Ecology and effects of metazoan parasites of fish in transitional waters.

Given the abundance, heterogeneity and ubiquity of parasitic organisms, understanding how they influence biodiversity, evolution, health and ecosystem functionality is crucial, especially currently when anthropogenic pressures are altering host­parasite balances. This review describes the features, roles and impacts of metazoan parasites of fish occurring in transitional waters (TW). These aquatic ecosystems are highly productive and widespread around the globe and represent most favourable theatres for parasitism given the availability of hosts (invertebrates, fishes and birds) and an increased probability of parasite transmission, especially of those having complex life cycles. Fascinating examples of how parasitism can influence different hierarchical levels of biological systems, from host individuals and populations to entire aquatic communities, through effects on food webs come from this kind of ecosystem. Edible fish of commercial value found in TW can harbour some parasite species, significantly reducing host health, marketability and food safety, with possible economic and public health consequences. Many TW are historically exploited by humans as sources of relevant ecosystem services, including fisheries and aquaculture, and they are highly vulnerable ecosystems. Alteration of TW can be revealed through the study of parasite communities, contributing, as bioindicators, for assessing environmental changes, health and restoration. Fish parasites can provide much information about TW, but this potential appears to be not fully exploited. More studies are necessary to quantify the ecological, economic and medical impacts fish parasites can have on these important ecosystems.

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.

Histological and ultrastructural study of Myxobolus mugchelo (Parenzan, 1966) with initial histopathology survey of the Liza ramada host intestine.

This paper provides histological and ultrastructural data on Myxobolus mugchelo (Myxozoa), a parasite of the intestinal wall of thinlip mullet Liza ramada from the Comacchio lagoons (Northern Adriatic Sea, Italy). About 80% of the examined mullets were infected with this myxozoan. In histological sections, the plasmodia appeared rounded to ovoid and spindle shaped, measured 181.61 ± 97 × 122.79 ± 53.16 µm (mean ± standard deviation, SD) in size. Early sporogenic plasmodia were located primarily in the muscle layer where no pronounced inflammatory response was detected. Plasmodia containing mature spores were situated closer to mucosal folds and were encircled by numerous immune cells, especially mast cells. Mature spores were generally oval and 6.06 ± 0.40 × 3.48 ± 0.92 µm in size. The spores showed symmetrical and smooth valves. The anterior end of the spores contained two equal pyriform polar capsules measuring 2.19 ± 0.47 × 1.59 ± 0.29 µm. Inside the polar capsules, an isofilar polar filament displayed 5-6 coils perpendicular or slightly oblique to the longitudinal axis. The wall of the polar capsule was filled with a hyaline substance contrasting with the very dense internal matrix. The mucus layer surrounded the spore surface. A thorough comparison with the Myxobolus species infecting intestine of fish is provided. This is the first report on occurrence of this myxozoan species in L. ramada population of the North Adriatic Sea.

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.

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.

Temporal and spatial changes in the composition and structure of helminth component communities in European eels Anguilla anguilla in an Adriatic coastal lagoon and some freshwaters in Italy.

The composition and diversity of the helminth component communities in eels Anguilla anguilla were determined in three separate localities in Italy: an Adriatic coastal lagoon, Comacchio and two freshwater localities, the River Po and the Lake Piediluco. Data from Comacchio lagoon were analysed over 15 years to determine whether community composition and diversity changed significantly overtime. The community was species rich (nine species, all marine except Proteocephalus macrocephalus) and was dominated by a suite of digeneans: Deropristis inflata, Helicometra fasciata, Lecithochirium musculus and Bucephalus anguillae. The community showed little change in composition over the period, but the relative abundance and dominance of the species did alter. By contrast, the component communities in the freshwater localities were species poor and the dominant species were freshwater acanthocephalans, Pomphorhyncus laevis in River Po and Acanthocephalus rhinensis in Lake Piediluco. The helminth community of Lake Piediluco with five species was richer than that of the River Po with only three species, but was poorer than that of Comacchio lagoons. Similarity indices between samples from Comacchio were high; between the lagoon and the freshwater localities and between the two freshwater localities, similarity indices were very low. Helminth component community structure in coastal lagoons was comparable across Europe. The helminth community in the River Po was similar to those in the River Tiber and other European rivers whilst that in Lake Piediluco was similar to that in other European lakes. Levels of the pathogenic Anguillicoloides crassus in swim bladders were consistently lower in prevalence and abundance in the coastal lagoons than in freshwater localities. This suggests that this parasite may have little impact on migrating eels if they are indeed primarily of marine origin and so it may be of little importance in the recent decline of eel populations throughout Europe.

Segmentation of Renal Thyroid Follicle Colloid in Common Carp: Insights into Perfluorooctanoic Acid-Induced Morphometric Alterations.

Perfluorooctanoic acid (PFOA) is a globally prevalent contaminant of concern recognised for its persistence and detrimental effects on both wildlife and humans. While PFOA has been established as a disruptor of thyroid function, limited data exist regarding its impact on thyroid morphology. The kidney of the common carp (Cyprinus carpio) harbours numerous thyroid follicles, rendering it a valuable biomarker organ for investigating PFOA-induced thyroid alterations. Renal tissue slides, stained with the Alcian blue/PAS method, were examined from carp in three experimental groups: unexposed, exposed to 200 ng L-1, and exposed to 2 mg L-1 of PFOA over 56 days. Thyroid follicle colloids were segmented, and related morphometric parameters, including perimeter, area, and shape descriptors, were obtained. Statistical analyses revealed significant reductions in thyroid follicle colloid perimeter and area in the 200 ng L-1 PFOA group compared to the unexposed and 2 mg L-1 PFOA groups. Additionally, the fish exposed to PFOA exhibited a significantly higher follicle count compared to the unexposed fish. These findings collectively suggest that PFOA induces thyroid folliculogenesis, emphasising its impact on thyroid morphology even at an environmentally relevant concentration (200 ng L-1).

Acidophilic granulocytes in the gills of gilthead seabream Sparus aurata: evidence for their responses to a natural infection by a copepod ectoparasite.

Immunohistochemical and ultrastructural studies were conducted on the gills of gilthead seabream, Sparus aurata L., naturally infected with the copepod ectoparasite Ergasilus lizae (Krøyer, 1863) in order to assess pathology and the host immune cell response. Gills of 56 gilthead seabream were screened for ectoparasites; 36 specimens (64.3%) harbored E. lizae. Intensity of infection was 32.7 ± 8.7 (mean ± SE). Pathological alterations to the gills of the host were more pronounced in close proximity to the copepod site of attachment. The parasite attached to the gills by means of its modified second antennae, occluded the arteries, provoked epithelial hyperplasia and hemorrhages and most often caused lamellar disruption. Numerous granular cells were encountered near the site of E. lizae attachment. In both infected and uninfected gills, the granular cells lay within the filaments and frequently occurred within the connective tissue inside and outside the blood vessels of the filaments. The type of granular cell was identified by immunohistochemical staining by using the monoclonal antibody G7 (mAb G7), which specifically recognizes acidophilic granulocytes (AGs) of S. aurata and with an anti-histamine antibody (as a marker for mast cells, MCs) on sections from 13 uninfected gills and 21 parasitized gills. The use of mAb G7 revealed that, in gills harboring copepods, the number of G7-positive cells (i.e., AGs; 32.9 ± 3.9, mean number of cells per 45,000 µm2 ± SE) was significantly higher than the density of the same cells in uninfected gills (15.3 ± 3.8; ANOVA, P < 0.05). Few histamine-positive granular cells (i.e., MCs) were found in the uninfected and parasitized gills. Here, we show, for the first time in S. aurata infected gills, that AGs rather than MCs are recruited and involved in the response to E. lizae infection in seabream.

Piscidins in the intestine of European perch, Perca fluviatilis, naturally infected with an enteric worm.

This study set out to determine how an enteric parasite, the thorny-headed worm Acanthocephalus lucii, affected the expression of antimicrobial peptides (piscidins) in its host population, the European perch (Perca fluviatilis) collected from Lake Piediluco in Central Italy. A total of 87 perch were examined; 44 (50.5%) were infected with A. lucii (1-18 worms fish(-1)). Pathological changes and immune response were assessed using histological, ultrastructural and immunohistochemical techniques. The acanthocephalans only penetrated the surficial zone of the intestinal wall and induced only slight inflammation. The main damage was destruction of the mucosal epithelium covering the villi adjacent to the parasite's attachment site, and included necrosis and degeneration. Infected intestine had numerous mast cells (MCs), often in close proximity to, and within, the capillaries, and were associated with fibroblasts of the submucosal layer. Mast cells were irregular in shape with a cytoplasm filled by numerous electron-dense, membrane-bounded granules. Immunostaining of intestine with antibodies against the antimicrobial peptides piscidin 3 and piscidin 4 showed subpopulations of MCs that were positive. Piscidin-positive MCs were mainly observed among the epithelial cells of the intestine, but also within the submucosa. In both uninfected and parasite-infected perch, the number of MCs positive for piscidin 4 was higher than those immunoreactive with piscidin 3 (p < 0.05). For both piscidins, there was no significant difference in the number of positive MCs between parasite-infected and uninfected intestine (p > 0.05). However, uninfected fish showed higher immunostaining intensity for piscidin 3 than infected conspecifics (p < 0.05).

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.

Teleost innate immunity, an intricate game between immune cells and parasites of fish organs: who wins, who loses.

Fish, comprising over 27,000 species, represent the oldest vertebrate group and possess both innate and adaptive immune systems. The susceptibility of most wild fish to parasitic infections and related diseases is well-established. Among all vertebrates, the digestive tract creates a remarkably favorable and nutrient-rich environment, which, in turn, renders it susceptible to microparasites and macroparasites. Consequently, metazoan parasites emerge as important disease agents, impacting both wild and farmed fish and resulting in substantial economic losses. Given their status as pathogenic organisms, these parasites warrant considerable attention. Helminths, a general term encompassing worms, constitute one of the most important groups of metazoan parasites in fish. This group includes various species of platyhelminthes (digeneans, cestodes), nematodes, and acanthocephalans. In addition, myxozoans, microscopic metazoan endoparasites, are found in water-dwelling invertebrates and vertebrate hosts. It is worth noting that several innate immune cells within the fish alimentary canal and certain visceral organs (e.g., liver, spleen, and gonads) play active roles in the immune response against parasites. These immune cells include macrophages, neutrophils, rodlet cells, and mast cells also known as eosinophilic granular cells. At the site of intestinal infection, helminths often impact mucous cells number and alter mucus composition. This paper presents an overview of the state of the art on the occurrence and characteristics of innate immune cells in the digestive tract and other visceral organs in different fish-parasite systems. The data, coming especially from studies employed immunohistochemical, histopathological, and ultrastructural analyses, provide evidence supporting the involvement of teleost innate immune cells in modulating inflammatory responses to metazoan and protozoan parasitic infections.

Ultrastructural effects of cisplatin on the inner ear and lateral line system of zebrafish (Danio rerio) larvae.

Zebrafish, Danio rerio, has been a prominent model vertebrate for the study of chemical toxicity and human disease. Zebrafish hair cells (HCs) show significant structural, functional and molecular similarities to the mammalian inner ear HCs. We examined the effects of cisplatin, an anti-cancer drug, on HCs of the inner ear and on HCs and support cells (SCs) of neuromasts in zebrafish using transmission and scanning electron microscopy. Forty-five zebrafish larvae, 12 days post-fertilization, were assessed: 15 unexposed controls, 15 exposed to 10 µM cisplatin solution, and 15 exposed to 50 µM cisplatin solution. Hair cells in the cristae and maculae of the inner ear and of neuromasts were extremely sensitive to cisplatin. The drug was associated with vacuolization and the presence of myelinoid bodies in HC cytoplasm and with a condensation of the nuclear chromatin. The predominant pattern of injury was widespread degeneration of mitochondria, which appeared swollen and less electron-dense with disorganized or reduced cristae. Severity of damage seemed to be concentration-dependent, and the inner ear suffered more damage than the lateral line. Alterations similar to those in HCs were also observed in SCs of the neuromasts. Scanning electron microscopy showed loss of kinocilia in neuromasts of fish exposed to the higher concentration of cisplatin.

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.

Mast cell responses to Ergasilus (Copepoda), a gill ectoparasite of sea bream.

Immunocytochemical, light microscopy and ultrastructural studies were conducted on gill of sea bream, Sparus aurata L., naturally parasitized with the important parasitic copepod Ergasilus sp. to assess pathology and cellular responses. Thirty-seven S. aurata were examined from a fish farm; 26 (70%) were parasitized, with infection intensity ranging from 3 to 55 parasites per fish. Hosts were divided into two groups, lightly infected fish (<15 parasites per fish) and heavily infected fish (>15 parasites per fish). In histological sections, the copepod encircled gill lamellae with its second antennae, compressed the epithelium, provoked hyperplasia and hemorrhage, occluded arteries and often caused lamellar disruption. Fusion of the secondary lamellae due to epithelial hyperplasia was common in all infected fish; heavily infected fish showed more intense branchial inflammation. In both healthy and infected fish, mast cells (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, mucous cells and rodlet cells (RCs). MCs were irregular in shape with a cytoplasm filled by numerous electron-dense, membrane-bound granules. Immunostaining of primary and secondary gill filaments with an antibody against the antimicrobial peptide (AMP) piscidin 3 (anti-piscidin 3 antibody, anti-HAGR) revealed a subpopulation of MCs that were positive. These MCs were more abundant in gills of heavily infected fish than in either lightly infected or uninfected fish (ANOVA, P<0.05). Our report documents the response of gill to ectoparasite infection and provides further evidence that mast cells and their AMPs may play a role in responding to branchial ectoparasite infections.

Survival of metazoan parasites in fish: Putting into context the protective immune responses of teleost fish.

Defence mechanisms of fish can be divided into specific and non-specific that act in concert and are often interdependent. Most fish in both wild and cultured populations are vulnerable to metazoan parasites. Endoparasitic helminths include several species of digeneans, cestodes, nematodes, and acanthocephalans. Although they may occur in large numbers, helminth infections rarely result in fish mortality. Conversely, some ectoparasites cause mass mortality in farmed fish. Given the importance of fish innate immunity, this review addresses non-specific defence mechanisms of fish against metazoan parasites, with emphasis on granulocyte responses involving mast cells, neutrophils, macrophages, rodlet cells, and mucous cells. Metazoan parasites are important disease agents that affect wild and farmed fish and can induce high economic loss and, as pathogen organisms, deserve considerable attention. The paper will provide our light and transmission electron microscopy data on metazoan parasites-fish innate immune and neuroendocrine systems. Insights about the structure and functions of the cell types listed above and a brief account of the effects and harms of each metazoan taxon to specific fish apparati/organs will be presented.

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.

Immunocytochemical localization of piscidin in mast cells of infected seabass gill.

Annual losses of approximately 5-10% of the juvenile stock of European seabass, Dicentrarchus labrax (L.) in the northern coast of the Adriatic Sea has been attributed to heavy infections of the gill monogenean Diplectanum aequans. Immunocytochemical, light and ultrastructural studies were carried out on seabass naturally parasitized with this monogenean. The site of the worm's attachment was marked by the common presence of haemorrhages and white mucoid exudate. In histological sections, infected gills showed hyperplasia, as well as proliferation of mucous cells and rodlet cells. Disruption and fusion of the secondary lamellae were common in all infected seabass, with several specimens also showing marked inflammation and erosion of the primary and secondary lamellar epithelium. Immunostaining of primary and secondary gill filaments with an antibody against the antimicrobial peptide piscidin 3 (anti-piscidin 3 antibody, anti-HAGR) revealed a subpopulation of mast cells that were positive. Mast cells were both within and outside the blood vessels of the primary and secondary lamellae, and often made intimate contact with vascular endothelial cells. Mast cells were irregular in shape with a cytoplasm filled by numerous electron-dense, membrane-bound granules. Our data provide evidence showing the presence of piscidin 3 in the cytoplasmic granules of an important group of fish inflammatory cells, the mast cells resident in seabass gill tissue. There was no significant difference in the number of HAGR-positive mast cells between infected and uninfected fish (ANOVA, p > 0.05). However, mast cells in parasitized gills usually showed much stronger immunostaining intensity compared to those in unparasitized gills. These data are the first to document a response of piscidins or any other antimicrobial peptide of fish to parasite infection and suggest that mast cells may play a role in certain inflammatory responses without a detectable increase in their numbers.

Temporal dynamics of species associations in the parasite community of European eels, Anguilla anguilla, from a coastal lagoon.

The resilience of biological communities is of central importance in ecology, but is difficult to investigate in nature. Parasite communities in individual hosts provide good model systems, as they allow a level of replication usually not possible with free-living communities. Here, using temporal data (2005-2017) on the communities of endohelminth parasites in European eels, Anguilla anguilla, from brackish-water lagoons in Italy, we test the resilience of interspecific associations to changes in the abundance of some parasite species and the disappearance of others. While most parasite species displayed changes in abundance over time, three trematodes that were present in the early years, two of which at high abundance, completely disappeared from the parasite community by the end of the study period. Possibly other host species required for the completion of their life cycles have declined in abundance, perhaps due to environmental changes. However, despite these marked changes to the overall community, pairwise correlations in abundance among the three most common parasite species (all trematodes) were stable over time and remained mostly unaffected by what happened to other species. We explore possible reasons for these resilient species associations within a temporally unstable parasite community inhabiting a mostly stable host population.

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.