Morphological and molecular study of Didymodiclinus marginati n. sp. (Trematoda: Didymozoidae) gill parasite of Epinephelus marginatus from the central and western Mediterranean Sea.

The current study provides a morphological and molecular characterization of a new species of Didymodiclinus (Trematoda: Didymozoidae) infecting the dusky grouper, Epinephelus marginatus (Teleostei: Serranidae) from the Mediterranean Sea. A total of 279 dusky grouper specimens were examined for didymozoid gill parasites from the Mediterranean Sea between 1998 and 2020. New species differs from the most similar congeneric species by the rudiments of female reproductive organs in functional male specimens, and the seminal receptacle, Mehlis gland and accessory gland cells in functional female specimens, not observed in Didymodiclinus branchialis (Yamaguti, 1970), Didymodiclinus epinepheli (Abdul-Salam, Sreelatha and Farah, 1990) and Didymodiclinus pacificus (Yamaguti, 1938), respectively. These species are also characterized by their different hosts and location within the host tissues, being from other geographical localities. Moreover, this is the first species reported in E. marginatus from the central and western Mediterranean Sea. Genetic analyses were performed on partial 28S and partial internal transcribed spacer-2 ribosomal RNA regions and the mitochondrial cytochrome oxidase 1 (cox1) gene by polymerase chain reaction. Comparison of genetic sequences of Didymodiclinus marginati n. sp. with the available deposited sequences of 28S revealed that the new isolates cluster with several unidentified didymozoids and groups as a sister clade of the Nematobothrinae subfamily. Moreover, 28S and cox1 phylogenetic trees evidenced that Didymodiclinae is well separated from Didymozoinae and other gonochoric didymozoids. Following both morphological and genetic results, a key of identification for the genus Didymodiclinus is proposed.

Diet and feeding behaviour of albacore Thunnus alalunga in the western Mediterranean Sea during the spawning period.

Mature albacore tuna (Thunnus alalunga) are expected to have high energy requirements at the time of breeding. However, there are no descriptions of the diet of albacore in the Mediterranean Sea that can help us to understand if such requirements can be obtained from feeding during reproduction. In this study, we analysed the stomach contents of reproductively active albacore captured from 2010 to 2015 in the oligotrophic waters of the western Mediterranean Sea, one of their main spawning grounds. Estimates of stomach fullness revealed intense feeding activity, and prey composition indicated important consumption of mesopelagic fish, including barracudinas, myctophids and small pelagic crustaceans. Plastic debris occurred in 25%-53% of the stomachs sampled across all years. Prey composition was not different between males and females. However, females fed at higher rates and had higher hepatosomatic index values than males, suggesting that increased feeding could contribute to meet their higher energy demand associated with offspring production. We observed a diet shift from small crustaceans to fish prey along fish size. During the spawning period, albacore showed a specialist feeding behaviour by preying on aggregations of vertically migrating myctophids and small crustaceans, probably when they were near the surface. This study provides information and biological data to support ecosystem modelling and to increase the understanding of albacore ecology.

Cardicola mediterraneus n. sp. (Trematoda, Aporocotylidae): a new species infecting the gilthead seabream, Sparus aurata L., from the Western Mediterranean Sea.

The genus Cardicola Short, 1953 has the highest number of species within the family Aporocotylidae (Trematoda: Digenea). Five Cardicola species have been reported to date in the Mediterranean Sea, one of them in the gilthead seabream Sparus aurata L. Analyses of infected S. aurata recovered from cultured fish off Sardinia (Italy) and from wild and cultured fish off the Levantine coast (Southeastern Spain) have revealed the presence of two species identified as Cardicola aurata Holzer, Montero, Repullés, Nolan, Sitjà-Bobadilla, Álvarez-Pellitero, Zarza and Raga, 2008 and Cardicola mediterraneus n. sp.. New morphological and molecular data are provided for both species. Features of C. aurata specimens differ slightly from those of the original description of the species, the most important differences being the longer extension of the metraterm and the central and posterior position of the female genital pore. Cardicola mediterraneus n. sp. can be easily distinguished from other Cardicola species by two unique specific characters: (i) the very unequal posterior caeca length and (ii) the shape of the testis, deeply notched at the anterior extremity. Cardicola spp. from sparids occupy a basal phylogenetic position respect the other congeneric species. The genus Cardicola has a complex taxonomy and shows high intrageneric differences for both 28S and ITS2 rDNAs, similar to the intergeneric differences among other aporocotylid genera, suggesting that it could be split. The presence of two Cardicola species could hamper treatment design and application; thus, data discriminating species herein reported can improve the infection management in fish farms.

Ecological and histopathological aspects of Didymodiclinus sp. (Trematoda: Didymozoidae) parasite of the dusky grouper, Epinephelus marginatus (Osteichthyes: Serranidae), from the western Mediterranean Sea.

The dusky grouper Epinephelus marginatus (Lowe) is an ecologically and commercially important fish species of the Atlantic and Mediterranean coastal rocky habitats. Despite records of didymozoid infections in several grouper species, the identification and pathogenesis of these parasites in E. marginatus are lacking. The aim of this study is to characterize the didymozoids of E. marginatus, particularly their mechanisms of infection and histopathological features. Dusky groupers (n = 205) were caught off Majorca Island (western Mediterranean Sea) and examined for parasites. Of the fish sampled, 45% were infected with white and yellow didymozoid capsules and brown nodules, found on the gills and pseudobranchs. Parasite abundance had a strong positive relationship with the fish length; only fish larger than 20 cm were infected, suggesting infection via consumption of an intermediate host, for which E. marginatus size was a limiting factor. The capsules contained two convoluted viable adult trematodes, identified as Didymodiclinus sp., in close contact with host capillary vessels, with no evidence of the tissue inflammatory response. Conversely, nodules containing degraded parasites were surrounded by an intense inflammatory infiltrate. The findings suggest that Didymodiclinus sp. have the potential to evade the host's immune system by inhibiting the inflammatory response.

Diagnostic accuracy of the light microscope method to detect the eggs of Cardicola spp. in the gill filaments of the bluefin tuna.

Trematode blood flukes of the genus Cardicola are potentially lethal in bluefin tuna cultures. The present study proposed a new method to detect aporocotylid eggs in tuna gills. Aporocotylid eggs were detected by analysing a pair of gill filaments of five transversal areas of the eight holobranches of one hundred Atlantic bluefin tuna and observed with glycerol and a stereomicroscope with an oblique brightfield. Data were gathered according to holobranches, transversal areas and their combination. Eggs were uniformly distributed among the holobranches, but they had the highest prevalence in the second and fifth transversal areas, which is controversial with respect to previous studies of egg distribution. An abbreviated method called the T-two test, which had the highest sensitivity (96.8%), is proposed for the detection of Cardicola spp. infections instead of the analysis all the holobranches. The T-two test limits the time and cost of the egg parasite screening analysis. The analyses of ten samples could be sufficient to detect the presence of parasites in farmed bluefin tuna; fish from the wild are expected to be less infected and more samples (45) would therefore be necessary.

Skoulekia erythrini n. sp. (Digenea: Aporocotylidae): a parasite of Pagellus erythrinus (L.) (Perciformes: Sparidae) from the western Mediterranean with an amendment of the generic diagnosis.

A new aporocotylid, Skoulekia erythrini n. sp., is described from the heart, cephalic kidney and gill blood vessels of the common pandora Pagellus erythrinus (L.) collected from several localities of the western Mediterranean off Spain. The new species differs from the type- and only species of the genus, S. meningialis Alama-Bermejo, Montero, Raga & Holzer, 2011 in possessing a symmetrical body (vs laterally curved), short tegumental spines (3-4 vs 7-10 µm) without hooked ends, diffuse (vs conspicuous) oesophageal gland-cells, a relatively longer oesophagus, a testis that is shorter in relation to body length and a much smaller seminal vesicle (17-34 × 10-26 vs 33-101 × 27-97 µm). Elongated ellipsoidal eggs of S. erythrini n. sp. were found trapped in gill vessels in histological sections. Phylogenetic analyses based on partial 28S rDNA and ITS2 sequences supported the placement of the new species within Skoulekia and the close relationships of this genus with Psettarium Goto & Ozaki, 1929 and Pearsonellum Overstreet & Køie, 1986. Skoulekia meningialis is described from a new sparid host, Diplodus puntazzo (Walbaum), collected off Santa Pola, Spain. The new morphological data for the two Skoulekia spp. and a re-examination of three paratypes of S. meningialis pinpointed features amending both the description of S. meningialis and the generic diagnosis of Skoulekia.

Ecology of the Atlantic black skipjack Euthynnus alletteratus (Osteichthyes: Scombridae) in the western Mediterranean Sea inferred by parasitological analysis.

Between 2008 and 2011, the head of 150 Euthynnus alletteratus (Osteichthyes: Scombridae) caught inshore off the southeastern Iberian coast (western Mediterranean Sea) were examined for parasites. Two monogeneans, four didymozoid trematodes and four copepods were found. Parasite abundance showed a positive relationship with the annual sea surface temperature, except for Pseudocycnus appendiculatus, but negative with the sea depth (Capsala manteri, Neonematobothrium cf. kawakawa and Caligus bonito). Prevalences and mean abundances differed significantly among sampling areas, except for C. manteri, Oesophagocystis sp. 2 and Ceratocolax euthynni, and sampling years (Melanocystis cf. kawakawa, N.cf. kawakawa, P. appendiculatus and Unicolax collateralis). Results indicate that the parasite abundances of E. alletteratus in the western Mediterranean Sea depend mainly on regional environmental variables, which can show interannual variations. The presence of pelagic parasites, i.e. didymozoids and P. appendiculatus, could indicate that E. alletteratus migrates between inshore and offshore pelagic domains. The different parasite faunas reported in E. alletteratus populations from the western Atlantic Ocean and the Mediterranean Sea appear to point out the geographical host isolation. These results suggest that E. alletteratus inhabiting the western Mediterranean Sea performs inshore-offshore small-scale migrations, and not transoceanic migrations between the western Atlantic Ocean and Mediterranean Sea.

Morphological characterisation and identification of four species of Cardicola Short, 1953 (Trematoda: Aporocotylidae) infecting the Atlantic bluefin tuna Thunnus thynnus (L.) in the Mediterranean Sea.

Blood flukes of the genus Cardicola Short, 1953 are considered the most potentially pathogenic parasites in bluefin tuna cultures. Morphological study and genetic analyses of the ribosomal internal transcribed spacer ITS-2 and the mitochondrial cytochrome c oxidase 1 (cox1) gene fragments revealed the occurrence of four aporocotylid species (C. forsteri Cribb, Daintith & Munday, 2000, C. orientalis Ogawa, Tanaka, Sugihara & Takami, 2010, C. opisthorchis Ogawa, Ishimaru, Shirakashi, Takami & Grabner, 2011 and Cardicola sp.) in 421 Thunnus thynnus (L.) from the Western Mediterranean (274 fished from the wild and 147 from sea-cages). Cardicola opisthorchis was the most abundant species, with higher prevalence in the cage-reared fish than in those fished in the wild (21 vs 6%, p < 0.05). Adults of three species were recovered: C. forsteri from both gills and heart, C. opisthorchis from heart and C. orientalis from gills. The secondary gill lamellae were profusely infected by eggs of C. orientalis. A fourth species was found in four tunas, based on the molecular analyses of eggs apparently indistinguishable in size and shape from the eggs of C. orientalis. The findings provided evidence that infections with Cardicola spp. differed in relation to locality, host origin (wild vs cage-reared) and site of infection. It is necessary to estimate the possible different pathogenic effects of each species of Cardicola in order to take appropriate control measures.

Metazoan gill parasites of the Atlantic bluefin tuna Thunnus thynnus (Linnaeus) (Osteichthyes: Scombridae) from the Mediterranean and their possible use as biological tags.

The gills of 63 specimens of the Atlantic bluefin tuna Thunnus thynnus (Linnaeus) (Osteichthyes: Scombridae) from three localities of the Mediterranean (Sardinian, Tyrrhenian and Levantine Seas) were examined for metazoan parasites. The parasite fauna of T. thynnus from the Sea of Sardinia included 11 species: five didymozoid trematodes, three capsalid and one hexostomid monogeneans, and one caligid and one pseudocycnid copepods. Four didymozoids were found in fish from the Levantine Sea and only one didymozoid was recorded in fish from the Tyrrhenian Sea. Dividing the hosts into four size-groups (small, medium-sized, large and extra large), the pairwise comparison of prevalence and mean abundance of the new and literary data) showed differences according to host size. The differences in the composition of the parasitic faunas and in the prevalence of parasites, observed between the small tunas from the Tyrrhenian Sea and the medium-sized tunas from the Adriatic Sea, Levantine Sea and the North-East (NE) Atlantic Ocean, indicated that these groups form discrete units. The parasite fauna of the large tunas from the Sea of Sardinia is the richest among the bluefin tuna populations of the Mediterranean and the NE Atlantic, due to the presence of species not found elsewhere in bluefin tunas, such as Caligus coryphaenae Steenstrup et Lütken, 1861, Capsala magronum (Ishii, 1936) and C. paucispinosa (Mamaev, 1968). This fact and the prevalence of some parasites of this group (lower than those of medium-sized fish from the NE Atlantic and higher than the small and medium-sized tunas from the Mediterranean) suggest that the large-sized tuna group in the western Mediterranean is formed by Mediterranean resident tunas (poorly infected), and by tunas migrating from the Atlantic Ocean (heavily infected).

Parasites of the head of Scomber colias (Osteichthyes: Scombridae) from the western Mediterranean Sea.

The metazoan parasite assemblage of the head of 30 specimens of the Atlantic chub mackerel (Scomber colias) from the western Mediterranean Sea was analysed. Eight species of parasites were found, four mazocraeid monogeneans: Grubea cochlear (prevalence = 10%), Kuhnia scombercolias (59%), K. scombri (52%), Pseudokuhnia minor (86%); three didymozoid trematodes: Nematobothrium cf. faciale (21%), N. filiforme (41%), N. scombri (7%); and one laerneopodid copepod: Clavelissa scombri (7%). Results were compared with previously published data from 14 localities of the eastern Mediterranean Sea and the Atlantic Ocean, using non-parametric univariate and multivariate analyses, and the whole parasite fauna of S. colias was compared with that of the congeners (S. australasicus, S. japonicus and S. scombrus). Parasites showed to reflect the biogeographical and phylogenetic history of host. From a methodological point of view, the use of both non-parametric univariate and multivariate techniques proved to be effective tools to detect dissimilarities between parasite assemblages.

Metazoan parasites on the gills of the skipjack tuna Katsuwonus pelamis (Osteichthyes: Scombridae) from the Alboran Sea (western Mediterranean Sea).

The gills of 31 skipjack tuna Katsuwonus pelamis (L.) caught in the Alboran Sea (western Mediterranean Sea) were examined for metazoan parasites, and the gills of 4 specimens from the Balearic Sea (also western Mediterranean Sea) were analysed for comparative purposes. Nine -species of parasites were found, including 8 didymozoid trematodes (Atalostrophion cf. bio-varium, Didymocylindrus filiformis, Didymocylindrus simplex, Didymocystis reniformis, Didymoproblema fusiforme, Didymozoon longicolle, Koellikeria sp. and Lobatozoum multisacculatum) and 1 caligid copepod (Caligus bonito). Koellikeria sp. and L. multisacculatum were not recorded in the Balearic Sea. Most of the parasites (79.2% of all specimens) were didymozoids. Didymozoon longicolle was the dominant species; A. cf. biovarium, D. simplex, D. fusiforme and L. multisacculatum are reported from the Mediterranean Sea for the first time. No correlation was found between the intensity of infection of any parasite species and host size or sex. Most of the parasites, particularly didymozoids, showed a high site-specificity. Significant differences were found between the parasite assemblages of K. pelamis from the Alboran Sea and from the Atlantic Ocean. D. fusiforme, D. longicolle and L. multisacculatum are suggested as potential tags to follow skipjack tuna migrations between the Atlantic Ocean and Mediterranean Sea.