Investigating the internal system of defense of Gastropoda Aplysia depilans (Gmelin, 1791): Focus on hemocytes.

The internal defense system of mollusks represents an efficient protection against pathogens and parasites, involving several biological immune processes, such as phagocytosis, encapsulation, cytotoxicity, and antigenic recognition of self/non-self. Mollusks possess professional, migratory, and circulating cells that play a key role in the defense of the organism, the hemocytes. Several studies have been performed on hemocytes from different mollusks, but, to date, these cells are still scarcely explored. Different hemocyte populations have been found, according to the presence or absence of granules, size, and the species of mollusks studied. Our study aims to deepen the knowledge of the hemocytes of the gastropod Aplysia depilans using morphological techniques and light and confocal microscopy, testing Toll-like receptor 2, inducible nitric oxide synthetase, and nicotinic acetylcholine receptor alpha 7 subunit. Our results show two hemocyte populations distinguishable by size, and presence/absence of granules in the cytoplasm, strongly positive for the antibodies tested, suggesting for the first time the presence of these receptors on the surface of sea hare hemocytes by immunohistochemistry. These data help in the understanding of the immune system of this gastropod, providing additional data for comprehending the evolution of the defense response in metazoan phylogenesis.

Expression of the Antimicrobial Peptide Piscidin 1 and Neuropeptides in Fish Gill and Skin: A Potential Participation in Neuro-Immune Interaction.

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 Antimicrobic Peptide Piscidin1 in Gills Mast Cells of Giant Mudskipper Periophthalmodon schlosseri (Pallas, 1770).

The amphibious teleost Giant mudskipper (Periophthalmodon schlosseri, Pallas 1770) inhabit muddy plains and Asian mangrove forests. It spends more than 90% of its life outside of the water, using its skin, gills, and buccal-pharyngeal cavity mucosa to breathe in oxygen from the surrounding air. All vertebrates have been found to have mast cells (MCs), which are part of the innate immune system. These cells are mostly found in the mucous membranes of the organs that come in contact with the outside environment. According to their morphology, MCs have distinctive cytoplasmic granules that are released during the degranulation process. Additionally, these cells have antimicrobial peptides (AMPs) that fight a variety of infections. Piscidins, hepcidins, defensins, cathelicidins, and histonic peptides are examples of fish AMPs. Confocal microscopy was used in this study to assess Piscidin1 expression in Giant Mudskipper branchial MCs. Our results demonstrated the presence of MCs in the gills is highly positive for Piscidin1. Additionally, colocalized MCs labeled with TLR2/5-HT and Piscidin1/5-HT supported our data. The expression of Piscidin1 in giant mudskipper MCs highlights the involvement of this peptide in the orchestration of teleost immunity, advancing the knowledge of the defense system of this fish.

Expression of acetylcholine, its contribution to regulation of immune function and O2 sensing and phylogenetic interpretations of the African butterfly fish Pantodon buchholzi (Osteoglossiformes, Pantodontidae).

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.

Structural Identification of the Pacemaker Cells and Expression of Hyperpolarization-Activated Cyclic Nucleotide-Gated (HCN) Channels in the Heart of the Wild Atlantic Cod, Gadus morhua (Linnaeus, 1758).

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.

Acute effects of neonicotinoid insecticides on Mytilus galloprovincialis: A case study with the active compound thiacloprid and the commercial formulation calypso 480 SC.

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 < 0.01) in electrolytes ions (Cl-, K+, Na+, Ca2+, S-phosphor), lactate dehydrogenase (LDH) enzyme activity and glucose concentration following exposure to both substances. The TH-exposed mussels showed significant imbalance (P < 0.05) in CAT activity in digestive gland and gills. CA caused significant decrease (P < 0.05) in SOD activity in gills and in CAT activity in both tissues. Results of histological analyses showed severe damage in both digestive gland and gills in a time- and concentration-dependent manner. This study provides useful information about the acute toxicity of a neonicotinoid compound and a commercial insecticide on mussels. Nevertheless, considering that neonicotinoids are still widely used and that mussels are very important species for marine environment and human consumption, further researches are needed to better comprehend the potential risk posed by such compounds to aquatic non-target species.

A Multi-screening Evaluation of the Nutritional and Nutraceutical Potential of the Mediterranean Jellyfish Pelagia noctiluca.

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's sex were highlighted for the other compound classes. Rather, the distribution of the investigated chemical classes varied depending on the jellyfish'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.

Mucosal immune parameters, immune and antioxidant defence related genes expression and growth performance of zebrafish (Danio rerio) fed on Gracilaria gracilis powder.

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-1ß), 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.

New Insights into the Culture Method and Antibacterial Potential of Gracilaria gracilis.

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.

Assessment of Electrolytes and Metals Profile of the Faro Lake (Capo Peloro Lagoon, Sicily, Italy) and Its Impact on Mytilus galloprovincialis.

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.

Evaluation of Functionality and Biological Responses of Mytilus galloprovincialis after Exposure to Quaternium-15 (Methenamine 3-Chloroallylochloride).

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.

Intra-population variability of the saccular, utricular and lagenar otoliths of the garfish Belone belone (Linnaeus, 1760) from South-Western Ionian Sea (Central Mediterranean Sea).

The garfish Belone belone represents the only valid endemic Belone species for the Mediterranean Sea and the eastern Atlantic Ocean. It shows a wide global distribution range, with a high commercial value and ecological relevance in the pelagic domain. Despite this, there needs to be more knowledge regarding the otoliths of this species, with the total absence of descriptions regarding asterisci and lapilli from Mediterranean populations and a lack of studies on the reliability of shape analysis on its sagittae. The present paper aims to provide the first main contours description of the three otoliths pairs from a Mediterranean population, providing an accurate investigation of morphology, morphometry, and intra-specific variability of sagittae, lapilli, and asterisci. Results showed (i) the absence of directional bilateral asymmetry and sexual asymmetry for the three otoliths pairs, (ii) a different morphology and morphometry of sagittae, lapilli and asterisci than those described in the literature, and (iii) an enhanced variability between sagittae morphometry and shape between the three investigated size classes. All these data confirmed the reliability of the studied species of shape analysis, showing a geographical and size-related variability of otoliths features probably related to genetics, environmental conditions, and life habits variations.

Investigating Development and Defense Systems in Early Reproductive Stages of Male and Female Gonads in Black Scorpionfish Scorpaena porcus (Linnaeus, 1758).

One of the most crucial biological indicators in tracking long-term variations in the reproductive cycle is sexual development. Scorpaena porcus (Linnaeus, 1758), commonly known as the black scorpionfish, is a small teleost from the family Scorpaenidae. Much is known about its ecology, but data on its reproductive and defense systems are still lacking. Antimicrobial peptides (AMPs), such as piscidins, are integral components of the innate immune system in fish. These peptides exhibit a wide range of activity against bacteria, fungi, viruses, and protozoa and act as the first line of host defense. This study aims to investigate the primary sexual development stages in male and female gonads of black scorpionfish, providing additional knowledge on the reproductive biology of this teleost while evaluating concomitant changes in the expression of a Piscidin-1 antimicrobial peptide. The results show a histological, morpho-structural change from the immature stage to the developing virgin stage. Immunohistochemical analyses show that germinal and somatic cells are strongly reactive to Piscidin-1 in both gonads at an early ontogeny stage. These data suggest that Piscidin-1 may play a key role in the local defense system of scorpionfish gonads at this delicate stage, which is critical for the continuation and maintenance of the species. The present findings are potentially useful for a better understanding of the reproductive cycle of this fish, improving our knowledge of the interaction between the immune system and reproduction.

Intraspecific variability of the saccular and utricular otoliths of the hatchetfish Argyropelecus hemigymnus (Cocco, 1829) from the Strait of Messina (Central Mediterranean Sea).

Mesopelagic species are enjoining increasing attention due to the growing impact of fisheries activities on deep marine biocenosis. Improving the knowledge base on mesopelagic species is required to enhance their conservation due to the knowledge gaps regarding many species and families. In this context, otoliths can be fundamental to assessing their life history, ecomorphological adaptation to the deep environment and stock composition. The present paper aims to explore the saccular and utricular otoliths morphology and intra-specific variability of the hatchetfish, Argyropelecus hemigymnus, from the Strait of Messina. Lapilli and sagittae were collected from 70 specimens and separated into four size classes. Morphometric, shape and SEM investigations were performed to describe their morphology, contours, and external structural organization, also studying their intraspecific variability related to sample sizes and differences between otolith pairs. Results showed an otolith morphology different from those reported in the literature with fluctuating asymmetry in sagittae and lapilli belonging to Class IV, and a high otolith variability between all the size classes. Data herein described confirm the otoliths singularity of the population from the Strait of Messina, shaped by a unique marine environment for oceanographic and ecological features.

Coelomocytes of the Oligochaeta earthworm Lumbricus terrestris (Linnaeus, 1758) as evolutionary key of defense: a morphological study.

Metazoans have several mechanisms of internal defense for their survival. The internal defense system evolved alongside the organisms. Annelidae have circulating coelomocytes that perform functions comparable to the phagocytic immune cells of vertebrates. Several studies have shown that these cells are involved in phagocytosis, opsonization, and pathogen recognition processes. Like vertebrate macrophages, these circulating cells that permeate organs from the coelomic cavity capture or encapsulate pathogens, reactive oxygen species (ROS), and nitric oxide (NO). Furthermore, they produce a range of bioactive proteins involved in immune response and perform detoxification functions through their lysosomal system. Coelomocytes can also participate in lithic reactions against target cells and the release of antimicrobial peptides. Our study immunohistochemically identify coelomocytes of Lumbricus terrestris scattered in the epidermal and the connective layer below, both in the longitudinal and in the smooth muscle layer, immunoreactive for TLR2, CD14 and α-Tubulin for the first time. TLR2 and CD14 are not fully colocalized with each other, suggesting that these coelomocytes may belong to two distinct families. The expression of these immune molecules on Annelidae coelomocytes confirms their crucial role in the internal defense system of these Oligochaeta protostomes, suggesting a phylogenetic conservation of these receptors. These data could provide further insights into the understanding of the internal defense system of the Annelida and of the complex mechanisms of the immune system in vertebrates.

Morpho-structural adaptations of the integument in different aquatic organisms.

The integument acts as a barrier to protect the body from harmful pathogenic infectious agents, parasites, UV rays, trauma, and germs. The integument of invertebrates and vertebrates are structurally different: while invertebrates usually have a simple monolayer epidermis frequently covered by mucus, cuticles, or mineralized structures, vertebrates possess a multilayered epidermis with several specialized cells. This study aims to describe by morphological, histological, and immunohistochemical analyses, the morpho-structural adaptations throughout evolution of the integument of gastropod Aplysia depilans (Gmelin, 1791), ascidian Styela plicata (Lesuer, 1823), myxine hagfish Eptatretus cirrhatus (Forster, 1801) and teleost Heteropneustes fossilis (Bloch, 1794) for the first time, with special reference to sensory epidermal cells. Different types of cells could be identified that varied according to the species; including mucous cells, serous glandular cells, clavate cells, club cells, thread cells, and support cells. In all integuments of the specimens analyzed, sensory solitary cells were identified in the epidermis, immunoreactive to serotonin and calbindin. Our study provided an essential comparison of integuments, adding new information about sensory epidermal cells phylogenetic conservation and on the structural changes that invertebrates and vertebrates have undergone during evolution.

Multi-characteristic toxicity of enantioselective chiral fungicide tebuconazole to a model organism Mediterranean mussel Mytilus galloprovincialis Lamarck, 1819 (Bivalve: Mytilidae).

The survey of available scientific literature shows a lack of data on the chronic effects of tebuconazole (TEB) on non-target aquatic organisms. Therefore, this study evaluates toxicity (10 and 20 days) of two considered concentrations 2 ng/L (E1) and 2 µg/L (E2) of TEB to bioindicator species Mytilus galloprovincialis. To this end, the TEB concentrations measured in soft mussel tissues showed a time-dependent increasing trend. The viability of haemocyte and digestive gland (DG) cells was higher than 95 % during the experiment. However, DG cells lost the ability to regulate their volume in both groups after 20-d. The E1 treatment increased Cl- and Na+ levels, and E2 decreased Na+ levels in the haemolymph. In addition, levels of superoxide dismutase (SOD) activity and oxidatively modified protein (OMP) increased after 10- and 20-d in both treatments. Histopathological findings showed abnormalities in the E2, e.g., haemocyte infiltration, hypertrophy, and hyperplasia in gills and DG. This study reveals the potential risks of TEB usage in the model organism M. galloprovincialis, primarily via bioaccumulation of TEB in food web links, and improves knowledge about its comprehensive toxicity.

Structural and Functional Aspects of the Spleen in Molly Fish Poecilia sphenops (Valenciennes, 1846): Synergistic Interactions of Stem Cells, Neurons, and Immune Cells.

In fish, the spleen is the prime secondary lymphoid organ. It has a role in the induction of adaptive immune responses, in addition to its significance in the elimination of immune complexes. This study was conducted on 18 randomly obtained adult molly fish (Poecilia sphenops) of both sexes using histological, immunohistochemical, and ultrastructural studies to highlight the cellular components of the spleen and their potential role in the immune system. The spleen of molly fish was characterized by the presence of well-distinct melanomacrophage centers, and other basic structures present in higher vertebrates including red and white pulps, blood vessels, and ellipsoids. Some mitotic cells could also be identified in the red pulp. Mast cells with characteristic metachromatic granules could be seen among the splenic cells. Rodlet cells were randomly distributed in the spleen and were also observed around the ellipsoids. The white pulp of the spleen expressed APG5. The expressions were well distinct in the melanomacrophages, leukocytes, and macrophages. Myostatin was expressed in leukocytes and epithelial reticular cells. IL-1ß showed immunoreactivity in monocytes and macrophages around the ellipsoids. NF-κB and TGF-ß were expressed in macrophages and epithelial reticular cells. Nrf2 expression was detected in stem cells and rodlet cells. Sox-9 had a higher expression in epithelial reticular cells and stem cells. The high frequency of immune cells in the spleen confirmed its role in the regulation of both innate and adaptive immunity, cell proliferation, and apoptosis.

Localization of vasoactive intestinal peptide and toll-like receptor 2 immunoreactive cells in endostyle of urochordate Styela plicata (Lesueur, 1823).

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.

Rodlet cells in kidney of goldfish (Carassius auratus, Linnaeus 1758): A light and confocal microscopy study.

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.