Genetic variation of Sparicotyle chrysophrii (Monogenea: Microcotylidae) from the gilthead sea bream Sparus aurata (Teleostei: Sparidae) in the Mediterranean Sea.

The gill monogenean Sparicotyle chrysophrii (Van Beneden & Hesse, 1863) Mamaev, 1984 is a specific and common parasite of wild and cultured gilthead sea bream Sparus aurata Linnaeus, 1758, able to cause disease and mortality in aquaculture systems. Few molecular studies have been carried out on this monogenean, and its population structure and genetic diversity are barely known. This study provides the first contribution to the population genetic variation of S. chrysophrii, based on two molecular markers - the structural ribosomal RNA (rRNA) for the large subunit (28S) and the cytochrome c oxidase subunit I (COI) gene. Samples were collected from the gills of farmed and wild S. aurata from Italy and the Spanish Mediterranean. The analysis included previously published sequences. The 28S rDNA analysis was consistent with previous studies of specimens isolated from S. aurata and confirmed the presence of only one species on the gills of this host in the Mediterranean Sea. The COI sequences analysis suggested that the samples isolated in a previous study from a different host species, wild Boops boops (Linnaeus, 1758) in the Adriatic Sea, may represent a new undescribed sister species of S. chrysophrii. The low nucleotide diversity of S. chrysophrii isolated only from S. aurata versus the high haplotype diversity revealed small differences between haplotypes. The haplotypes shared between wild and farmed hosts from Spain provided the first molecular evidence of the possible transfer of S. chrysophrii between wild and farmed populations of S. aurata. The mtDNA COI analysis did not show a clear genetic structure, probably the result of several factors including coevolution, wild and farmed host interactions, and host population structure in space and time.

New insights into the coexistence of Contracaecum rudolphii A and Contracaecum rudolphii B (Nematoda: Anisakidae) in Phalacrocorax carbo sinensis from Sardinia: genetic variability and phylogenetic analysis.

Contracaecum sp. nematodes are important parasites of fish eating birds that can cause animal health problems. In the present study, specimens of Contracaecum rudolphii sensu lato, from the great cormorant Phalacrocorax carbo sinensis from Sardinia, were characterized based on morphological and molecular data. The morphological analysis allowed to identify all the fourth stage larvae (n = 1918) as Contracaecum sp., and adults, male (n = 5845) and female (n = 8312), as C. rudolphii sensu lato. Population genetics and phylogenetic relationships were inferred based on mitochondrial and nuclear markers. Multiple sequence alignment of the ribosomal internal transcribed spacer showed the coexistence of C. rudolphii A (n = 157), C. rudolphii B (n = 22) and a rare heterozygote of these species. Moreover, mitochondrial markers, namely NADH dehydrogenase subunits I (nad1), cytochrome c oxidase subunit (cox1 and cox2) and small subunit of rRNA (rrnS), showed that the studied C. rudolphii A populations had undergone bottleneck, or founder effect event, subsequent to a rapid population growth and expansion. The observed heterozygote is with a mitochondrial pattern of C. rudolphii B. Although, both Contracaecum species showed high genetic diversity, no genetic structure between localities was detected. Phylogenetic reconstructions supported the paraphyly of the avian Contracaecum species including C. ogmorhini (parasite of otariids).

New insight into genetic variation and haplotype diversity of Fasciola hepatica from Algeria.

The liver fluke Fasciola hepatica is the main cause of fasciolosis in North Africa leading to significant economic losses and public health problems. In this study, the ribosomal internal transcribed spacer (ITS), cytochrome c oxidase I (COI), the mitochondrial region spanning the COI-trnT-rrnL, and the NADH dehydrogenase subunit I (NADI) markers were used to characterize Fasciola flukes from Algeria. Fasciola appeared widespread from the east to the west of Algeria. Among 1701 sampled cattle from 8 Algerian provinces, 5% were infected. Using morphological and morphometric analysis, one morphotype of Fasciola was observed. Nuclear ITS marker indicated that all collected flukes belong to F. hepatica. Multiple alignments of ITS dataset revealed two haplotypes, one described here for the first time. Analysis of molecular variance (AMOVA) of mitochondrial markers revealed weak population structure in Algeria. Mismatch distributions, neutrality tests, and median-joining network analysis all were compatible with a recent expansion of Algerian F. hepatica population. Fasciolosis appeared common in Algerian cattle, it seems that the absence of control strategy coupled to the favorable Mediterranean climate may lead to a reconstruction and dispersion of its populations. This study provides important results concerning the genetic characterization and variability of F. hepatica in Algeria as well as the significant role of cattle importation in shaping its dispersal route worldwide.

Molecular characterization of Fasciola hepatica from Sardinia based on sequence analysis of genomic and mitochondrial gene markers.

The aim of the present study is to investigate for the first time the genetic diversity of samples identified morphologically as Fasciola hepatica (Platyhelminthes: Trematoda: Digenea) (n=66) from sheep and cattle from two localities of Sardinia and to compare them with available data from other localities by partial sequences of the first (ITS-1), the 5.8S, and second (ITS-2) Internal Transcribed Spacers (ITS) of nuclear ribosomal DNA (rDNA) genes, the mitochondrial cytochrome c oxidase subunit I (COI), and nicotinamide adenine dinucleotide dehydrogenase subunit I (ND1) genes. Comparison of the sequences from Sardinia with sequences of Fasciola spp. from GenBank confirmed that all samples belong to the species F. hepatica. The nucleotide sequencing of ITS rDNA showed no nucleotide variation in the ITS-1, 5.8S and ITS-2 rDNA sequences among all Sardinian samples, comparing with two ITS-2 haplotypes in standard F. hepatica, showing a substitution C/T in 20 position 859, reported previously from Tunisia, Algeria, Australia, Uruguay and Spain. The present study shows that in Sardinian sheep and cattle there is the most frequent haplotype (FhITS-H1) of F. hepatica species from South Europe. Considering NDI sequences, the phylogenetic trees showed reliable grouping among the haplotypes of F. hepatica from Sardinia and the mitochondrial lineage I, including the main N1 haplotype, observed previously from Europe (Russia, Belarus, Ukraine and Bulgaria), Armenia, West Africa (Nigeria), America (Uruguay and USA), Asia (Turkey, Japan, and China), Georgia, Turkmenistan, Azerbaijan and Australia. Furthermore, common haplotypes FhCOI-H1 and FhCOI-H2 of F. hepatica from Sardinia also corresponded mostly to the first lineage including the main C1 haplotype reported previously from Eastern European and Western Asian populations, they belonged just to a phylogenically distinguishable clade, as F. hepatica from Australia, France, Turkey, Uruguay, Russia, Armenia, Ukraine, Belarus, Turkmenistan, USA, Tunisia and Algeria, indicating that this is the main haplotype involved in the spread of F. hepatica throughout all continents.

Molecular characterization of Fasciola spp. from the endemic area of northern Iran based on nuclear ribosomal DNA sequences.

Fasciolosis caused by Fasciola spp. (Platyhelminthes: Trematoda: Digenea) is considered as the most important helminth infection of ruminants in tropical countries, causing considerable socioeconomic problems. In the endemic regions of the North of Iran, Fasciola hepatica and Fasciola gigantica have been previously characterized on the basis of morphometric differences, but the use of molecular markers is necessary to distinguish exactly between species and intermediate forms. Samples from buffaloes and goats from different localities of northern Iran were identified morphologically and then genetically characterized by sequences of the first (ITS-1) and second (ITS-2) Internal Transcribed Spacers (ITS) of nuclear ribosomal DNA (rDNA). Comparison of the ITS of the northern Iranian samples with sequences of Fasciola spp. from GenBank showed that the examined specimens had sequences identical to those of the most frequent haplotypes of F. hepatica (n=25, 48.1%) and F. gigantica (n=20, 38.45%), which differed from each other in different variable nucleotide positions of ITS region sequences, and their intermediate forms (n=7, 13.45%), which had nucleotides overlapped between the two Fasciola species in all the positions. The ITS sequences from populations of Fasciola isolates in buffaloes and goats had experienced introgression/hybridization as previously reported in isolates from other ruminants and humans. Based on ITS-1 and ITS-2 sequences, flukes are scattered in pure F. hepatica, F. gigantica and intermediate Fasciola clades, revealing that multiple genotypes of Fasciola are able to infect goats and buffaloes in North of Iran. Furthermore, the phylogenetic trees based upon the ITS-1 and ITS-2 sequences showed a close relationship of the Iranian samples with isolates of F. hepatica and F. gigantica from different localities of Africa and Asia. In the present study, the intergenic transcribed spacers ITS-1 and ITS-2 showed to be reliable approaches for the genetic differentiation of Fasciola spp., providing bases for further studies on F. hepatica, F. gigantica and their intermediate forms in the endemic areas in Asia.

Molecular characterization of Fasciola gigantica from Mauritania based on mitochondrial and nuclear ribosomal DNA sequences.

Fasciolosis caused by Fasciola hepatica and Fasciola gigantica (Platyhelminthes: Trematoda: Digenea) is considered the most important helminth infection of ruminants in tropical countries, causing considerable socioeconomic problems. From Africa, F. gigantica has been previously characterized from Burkina Faso, Senegal, Kenya, Zambia and Mali, while F. hepatica has been reported from Morocco and Tunisia, and both species have been observed from Ethiopia and Egypt on the basis of morphometric differences, while the use of molecular markers is necessary to distinguish exactly between species. Samples identified morphologically as F. gigantica (n=60) from sheep and cattle from different geographical localities of Mauritania were genetically characterized by sequences of the first (ITS-1), the 5.8S, and second (ITS-2) Internal Transcribed Spacers (ITS) of nuclear ribosomal DNA (rDNA) genes and the mitochondrial Cytochrome c Oxidase I (COI) gene. Comparison of the sequences of the Mauritanian samples with sequences of Fasciola spp. from GenBank confirmed that all samples belong to the species F. gigantica. The nucleotide sequencing of ITS rDNA of F. gigantica showed no nucleotide variation in the ITS-1, 5.8S, and ITS-2 rDNA sequences among all samples examined and those from Burkina Faso, Kenya, Egypt and Iran. The phylogenetic trees based on the ITS-1 and ITS-2 sequences showed a close relationship of the Mauritanian samples with isolates of F. gigantica from different localities of Africa and Asia. The COI genotypes of the Mauritanian specimens of F. gigantica had a high level of diversity, and they belonged to the F. gigantica phylogenically distinguishable clade. The present study is the first molecular characterization of F. gigantica in sheep and cattle from Mauritania, allowing a reliable approach for the genetic differentiation of Fasciola spp. and providing basis for further studies on liver flukes in the African countries.

Molecular characterization of Hysterothylacium aduncum (Nematoda: Raphidascaridae) from different fish caught off the Tunisian coast based on nuclear ribosomal DNA sequences.

Larval forms of the genus Hysterothylacium have been previously reported in teleost fish from the North African coasts of central Mediterranean Sea by morphological analysis. In the present study, samples identified morphologically as Hysterothylacium aduncum (n = 62), from Merluccius merluccius, Trachurus mediterraneus and Pagellus erythrinus from different geographical locations of the Tunisian coasts, were genetically characterised by sequences of the first (ITS-1), the 5.8S and second (ITS-2) Internal Transcribed Spacers (ITS) of nuclear ribosomal DNA (rDNA). Comparison of the sequences obtained with those available in public gene databases confirmed that all the samples from the Tunisian coasts belong to a single species, namely H. aduncum. All specimens from the Tunisian coasts showed one indel in position 787 in ITS-2 sequences not reported by any of the previously published sequences from the Atlantic and Pacific Oceans, Adriatic Sea (Mediterranean Sea) and the East Greenland Sea, suggesting the existence of a population-specific pattern exhibiting a low differentiation of this parasite in this area. This is the first molecular characterization of H. aduncum from the Tunisian coasts using ITS rDNA sequences which allows the definition of genetic markers for their unequivocal identification, and provides further biological data on these nematodes in marine fish off the Tunisian coasts, improving the picture of the occurrence of these taxa in the North African coasts of central Mediterranean Sea.

Multilocus approach reveals discordant molecular markers and corridors for gene flow between North African populations of Fasciola hepatica.

Fasciolosis is a foodborne trematodosis characterised by a worldwide distribution. Various approaches have been developed for the study of the causative agents of this parasitic infection: Fasciola hepatica, Fasciola gigantica and the aspermic intermediated forms (hybrid and introgressed). In the present study, novel and common molecular markers (pepck and pold, ITS, CO1, ND1 and CO1-trnT-rrnL) were used to characterise Fasciola flukes from the Tunisian-Algerian border, to estimate the gene flow between these populations and to evaluate the reliability of different molecular markers. All nuclear and mitochondrial markers, apart from pepck, supported the monophyly of the studied flukes identified as F. hepatica. Multiplex PCR for pepck revealed three different genotypes corresponding to F. hepatica (pepck-Fh), F. gigantica (pepck-Fg) and the aspermic Fasciola flukes (pepck-Fh/Fg). Sequence analysis of pepck revealed high polymorphism, length variation, within this intronic marker. The observed inconsistencies were due to the position of the forward primer within the intronic region. Pepck sequences showed different level of heterozygosity and homozygosity with length polymorphisms in the introns. Pepck multiplex PCR patterns could not differentiate between Fasciola species. All studies based on only pepck multiplex PCR with mitochondrial markers should be revised. Nuclear and mitochondrial markers revealed an important gene flow between Tunisian and Algerian populations of F. hepatica. The combination of nuclear and mitochondrial sequence analysis is still the best method to distinguish these taxa. Effective measures are needed in order to better control cross-country illegal trade of vector.

Genetic characterization of Fasciola hepatica from Tunisia and Algeria based on mitochondrial and nuclear DNA sequences.

Fasciolosis caused by Fasciola spp. (Platyhelminthes: Trematoda: Digenea) is considered the most important helminth infection of ruminants in tropical countries, causing considerable socioeconomic problems. In the present study, samples identified morphologically as Fasciola hepatica from sheep and cattle from different geographical locations of Tunisia and Algeria were genetically characterised by sequences of the first (ITS-1), the 5.8S and second (ITS-2) Internal Transcribed Spacers (ITS) of nuclear ribosomal DNA (rDNA) and mitochondrial Cytochrome c Oxidase subunit I (COI) gene. Comparison of the ITS and COI sequences of the North African samples with sequences of Fasciola spp. from GenBank confirmed that all samples from Tunisia and Algeria samples belong to a single species, namely F. hepatica. Several specimens from Tunisia and Algeria showed a substitution C/T in position 859 in the ITS-2 sequences, previously reported from Spain, suggesting that the above mentioned variant may have a common origin and spread recently throughout the three countries because of movement of infected animals. This is the first molecular characterization of F. hepatica in North Africa which provides a foundation for further studies on Fasciola spp. in Tunisia and Algeria.

Molecular characterization of larval anisakid nematodes from marine fishes off the Moroccan and Mauritanian coasts.

A total of 242 larval forms of Anisakis collected from marine fishes at different sites off the Moroccan and Mauritanian coasts, recognised as belonging to Type I and Type II larvae, were identified by PCR-RFLP (Polymerase Chain Reaction-Restriction Fragment Length Polymorphisms) of the ITS (Internal Transcribed Spacers) region (ITS-1, 5.8 subunit rRNA gene and ITS-2), using a previously established molecular key. The Type I larvae were found with a frequency of 98.34% and were identified as belonging to the following species: A. simplex s.str., A. pegreffii, A. simplex s.str/A. pegreffii heterozygote genotypes, A. typica, A. ziphidarum and Anisakis sp. A. The Type II larvae were found to belong to A. physeteris, with the frequency of 1.65%. The results reported in the present study provide further epizootiological and biological data on the Anisakis spp. in marine fishes off the Moroccan and Mauritanian coasts, improving the picture of the occurrence of these species in the central Atlantic coasts.

Molecular evidence for the occurrence of Contracaecum rudolphii A (Nematoda: Anisakidae) in shag Phalacrocorax aristotelis (Linnaeus) (Aves: Phalacrocoracidae) from Sardinia (western Mediterranean Sea).

Specimens of Contracaecum rudolphii Hartwich, 1964 (Nematoda: Anisakidae) from Phalacrocorax aristotelis (Linnaeus) from the Archipelago of La Maddalena (Sardinia, western Mediterranean Sea) were characterised genetically and compared with C. rudolphii A sensu D'Amelio et al. 1990 and C. rudolphii B sensu D'Amelio et al. 1990 from Phalacrocorax carbo sinensis (Blumenbach) from north-eastern Italy, and with C. rudolphii C sensu D'Amelio et al. 2007 from Phalacrocorax auritus (Lesson) from west-central Florida, USA. The sequencing of the small subunit of the mitochondrial ribosomal RNA gene (rrnS) and by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the same gene and of the internal transcribed spacers (ITS) of nuclear ribosomal DNA (rDNA) allowed the identification of all specimens of C. rudolphii from P. aristotelis as C. rudolphii A. The results confirmed that the definition of genetic markers, following the analysis of nuclear ribosomal and mitochondrial DNA, provides quick and practical diagnostic tools for the detection of the 3 sibling species of C. rudolphii. The occurrence of C. rudolphii in P. aristotelis is reported for the first time from the Mediterranean area, improving the picture of the dispersal patterns of the populations of these piscivorous birds, and confirming the existence of different and isolated populations between the North and South European waters.

Occurrence and molecular identification of Anisakis spp. from the North African coasts of Mediterranean Sea.

Larval forms of the genus Anisakis were reported infecting several fish species from the North African coasts of central Mediterranean Sea. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis was used to investigate the occurrence of larval forms of different Anisakis species in teleost fishes and squid from North African coasts of the Mediterranean Sea and to establish the geographical and host range of these parasites in this area. A total of 282 Anisakis larvae were identified by PCR-RFLP from 13 teleost fish species and one cephalopod species captured at different sites off the Algerian, Tunisian and Libyan coasts. The type I larvae were found with a frequency of 93.62% and were identified as belonging to the following species: Anisakis simplex s.str., Anisakis pegreffii, A. simplex s.str/A. pegreffii hybrids and Anisakis typica. The type II larvae were found to belong to Anisakis physeteris, with the frequency of 6.38%. The record of A. simplex s.str/A. pegreffii hybrids, previously recorded from the Spanish and Portuguese Atlantic coasts and the Alboran Sea, extends their geographic distribution to the Tunisian coasts. The occurrence of A. simplex s.str. and hybrids away from their known area of distribution may predict the successful use of Anisakis larvae for tagging Scomber scombrus fish stocks for fisheries management purposes. Moreover, the results reported provide valuable information regarding the diversity of Anisakis species in the study area, indicating that several Anisakis sibling and morphospecies coexist in the North African coasts of the Mediterranean Sea.

Anisakid parasites of two forkbeards (Phycis blennoides and Phycis phycis) from the eastern Mediterranean coasts in Tunisia.

Two gadiform species with a successive bathymetric and an ecological and economical importance in the Mediterranean fishing industry, Phycis blennoides and Phycis phycis, were selected for the present study. A total of 592 fresh specimens belonging to the Gadiformes genus were obtained from local commercial fisheries. The investigation was centred on anisakid parasites of 272 specimens of the greater forkbeard (P. blennoides) and 320 of the forkbeard (P. phycis) captured off the Mediterranean coasts of Tunisia (eastern Mediterranean Sea). Four species of nematodes were identified: Anisakis simplex s.1., Anisakis physeteris, Hysterothylacium aduncum and Hysterothylacium fabri. The total prevalence was 53.75% in the forkbeard and 51.47% in the greater forkbeard. The highest values of prevalence (38.75%, 2-14), mean intensity (6.74+/-3.4) and mean abundance (2.61) were all obtained for H. fabri L4 in the forkbeard. The most frequent parasite in the greater forkbeard was H. aduncum L3 with 32.35% (1-3) prevalence and values of 1.21+/-0.58 and 0.39 for mean intensity and mean abundance, respectively. The infestation parameters were also analysed according to the host length, and prevalence was highest in P. blennoides longer than 35 cm in respect of all anisakid species. Whilst in P. phycis, the highest prevalence, conditioned by H. fabri parasitisation, was found in fish with length reaching a maximum of more than 40 cm. When the data were grouped seasonally, clear patterns were observed for P. blennoides and P. phycis species, with prevalence and mean intensity of all the anisakid species peaking in spring and summer.