Infection of endemic chub Squalius tenellus with the intestinal tapeworm Caryophyllaeus brachycollis (Cestoda): histopathology and ultrastructural surveys.

The endemic chub Squalius tenellus (Heckel, 1843) was introduced more than 100 years ago to Lake Blidinje (Bosnia-Herzegovina). Only 1 species of enteric helminth was found in a sample of 35 chubs, the tapeworm Caryophyllaeus brachycollis (Janiszewska, 1953). The paper includes histopathological investigation with identification of innate immune cells involved in host reaction and molecular data allowed correct designation of the cestode species. Of 35 specimens of chub examined, 21 (60%) harboured individuals of C. brachycollis and a total of 1619 tapeworms were counted, the intensity of infection ranged from 1 to 390 worms per fish (46.2 ± 15.3, mean ± s.e.). Histopathological and ultrastructural investigations showed strict contact between the worm's body and the epithelia and increase in the number of mucous cells, rodlet cells among the epithelial cells. Within the tunica propria-submucosa, beneath the site of scolex attachment, numerous neutrophils and mast cells were noticed. This is the first study of the occurrence of C. brachycollis in chub from Lake Blidinje and on the response of the innate immune cells of S. tenellus to this tapeworm. Interestingly, in 3 very heavily infected chubs, perforation of the intestinal wall was documented; this is uncommon among cestodes which use fish as a definitive host.

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.

Cast away in the Adriatic: Low degree of parallel genetic differentiation in three-spined sticklebacks.

The three-spined stickleback (Gasterosteus aculeatus) has repeatedly and independently adapted to freshwater habitats from standing genetic variation (SGV) following colonization from the sea. However, in the Mediterranean Sea G. aculeatus is believed to have gone extinct, and thus the spread of locally adapted alleles between different freshwater populations via the sea since then has been highly unlikely. This is expected to limit parallel evolution, that is the extent to which phylogenetically related alleles can be shared among independently colonized freshwater populations. Using whole genome and 2b-RAD sequencing data, we compared levels of genetic differentiation and genetic parallelism of 15 Adriatic stickleback populations to 19 Pacific, Atlantic and Caspian populations, where gene flow between freshwater populations across extant marine populations is still possible. Our findings support previous studies suggesting that Adriatic populations are highly differentiated (average FST  ≈ 0.45), of low genetic diversity and connectivity, and likely to stem from multiple independent colonizations during the Pleistocene. Linkage disequilibrium network analyses in combination with linear mixed models nevertheless revealed several parallel marine-freshwater differentiated genomic regions, although still not to the extent observed elsewhere in the world. We hypothesize that current levels of genetic parallelism in the Adriatic lineages are a relic of freshwater adaptation from SGV prior to the extinction of marine sticklebacks in the Mediterranean that has persisted despite substantial genetic drift experienced by the Adriatic stickleback isolates.

Biological and trophic consequences of genetic introgression between endemic and invasive Barbus fishes.

Genetic introgression with native species is recognized as a detrimental impact resulting from biological invasions involving taxonomically similar invaders. Whilst the underlying genetic mechanisms are increasingly understood, the ecological consequences of introgression are relatively less studied, despite their utility for increasing knowledge on how invasion impacts can manifest. Here, the ecological consequences of genetic introgression from an invasive congener were tested using the endemic barbel populations of central Italy, where the invader was the European barbel Barbus barbus. Four populations of native Barbus species (B. plebejus and B. tyberinus) were studied: two purebred and two completely introgressed with alien B. barbus. Across the four populations, differences in their biological traits (growth, body condition and population demographic structure) and trophic ecology (gut content analysis and stable isotope analysis) were tested. While all populations had similar body condition and were dominated by fish up to 2 years of age, the introgressed fish had substantially greater lengths at the same age, with maximum lengths 410-460 mm in hybrids versus 340-360 mm in native purebred barbel. The population characterized by the highest number of introgressed B. barbus alleles (81 %) had the largest trophic niche and a substantially lower trophic position than the other populations through its exploitation of a wider range of resources (e.g. small fishes and plants). These results attest that the genetic introgression of an invasive congener with native species can result in substantial ecological consequences, including the potential for cascading effects. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10530-021-02577-6.

The impact of Anguillicoloides crassus (Nematoda) on European eel swimbladder: histopathology and relationship between neuroendocrine and immune cells.

The swimbladder functions as a hydrostatic organ in most bony fishes, including the European eel, Anguilla anguilla. Infection by the nematode Anguillicoloides crassus impairs swimbladder function, significantly compromising the success of the eel spawning migration. Swimbladders from 32 yellow eels taken from Lake Trasimeno (Central Italy) were analysed by histopathology- and electron microscopy-based techniques. Sixteen eels (50%) harboured A. crassus in their swimbladders and intensity of infection ranged from 2 to 17 adult nematodes per organ (6.9 ± 1.6, mean ± s.e.). Gross observations of heavily infected swimbladders showed opacity and histological analysis found a papillose aspect to the mucosa and hyperplasia of the lamina propria, muscularis mucosae and submucosa. Inflammation, haemorrhages, dilation of blood vessels and epithelial erosion were common in infected swimbladders. In the epithelium of parasitized swimbladders, many empty spaces and lack of apical junctional complexes were frequent among the gas gland cells. In heavily infected swimbladders, we observed hyperplasia, cellular swelling and abundant vacuolization in the apical portion of the gas gland cells. Numerous mast cells and several macrophage aggregates were noticed in the mucosal layer of infected swimbladders. We found more nervous and endocrine elements immunoreactive to a panel of six rabbit polyclonal antibodies in infected swimbladders compared to uninfected.

Cryptic diversity within endemic Italian barbels: revalidation and description of new Barbus species (Teleostei: Cyprinidae).

Two fluviolacustrine lineages (SI1 Barbus and SI2 Barbus) of the Barbus genus have been recently detected in the Apulia-Campania ichthyogeographic district (southern Italy). The aim of this study was to determine the taxonomic status of these lineages by comparing them with the two already-established Italian fluviolacustrine species Barbus plebejus and Barbus tyberinus through a more in-depth molecular and morphological investigation. Genetic analyses were performed on both mitochondrial (cytochrome b) and nuclear (growth hormone paralog 2) DNA markers, and morphological analyses were carried out on specific age classes and purebred populations. Molecular analyses detected four evolutionary lineages at the mitochondrial level, whereas the nuclear data set highlighted the strict evolutionary relation between B. plebejus sensu stricto and the new lineages, converged in the B. plebejus complex clade. The morphological analyses allowed us to discriminate SI1 Barbus and SI2 Barbus from both B. plebejus and B. tyberinus. The new taxa could be discriminated by the greatest maximum body height and the longest pre-orbital distance, respectively. Both the new lineages have longer ventral and pectoral fins than B. plebejus and B. tyberinus, a larger caudal fin than B. tyberinus and a lower number of scales along the lateral line than B. plebejus. Both molecular and morphological results suggested the two southern Italian lineages could be considered as distinct endemic species: the formal description of the new species Barbus samniticus sp. nov. (i.e., SI1 Barbus) and the revalidation of Barbus fucini Costa, 1853 (i.e., SI2 Barbus) were thus proposed, and, for both species, molecular and morphological diagnosis were provided.

Mucosal Hallmarks in the Alimentary Canal of Northern Pike Esox lucius (Linnaeus).

On the basis of trophic behavior, fish are classified as herbivores, carnivores, omnivores, or detritivores. Epithelial mucous cells secrete mucin types specific to diet and digestive function. Mucus secretion is regulated mainly by molecular modulators produced by epithelial endocrine cells in response to luminal or tissue stimuli. These modulators are involved in control of food intake and digestive functions. Immunohistochemical and immunofluorescence studies were conducted on 10 adult northern pike (Esox lucius Linnaeus, 1758) from Lake Piediluco (Central Italy) to quantify distribution of sub-types of mucous and endocrine cells in alimentary mucosal epithelium. Neutral mucins predominated in the esophagus, and mixed and acidic mucins predominated in stomach and intestine. The gastric epithelium contained endocrine cells secreting somatostatin, tyrosine hydroxylase, and substance P. Mucous cells secreting neutral mucins increased in number from proximal to distal intestine, with endocrine cells containing substance P in the proximal intestine and those containing Leu-enkephalin throughout the intestine. Lectin histochemistry of gut sections revealed an abundance of N-acetyl-glucosamine and N-acetyl-galactosamine as carbohydrate residues on the mucin chain. The quantity and content of endocrine and mucous cells in the alimentary canal of E. lucius showed a direct relationship with its diet.

Population status and ecology of the Salmo trutta complex in an Italian river basin under multiple anthropogenic pressures.

Salmonids inhabiting Mediterranean rivers are of particular concern for biodiversity conservation, as they are threatened by various stressors, including habitat alterations, overfishing, climate change, and introgressive hybridization with alien species. In the Tiber River basin (Central Italy), genetic introgression phenomena of the native Salmo cettii with the non-native Salmo trutta hinder the separate analysis of the two species, which are both included in the S. trutta complex. Little is known about the factors currently limiting the trout populations in this area, particularly with respect to climate change. With the intention of filling this gap, the aims of the current study were to (a) quantify changes in the climate and (b) analyze the distribution, status, and ecology of trout populations, in the context of changing abiotic conditions over the last decades. Fish stock assessments were carried out by electrofishing during three census periods (1998-2004, 2005-2011, and 2012-2018) at 129 sites. The trend over time of meteorological parameters provided evidence for increased air temperature and decreased rainfall. Multivariate analysis of trout densities and environmental data highlighted the close direct correlation of trout abundance with water quality, altitude, and current speed. Climate-induced effects observed over time in the sites where trout were sampled have not yet led to local extinctions or distribution shifts, indicating a marked resilience of trout, probably due to the buffering effect of intrinsic population dynamics. Decreasing body conditions over time and unbalanced age structures support the hypothesis that variations in hydraulic regime and water temperature could overcome these compensatory effects, which may lead to a severe decline in trout populations in the near future. In a climate change context, habitat availability plays a key role in the distribution of cold-water species, which often do not have the possibility to move upstream to reach their thermal optimum because of water scarcity in the upper river stretches.

Genetic and morphological analyses reveal a complex biogeographic pattern in the endemic barbel populations of the southern Italian peninsula.

The Italian peninsula is a biodiversity hotspot, with its freshwater fish fauna characterized by high levels of local endemism. Two endemic fluvio-lacustrine fishes of the genus Barbus (barbel, family Cyprinidae) have allopatric distributions in the Tyrrhenian and Adriatic basins of Italy. Barbus plebejus inhabits the mid- to northern Adriatic basins, while B. tyberinus is widespread in all central-northern basins draining into the Tyrrhenian Sea. For basins in Southern Italy draining into the southern parts of these seas, there remains a knowledge gap on their barbel populations due to no previous genetic and morphological studies, despite their apparent biogeographic isolation. Correspondingly, this study quantified the presence and distribution of barbels in the Adriatic and Tyrrhenian basins of Southern Italy through genetic and morphological analyses of 197 fish sampled across eight populations. Testing of how local isolation has influenced the evolution and persistence of these populations was completed by examining sequence variation at two mitochondrial loci (cytochrome b and D-loop) and performing geometric morphometric analyses of body shape, plus measuring 11 morphometric and meristic characters. Phylogenetic and morphological analyses revealed the presence of two genetically distinct lineages that differed significantly from adjacent B. tyberinus and B. plebejus populations. These two new taxa, here described as SI1 and SI2 Barbus lineages, are highly structured and reflect a complex mosaic biogeographic pattern that is strongly associated with the underlying hydrographical scenarios of the basins. The geographic isolation of these basins thus has high evolutionary importance that has to be considered for maintaining endemism.

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.