Functional insights into the infective larval stage of Anisakis simplex s.s., Anisakis pegreffii and their hybrids based on gene expression patterns.

BACKGROUND: Anisakis simplex sensu stricto and Anisakis pegreffii are sibling species of nematodes parasitic on marine mammals. Zoonotic human infection with third stage infective larvae causes anisakiasis, a debilitating and potentially fatal disease. These 2 species show evidence of hybridisation in geographical areas where they are sympatric. How the species and their hybrids differ is still poorly understood. RESULTS: Third stage larvae of Anisakis simplex s.s., Anisakis pegreffii and hybrids were sampled from Merluccius merluccius (Teleosti) hosts captured in waters of the FAO 27 geographical area. Specimens of each species and hybrids were distinguished with a diagnostic genetic marker (ITS). RNA was extracted from pools of 10 individuals of each taxon. Transcriptomes were generated using Illumina RNA-Seq, and assembled de novo. A joint assembly (here called merged transcriptome) of all 3 samples was also generated. The inferred transcript sets were functionally annotated and compared globally and also on subsets of secreted proteins and putative allergen families. While intermediary metabolism appeared to be typical for nematodes in the 3 evaluated taxa, their transcriptomes present strong levels of differential expression and enrichment, mainly of transcripts related to metabolic pathways and gene ontologies associated to energy metabolism and other pathways, with significant presence of excreted/secreted proteins, most of them allergens. The allergome of the 2 species and their hybrids has also been thoroughly studied; at least 74 different allergen families were identified in the transcriptomes. CONCLUSIONS: A. simplex s.s., A. pegreffi and their hybrids differ in gene expression patterns in the L3 stage. Strong parent-of-origin effects were observed: A. pegreffi alleles dominate in the expression patterns of hybrids albeit the latter, and A. pegreffii also display significant differences indicating that hybrids are intermediate biological entities among their parental species, and thus of outstanding interest in the study of speciation in nematodes. Analyses of differential expression based on genes coding for secreted proteins suggests that co-infections presents different repertoires of released protein to the host environment. Both species and their hybrids, share more allergen genes than previously thought and are likely to induce overlapping disease responses.

Invasive anisakiasis by the parasite Anisakis pegreffii (Nematoda: Anisakidae): diagnosis by real-time PCR hydrolysis probe system and immunoblotting assay.

BACKGROUND: Anisakiasis is a fish-borne zoonosis caused by Anisakis spp. larvae. One challenging issue in the diagnosis of anisakiasis is the molecular detection of the etiological agent even at very low quantity, such as in gastric or intestinal biopsy and granulomas. Aims of this study were: 1) to identify three new cases of invasive anisakiasis, by a species-specific Real-time PCR probe assay; 2) to detect immune response of the patients against the pathogen. METHODS: Parasite DNA was extracted from parasites removed in the three patients. The identification of larvae removed at gastric and intestinal level from two patients was first obtained by sequence analysis of mtDNA cox2 and EF1 α-1 of nDNA genes. This was not possible in the third patient, because of the very low DNA quantity obtained from a single one histological section of a surgically removed granuloma. Real-time PCR species-specific hydrolysis probe system, based on mtDNA cox2 gene, was performed on parasites tissue of the three cases. IgE, IgG4 and IgG immune response against antigens A. pegreffii by Immunoblotting assay was also studied. RESULTS: According to the mtDNA cox2 and the EF1 α - 1 nDNA sequence analysis, the larvae from stomach and intestine of two patients were assigned to A. pegreffii. The Real-time PCR primers/probe system, showed a fluorescent signal at 510 nm for A. pegreffii, in all the three cases. In Immunoblotting assay, patient CC1 showed IgE, IgG4 reactivity against Ani s 13-like and Ani s 7-like; patient CC2 revealed only IgG reactivity against Ani s 13-like and Ani s 7-like; while, the third patient showed IgE and IgG reactivity against Ani s 13-like, Ani s 7-like and Ani s 1-like. CONCLUSION: The Real-time PCR assay, a more sensitive method than direct DNA sequencing for the accurate and rapid identification of etiological agent of human anisakiasis, was successfully assessed for the first time. The study also highlights the importance to use both molecular and immunological tools in the diagnosis of human anisakiasis, in order to increase our knowledge about the pathological findings and immune response related to the infection by zoonotic species of the genus Anisakis.

Anisakis (Nematoda: Ascaridoidea) from Indonesia.

Despite Indonesia's high marine biodiversity, there is a lack of information regarding fish parasites in Indonesian waters. During a sampling of 136 teleost species from Indonesian waters, 22 of them were infected with larvae of Anisakis Dujardin, 1845, a genus with zoonotic potential. We genetically identified 118 worms, provide a revision of all available sequences of the ITS-1-5.8S-ITS-2 marker from Indonesian Anisakis in GenBank (n = 125), and establish 16 new host records. So far, 53 Indonesian teleosts harbour Anisakis spp., 32 of them with known sequence data, increasing the worldwide teleosts with genetically identified Anisakis from 155 to 177. Sequence analyses of this marker in the 243 Anisakis specimens identified 3 Anisakis sp. HC-2005 and 39 (16%) A. typica (sensu stricto). A. berlandi and A. pegreffii are reported for the first time from teleosts in the equatorial region and A. physeteris from the Pacific Ocean. The latter 3 species were exclusively found in the migratory scombrid Auxis rochei. Most common infection sites were the body cavity, with 299 (of 848) worms in the mesenteries surrounding the liver, and 129 unattached. Musculature infection was very low, demonstrating minor risk of anisakiasis for human consumers. A total of 193 worms (~79%) had a distinct genotype distinguished from A. typica by 4 positions in the ITS-1 region. This genotype is reported since 2008 as 'A. typica', 'sibling', 'Anisakis sp./type 1', 'sp. I', 'sp. 2' or 'sp. II'. To avoid further misleading identification, we hereby apply the subspecific entity Anisakis typica var. indonesiensis until description of the adults becomes available.

Recombinase polymerase amplification combined with lateral flow dipstick assay for rapid visual detection of A.simplex (s. s.) and A.pegreffii in sea foods.

Anisakiasis is a food-borne parasitic disease mainly caused by the third stage of Anisakis simplex (s. s.) and Anisakis pegreffii. Traditional methods for detecting of Anisakis involve morphology identification such as visual inspection, enzyme digestion, and molecular methods based on PCR, but they have certain limitations. In this study, the internal transcribed spacer 1 (ITS 1) regions of Anisakis were targeted to develop a visual screening method for detecting A. simplex (s. s.) and A. pegreffii in fish meat based on recombinase polymerase amplification (RPA) combined with lateral flow dipstick (LFD). Specific primers and probes were designed and optimized for temperature, reaction time, and detection threshold. LFD produced clear visual results that were easily identifiable after a consistent incubation of 10-20 min at 37 °C. The whole process of DNA amplification by RPA and readout by LFD did not exceed 30 min. In addition, the detection limit is up to 9.5 × 10-4 ng/µL, and the detection of the artificially contaminated samples showed that the developed assay can effectively and specifically detect A. simplex (s. s.) and A. pegreffii, which fully meet the market's requirements for fish food safety supervision.

Quantitative Proteomics Comparison of Total Expressed Proteomes of Anisakis simplex Sensu Stricto, A. pegreffii, and Their Hybrid Genotype.

The total proteomes of Anisakis simplex s.s., A. pegreffii and their hybrid genotype have been compared by quantitative proteomics (iTRAQ approach), which considers the level of expressed proteins. Comparison was made by means of two independent experiments considering four biological replicates of A. simplex and two each for A. pegreffii and hybrid between both species. A total of 1811 and 1976 proteins have been respectively identified in the experiments using public databases. One hundred ninety-six proteins were found significantly differentially expressed, and their relationships with the nematodes' biological replicates were estimated by a multidimensional statistical approach. Results of pairwise Log2 ratio comparisons among them were statistically treated and supported in order to convert them into discrete character states. Principal component analysis (PCA) confirms the validity of the method. This comparison selected thirty seven proteins as discriminant taxonomic biomarkers among A. simplex, A. pegreffii and their hybrid genotype; 19 of these biomarkers, encoded by ten loci, are specific allergens of Anisakis (Ani s7, Ani s8, Ani s12, and Ani s14) and other (Ancylostoma secreted) is a common nematodes venom allergen. The rest of the markers comprise four unknown or non-characterized proteins; five different proteins (leucine) related to innate immunity, four proteolytic proteins (metalloendopeptidases), a lipase, a mitochondrial translocase protein, a neurotransmitter, a thyroxine transporter, and a structural collagen protein. The proposed methodology (proteomics and statistical) solidly characterize a set of proteins that are susceptible to take advantage of the new targeted proteomics.

A novel nuclear marker and development of an ARMS-PCR assay targeting the metallopeptidase 10 (nas 10) locus to identify the species of the Anisakis simplex (s. l.) complex (Nematoda, Anisakidae).

The genus Anisakis represents one of the most widespread groups of ascaridoid nematodes in the marine ecosystem. Three closely related taxa are recognized in the Anisakis simplex (s. l.) complex: A. pegreffii, A. simplex (s. s.) and A. berlandi. They are widely distributed in populations of their intermediate/paratenic hosts (fish and squids) and definitive hosts (cetaceans). A novel nuclear gene locus, metallopeptidase 10 (nas 10) (451 bp), was sequenced and validated on a total of 219 specimens of the three species of Anisakis, collected in fish and cetacean hosts from allopatric areas included in their ranges of distribution. The specimens of Anisakis were first identified by allozymes and sequence analysis of the mtDNA cox2 and EF1α-1 nDNA. The novel nuclear marker has shown fixed alternative nucleotide positions in the three species, i.e. diagnostic at 100%, permitting the species determination of a large number of specimens analyzed in the present study. In addition, primers to be used for amplification-refractory mutation system (ARMS) PCR of the same gene locus were designed at these nucleotide positions. Thus, direct genotyping determination, by double ARMS, was developed and validated on 219 specimens belonging to the three species. Complete concordance was observed between the tetra-primer ARMS-PCR assays and direct sequencing results obtained for the nas 10 gene locus. The novel nuclear diagnostic marker will be useful in future studies on a multi-locus genotyping approach and also to study possible hybridization and/or introgression events occurring between the three species in sympatric areas.

TITLE: Un nouveau marqueur nucléaire et développement d'un test ARMS-PCR ciblant le locus de la métallopeptidase 10 (nas 10) pour identifier les espèces du complexe Anisakis simplex (s. l.) (Nematoda, Anisakidae). ABSTRACT: Le genre Anisakis représente l'un des groupes de nématodes ascaridoïdes les plus répandus dans l'écosystème marin. Trois taxons étroitement apparentés sont reconnus dans le complexe Anisakis simplex (s. l.) : A. pegreffii, A. simplex (s. s.) et A. berlandi. Ils sont largement répartis dans les populations de leurs hôtes intermédiaires/paraténiques (poissons et calmars) et définitifs (cétacés). Un nouveau locus de gène nucléaire, la métallopeptidase 10 (nas 10) (451 pb), a été séquencé et validé sur un total de 219 spécimens des trois espèces d'Anisakis, collectés chez des hôtes poissons et cétacés de zones allopatriques incluses dans leur aire de répartition. Les échantillons d'Anisakis ont d'abord été identifiés par des allozymes et une analyse des séquences de l'ADNmt cox2 et de l'ADNn EF1α-1. Le nouveau marqueur nucléaire a montré des positions de nucléotides alternatives fixes dans les trois espèces, c'est-à-dire qu'il a permis un diagnostic à 100%, permettant la détermination de l'espèce d'un grand nombre d'échantillons analysés dans la présente étude. De plus, des amorces à utiliser pour la PCR par système de mutation réfractaire à l'amplification (ARMS) du même locus génique ont été conçues à ces positions nucléotidiques. Ainsi, la détermination directe du génotypage, par double ARMS, a été développée et validée sur 219 spécimens appartenant aux trois espèces. Une concordance complète a été observée entre les dosages ARMS PCR tétra-amorces et les résultats de séquençage direct obtenus pour le locus du gène nas 10. Le nouveau marqueur de diagnostic nucléaire sera utile dans les travaux futurs d'une approche de génotypage multi-locus et également pour étudier les éventuels événements d'hybridation et/ou d'introgression se produisant entre les trois espèces dans des zones sympatriques.

Population parameters and mito-nuclear mosaicism of Anisakis spp. in the Adriatic Sea.

Recombinant genotypes of A. simplex sensu stricto (s.s.) and A. pegreffii, two species of Anisakis simplex complex found in sympatric waters of the Mediterranean Sea, are believed to be a product of interspecific hybridisation and/or DNA introgression. In contrast, such events within an allopatric area as the Adriatic Sea are unlikely to occur and therefore observed recombination should be assessed more closely. We have genotyped 525 anisakids collected from migratory and non-migratory fish of the southern part of the Adriatic Sea, inferring its omniparentage at nuclear (ITS locus) and matrilineage at mitochondrial locus (cox2). The aim was to address the presence and cause of the recombination within the population and to test its genetic structure under admixture theory. Population parameters, i.e. prevalence, and mean abundance and intensity of anisakids were also evaluated to contribute for future epidemiological risk assessments. As a result, we have inferred the presence of A. pegreffii, A. typica and A. ziphidarum in the Adriatic, lacking type species A. simplex s.s. at both nuclear and mitochondrial locus. A. pegreffii population shows a high level of admixture and heterogeneity and a recent demographic expansion from a small population size. We argue that the observed recombinant genotypes in the Adriatic are a product of ancestral polymorphism and consequent remote genetic introgression.

Molecular characterization and comparison of four Anisakis allergens between Anisakis simplex sensu stricto and Anisakis pegreffii from Japan.

It remains unclear whether allergens are the same among the sibling species of Anisakis simplex sensu lato. This study was carried out to compare the amino acid sequences of three major (Ani s 1, 2 and 12) and one minor (Ani s 9) Anisakis allergens between A. simplex s.s. and Anisakis pegreffii. We found 2 (out of 163), 1 (out of 869), and 29 (out of 266) amino acid variable sites for Ani s 1, 2, and 12, respectively. However, as both intra- and inter-species variations were present at the same amino acid positions, no amino acid variations clearly distinguished the two sibling species. IgE-binding epitopes (Ani s 1) and a binding motif of human leukocyte antigen (Ani s 2 and 9) demonstrated by previous studies were conserved. The similarities of the amino acid sequences of the allergens indicate possible similar allergy-associated health risks in humans infected with or accidentally ingesting either Anisakis species.