Toxicological Evaluation of Acetylsalicylic Acid in Non-Target Organisms: Chronic Exposure on Mytilus galloprovincialis (Lamarck, 1819).

Pharmaceuticals are now considered to be established contaminants, and their presence in water poses a real risk not only to the marine ecosystem, as they may adversely affect non-target organisms that are exposed to them, but also indirectly to humans. This is particularly true for the model organism considered in this work, Mytilus galloprovincialis (Lamarck, 1819), a suspensivore and bioaccumulating organism that enters the human food chain. Among the most commonly used over-the-counter medicines, anti-inflammatory drugs certainly feature prominently, with acetylsalicylic acid (ASA) at the top. In this work, M. galloprovincialis specimens were exposed to two concentrations of ASA (10 and 100 µg/L) for 10 and 20 days to evaluate possible alterations in the decrease in regulatory volume (RVD) in digestive gland cells and cell viability of both these cells and hemocytes. In addition, the histopathological condition index of the gills and digestive gland was evaluated. The data obtained showed that chronic exposure to ASA did not alter the cell viability of hemocytes and digestive gland cells but alters the physiological mechanisms of volume regulation in the digestive gland and, in addition, a time-dose reaction to ASA in the gills and digestive gland showing numerous alterations such as lipofuscin deposits and hemocyte infiltration was found. These results confirm the potential toxicity to the marine biota, highlighting the necessity to deepen the knowledge regarding the link between over-the-counter pharmaceuticals and non-target organisms.

Effect of light on the calretinin and calbindin expression in skin club cells of adult zebrafish.

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.

Expression of Langerin/CD207 in airways, lung and associated lymph nodes of a stranded striped dolphin (Stenella coeruleoalba).

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.

Merkel cells immunohistochemical study in striped dolphin (Stenella coeruleoalba) skin.

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.

Intestinal immunity of dogfish Scyliorhinus canicula spiral valve: A histochemical, immunohistochemical and confocal study.

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 α5ß1 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.

Microplastics occurrence in the Tyrrhenian waters and in the gastrointestinal tract of two congener species of seabreams.

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.

Neuromast hair cells retain the capacity of regeneration during heavy metal exposure.

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.

Immunohistochemical characterization of epidermal dendritic-like cells in giant mudskipper, Periophthalmodon schlosseri.

Giant Mudskipper, Periophthalmodon schlosseri (Pallas, 1770), is euryhaline, amphibious, and air-breathing fish. These fishes live in close association to mangrove forests and often spend over 90% of time out of water, in adjacent mudflats. They have developed morphological and physiological adaptations to satisfy their unique lifestyles. The skin is the primary interface between the body and the environment, and has a central role in host defence. The initiation of immune responses to antigens in the vertebrate skin has often been attributed to epidermal Langerhans'cells (LC) that are dendritic cells (DC), antigen-presenting cells (APC) which reside in the epidermis. Dendritic cells have been characterized morphologically and functionally in the teleost fish tissues such as rainbow trout, salmonids, medaka, African catfish and zebrafish. However, there is no evidence of the presence of DCs and their role in mudskippers immunity. The aim of this preliminary study was to characterize, through use of specific antibodies: Toll-like receptor 2, S100, serotonin (5-HT), and Vesicular acetylcholine transporter VAChT, a specific DC-like subpopulation in Pn. schlosseri's epidermis.

Immunohistochemical colocalization of G protein alpha subunits and 5-HT in the rectal gland of the cartilaginous fish Scyliorhinus canicula.

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.

Immunohistochemical characterization of Toll-like receptor 2 in gut epithelial cells and macrophages of goldfish Carassius auratus fed with a high-cholesterol diet.

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.