Texture Analysis as a Discriminating Tool: Unmasking Rodlet Cell Degranulation in Response to a Contaminant of Emerging Concern.

BACKGROUND: Contaminants of emerging concern (CECs) have garnered significant attention due to their potential impacts on ecology, wildlife, and human health. The interest in these contaminants arises from their inadequate regulation or lack of routine monitoring in natural environments. Among them, per- and polyfluoroalkyl substances (PFAS) are of particular concern due to their notable propensity to accumulate within the kidney, significantly influencing the excretion of these pollutants. Rodlet cells (RCs) have emerged as promising indicators of immunotoxicity in response to chemical stressors. A prior comprehensive study extensively detailed the effects of sub-chronic exposure to perfluorooctanoic acid (PFOA), a well-known PFAS compound, on RCs located in the hematopoietic tissue of the common carp kidney. Even at concentrations commonly found in the environment, PFOA exhibited a significant impact on the distribution patterns of RCs, concurrently enhancing exocytosis activity. METHODS: The assessment of PFOA-induced RC degranulation employed texture analysis combined with linear discriminant analysis (LDA) to differentiate between various experimental exposure groups. The investigation encompassed three fish groups: an unexposed group, a group exposed to an environmentally relevant PFOA concentration (200 ng L-1), and a group exposed to a higher PFOA concentration (2 mg L-1). Texture analysis was conducted on high-resolution color (RGB) images obtained from light microscopy of ultrathin sections from five fish per experimental group, stained with toluidine blue. RESULTS: This analysis facilitated the quantification of potential cytoplasmic alterations associated with degranulation, encompassing all three RGB channels. The data subjected to LDA enabled the identification of the most distinctive texture characteristics, providing a reliable, objective, and reproducible method to differentiate between experimental groups. Remarkably, 98.0% of both the original and cross-validated cases were correctly classified. However, only one unexposed case was misclassified as a fish exposed to a 200 ng L-1 PFOA concentration, constituting the single false positive in the analysis. CONCLUSIONS: Utilizing texture analysis and LDA to quantify RC degranulation offers a dependable approach for assessing immunotoxicity within experimental models of toxicological and environmental pathology. This underscores the scientific significance of employing a morphological approach in such investigations.

Perfluorooctanoic Acid Promotes Recruitment and Exocytosis of Rodlet Cells in the Renal Hematopoietic Tissue of Common Carp.

Per- and polyfluoroalkyl substances (PFAS) are ubiquitous environmental contaminants, with perfluorooctanoic acid (PFOA) being a prominent member. PFOA poses a risk to aquatic ecosystems and human health due to its presence in water, environmental persistence, and bioaccumulation. Since rodlet cells (RCs) have emerged as potential biomarkers for chemical stressors, this study aimed to investigate the effects of sub-chronic PFOA exposure on RCs in the renal hematopoietic tissue of common carp. Three groups of fish were used: an unexposed control group and two groups exposed to environmentally relevant (200 ng L-1) and elevated (2 mg L-1) PFOA concentrations. Light and transmission electron microscopy were employed to assess RCs' distribution patterns and exocytosis, while biometry quantified RCs in the hematopoietic tissue. The results showed that, even at environmentally relevant concentrations, PFOA significantly influenced RCs' distribution patterns, leading to increased occurrence and cluster formation, as well as heightened exocytosis activity. This research highlights PFOA's immunotoxicity in fish and suggests the potential of RCs as sentinel cells in the immunological response to environmental contaminants. These findings enhance our understanding of PFAS toxicity and emphasise the importance of monitoring their impact on fish as representative vertebrates and reliable animal models.

Ultrastructural Alterations of the Glomerular Filtration Barrier in Fish Experimentally Exposed to Perfluorooctanoic Acid.

Per- and polyfluoroalkyl substances can be referred to as the most critical group of contaminants of emerging concern. They can accumulate in high concentration in the kidney and are known to potentially affect its function. Nonetheless, there is a lack of knowledge about their morphopathological effect on the glomerular filtration barrier. Since previous research suggests perfluorooctanoic acid (PFOA) induces glomerular protein leakage, the glomerular filtration barrier of 30 carp from the same parental stock (10 unexposed; 10 exposed to 200 ng L-1 of PFOA; and 10 exposed to 2 mg L-1 of PFOA for 56 days) was screened for possible PFOA-induced ultrastructural lesions in order to shed light on the related pathophysiology. PFOA exposure affected the glomerular filtration barrier in carp experimentally exposed to 2 mg L-1, showing ultrastructural alterations compatible with glomerulonephrosis: podocyte effacement, reduction of filtration slits and filtration slit diaphragms, basement membrane disarrangement, and occurrence of proteinaceous material in the urinary space. The results of the present research confirm the glomerular origin of the PFOA-induced protein leakage and can contribute to the mechanistic comprehension of PFOA's impact on renal function and to the assessment of the exposure effect of environmental pollutants on animals and humans, according to the One Health approach.

Perfluorooctanoic Acid Affects Thyroid Follicles in Common Carp (Cyprinus carpio).

Carp kidney is comprised of nephrons, hemopoietic tissue, and also hormonally-active thyroid follicles. Given this anatomical trait, it has been used to assess the thyroid disrupting potential of perfluorooctanoic acid (PFOA), a widespread and feared per- poly-fluoroalkyl substance and a persistent organic pollutant capable of interfering with the endocrine system in animals and humans. The occurrence and morphology of thyroid follicles in kidneys of carp experimentally exposed to 200 ng L-1 or 2 mg L-1 waterborne PFOA for 56 days were studied. The abundance of thyroid follicles was significantly higher and vesiculation increased in exposed fish as compared to controls. The number of vesiculated follicles/total number of follicles was positively correlated with PFOA blood concentration in fish exposed to the highest dose (2 mg L-1). The structure and ultrastructure of thyroid follicles were affected by PFOA also at the lower, environmentally relevant, concentration (200 ng L-1). Increased cellular projections, enhanced colloid endocytosis, rough endoplasmic reticulum enlargement and fragmentation and cytoplasm vacuolation were the main features displayed by PFOA-exposed carp. These results show that PFOA affects the occurrence and status of follicles and suggest the utility of fish kidney as a multipurpose biomarker organ in environmental pathology research, according to the One Health approach.

Effect of waterborne exposure to perfluorooctanoic acid on nephron and renal hemopoietic tissue of common carp Cyprinus carpio.

Per- and poly-fluoroalkyl substances (PFAS) are synthetic contaminants of global concern for environmental and public health. Perfluorooctanoic acid (PFOA) is an important PFAS, and considerable attention has been paid to its hepatotoxicity and reproductive and developmental impact, while potential nephrotoxic effects are largely ignored, especially in fish. This study documents the structural and ultrastructural effects on kidney of common carp Cyprinus carpio exposed to waterborne PFOA at an environmentally relevant concentration of 200 ng L-1 and at 2 mg L-1. Dilation of the glomeruli capillary bed, increased vesiculation in the proximal tubular segment, compromised mitochondria, apical blebbing, and sloughing of collecting duct cells occurred in exposed fish, primarily at 2 mg L-1. Perfluorooctanoic acid exposure resulted in higher numbers of rodlet cells (RC), putative immune cells exclusive to fish, mainly in the renal interstitium, than seen in controls, increased association with cells of myeloid lineage and modifications to ultrastructure. No differences in other cells of innate immunity were observed. Despite the absence of severe histological lesions, PFOA was shown to affect both nephron and hemopoietic interstitium at high concentration, raising concern of the impact on renal and immune function in fish. The response of RCs to PFOA concentration of 200 ng L-1 suggests a potential role as a biomarker of PFOA exposure.

Perspectives on Complexity, Chaos and Thermodynamics in Environmental Pathology.

Though complexity science and chaos theory have become a common scientific divulgation theme, medical disciplines, and pathology in particular, still rely on a deterministic, reductionistic approach and still hesitate to fully appreciate the intrinsic complexity of living beings. Herein, complexity, chaos and thermodynamics are introduced with specific regard to biomedical sciences, then their interconnections and implications in environmental pathology are discussed, with particular regard to a morphopathological, image analysis-based approach to biological interfaces. Biomedical disciplines traditionally approach living organisms by dissecting them ideally down to the molecular level in order to gain information about possible molecule to molecule interactions, to derive their macroscopic behaviour. Given the complex and chaotic behaviour of living systems, this approach is extremely limited in terms of obtainable information and may lead to misinterpretation. Environmental pathology, as a multidisciplinary discipline, should grant privilege to an integrated, possibly systemic approach, prone to manage the complex and chaotic aspects characterizing living organisms. Ultimately, environmental pathology should be interested in improving the well-being of individuals and the population, and ideally the health of the entire ecosystem/biosphere and should not focus merely on single diseases, diseased organs/tissues, cells and/or molecules.

Intestinal Histopathology due to an Acanthocephalan in Two Corvid Species from Northern Italy.

Carnivorous birds maintain parasites in the sylvatic cycle and have a role in their diffusion. The histopathology and ultrastructure of the intestine of 29 Hooded Crows (Corvus corone cornix) and 51 Eurasian Magpies (Pica pica), from the Province of Ferrara (Northern Italy), naturally infected with Sphaerirostris picae (Acanthocephala), were investigated. In both bird species, the prevalence of infection was around 10%, and the intensity of the infection in the Hooded Crows ranged from two to 12 acanthocephalans per host, whereas in the Eurasian Magpies it ranged from one to nine worms per bird. Previous records on the histopathology of acanthocephalans in birds do not provide information on the type of cells involved in the host's reaction. We aimed to gain information on the effects of acanthocephalans on the structural integrity of the birds' intestine and to describe the type of immune cells in the hosts against the parasite. Our results showed that S. picae disrupted the intestinal wall at the site of attachment by means of its neck and proboscis, and three main types of bird intestinal reactions were noticed. The most severe response of the hosts was against the proboscis because of the action of its hooks with recruitment of macrophages, giant cells, eosinophils, and heterophils. Sphaerirostris picae perforated the birds' entire intestinal wall, reaching the peritoneal visceral serosa, but it did not provoke a diffuse peritonitis.

Exploratory Factor Analysis of Rainbow Trout Serum Chemistry Variables.

Clinical chemistry offers a valuable, affordable, moderately invasive, and nondisruptive way to assess animal physiological status and wellness within defined ranges and is widely used as a diagnostic clinical tool. Because of physiological differences between mammals, clinical correlates of blood chemistry variables are not known in detail in fish, in which tissue/organ function tests are inferred from mammal-derived clinical chemistry data. The aim of the present study was to apply exploratory factor analysis on a serum chemistry dataset from clinically healthy, reared rainbow trout Oncorhynchusmykiss (Walbaum, 1792) to select the most correlated variables and to test for possible underlying factors explaining the observed correlations as possible physiological status estimates in trout. The obtained factors were tested for correlation with hepatosomatic and splenosomatic indexes. Thirteen highly correlated variables were selected out of 18 original serum chemistry variables, and three underlying factors (Factors 1, 2, and 3) were identified that explained the observed correlations among variables. Moreover, Factor 1 correlated negatively with the hepatosomatic index and Factors 2 and 3 negatively with the splenosomatic index. The obtained factors were tentatively associated with: protein (liver) metabolism (Factor 1), cell turnover (Factor 2), and lipid (muscle) metabolism (Factor 3).

Perfluorooctanoic acid-induced cellular and subcellular alterations in fish hepatocytes.

Liver perfluorooctanoic acid (PFOA) pathophysiology and related morphofunction disturbances were studied in common carp at the cellular and subcellular level and with box-counting fractal analysis of ultrathin sections to assess the effect of PFOA exposure on hepatocyte structure complexity and heterogeneity. Three experimental groups were investigated: unexposed; low exposure (200 ng L-1 PFOA); high exposure (2 mg L-1 PFOA). PFOA-exposed cells showed differences from controls at both tested concentrations, manifested mainly as cloudy swelling and reversible vacuolar degeneration. Subcellular modifications primarily involved mitochondria and secondarily endoplasmic reticulum, with evidence of increased subcellular turnover. The alterations were consistent with oxidative stress related pathophysiology. Fractal analysis discriminated exposed from unexposed fish and low from high PFOA exposure based on lacunarity and fractal dimension, respectively. The absence of irreversible organelle alterations and apoptosis/necrosis, along with the increase of cellular complexity, led to the conclusion that the patterns observed represented an adaptive recovery response.

Perfluorooctanoic Acid Exposure Assessment on Common Carp Liver through Image and Ultrastructural Investigation.

Perfluorooctanoic acid (PFOA) poses particular concern as an emerging pollutant in both surface and ground waters. Fish, as a natural inhabitant of these waters and being highly representative of vertebrates, represents an ideal animal model to assess the toxic effects of PFOA. Hereby, liver microscopic texture was comparatively evaluated in individuals of common carp subchronically exposed to PFOA using grayscale differential box counting, a fractal analysis method. Furthermore, liver cytoplasmic glycogen areas and ultrastructure were also evaluated and compared to the image analysis findings. Redundancy Analysis was performed to assess, in summary, how much the variation of fractal dimension and lacunarity was explained by the concentration of PFOA in liver, the mass of liver and the number of proliferating cell nuclear antigen (PCNA)-immunoreactive nuclei. Treatment group ordination was better determined by fractal dimension than lacunarity. Interestingly, a significant complexity increase was associated with the modification of liver microscopic texture due to PFOA exposure. This complexity increase was related to "cloudy swelling", possibly representing a primarily adaptive strategy against PFOA challenge, rather than a slight, reversible form of degeneration as traditionally proposed. The occurrence of endoplasmic reticulum stress, unfolded protein reaction and hormetic response was proposed and discussed.

Description of epithelial granular cell in catshark spiral intestine: Immunohistochemistry and ultrastructure.

We evaluated the histology of the spiral intestine of the blackmouth catshark (Galeus melastomus), a small shark distributed in the eastern Atlantic and Mediterranean Sea basin. Entire digestive tracts of 10 G. melastomus were studied using histochemical, immunohistochemical, and ultrastructural methods. Our studies identified a unique, large granular cell type in the intestinal epithelium. Transmission electron microscopy showed that the epithelial granular cell type made intimate contact, by means of junctional complexes, with adjacent epithelial and mucous cells. Several histochemical staining methods showed that the cytoplasmic granules were strongly eosinophilic. Immunostaining of intestinal sections revealed immunoreactivity of the granular cell to tumor necrosis factor-α (TNF-α) antibody. However, no reactivity to inducible-nitric oxide synthase (i-NOS), interleukin-6 (IL-6), interleukin IL-1ß, lysozyme, serotonin 5-HT antibodies was detected.

Pike intestinal reaction to Acanthocephalus lucii (Acanthocephala): immunohistochemical and ultrastructural surveys.

BACKGROUND: The Northern pike, Esox lucius, is a large, long-lived, top-predator fish species and occupies a broad range of aquatic environments. This species is on its way to becoming an important model organism and has the potential to contribute new knowledge and a better understanding of ecology and evolutionary biology. Very few studies have been done on the intestinal pathology of pike infected with helminths. The present study details the first Italian record of adult Acanthocephalus lucii reported in the intestine of E. lucius. RESULTS: A total of 22 pike from Lake Piediluco (Central Italy) were examined, of which 16 (72.7%) were infected with A. lucii. The most affected areas of gastrointestinal tract were the medium and distal intestine. The intensity of infection ranged from 1 to 18 parasites per host. Acanthocephalus lucii penetrated mucosal and submucosal layers which had a high number of mast cells (MCs) with an intense degranulation. The cellular elements involved in the immune response within the intestine of pike were assessed by ultrastructural techniques and immunohistochemistry using antibodies against met-enkephalin, immunoglobulin E (IgE)-like receptor (FCεRIγ), histamine, interleukin-6, interleukin-1ß, substance P, lysozyme, serotonin, inducible-nitric oxide synthase (i-NOS), tumor necrosis factor-α (TNF-α) and the antimicrobial peptide piscidin 3 (P3). In intestines of the pike, several MCs were immunopositive to 9 out of the 11 aforementioned antibodies and infected fish had a higher number of positive MCs when compared to uninfected fish. CONCLUSIONS: Pike intestinal tissue response to A. lucii was documented. Numerous MCs were seen throughout the mucosa and submucosal layers. In infected and uninfected intestines of pike, MCs were the dominant immune cell type encountered; they are the most common granulocyte type involved in several fish-helminth systems. Immunopositivity of MCs to 9 out of 11 antibodies is of great interest and these cells could play an important key role in the host response to an enteric helminth. This is the first report of A. lucii in an Italian population of E. lucius and the first account on positivity of MCs to piscidin 3 and histamine in a non-perciform fish.

Pigmented macrophages and related aggregates in the spleen of european sea bass dosed with heavy metals: Ultrastructure and explorative morphometric analysis.

The ultrastructure and morphometrics of pigmented macrophages (PMs) were assessed in the spleen of European sea bass experimentally dosed with Cd and Hg. PMs occurred either as solitary cells or as variably structured aggregations, defined as macrophage aggregates (MAs). Light microscopy revealed a high degree of morphological heterogeneity amongst MAs of all experimental groups. At the ultrastructural level, MAs showed a heterogeneous pigment content that was not influenced by the treatment. Cytoplasm rarefaction/vacuolation and euchromatic nuclei, were observed in PMs of dosed fish. Undosed and Cd-dosed samples differ significantly with regard to the following morphometric features: the Minor axis of the best fitting ellipse, Aspect Ratio, and Roundness. In Cd-dosed fish, MAs showed reduced size and complexity. Lacunarity showed significant differences between undosed and both Cd and Hg-dosed samples. These results suggest that heavy metals, and especially Cd, may influence the dynamics of PM aggregation/disaggregation. Variability in splenic MAs was observed both by light and electron microscopy. However, only the morphometric techniques adequately and objectively described the phenomenon, allowing a quantitative/statistical comparison of morphology among experimental groups. These morphometric analyses could be usefully applied in toxicological and ecotoxicological, as well as morpho-functional studies.

Nematode infection in liver of the fish Gymnotus inaequilabiatus (Gymnotiformes: Gymnotidae) from the Pantanal Region in Brazil: pathobiology and inflammatory response.

BACKGROUND: A survey on endoparasitic helminths from freshwater fishes in the Pantanal Region (Mato Grosso do Sul, Brazil) revealed the occurrence of third-larval stage of the nematode Brevimulticaecum sp. (Heterocheilidae) in most organs of Gymnotus inaequilabiatus (Gymnotidae) also known by the local name tuvira. The aim of the present study was to examine Brevimulticaecum sp.-infected tuvira liver at the ultrastructural level and clarify the nature of granulomas and the cellular elements involved in the immune response to nematode larvae. METHODS: Thirty-eight adult specimens of tuvira from Porto Morrinho, were acquired in January and March 2016. Infected and uninfected liver tissues were fixed and prepared for histological and ultrastructure investigations. RESULTS: The prevalence of infection of tuvira liver by the nematode larvae was 95 %, with an intensity of infection ranging from 4 to 343 larvae (mean ± SD: 55.31 ± 73.94 larvae per liver). In livers with high numbers of nematode larvae, almost entire hepatic tissue was occupied by the parasites. Hepatocytes showed slight to mild degenerative changes and accumulation of pigments. Parasite larvae were surrounded by round to oval granulomas, the result of focal host tissue response to the infection. Each granuloma was typically formed by three concentric layers: an outer layer of fibrous connective tissue with thin elongated fibroblasts; a middle layer of mast cells entrapped in a thin fibroblast-connective mesh; and an inner layer of densely packed epithelioid cells, displaying numerous desmosomes between each other. Numerous macrophage aggregates occurred in the granulomas and in the parenchyma. CONCLUSIONS: Our results in tuvira showed that the larvae were efficiently sequestered within the granulomas, most of the inflammatory components were confined within the thickness of the granuloma, and the parenchyma was relatively free of immune cells and without fibrosis. Presumably this focal encapsulation of the parasites permits uninfected portions of liver to maintain its functions and allows the survival of the host.

Multivariate approach to gill pathology in European sea bass after experimental exposure to cadmium and terbuthylazine.

The combined use of guided quantitative expert analysis and of multivariate exploratory data analysis is reported as a robust, sensitive and sufficiently specific approach to study European sea bass gill secondary lamellar pathology after exposure to incremental doses of cadmium and terbuthylazine up to 48h. The following elementary pathological findings were considered: "epithelial lifting", "epithelial shrinkage", "epithelial swelling", "pillar cells coarctation", "pillar cells detachment", "channels fusion", "chloride cells swelling", and "chloride cells invasion". The relative spatial extension was determined according to exposure class and data were analyzed by means of canonical correspondence analysis (CCA), linear discriminant analysis (LDA) and canonical variates analysis (CVA). Histologically and ultrastructurally, cellular shrinkage/coarctation prevailed in cadmium exposed lamellae, whereas cellular swelling and epithelial lifting were predominant in terbuthylazine exposed lamellae compared to unexposed fish. Both CCA and CVA permit a good graphical data grouping according to exposure classes by means of the convex hull minimum polygons. This also reveals exposure dose and time gradients in CCA plot. Accordingly, epithelial swelling and epithelial shrinkage were comparatively associated to higher exposure time, whereas epithelial shrinkage and pillar cells coarctation were comparatively associated to higher exposure dose. LDA with only "epithelial shrinkage", "epithelial swelling" and "pillar cells coarctation" in the model classified correctly 87.5% of the cross-validated cases. A possible pathogenetic relationship between the discriminant elementary lesions and the toxic mode of action at the cellular level of both cadmium and terbuthylazine is also discussed.

Local connected fractal dimension analysis in gill of fish experimentally exposed to toxicants.

An operator-neutral method was implemented to objectively assess European seabass, Dicentrarchus labrax (Linnaeus, 1758) gill pathology after experimental exposure to cadmium (Cd) and terbuthylazine (TBA) for 24 and 48h. An algorithm-derived local connected fractal dimension (LCFD) frequency measure was used in this comparative analysis. Canonical variates (CVA) and linear discriminant analysis (LDA) were used to evaluate the discrimination power of the method among exposure classes (unexposed, Cd exposed, TBA exposed). Misclassification, sensitivity and specificity, both with original and cross-validated cases, were determined. LCFDs frequencies enhanced the differences among classes which were visually selected after their means, respective variances and the differences between Cd and TBA exposed means, with respect to unexposed mean, were analyzed by scatter plots. Selected frequencies were then scanned by means of LDA, stepwise analysis, and Mahalanobis distance to detect the most discriminative frequencies out of ten originally selected. Discrimination resulted in 91.7% of cross-validated cases correctly classified (22 out of 24 total cases), with sensitivity and specificity, respectively, of 95.5% (1 false negative with respect to 21 really positive cases) and 75% (1 false positive with respect to 3 really negative cases). CVA with convex hull polygons ensured prompt, visually intuitive discrimination among exposure classes and graphically supported the false positive case. The combined use of semithin sections, which enhanced the visual evaluation of the overall lamellar structure; of LCFD analysis, which objectively detected local variation in complexity, without the possible bias connected to human personnel; and of CVA/LDA, could be an objective, sensitive and specific approach to study fish gill lamellar pathology. Furthermore this approach enabled discrimination with sufficient confidence between exposure classes or pathological states and avoided misdiagnosis.

Occurrence of immune cells in the intestinal wall of Squalius cephalus infected with Pomphorhynchus laevis.

A sub-population of 34 specimens of chub, Squalius cephalus, was sampled from the River Brenta (Northern Italy) and examined for ecto- and endo-parasites. Pomphorhynchus laevis (Acanthocephala) was the only enteric helminth encountered. Immunofluorescence and ultrastructural studies were conducted on the intestines of chub. Near the site of parasite's attachment, mucous cells, mast cells (MCs), neutrophils and rodlet cells (RCs) were found to co-occur within the intestinal epithelium. The numbers of mucous cells, MCs and neutrophils were significantly higher in infected fish (Mann-Whitney U test, p < 0.05). Dual immunofluorescence staining with the lectin Dolichos Biflorus Agglutinin (DBA) and the macrophage-specific MAC387 monoclonal antibody, with parallel transmission electron microscopy, revealed that epithelial MCs often made intimate contact with the mucous cells. Degranulation of a large number of MCs around the site of the acanthocephalan's attachment and in proximity to mucous cells was also documented. MCs and neutrophils were abundant in the submucosa. Immune cells of the intestinal epithelium have been described at the ultrastructural level and their possible functions and interactions are discussed.

Histopathology and the inflammatory response of European perch, Perca fluviatilis muscle infected with Eustrongylides sp. (Nematoda).

BACKGROUND: The European perch, Perca fluviatilis L. is a common paratenic host of dioctophymatid nematodes belonging to the genus Eustrongylides. In this host, once infected oligochaetes, which serve as the first intermediate host, are ingested, Eustrongylides migrates through the intestine and is frequently encountered within the musculature, free within the body cavity, or encapsulated on the viscera. The current study details the first Italian record of Eustrongylides sp. with larvae reported in the muscle of P. fluviatilis. METHODS: Uninfected and nematode-infected muscle tissues of perch were fixed and prepared for histological evaluation and electron microscopy. Some sections were subjected to an indirect immunohistochemical method using anti-PCNA, anti-piscidin 3 and anti-piscidin 4 antibodies. RESULTS: A total of 510 P. fluviatilis (TL range 15-25 cm) from Lake Trasimeno, Perugia were post-mortemed; 31 individuals had encysted nematode larvae within their musculature (1-2 worms fish(-1)). Histologically, larvae were surrounded by a capsule with an evident acute inflammatory reaction. Muscle degeneration and necrosis extending throughout the sarcoplasm, sarcolemmal basal lamina, endomysial connective tissue cells and capillaries was frequently observed. Within the encapsulating reaction, macrophage aggregates (MAs) were seen. Immunohistochemical staining with the proliferating cell nuclear antigen (PCNA) revealed numerous PCNA-positive cells within the thickness of the capsule and in the immediate vicinity surrounding Eustrongylides sp. larvae (i.e. fibroblasts and satellite cells), suggesting a host response had been initiated to repair the nematode-damaged muscle. Mast cells (MCs) staining positively for piscidin 3, were demonstrated for the first time in response to a muscle-infecting nematode. The piscidin 3 positive MC's were seen principally in the periphery of the capsule surrounding the Eustrongylides sp. larva. CONCLUSIONS: A host tissue response to Eustrongylides sp. larvae infecting the musculature of P. fluviatilis was observed. Numerous fibroblasts, MAs and MCs were seen throughout the thick fibroconnectival layer of the capsule enclosing larvae. PCNA positive cells within the capsule suggest that host repair of nematode damaged muscle does occur, while the presence of the antimicrobial peptide piscidin 3 is shown for the first time. This is first report of Eustrongylides sp. in an Italian population of P. fluviatilis.

Lead, cadmium and chromium in raw and boiled portions of Norway lobster.

Lead, cadmium and chromium levels were determined in different raw and boiled portions of Norway lobster caught in the central Adriatic Sea (Italy). In raw specimens, the lowest concentrations were always detected in the white meat. Lead and cadmium content in the edible portion never exceeded the maximum levels set by European legislation. The highest cadmium and chromium values (0.47 ± 0.04 and 0.62 ± 0.13 mg/kg wet weight, respectively) were detected in the brown meat, while the highest lead concentrations were found in the exoskeleton (0.21 ± 0.01 mg/kg wet weight). Also, the boiled samples showed the lowest metal levels in the white meat, even if a significant increase (p < 0.01) was found for lead and cadmium compared to the corresponding raw portions. Among metals, chromium showed the highest concentrations in both raw and boiled portions, but up to now, the European legislation did not envisage any limits in seafood.

Heavy metal (As, Cd, Hg, Pb, Cu, Zn, Se) concentrations in muscle and bone of four commercial fish caught in the central Adriatic Sea, Italy.

Heavy metal (As, Cd, Cu, Pb, Zn, Hg and Se) concentrations in the muscle and bone of four fish species (Mullus barbatus, Merluccius merluccius, Micromesistius poutassou, and Scomber scombrus) from the central Adriatic Sea were measured and the relationships between fish size (length and weight) and metal concentrations in the tissues were investigated. Samples were analyzed by inductively coupled plasma-atomic emission spectrophotometry with automatic dual viewing. In the muscle, results of linear regression analysis showed that, except for mercury, significant relationships between metal concentrations and fish size were negative. Only mercury levels were positively correlated with Atlantic mackerel size (p < 0.05). No significant variations of heavy metal concentrations were observed in muscles of the examined species, but a significant difference (p < 0.01) was found for As, Cd, Pb, and Se concentrations in bone. All the investigated metals showed higher values in the muscle than in bone, except for lead and zinc. Regarding cadmium, lead, and mercury maximum levels, set for the edible portion by European legislation, several samples exceeded these values, confirming the heavy metal presence in species caught near the Jabuka Pit.