PFAS levels in fish species in the Po River (Italy): New generation PFAS, fish ecological traits and parasitism in the foreground.

Per- and polyfluoroalkyl substances (PFAS) are resistant to breakdown and are now considered ubiquitous and concerning contaminants. Although scientific and legislative interest in these compounds has greatly increased in recent decades, our knowledge about their environmental fate and their effects on organisms is still incomplete, especially those of the new generation PFAS. In this study, we analysed the level of PFAS contamination in the fish fauna of the Po River, the most important waterway in Italy, to evaluate the influence of different factors (such as fish ecological traits and parasitism) on the accumulation of 17 PFAS. After solvent extraction and purification, hepatic or intestinal tissues from forty specimens of bleak, channel catfish, and barbel were analysed by liquid chromatography coupled with mass spectrometry (LOQ = 2.5 ng/g w.w.). The prevalent PFAS were perfluorooctane sulfonate (PFOS), present in all samples at the highest concentration (reaching a maximum of 126.4 ng/g and 114.4 ng/g in bleak and channel catfish, respectively), and long-chain perfluoroalkyl carboxylic acids (PFDA and PFUnDA). Perfluorooctanoic acid and new generation PFAS (Gen X and C6O4) were not detected. Comparison of the hepatic contamination between the benthic channel catfish and the pelagic bleak showed similar concentrations of PFOS (p > 0.05) but significantly higher concentrations of other individual PFAS and of the sum of all measured PFAS (p < 0.05) in bleak. No correlation was found between the hepatic level of PFAS and fish size in channel catfish. For the first time, PFAS partitioning in a parasite-fish system was studied: intestinal acanthocephalans accumulated PFOS at lower levels than the intestinal tissue of their host (barbel), in contrast to what has been reported for other pollutants (e.g., metals). The infection state did not significantly alter the level of PFAS accumulation in fish, and acanthocephalans do not appear to be a good bioindicator of PFAS pollution.

Challenge for macrophages and mast cells of Chelon ramada to counter an intestinal microparasite, Myxobolus mugchelo (Myxozoa).

Thinlip mullet Chelon ramada is the most abundant mullet species found in the Comacchio lagoons (northern Adriatic Sea, Italy). Histological and ultrastructural sections of the intestine of C. ramada showed that over 83% of 48 mullets were infected with the intestinal parasite Myxobolus mugchelo (Myxozoa). In histological sections, plasmodia of M. mugchelo containing mature spores were situated closer to mucosal folds and were surrounded by numerous mast cells (MCs). Mature spores, generally oval in shape, were observed in the paracellular space among the enterocytes or within them. Near the infected epithelial cells, several MCs, rodlet cells and few neutrophils occurred. In intestinal epithelium, large cells resembling macrophages, some with spores of M. mugchelo inside, were observed. These macrophage-like cells were foamy and possessed elongate striated granules. The number of MCs and macrophages in the intestinal epithelium was significantly higher in parasitized fish. In some parasitized intestines, portions of epithelium were displaced by spores, or the spores were observed inside the damaged enterocytes. Immunohistochemical analysis of C. ramada infected or uninfected intestinal tissue revealed the presence of histamine, serotonin (5-HT), leu-enkephalin and inducible-nitric oxide synthase in epithelial macrophages. Several epithelial cells positive to proliferating cell-nuclear antigen were also observed in the proximity of the macrophages. The current study is the first to record the occurrence of intraepithelial macrophages which engulf myxozoan spores. A hypothesis on migration of spores from pancreas via intestinal wall to gut lumen is presented.

Proposing priorities of intervention for the recovery of native fish populations using hierarchical ranking of environmental and exotic species impact.

The freshwater populations of native fish species (Ns) have reached critical levels in many parts of the world due to combined habitat deterioration by human interventions and exotic fish species (Es) invasions. These alarming conditions require combined and well-designed interventions for restoring environmental quality and restricting Es invasion. The aim of the study is to propose a method to design spatially explicit priorities of intervention for the recovery of Ns populations in highly impacted freshwater systems by exotic multi-species invasion and water quality (WQ) degradation. WQ and Es are used as Ns descriptors, which require intervention. The method uses gradient analysis (ordination method of Canonical Correspondence Analysis) for assessing the weights of Ns descriptors' effects, which are further used to develop weighted severity indices; the severity index of WQ (Swq) and Es invasion (Se), respectively. Swq and Se are further merged to one combined total severity index St. The proposed method provides a) a ranking of the sites, based on the values of St, which denotes the priority for combined intervention in space and can be visualized in maps, b) a ranking of the most important Ns descriptors for each site to perform site-specific interventions, and c) Es rankings based on their potential threat on Ns for species-specific interventions. WQ, Es and Ns data from 208 sampling sites located in the Emilia-Romagna Region (Northern Italy) were used as a case study for the presentation of the proposed method. The application of the method showed that the north and northwestern lowland areas of Emilia-Romagna region presented the higher priority for intervention since the Ns of these areas are the most impacted from combined Es invasions and WQ degradation. Specific Es belonging to cyprinids, which are mostly responsible for the decline of aquatic vegetation and the increase of water turbidity, and a top Es predator (Wels catfish) were mostly present in these areas. Additionally, the most important WQ stressors of Ns were found to be COD, BOD and temperature that are all connected to oxygen depletion. The aforementioned conditions in the areas described by high priority for intervention can be used as a basis for the development of specific Ns conservation practices targeting the containment of the most harmful Es, the restoration of aquatic vegetation and the improvement of oxygen conditions.

Histochemical and immunohistochemical characterization of rodlet cells in the intestine of two teleosts, Anguilla anguilla and Cyprinus carpio.

Rodlet cells (RC) are characterized by a distinctive cell cortex and conspicuous inclusions named "rodlets." These cells are particularly abundant and large in size in intestine of eels. Histochemical, immunohistochemical and ultrastructural investigations were carried out on European eel Anguilla anguilla and Common carp Cyprinus carpio from Northern Italy. Eight biotinylated lectins were used to probe for specific carbohydrate residues in deparaffinized, hydrated intestinal sections of eel and carp. Five antibodies were tested on intestinal sections of both fish species: inducible nitric oxide synthase (i-NOS), leu-enkephalin, lysozyme, serotonin and tumour necrosis factor-α. Lectin histochemistry revealed rodlet cells (RCs) of the eel intestine to react with two of the eight lectins tested, specifically Concanavalin A (ConA) and Sambucus Nigra Agglutinin (SNA). This contrasted to lectin staining of RCs in the intestine of common carp, where four of the eight lectins showed a positive reaction; Dolichos Biflorus Agglutinin (DBA), Wheat Germ Agglutinin (WGA), SNA and ConA. RCs in eel and carp intestine were immunoreactive with antibodies to lysozyme and i-NOS. The occurrence of the inflammatory peptides lysozyme and i-NOS in RCs of the eel and common carp poses in favour that these cells are involved in the mechanism of defence against pathogens.

Histopathological and ultrastructural assessment of two mugilid species infected with myxozoans and helminths.

The histopathology and ultrastructure of the intestine of mullets, Liza ramada and Liza saliens, from Comacchio lagoons (northern Italy) naturally infected with myxozoans and helminths were investigated and described. Sixty-two (80.5%) of 77 mullets harboured one or more of the following parasites species: Myxobolus mugchelo (Myxozoa), Neoechinorhynchus agilis (Acanthocephala), Haplosplanchnus pachysomus and Dicrogaster contractus (Digenea). Co-occurrence of helminths with myxozoans was common. The main damage caused by digeneans was destruction of the mucosal epithelium of the villi, necrosis and degeneration of intestinal epithelial cells. More severe intestinal damage was caused by acanthocephalans which reach the submucosa layer with their proboscis. At the site of helminths infection, several mast cells (MCs), rodlet cells (RCs), mucous cells and few neutrophils and macrophages were observed in the epithelium. RCs and mucous cells exhibited discharge activity in close vicinity to the worm's tegument. M. mugchelo conspicuous plasmodia were encysted mainly in muscle and submucosa layers of the intestine. Indeed, spores of M. mugchelo were documented within the epithelial cells of host intestine and in proximity to MCs. Degranulation of the MCs near the myxozoans was very frequent.

A fish model for the study of the relationship between neuroendocrine and immune cells in the intestinal epithelium: Silurus glanis infected with a tapeworm.

Immunohistochemical, immunofluorescence and ultrastructural studies were conducted on a sub-population of 20 wels catfish Silurus glanis from a tributary of the River Po (Northern Italy). Fish were examined for the presence of ecto- and endo-parasites; in the intestine of 5 fish, 11 specimens of cestode Glanitaenia osculata were noted and was the only helminth species encountered. The architecture of intestine and its cellular features were nearly identical in either the uninfected S. glanis or in those harboring G. osculata. Near the site of worm's attachment, mucous cells, several mast cells (MCs), few neutrophils and some endocrine cells (ECs) were found to co-occur within the intestinal epithelium. MCs and neutrophils were abundant also in the submucosa. Immunohistochemical staining revealed that enteric ECs were immunoreactive to met-enkephalin, galanin and serotonin anti-bodies. The numbers of ECs, mucous cells and MCs were significantly higher in infected wels catfish (Mann-Whitney U test, p < 0.05). Dual immunofluorescence staining with the biotinylated lectin Sambucus nigra Agglutinin and the rabbit polyclonal anti-met-enkephalin or anti-serotonin, with parallel transmission electron microscopy, showed that ECs often made intimate contact with the mucous cells and epithelial MCs. The presence of numerous MCs in intestinal epithelium shows S. glanis to be an interesting model fish to study processes underlying intestinal inflammation elicited by an enteric worm. Immune cells, ECs and mucous cells of the intestinal epithelium have been described at the ultrastructural level and their possible functions and interactions together will be discussed.

Anguilla anguilla intestinal immune response to natural infection with Contracaecum rudolphii A larvae.

The European eel, Anguilla anguilla, is a major warm-water fish species cultured in North and South Europe. Seventy-one A. anguilla collected between 2010 and 2015 from the Comacchio lagoons were examined. Fish were infected and damaged by larvae (L3) of the nematode Contracaecum rudolphii A, which were encapsulated within the thickness of the intestinal wall and within the external visceral peritoneum (serosa). Conspicuous granulomas, visible at sites of infection, were arranged in a trilayer, formed by a series of concentric whorls. The cells involved in the immune response and their distribution in the granuloma layers were assessed by immunohistochemical, immunofluorescence, and ultrastructural techniques. The outer part of the granuloma contained macrophages, macrophage aggregates, and mast cells (MCs) scattered among fibroblasts. This layer was vascularized, with degranulation of MCs occurring in close proximity to the capillaries. The middle layer was rich in MCs and fibroblasts. The inner layer, closest to the parasite larva, consisted mainly of dark epithelioid cells, some of which were necrotic. Non-necrotic epithelioid cells formed desmosomes between themselves or with fibroblasts. Within the granulomas, numerous cells of different types were positive to proliferative cell nuclear antigen antibody, indicating a high degree of cellular proliferation around the larvae.

Onsite and online FT-NIR spectroscopy for the estimation of total nitrogen and moisture content in poultry manure.

The nitrogen and moisture of manure are highly variable parameters and depend on animal type, husbandry techniques, environmental conditions and storage time. The precision in manure dose estimation for crops fertilization depends on the total nitrogen and moisture content just before its incorporation in the field. The aim of the study is to develop a Fourier Transform Near Infrared (FT-NIR) spectroscopy method to determine the total Kjeldhal nitrogen (TKN%) and moisture (M%) of different types of poultry manure prior to land application. Samples covering a wide range of poultry types and different husbandry conditions were obtained from farms of North-Eastern Italy in order to develop the method. The method was calibrated (R(2) = 0.94 for TKN%, R(2) = 0.99 for M%) and validated (R(2) = 0.82 for TKN%, R(2) = 0.95 for M%) in the laboratory. An external validation was also performed in situ with independent samples, of similar origin to the previous data set, which were collected just before application in the field. Spectra acquisitions for these samples were carried out using the same instrumentation which was placed in a special vehicle for monitoring campaigns. The results showed satisfactory prediction accuracy (R(2) = 0.82 for TKN%, R(2) = 0.93 for M%). Finally, an additional analysis was performed to discriminate the different types of poultry effluents. The TKN and M measurements in the disposal areas indicated that current agronomic practices lead to more than double poultry manure oversupply. The proposed FT-NIR methodology aims to improve the current fertilization management and environmental protection by providing fast and precise estimations of poultry manure doses prior to land application.

Innate immune defence mechanisms of tench, Tinca tinca (L.), naturally infected with the tapeworm Monobothrium wageneri.

A histochemical and ultrastructural investigation of the cellular inflammatory response within the intestines of tench Tinca tinca L. naturally infected with the caryophyllidean cestode Monobothrium wageneri was conducted and the data obtained compared to those in uninfected counterparts. Cestode infections within the intestines were evident through the appearance of raised inflammatory swellings induced by the deep penetration of their scolices into the intestinal wall. Cestodes typically attached in tight clusters, inducing a massive hyperplastic granulocyte response of mast cells and neutrophils, which were significantly more numerous (P < 0·01) in the intestines of infected (n = 14) than of uninfected (n = 9) tench. Neutrophils were more abundant than mast cells (P < 0·01) in host tissues in close proximity to the parasite tegument. In transmission electron microscopy sections, mast cells and neutrophils were frequently observed in contact with or inside capillaries, and in close proximity to the cestode. Degranulation of both cell types was seen in the submucosa and lamina muscularis, notably in the immediate tissues surrounding the scolex of M. wageneri. No tegumental secretions were seen at the host-parasite interface. Occasional rodlet cells were encountered in the submucosa of infected fish.

Infiltration and activation of acidophilic granulocytes in skin lesions of gilthead seabream, Sparus aurata, naturally infected with lymphocystis disease virus.

Light, ultrastructural and immunocytochemical investigations were carried out on the skin of gilthead seabream, Sparus aurata L., naturally infected with lymphocystis iridovirus, to assess pathology and host cellular responses. Of 220,000 young seabream examined, 32,400 (14.7%) had clinical signs of lymphocystis and within 6 months of disease appearance, 45% of clinically affected fish had died. A subsample of 20 S. aurata (80.0 ± 12.5mm total length, mean ± S.D.), including 10 with lymphocystis on the skin and 10 clinically normal, were examined via immunohistochemistry. Affected skin displayed macroscopic, wart-like clusters of hypertrophic fibroblasts which arose from the dermis and were covered by the epithelium. Clusters were encountered on the head, trunk and fins, but there was no evidence of visceral lymphocystis. The lymphocysts were surrounded by numerous granular cells that were positive for the antimicrobial peptide (AMP) piscidin 3 and underwent intense degranulation. To identify the type of granular cells involved in this viral disease, a double immunohistochemical staining with the monoclonal antibody G7 (mAb G7), which is specific for seabream acidophilic granulocytes (AGs), and with anti-histamine (as a marker for mast cells, MCs) was applied to the skin sections of the 10 clinically normal fish and 10 fish with lymphocystis. In infected skin, the number of G7-positive cells (i.e., AGs) (18.5 ± 10.5, mean number of cells per 20,000 µm(2) ± S.D.) was significantly higher compared to their density in uninfected skin (1.4 ± 2.2) (t test, p<0.01). Notably, the AGs that infiltrated the skin lesions of infected animals were found to be degranulated and to produce the pro-inflammatory cytokine interleukin-1ß. No histamine-positive granular cells (i.e., MCs) were encountered in the lymphocystis lesions. The present study shows the response of skin to lymphocystis disease virus (LCDV) and provides evidence that AGs, but not MCs, are recruited and activated in response to this skin infection.

The impact of an oil spill on organs of bream Abramis brama in the Po River.

An oil spill into the River Lambro occurred on 23 February 2010 and reached the Po River the following day. Breams captured here on 1 March 2010, along with a sample from a control site, were examined by light and electron microscopy. The main affected organs were skin and gill with slight or no damage to liver, kidney, and intestine. The gills exhibited lamellar aneurisms, fusion of secondary lamellae, edema with epithelial lifting, mucous cell hypertrophy, and mucus hypersecretion. Significantly higher mucous cell density was observed in the skin of exposed fish. Histochemical staining revealed that acid glycoconjugates were prevalent in epidermal mucous cells in the exposed Abramis brama, whereas neutral and mixed glycoconjugates were dominant in the control fish. Rodlet cells were significantly more abundant in the kidney of exposed fish and showed ultrastructural differences compared to controls. These histopathologic effects were indicators of chemical stress due to exposure to oil. The present study is one of the first which explores the acute effects of this incident and makes part of a few reports focused on freshwater oil spill.

Intestinal immune response of Silurus glanis and Barbus barbus naturally infected with Pomphorhynchus laevis (Acanthocephala).

Immunopathological and ultrastructural studies were conducted on the intestine of barbel Barbus barbus and sheatfish Silurus glanis that were naturally infected with the acanthocephalan Pomphorhynchus laevis. Enteric helminths often cause inflammation of the digestive tract, inducing the recruitment of different types of immune cells at the site of infection. The results of our study clearly demonstrated that mast cells (MC) were the dominant immune cells which occur at the site of inflammation in both hosts. MC were associated with fibroblasts and were found in close proximity to, and inside, the capillaries of the intestine, thus, migration of mast cells via the bloodstream was suggested. Significant degranulation of MC was present. Immunohistochemical staining revealed met-enkephalin and serotonin (5-HT) in intestinal MC of both uninfected and infected barbel and the absence of the antimicrobial peptides piscidin 3 and piscidin 4 in both species. Data are discussed with respect to host immune response to an intestinal helminth and compared with other host-parasite systems.

Cellular response in semi-intensively cultured sea bream gills to Ergasilus sieboldi (Copepoda) with emphasis on the distribution, histochemistry and fine structure of mucous cells.

Light and ultrastructure studies were carried out on gill of sea bream, Sparus aurata L., naturally infected with Ergasilus sieboldi (Copepoda) to assess pathology and host cell responses. Thirty S. aurata were examined, and 23 (74%) were infected, the intensity of infection ranging from 3 to 50 parasites per host. The copepod encircled gill lamellae with its second antennae, occluded arteries, compressed the epithelium, provoked hyperplasia and haemorrhage, and often caused tissue disruption. Adjacent to the site of attachment, rodlet cells (RCs), mast cells (MCs) and mucous cells were observed. In parasitized fish, mucous cells were more abundant in infected gills than in uninfected (t-test, P<0.01), while no significant differences were encountered in the numbers of RCs and MCs between gill of infected and uninfected fish (t-test, P>0.01). In both infected and uninfected gill, the RCs were within the primary lamella and also sometimes occurred in secondary lamella. In healthy and infected gill, MCs were free within the connective tissue inside and outside the blood vessels of the primary lamellae and made close contact with vascular endothelial cells. Infected and uninfected gill mucous cells stained positively for neutral muco-substances (PAS positive). In all sea bream, gill mucous cells presented a central or eccentric electron-dense core within the mucus granules.