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Epidermal rodlet cells were evaluated after treatment with the tyrosine phosphatase inhibitor pervanadate. Treatment of sunfish explant cell cultures with the inhibitor triggered a contraction of the rodlet cells and expulsion of cell contents. Time-lapse video differential interference contrast (DIC) microscopy was used to evaluate rodlet cell contraction and rodlet discharge. Three general steps in pervanadate triggered discharge were identified. First the rodlet cell undergoes a constriction of the midsection. Constriction is followed by a rapid forward movement of rodlets and sacs to the apical end of cell, culminating in discharge of rodlets and other cellular contents, including the nucleus. A ring-shaped structure around the apical pore was identified with DIC microscopy. Fluorescent-labeled phalloidin and antibodies to alpha-actinin and phosphotyrosine strongly stained the apical ring. A diffuse granular staining for both antibodies was also observed throughout the fibrous capsule. The results suggest that tyrosine kinases play a role in rodlet cell contraction. Alpha-actinin is a known substrate for tyrosine kinases and is a potential target for triggering rodlet cell contraction and rodlet ejection. Modification of alpha-actinin tyrosines could also be a mechanism for regulating the structural integrity of the fibrous capsule. © 2012 The Royal Swedish Academy of Sciences. Source

Manera M.,University of Teramo | Giari L.,University of Ferrara | De Pasquale J.A.,Morphogenyx Inc | Sayyaf Dezfuli B.,University of Ferrara
Aquatic Toxicology | Year: 2016

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 48 h. 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. © 2016 Elsevier B.V. Source

Manera M.,University of Teramo | Giari L.,University of Ferrara | Depasquale J.A.,Morphogenyx Inc | Dezfuli B.S.,University of Ferrara
Journal of Microscopy | Year: 2016

The objective of this study was to compare expert versus fractal analysis as new methods to evaluate branchial lamellar pathology in European sea bass Dicentrarchus labrax (Linnaeus, 1758) experimentally exposed to cadmium and to terbuthylazine. In particular, guided expert quantitative and fractal analysis were performed on selected images from semithin sections to test possible differences according to exposure class (unexposed, cadmium exposed, or terbuthylazine exposed) and the discrimination power of the two methods. With respect to guided expert quantitative analysis, the following elementary pathological features were assessed according to pre-determined cover classes: 'epithelial lifting', 'epithelial shrinkage', 'epithelial swelling', 'pillar cells coarctation', 'pillar cells detachment', 'channels fusion', 'chloride cells swelling' and 'chloride cells invasion'. Considering fractal analysis, DB (box dimension), DM (mass dimension), (mean fractal dimension) as fractal dimensions and lacunarity from DM and scan types were calculated both from the outlined and skeletonized (one pixel wide lines) images. Despite significant differences among experimental classes, only expert analysis provided good discrimination with correct classification of 91.7 % of the original cases, and of 87.5 % of the cross-validated cases, with a sensitivity of 95.45 % and 91.3 %, respectively, and a specificity of 75 % in both cases. Guided expert quantitative analysis appears to be a reliable method to objectively characterize fish gill pathology and may represent a powerful tool in environmental biomonitoring to ensure proper standardization and reproducibility. Though fractal analysis did not equal the discrimination power of the expert method, it certainly warrants further study to evaluate local variations in complexity or possible multiple scaling rules. © 2015 Royal Microscopical Society. Source

Dezfuli B.S.,University of Ferrara | Manera M.,University of Teramo | Giari L.,University of Ferrara | DePasquale J.A.,Morphogenyx Inc | Bosi G.,University of Milan
Fish and Shellfish Immunology | Year: 2015

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. © 2015 Elsevier Ltd. Source

Manera M.,University of Teramo | Sayyaf Dezfuli B.,University of Ferrara | DePasquale J.A.,Morphogenyx Inc | Giari L.,University of Ferrara
Ecotoxicology and Environmental Safety | Year: 2016

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 48 h. 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. © 2016 Elsevier Inc. Source

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