Mediterranean Seas, information is outdated and fragmentary. In all investigated sub areas, the data showed that G. melastomus is an opportunistic demersal supra benthic predator, benthic feeder, and scavenger, that adapts its diet to the seasonal and geographical fluctuations of the prey availability. It occupies a generalist niche showing individual specialization. In all reviewed Mediterranean sub areas, the most important prey groups were crustaceans, cephalopods, and teleost fishes. Taxa percentage in its diet composition can vary depending on different habitats with ontogenetic development of individuals, depth (that is correlated with the ontogenetic development), seasonal availability, and distribution of different prey groups. Widening knowledge of G. melastomus feeding habits is a fundamental tool for better understand meso and bathy-pelagic ecosystems.

Data on the biology and ecology of Galeus melastomus are old/absent for the Southern Tyrrhenian Sea, despite there being numerous studies in the wider area. A total of 127 specimens of G. melastomus from the southern Tyrrhenian Sea, collected in 2018–2019 using trawling nets, were analyzed to investigate size at sexual maturity, sex ratio, length–weight relationships, and feeding habits. To our best knowledge, this is the first time in which all these features were investigated in the Southern Tyrrhenian Sea for G. melastomus. The stomach content analysis showed that G. melastomus had intermediate feeding habits, preying on a great variety of species, especially Cephalopoda, Osteichthyes, and Crustacea. The Levin’s index value (Bi) was 0.53. Sex ratio was 0.92:1, with females slightly more abundant and bigger than males. The results also showed a decrease (33.7 cm for females, 31.1 cm for males) in length at 50% maturity (L50). This could be a result of anthropogenic stressors, such as overfishing and/or and environmental changes, which can induce physiological responses in several species. Our results highlighted the differences related to sexual maturity, growth, and feeding habits of the blackmouth catshark in the studied area, providing reference data to allow comparison with future studies on this species adaptations to this and other deep-sea areas in the Mediterranean Sea.

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.

The Strait of Messina (Sicily, Italy) attracts the interest of marine ecologists for the presence of a large variety of habitat and mutually-interacting communities. Among them, beachrock formations, despite their wide geographic distribution, which also includes the Mediterranean area, have been poorly investigated from the biotic viewpoint. In this paper, the spatial and seasonal variability of benthic megafauna from the Messina microtidal beachrock is described. Combining in situ collected data (measurements of abiotic parameters and underwater visual census) with theoretical post-processing analyses (analysis of similarity percentages and cluster analysis), we deduced the possibility to model the dynamics observed between the most dominant species, a top snail, Phorcus turbinatus (Born, 1778), and a hermit crab, Clibanarius erythropus (Latreille, 1818), in terms of a prey-predator interaction. These species gave rise to different intriguing dynamical regimes (including periodic oscillations) that were qualitatively captured by a mathematical model focused on the respective trophic chain levels. The identification of all model parameters and the use of numerical simulations complemented the above analysis and allowed to gain more insights into the complex dynamics of these oligotypic communities and on the most relevant factors determining the ecosystem equilibria.

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 o...

In fishes, exploitation of aerial gas exchange has evolved independently many times, involving a variety of air-breathing organs. Indeed, air-breathing occurs in at least 49 known families of fish (Graham, 1997). Many amphibious vertebrates, at some stage of their development are actually trimodal breathers that use various combinations of respiratory surfaces to breath both water (skin and/or gill) and air (skin and/or lung). The present review examines the evolutionary implications of air-breathing organs in fishes and the morphology of the peripheral receptors and the neurotransmitter content of the cells involved in the control of air-breathing. Control of breathing, whether gill ventilation or air-breathing, is influenced by feedback from peripheral and/or central nervous system receptors that respond to changes in PO2, PCO2 and/or pH. Although the specific chemoreceptors mediating the respiratory reflexes have not been conclusively identified, studies in water-breathing teleosts have implicated the neuroepithelial cells (NECs) existing in gill tissues as the O2 sensitive chemoreceptors that initiate the cardiorespiratory reflexes in aquatic vertebrates. Some of the air-breathing fishes, such as Protopterus, Polypterus and Amia have been shown to have NECs in the gills and/or lungs, although the role of these receptors and their innervation in the control of breathing is not known. NECs have been also reported in the specialized respiratory epithelia of accessory respiratory organs (ARO's) of some catfish species and in the gill and skin of the mudskipper Periophthalmodon schlosseri. Unlike teleosts matching an O2-oriented ventilation to ambient O2 levels, lungfishes have central and peripheral H+/CO2 receptors that control the acid-base status of the blood.

In this study, the vitellogenin (Vtg) modulation by genistein and polychlorinated biphenyl-126 (PCB-126) exposure in zebrafishes has been investigated. Both PCB-126 and genistein have been identified as aquatic pollutants and can further increase estrogenicity of waterways. Vtg is egg yolk precursor protein release by the hepatocytes during vitellogenesis. This process occurs normally in the hepatocytes in response to the activation with the estrogens such as 17-β-estradiol. Our immunohistochemical findings showed a Vtg expression that increases at 12 h and at 72 h in the liver of treated fishes with both PCB-126 and genistein, individually and in combination. Furthermore, for the first time, also hepatic stellate cells (HSC) in the liver parenchyma were strongly positive for vitellogenin.

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.

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.

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.

In the present study, it has been evaluated how 10 µm of polyethylene microspheres can be ingested by Artemia salina (Linnaeus, 1758) larvae within the first 7 days of the life cycle, and the impact on their health. Twelve A. salina larvae (instar I) groups were exposed to different microplastics (MPs) concentrations (0-1-10-102-103-104 MPs/mL), with and without Dunaliella salina as a food source. The results highlighted that A. salina larvae ingest MPs in relation to the exposure times in a dose-dependent manner and are significantly influenced by food availability. The highest contamination found was 306.2 MPs/individual at 104 MPs/mL exposure without a food source. No MPs were found in the presence of the food source from 1 to 102 MPs/mL, while contamination was detected at all concentrations of MPs without a food source. The worst effect on the developmental stages was evaluated at 168 h with a food source, with a delay compared to the control of I and II instars at 103 and 104 MPs/mL, respectively. Furthermore, microalgal feeding was significantly reduced for about 50% in the presence of 104 MPs/mL. These results highlight that aquatic microplastics pollution could affect the A. salina’s feeding behavior and life cycle.

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 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.

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.

Enormous marine biodiversity offers an endless reservoir of chemicals for many applications. In this scenario, the extraction of seaweeds represents an interesting source of compounds displaying antimicrobial activity. In particular, among the different red algae, Gracilaria gracilis plays an important role due to the presence of important bioactives in its composition. In spite of these features, an efficient culture system is still absent. In the present study, a novel algal culture method was developed and compared to another more common cultural practice, widely reported in literature. A higher efficiency of the new method, both for daily growth rate and biomass, was assessed. Furthermore, the growth inhibitory activity of five extracts, obtained using ethanol, methanol, acetone, chloroform or diethyl ether as a solvent, from the cultured G. gracilis was tested against Gram-positive and Gram-negative pathogens. Algal extracts exhibited a considerable inhibitory activity against B. subtilis strains, while a slight inhibition was observed against V. fischeri. The different extracts showed significant differences in bacterial growth inhibition, with the highest activity that was recorded for the ethanol extract, followed by that of methanol. Based on the chemical characterization, these findings could be related to the antimicrobial activity played by the combination of total carbohydrates and polyphenols, which were determined at high levels in ethanol and methanol extracts, as well as by the highest number and levels of single polyphenols. Conversely, the lower growth inhibitory activities found in chloroform and diethyl ether extracts could be related to the isolation of minor lipid classes (e.g., neutral and medium polar lipids) composed by fatty acids, such as stearic, oleic and arachidonic acids, typically characterized by antimicrobial activity. In consideration of the results obtained, the present study has a double implication, involving both the field of cultural practices and the exploitation of natural sources for the isolation of antimicrobial agents useful both in pharmaceutical and food applications.

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.

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.

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.

We report here on the macroscopic, light microscopic, and electron microscopic structure of the gas bladder (GB) of the spotted gar, Lepisosteus oculatus. The GB opens into the pharynx, dorsal to the opening of the oesophagus, through a longitudinal slit bordered by two glottal ridges. Caudal to the ridges, the GB is an elongated sac divided into a central duct and right and left lobes. The lobes are formed by a cranio-caudal sequence of large air spaces that open into the central duct. The structure of the GB is that of a membranous sac supported by a system of septa arising from the walls of a central duct. The septa contain variable amounts of striated and smooth muscle might function to maintain the bladder shape and in providing contractile capabilities. The presence of muscle cells, nerves, and neuroepithelial cells in the wall of the GB strongly suggests that GB function is tightly regulated. The central duct and the apical surface of the thickest septa are covered by mucociliated epithelium. Most of the rest of the inner bladder surface is covered by a respiratory epithelium which contains goblet cells and a single type of pneumocyte. These two cell types produce surfactant. The respiratory barrier contains thick areas with fibrillar material and cell prolongations, and thin areas that only contain basement membrane material between the capillary wall and the respiratory epithelium. Lungs and GBs share many anatomical and histological features. There appears to be no clear criterion for structural distinction between these two types of respiratory organs. J. Morphol. 276:90–101, 2015. © 2014 Wiley Periodicals, Inc.

An immunohistochemical study of the cutaneous glands of the caecilian Typhlonectes natans was conducted. Analyses of nerve fibres revealed that adrenergic and galanin-positive axons innervate the MECs and mediate their contraction. These glands may represent one of the main targets of the adrenergic ganglion cells and reflect the prominent preganglionic cell columns of the species studied. But neurochemical features of the sympathetic ganglia and retrograde tract-tracing studies are necessary to study the morphology and organization of the sympathetic nervous system of studied species.

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.

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.

Zaccone, G., Fudge, D.S., Winegard, T.M., Capillo, G., Kuciel, M., Funakoshi, K. and Lauriano, E.R. 2014. Confocal imaging and phylogenetic considerations of the subcutaneous neurons in the Atlantic hagfish Myxine glutinosa .— Acta Zoologica (Stockholm) 00: 000–000. We used confocal microscopy and immunohistochemistry to characterize the morphology of the subcutaneous neurons and the innervation of the slime glands and striated muscles in the hagfish Myxine glutinosa. A rich plexus of 5HT-, ChAT- and TH-positive neurons is described in the capsule of the slime glands. These neurons, like those of the subcutaneous plexus, receive pericellular terminations from the axons of central cells. Capsular neurons receive innervation from 5HT-positive and nNOS-positive nerve fibres. Other nerve endings belonging to two separate nerve populations are identified in the striated muscles. They contain TH and nNOS immunoreactivity. Due to the lack of any topographical labelling, the cell origin and the projections of the neurons into the cranial and spinal nerves are unknown. This study provides anatomical evidence of multiple (5HT and nNOS) peripheral innervation of the neurons. However, it does not provide information about the function of these neurons in the hagfish. We suggest that hagfish neurons have a phylogenetic relationship with the spinal group of the dorsal cells of lampreys and the supramedullary cells of teleosts.

Comprehensive two-dimensional gas chromatography-mass spectrometry was exploited for the obtainment of fatty acid fingerprints in fish and molluscs from South Italy. Scope of the lipidomic investigation was to highlight differences in the fatty acid profiles of samples from aquaculture and from the wild. The fish species under investigation were Sparus aurata and Dicentrarchus labrax, typically cultivated in Italy and commonly found in the fish market. Another aim was to find a correlation between the diet fed to fish and the expression of specific fatty acids in muscle composition. Beyond fish, two species of bivalves were analyzed to assess their lipidomic composition: Mytilus galloprovincialis (mussels) and Venerupis aurea var. laeta (clams). Both species originated from two salt lakes in South Italy. GC × GC–MS analysis showed that omega-3 fatty acids were naturally more concentrated in wild fish, whereas their presence in cultivated fish was lower and in favor of omega-6 and oleic acids, as an evident consequence of food supplementation. Bivalves reported a consistent amount of PUFA, although presenting some differences in the quality of fatty acids. As an additional parameter of investigation, the content of mercury was determined in all the samples by means of a Direct Mercury Analyzer, based on Atomic Absorption Spectroscopy. Wild fish reported a level of mercury that was ten times higher than in aquafarmed fish, and the maximum amount tolerated by the EC regulation. On the contrary, the level of mercury in molluscs was in general very low, particularly when compared with samples from other Italian sites of collection. Practical applications: One of the possible applications of the GC × GC methodology here presented could be the detection of a common food fraud. In the Mediterranean Sea, gilthead sea bream and sea bass are today rarely found in sea open waters. Basically, fish sold in the market comes from cultivation. Due to the fact that wild fish is more appreciated by the consumer, quite often fish sellers are encouraged to lie about the real origin of fish. The bidimensional plots shown in this study represent an extraordinary tool for differentiating the two types of fish. Also, this study provides substantial support to aquafarmers, who can draw useful advice on how to tune optimal diets to be fed to fish. In fact, it was demonstrated that fish feed highly affects fatty acid composition. The fatty acid composition of aquafarmed and wild caught fish and shellfish from the Mediterranean Sea was investigated. Comprehensive gas chromatography (GC × GC) was the analytical technique that allowed obtaining highly structured fingerprints of fatty acids.