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Abstract The family Acuariidae is a speciose group of parasitic nematodes, infecting mostly birds as definitive hosts. This study focused on the characterization of two species of acuariids, collected in two different species of piscivorous birds, the European great cormorant Phalacrocorax carbo sinensis from Italy, and the pygmy cormorant Microcarbo pygmaeus from Israel. Parasites were analyzed using light and scanning electron microscopy and by amplification and sequencing of the 28S rDNA. The results of morphological and molecular analyses showed that Ph. carbo sinensis was infected by the acuariid Syncuaria squamata (12 females) and Cosmocephalus obvelatus (1 female), whereas M. pygmaeus was infected by C. obvelatus (2 males, 12 females). The present results provide new data on the distribution of acuariid parasites of piscivorous birds, the first report of Acuariidae in Israel, and the first molecular data on S. squamata and C. obvelatus, which will be useful in future epidemiological and phylogenetic studies of these widely distributed, but less molecularly studied parasites.

The distribution of parasites is shaped by a variety of factors, among which are the migratory movements of their hosts. Israel has a unique position to migratory routes of several bird species leaving Europe to winter in Africa, however, detailed studies on the parasite fauna of birds from this area are scarce. Our study investigates occurrence and distribution of sibling species among Contracaecum rudolphii complex in Phalacrocorax carbo sinensis from Italy and Israel, to acquire further information on the geographical range of these species to gain deeper knowledge on the ecology of these parasites and their bird host. A total of 2383 Contracaecum were collected from the gastric mucosa of 28 great cormorants (18 from Israel and 10 from Italy). A subsample was processed for morphological analyses in light and scanning electron microscopy (SEM), and for molecular analyses through amplification and sequencing of the ITS rDNA and the cox2 mtDNA, and through PCR-RFLP. All the 683 Contracaecum subjected to molecular identification belonged to C. rudolphii s.l., (300 C. rudolphii A and 383 C. rudolphii B). SEM micrographs provided, for the first time, details of taxonomic structures in male specimens from both sibling species, and the first SEM characterization of C. rudolphii B. This work presents the first data on the occurrence of sibling species of C. rudolphii in Israel and provides additional information on the distribution of C. rudolphii A and B in Italy, confirming the high prevalence and intensity of infection observed in Ph. carbo sinensis from other Italian areas.

Abstract Very little information is reported for parasites of cnidarians, therefore, the present work aimed to investigate parasitic infections in one of the most widespread jellyfish in the Mediterranean Sea, Rhizostoma pulmo. The goals were to determine prevalence and intensity of parasites in R. pulmo, identify the species involved through morphological and molecular analysis, test whether infection parameters differ in different body parts and in relation to jellyfish size. 58 individuals were collected, 100% of them infected with digenean metacercariae. Intensity varied between 18.7 ± 6.7 per individual in 0–2 cm diameter jellyfish up to 505 ± 50.6 in 14 cm ones. Morphological and molecular analyses suggest that the metacercariae belonged to the family Lepocreadiidae and could be possibly assigned to the genus Clavogalea. Prevalence values of 100% suggest that R. pulmo is an important intermediate host in the life cycle of lepocreadiids in the region. Our findings also support the hypothesis that R. pulmo is an important part in the diet of teleost fish, which are reported as definitive hosts of lepocreadiids, since trophic transmission is necessary for these parasites to complete their life cycles. Parasitological data may therefore be useful to investigate fish-jellyfish predation, integrating traditional methods such as gut contents analysis.

Freshwater and marine ecosystems are a suitable habitat for parasitic nematodes of the genus Contracaecum (family: Anisakidae) to complete their complex life cycle. Several fish species of the Sea of Galilee (Lake Kinneret) were reported in 1964 as second intermediate/paratenic hosts of Contracaecum spp. larvae. The lack of taxonomically relevant morphological features of these larvae hindered their proper identification. Here we report the results of a 1-year survey conducted in 2021, 57 years after the first (and only) such survey. We analyzed 352 specimens from 10 fish species (native and non-native) of the Sea of Galilee (Israel) ichthyofauna. We compared our results with those of the first parasitological survey conducted by Paperna in 1964; the overall prevalence of nematodes referable to Contracaecum larvae was 16.8% and 0.85% in 1964 and in 2021, respectively. Different from the first survey that identified Contracaecum larvae morphologically, we used both morphological and molecular tools. Two wild native cyprinids—Jordan himri (Carasobarbus canis) and Jordan barbel (Luciobarbus longiceps)—were infected (a single specimen each) with Contracaecum quadripapillatum larvae in their abdominal cavity. A single specimen of blue tilapia (Oreochromis aureus) was infected with two larvae of Contracaecum multipapillatum E, localized in the pericardial cavity. The findings of our study, which is part of a large project focused on Contracaecum spp. infecting both piscivorous birds and fish collected in Israel, advance our knowledge about the distribution and host range of this potentially zoonotic parasite in fishery products of the Sea of Galilee.

Ascocotyle (Phagicola) trentinii n. sp. is described based on adults from experimentally infected ducklings (Anas platyrhynchos domesticus) fed with metacercariae from the visceral serosa of the Mediterranean banded killifish, Aphanius fasciatus (Cyprinodontiformes: Aphaniidae), from coastal lagoons in northeastern Italy (Emilia-Romagna Region). The new species is placed into the subgenus Phagicola because of the presence of a single row of circumoral spines, vitelline follicles being confined between the ventral sucker and testes, and uterine loops not reaching anterior to the ventral sucker. Ascocotyle (P.) trentinii n. sp. differs from other members of the subgenus Phagicola, as well as other species of Ascocotyle, by the number (27–33) of circumoral spines which are 13.5–17 μm long and 3.5–5 μm wide, and by the morphology of a gonotyl which is composed of about 8 large refractile pockets. The occurrence of metacercariae in A. fasciatus indicates that the life cycle of the new species is completed in brackish water lagoons. It is the fourth species of Ascocotyle described in Europe and may be endemic to the Mediterranean region because its second (fish) intermediate host is endemic to this region.

The present investigation reports a monogenean outbreak in broodstock of meagre Argyrosomus regius (Asso, 1801) from a Croatian farm. The parasites were visible by naked eye and were found in large numbers in the oral cavity and over the body surface. Morphological and molecular analyses allowed to identify the parasites as Ktariella polyorchis Vala & Euzet, 1977 (Monogenea: Calceostomatidae), here reported for the first time in farmed A. regius. Calceostomatids are known to parasitize different fish species of the family Sciaenidae worldwide: particularly, K. polyorchis had been described infecting the gills of wild A. regius caught off Tunisia and Egypt. This work provides the first detailed description of the parasite surface by SEM analysis, and the first molecular data (complete ITS and partial 28 S rDNA sequences), useful in future taxonomical and phylogenetic studies. Furthermore, we provide a synthesis of previous records of calceostomatid parasites of wild and cultured fish, highlighting their role as potential emerging parasites in the farming of sciaenid fish worldwide.

Nematodes belonging to the genus Contracaecum (family: Anisakidae) are heteroxenous parasites with a complex life cycle. Contracaecum larvae infecting farmed fish and fishery products are economically important causing market rejection in massive infection and may have zoonotic potential. In Israel, Contracaecum larvae have been described morphologically in several fish species; however, none of these descriptions were supported by molecular tools. In 2019–2020, hybrid tilapia (Oreochromis aureus x Oreochromis niloticus) and red drum (Sciaenops ocellatus), farmed in polyculture were found to be heavily infected with nematodes referable to Contracaecum larvae. Prevalence of infection in hybrid tilapia and red drum was 53.8% and 40.9%, respectively. A combined (morphological and molecular) approach revealed that both infected fish species were parasitized by the same species of Contracaecum, although larvae in hybrid tilapia were localized in the pericardial cavity whereas in red drum, they were observed in the abdominal cavity. Genetic analysis of internal transcribed spacer rDNA and cox2 mtDNA showed high similarity to unidentified Contracaecum larvae detected in several fish species in Ethiopia, Egypt and Kenya. In this study, molecular and morphological analyses place the possible new species in the C. multipapillatum complex and was provisionally named C. multipapillatum E. Further analyses combining morphological and molecular approaches are required on adult specimens collected from piscivorous birds living in the same area to support the identification of a potentially new species.

Occurrence of metacercariae of potentially zoonotic trematodes (Platyhelminthes: Digenea) in the musculature of common carp (Cyprinus carpio L. 1758) was monitored in four Hungarian aquacultures. Four geographically distinct fish farms (located in the Northwestern, Southwestern, Northeastern and Southeastern parts of Hungary) were selected for the investigation. From each farm, a total of 258 one-summer-old fingerlings were sampled and examined in the years 2016 and 2017. In addition, in 2017, we examined 60 market size specimens (30 two-summers and 30 three-summers) sampled from the most infected aquaculture in the Northeastern part of Hungary. The fish were euthanized and decapitated whereafter their musculature (fillets) was digested in a pepsin solution to isolate metacercariae from the tissue whereafter morphological and molecular analyses (PCR and sequencing of ITS region) were performed. Opisthorchiid metacercariae were not recovered but in one of the farms numerous metacercariae were detected in the musculature of carp. They were identified as cyathocotylid trematodes based on their morphological characteristics and by sequencing the ITS region. The infection levels proved to be remarkably different among the four fish farms. Carps from the Northeastern farm were infected by large numbers of cyathocotylid metacercariae, while 8 Posthodiplostomum cuticola metacercariae were detected in the Northwestern aquaculture. In the other two farms (Southwestern and Southeastern) no infection was recorded. The infected farm is located close to a protected natural wetland habitat populated by a rich fauna of aquatic birds (potential final hosts) and snails (first intermediate host) which may create a higher risk of infection in the neighbouring fish farms.

The fish parasites Saprolegnia spp. (Oomycota) and Amyloodinium ocellatum (Dinophyceae) cause important losses in freshwater and marine aquaculture industry, respectively. The possible adverse effects of compounds used to control these parasites in aquaculture resulted in increased interest on the search for natural products with antiparasitic activity. In this work, eighteen plant-derived compounds (2′,4′-Dihydroxychalcone; 7-Hydroxyflavone; Artemisinin; Camphor (1R); Diallyl sulfide; Esculetin; Eucalyptol; Garlicin 80%; Harmalol hydrochloride dihydrate; Palmatine chloride; Piperine; Plumbagin; Resveratrol; Rosmarinic acid; Sclareolide; Tomatine, Umbelliferone, and Usnic Acid) have been tested in vitro. Sixteen of these were used to determine their effects on the gill cell line G1B (ATCC®CRL-2536™) and on the motility of viable dinospores of Amyloodinium ocellatum, and thirteen were screened for inhibitory activity against Saprolegnia spp. The cytotoxicity results on G1B cells determined that only two compounds (2′,4′-Dihydroxychalcone and Tomatine) exhibited dose-dependent toxic effects. The highest surveyed concentrations (0.1 and 0.01 mM) reduced cell viability by 80%. Upon lowering the compound concentration the percentage of dead cells was lower than 20%. The same two compounds revealed to be potential antiparasitics by reducing in a dose-dependent manner the motility of A. ocellatum dinospores up to 100%. With respect to Saprolegnia, a Minimum Inhibitory Concentration was found for Tomatine (0.1 mM), Piperine and Plumbagin (0.25 mM), while 2′,4′-Dihydroxychalcone considerably slowed down mycelial growth for 24 h at a concentration of 0.1 mM. Therefore, this research allowed to identify two compounds, Tomatine and 2′,4′-Dihydroxychalcone, effective against both parasites. These compounds could represent promising candidates for the treatment of amyloodiniosis and saprolegniosis in aquaculture. Nevertheless, further in vitro and in vivo tests are required in order to determine concentrations that are effective against the considered pathogens but at the same time safe for hosts, environment and consumers.

The nematode Eustrongylides excisus is a parasite of freshwater fish- and fish-eating birds, with known differences on prevalence values among fish species. Thus, the present study aims to explore the hypothesis that the feeding behavior and the size of fish belonging to different trophic levels could explain such differences. For that, 14 sampling sites were selected to perform a fish parasitological survey on Lake Garda (Italy) during spring-summer 2020. Amplification of nuclear ribosomal internal transcribed spacer (ITS) rDNA sequences of nematodes morphologically ascribable to the genus Eustrongylides allowed to identify them as E. excisus. From the five studied fish species (Perca fluviatilis, Lepomis gibbous, Coregonus lavaretus, Alosa fallax lacustris and Micropterus salmoides), only three presented the parasite E. excisus: P. fluviatilis, L. gibbous and M. salmoides, with significant differences in prevalence values among species (p = 0.002). Additionally, there were differences in prevalence values within the same fish species captured from different sampling sites. Findings showed that mainly piscivorous fish were positive for E. excisus and how the prevalence was highest in M. salmoides. As regard the fish size, a negative correlation between body size and E. excisus was found in P. fluviatilis due to the feeding habit of juvenile perch which feed mainly zooplankton and benthic invertebrates (i.e., oligochaetes, which are the first intermediate hosts of E. excisus). The study findings advance novel knowledge in the field of pathogens of zoonotic importance in the aquatic environment.

Monogenean capsalids of the genus Neobenedenia are widespread parasites of wild and farmed marine fish, and represent a potential threat to mariculture due to their pathogenicity and ability to cause mortality in fish maintained in controlled conditions. The identification of Neobenedenia species and, consequently, the definition of their host specificity is often problematic due to their highly conserved morphology; therefore, in order to establish their specific identity, microscopic observation should be complemented with molecular analysis. The present work aims at characterizing Neobenedenia specimens infecting the skin of cage reared gilthead seabream Sparus aurata from Portugal. Parasite samples obtained from caged fish were processed for morphological analysis, through observation in light and scanning electron microscopy, and for molecular analysis, through amplification and sequencing of 28S rDNA and cytB, aimed at identifying them to the species level. Our results showed that the collected parasites belonged to the species Neobenedenia girellae; the susceptibility of S. aurata towards this pathogenic capsalid monogenean highlighted in the present work represents an important risk in the farming of this valuable fish species.

Saprolegnia infections are among the main parasitic diseases affecting farmed salmonids. The distribution and potential transfer of Saprolegnia spp. between farms and the natural environment has been scarcely investigated. Therefore, this work aimed to study the diversity and abundance of oomycete species in salmonid farms, tributary water, and effluent water systems. Four trout farms in Italy and two Atlantic salmon farms in Scotland were considered. In Italian farms, 532 isolates of oomycetes were obtained from fish and water, at upstream, inside, and downstream the farms. In Scottish farms, 201 oomycetes isolates were obtained from water outside the farm and from fish and water inside the farming units. Isolates were identified to the species level through amplification and sequencing of the ITS rDNA region. In Italy, S. parasitica was significantly more present in farmed than in wild fish, while in water it was more frequently isolated from the wild, particularly in effluent systems, not associated with more frequent isolation of S. parasitica in wild fish downstream the farm. In Scotland, S. parasitica was the most prevalent species isolated from fish, while isolates from water were mostly Pythium spp. with few S. parasitica isolates from upstream and downstream the farms.

We provide new data on the presence of helminth parasites in 64 individual loggerhead sea turtles Caretta caretta stranded along the coasts of Sicily and the northwest Adriatic Sea between June 2014 and August 2016. The necropsy examination revealed 31 individuals (48.4%) positive for endoparasites, showing a greater prevalence of trematodes than nematodes. In particular, seven species and a single genus of Trematoda (Hapalotrema) and a single species and genus of Nematoda (Kathlania) were identified. Among the Digenea flukes the species with the highest prevalence of infection were Rhytidodes gelatinosus (34.6%) and Hapalotrema sp. (33.3%), while among the Nematoda they were Kathlania sp. (33.3%) and Sulcascaris sulcata (33.3%). Analysis of variance (ANOVA) was applied among the recovery sites of the stranded loggerhead sea turtles and prevalence of endoparasites was used to highlight any relationship between the parasites and the origin of the hosts. ANOVA showed significant differences (p < 0.001) among the data used.

Cephalopods are intermediate/paratenic hosts in the life cycle of elasmobranch tapeworms, nevertheless most records of infection in this group of mollusks are outdated and fragmentary. The present work aimed to investigate the cestode fauna of the common octopus Octopus vulgaris from the Tyrrhenian Sea (Central Mediterranean). The parasitic stages were characterized by light and Scanning Electron Microscopy (SEM) and sequencing of 28S rDNA. Three cestode taxa were identified to the genus level: the onchoproteocephalidean Acanthobothrium sp. (prevalence 28%), the “tetraphyllidean” Anthobothrium sp. (prevalence 13%) and the trypanorhynch Nybelinia sp. (prevalence 3%). The remarkable prevalence observed for gastrointestinal cestodes highlight a possible important role of O. vulgaris in the transmission of elasmobranch tapeworms, particularly Onchoproteocephalideans. Furthermore, the present work provides, for the first time, detailed morphological (SEM) and molecular support to confirm the occurrence of Anthobothrium sp. in cephalopod hosts. In order to gain higher taxonomic resolution for the identified taxa, we stress the need to collect further morphological and molecular data of adult cestodes infecting their elasmobranch definitive hosts.

Mycobacterium pseudoshottsii, a slow-growing mycobacterium closely related to M. marinum, has been isolated only in wild fish in the United States and in Japanese fish farms to date. Here, we report cases of mortality in three farmed fish species (Dicentrarchus labrax, Sparus aurata, and Sciaenops ocellatus) caused by M. pseudoshottsii in Italy. Samples underwent necropsy, histology, and culture with pathogen identification based on PCR and sequencing of housekeeping genes (16S rRNA, hsp65, rpoB). Multifocal to coalescing granulomatous and necrotizing inflammation with acid-fast bacilli were observed in the parenchymatous organs, from which M. pseudoshottsii was isolated and identified. Phylogenetic analysis confirmed the results of gene sequencing and allowed subdivision of the isolates into three distinct groups. M. pseudoshottsii poses a potential threat for Mediterranean aquaculture. Its origin in the area under study needs to be clarified, as well as the threat to the farmed fish species.