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Simple Summary: The nematodes of the genus Anisakis are among the most prevalent parasites found in fishes and marine mammals and they are the main cause of human anisakiasis. Genetic studies have described two species, A. simplex and A. pegreffii, in European waters parasitizing several hosts, being the European hake, by far, the fish with higher infection values. The aim of this study is to enhance the knowledge of the distribution and population structure of the Anisakis species infecting hakes from the major European fishing areas through the use of genetic analysis. This study provides useful information about the genetic diversity of these parasites in the different fishing areas and fish tissues, valuable findings to understand the parasite speciation to different hake tissues and how they are structured along European waters. The European hake, one of the most commercially valuable species in ICES fishing areas, is considered an important neglected source of zoonotic risk by nematode parasites belonging to the genus Anisakis. Merluccius merluccius is, by far, the most important host of Anisakis spp. at the European fishing grounds, in terms of demographic infection values, and carries the highest parasite burden. These high parasite population densities within an individual fish host offer a chance to explore new sources of variations for the genetic structure of Anisakis spp. populations. A total of 873 Anisakis spp. third-stage larvae, originally sampled from viscera and muscular sections of hake collected at ten fishing grounds, were primarily identified using ITS rDNA region as molecular marker. After that, we used mtDNA cox2 gene to reveal the high haplotype diversity and the lack of genetic structure for A. simplex. Dominant haplotypes were shared among the different fishing areas and fish sections analyzed. Results indicate a clear connection of A. simplex from European hake along the Northern North Sea to the Portuguese coast, constituting a single genetic population but revealing a certain level of genetic sub-structuring on the Northwest coast of Scotland. This study also provides useful information to advance the understanding of parasite speciation to different fish host tissues or microenvironments. [ABSTRACT FROM AUTHOR]

Abstract Backgrounds Geographic regions, where two closely related taxa with different migration routes come into contact, are known as migratory divides. Hybrids originating from migratory divides are hypothesized to migrate intermediately relative to the parental populations. Few studies have tested this hypothesis in wild birds, and only in hybrids that have completed the migration back to the breeding grounds. Here, we make use of the well-established migration routes of willow warblers (Phylloscopus trochilus), for which the subspecies trochilus and acredula have migration-associated genetic markers on chromosomes 1 and 5. The genetic approach enabled us to analyze the geographic distribution of juveniles during their first autumn migration, predicting that hybrids should be more frequent in the central flyway over Italy than along the typical SW routes of trochilus and SE routes of acredula. Methods Blood and feather samples were collected from wintering birds in Africa (n = 69), and from juveniles during autumn migration in Portugal (n = 33), Italy (n = 38) and Bulgaria (n = 32). Genotyping was carried out by qPCR SNP assays, on one SNP each on chromosome 1 (SNP 65) and chromosome 5 (SNP 285). Both these SNPs have alternative alleles that are highly fixed (> 97%) in each of the subspecies. Results The observed combined genotypes of the two SNPs were associated with the known migration routes and wintering distributions of trochilus and acredula, respectively. We found hybrids (HH) among the juveniles in Italy (5/38) and in Portugal (2/33). The proportion of hybrids in Italy was significantly higher than expected from a background rate of hybrid genotypes (1.5%) in allopatric populations of the subspecies. Conclusions Our genetic approach to assign individuals to subspecies and hybrids allowed us to investigate migration direction in juvenile birds on their first migration, which should better reflect the innate migratory direction than studies restricted to successful migrants. The excess of hybrids in Italy, suggests that they employ an intermediate route relative to the parental populations. Our qPCR SNP genotyping method is efficient for processing large sample sizes, and will therefore be useful in migration research of species with known population genetic structure.

The European hake, one of the most commercially valuable species in ICES fishing areas, is considered an important neglected source of zoonotic risk by nematode parasites belonging to the genus Anisakis. Merluccius merluccius is, by far, the most important host of Anisakis spp. at the European fishing grounds, in terms of demographic infection values, and carries the highest parasite burden. These high parasite population densities within an individual fish host offer a chance to explore new sources of variations for the genetic structure of Anisakis spp. populations. A total of 873 Anisakis spp. third-stage larvae, originally sampled from viscera and muscular sections of hake collected at ten fishing grounds, were primarily identified using ITS rDNA region as molecular marker. After that, we used mtDNA cox2 gene to reveal the high haplotype diversity and the lack of genetic structure for A. simplex. Dominant haplotypes were shared among the different fishing areas and fish sections analyzed. Results indicate a clear connection of A. simplex from European hake along the Northern North Sea to the Portuguese coast, constituting a single genetic population but revealing a certain level of genetic sub-structuring on the Northwest coast of Scotland. This study also provides useful information to advance the understanding of parasite speciation to different fish host tissues or microenvironments.

The sibling species Anisakis simplex (s.s.) and Anisakis pegreffii are parasites of marine mammals and fish worldwide and the main causative agents of human anisakiasis. In sympatric areas, a hybrid genotype between the two species has been identified, mainly in third-stage larvae, but rarely in fourth-stage and adult forms. The aim of this study was to confirm the presence of hybrid genotypes in larvae parasitizing fish caught in sympatric and allopatric Spanish marine waters, the North-East Atlantic and West Mediterranean, respectively, and to study possible differences in the growth behaviour between genotypes. Of the 254 molecularly analysed larvae, 18 were identified as hybrids by PCR-RFLP analysis of the rDNA ITS region, 11 of which were subsequently confirmed by EF1 α-1 nDNA gene sequencing. These results therefore indicate an overestimation of hybrid genotypes when identification is based only on the ITS region. We also report the detection of a hybrid specimen in a host from the West Mediterranean, considered an allopatric zone. Additionally, fourth-stage larvae with a hybrid genotype were obtained in vitro for the first time, and no differences were observed in their growth behaviour compared to larvae with A. simplex (s.s.) and A. pegreffii genotypes.

In this study, 1029 fish and cephalopod samples came from Central-Western Mediterranean (FAO 37.1.1 and FAO 37.1.3) were analysed for Anisakidae larvae research with the aim to identify possible hybridisations between Anisakis pegreffii and Anisakis simplex s.s. species. A total of 1765 larvae were detected, with prevalence values between 8.1% and 100%. The morphologic analysis revealed characters attributable to morphotype I of Anisakis in 98.5% of the examined larvae, while 1.5% belonged to the morphotype II. PCR-based Restriction Fragment Length Polymorphism (PCR–RFLP) analysis of the entire ITS region (ITS1, 5.8S and ITS2) of nuclear ribosomal DNA (rDNA) was performed with Hinf I and Hha I restriction enzymes. The majority of the larvae examined by PCR-RFLP were identified as A. pegreffii (71%), with a prevalence on horse mackerel from FAO 37.1.3, while 10% were identified as A. simplex s.s., 2% as A. physeteris and 17% as A. pegreffii × A. simplex s.s. hybrid genotype. The sequence analysis confirmed the hybridisation in the 85% of the larvae recognised as hybrid forms by PCR- RFLP, suggesting this form as the product of natural interspecific recombination due to the presence of sympatry areas. The presence of hybrid forms were mostly found in fish samples from FAO subzone 37.1.1. This is the first report of A. pegreffii x A. simplex s.s. hybrid genotype in fishes caught off the coasts of Sicily (Southern Italy). Finally, this study provided substantial information about the geographical distribution of Anisakidae family in Central-Western Mediterranean Sea. [ABSTRACT FROM AUTHOR]

Abstract: Approximately 10% of the Trypanosoma cruzi genome is formed by a satellite DNA, composed by 195-bp repeats organized in 30±10kb clusters in some, but not all chromosomes. Here, the satellite DNA of six representative T. cruzi strains was sequenced and used for phylogenetic inference. The results show that CL Brener contains satellite repeats from T. cruzi I and T. cruzi II strains, although type II sequences are more abundant. The presence of types I and II sequences extends previous propositions that genetic exchange between the two major T. cruzi lineages have occurred in CL Brener, although our data accommodate alternative scenarios of hybridization within T. cruzi II, as proposed by others. Altogether, present data suggest a complex origin for CL Brener. Sequence analysis of satellites isolated from chromosomal bands indicates that satellite DNA sequences are not chromosome specific. Neighbor analysis of in tandem satellite DNAs containing up to five repeats shows that each cluster contains only one type of sequence. Consequently, clusters with intercalated types I and II repeats were not found. We propose that the CL Brener genome contains large pieces of satellite DNA originated mainly from chromosomes of T. cruzi II with introgression of T. cruzi I lineage. [Copyright &y& Elsevier]

A set of 65 Trypanosoma cruzi stocks from dogs, opossums, insect vectors and humans was isolated in a geographically restricted endemic area for Chagas’ disease in Argentina and was analysed by multilocus enzyme electrophoresis for 15 loci. The results show that at least five multilocus genotypes (clonets) circulate in the study area, one belonging to T. cruzi IIe, one to T. cruzi IId and three clonets belonging to T. cruzi I; and they confirm the presence of these lineages in the country. The three clonets attributed to T. cruzi I were identical to each other for all loci except for Sod-2, where three different patterns were identified. These patterns suggest the presence of two homozygous genotypes and one heterozygous genotype. Our results also suggest association of clonet IIe with dogs, clonet IId with humans and the three T. cruzi I clonets with Didelphis albiventris. On the other hand, there was no significant association between Triatoma infestans and any particular clonet circulating in the area. These findings are consistent with the hypothesis of natural selection, from mixed populations of T. cruzi in vectors, toward more restricted populations in mammals. The epidemiological implications of the possible selection of different clonets by different mammal hosts and the significance of two homozygous genotypes and one heterozygous genotype for the Sod-2 locus are discussed. [Copyright &y& Elsevier]

Simple Summary: The nematode species Anisakis simplex sensu stricto (s.s.) and Anisakis pegreffii are wormlike parasites found in commonly consumed fish and are the main cause of human anisakiasis. Outwardly, the two nematodes are extremely similar and difficult to distinguish, especially in their larval forms. Genetic analysis has discovered the existence of a hybrid between these two "sibling species", but its identification is a controversial matter, as results differ according to the specific region of the DNA analysed. The aim of our work was to confirm the presence of this hybrid genotype in fish off the Spanish coast and to obtain fourth-stage larvae in the laboratory to study if different genotypes are associated with different growth behaviour. Our results confirm that hybrid genotypes can be overestimated if identification is based on a particular molecular marker. We also obtained fourth-stage larvae with a hybrid genotype, which has not been reported previously. These findings are valuable for the taxonomic classification of Anisakis species, and for further epidemiological and biomedical research. The sibling species Anisakis simplex (s.s.) and Anisakis pegreffii are parasites of marine mammals and fish worldwide and the main causative agents of human anisakiasis. In sympatric areas, a hybrid genotype between the two species has been identified, mainly in third-stage larvae, but rarely in fourth-stage and adult forms. The aim of this study was to confirm the presence of hybrid genotypes in larvae parasitizing fish caught in sympatric and allopatric Spanish marine waters, the North-East Atlantic and West Mediterranean, respectively, and to study possible differences in the growth behaviour between genotypes. Of the 254 molecularly analysed larvae, 18 were identified as hybrids by PCR-RFLP analysis of the rDNA ITS region, 11 of which were subsequently confirmed by EF1 α-1 nDNA gene sequencing. These results therefore indicate an overestimation of hybrid genotypes when identification is based only on the ITS region. We also report the detection of a hybrid specimen in a host from the West Mediterranean, considered an allopatric zone. Additionally, fourth-stage larvae with a hybrid genotype were obtained in vitro for the first time, and no differences were observed in their growth behaviour compared to larvae with A. simplex (s.s.) and A. pegreffii genotypes. [ABSTRACT FROM AUTHOR]

We present a case of the 42 year old pig farmer from the province of Cuneo in Northwest Italy who was infected by the soil-transmitted nematode Ascaris sp. In November 2010 the patient found one worm in his stool, subsequently identified as female specimen of /Ascaris sp. After a first anthelmintic treatment, another worm was found in his stool, that was later identified as male Ascaris sp. Blood tests prescribed by the patient's family physician, as suggested by a parasitologist, found nothing abnormal. A chest x-ray was negative for Loeffler's syndrome and an ultrasound of the abdomen was normal with no evidence of hepatic problems. The nematode collected from the patient was genetically characterized using the ribosomal nuclear marker ITS. The PCR-RFLP analysis showed a hybrid genotype, intermediate between A. suum/lumbricoides. It was subsequently ascertained that some pigs on the patient's farm had A. suum infection; no other family member was infected. A cross-infestation from the pigs as source was the likely way of transmission. This conclusion is further warranted by the fact, that the patient is a confirmed nail-biter, a habit which facilitates oral-fecal transmission of parasites and pathogens. [ABSTRACT FROM AUTHOR]

Globalization opens new market areas and affects food consumption habits, resulting in rapid and remarkable cultural change. Food habits such as consumption of raw fish meat have become popular, resulting in increased risk of emerging infectious diseases. Anisakis simplex sensu stricto (s.s) and A. pegreffii are the most common and important fish-borne zoonotic nematodes responsible for human anisakiasis, which occurs through the consumption of raw or undercooked fish as well as cooked fish due to their heat-stable allergens. Here, we investigated the prevalence, intensity, and abundance of Anisakis larvae in imported fish and ready-to-eat local fish products in Turkey. A total of 205 ready-to-eat fish products, 100 imported frozen Atlantic salmon (Salmo salar) fillets, and 100 imported frozen whole Atlantic mackerel (Scomber scombrus) were sampled from supermarkets, sushi restaurants, and fish markets. All samples were individually examined using a pepsin digestion technique. In total, 602 Anisakis type I larvae were recovered from 98/100 mackerel. No larvae were found in ready-to-eat products or frozen Atlantic salmon fillets. Overall, 8.8% of the larvae were found in the muscle tissue. The overall mean intensity and abundance of infection in mackerel were 6.14 and 6.02, respectively. The larvae were molecularly identified and their phylogenetic relationships with the relevant Anisakis sequences in GenBank were investigated. For this purpose, a subsample of randomly selected 100 Anisakis larvae were analyzed with PCR-RFLP of the ITS region. The larvae were identified as A. simplex (s.s.) (n = 87) and hybrids (n = 13). ITS and cox2 gene regions of all hybrids and randomly selected 50 A. simplex (s.s.) larvae were sequenced for species confirmation and phylogenetic analyses. No intraspecific nucleotide variation was found among the ITS sequences of either species. Seven and three haplotypes, respectively, were identified for A. simplex (s.s.) and hybrid species according to DNA polymorphism of the cox2 gene. Hybrids in our study clustered within the common A. simplex (s.s.) clade in the cox2 phylogenetic tree indicating the dominance of A. simplex (s.s) in the catching area of Atlantic mackerel. Consequently, our study indicates high occurrence of A. simplex (s.s.) larvae with an overall 98.0% prevalence in imported Atlantic mackerel, and highlights the importance of these fish as potential reservoirs for human allergic anisakiasis in Turkey and possibly in other countries. • Ready to eat fish products and imported frozen fish in Turkey were analyzed. • Anisakis larvae were only found in Atlantic mackerel specimens with high prevalence. • A total of 602 larvae were collected from 98 infected mackerels. • Larvae were genetically identified as A. simplex (s.s.) and hybrid species. • The imported Atlantic mackerel may cause significant risk for public health. [ABSTRACT FROM AUTHOR]