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Parasites of the nematode genus Anisakis are associated with aquatic organisms. They can be found in a variety of marine hosts including whales, crustaceans, fish and cephalopods and are known to be the cause of the zoonotic disease anisakiasis, a painful inflammation of the gastro-intestinal tract caused by the accidental consumptions of infectious larvae raw or semi-raw fishery products. Since the demand on fish as dietary protein source and the export rates of seafood products in general is rapidly increasing worldwide, the knowledge about the distribution of potential foodborne human pathogens in seafood is of major significance for human health. Studies have provided evidence that a few Anisakis species can cause clinical symptoms in humans. The aim of our study was to interpolate the species range for every described Anisakis species on the basis of the existing occurrence data. We used sequence data of 373 Anisakis larvae from 30 different hosts worldwide and previously published molecular data (n = 584) from 53 field-specific publications to model the species range of Anisakis spp., using a interpolation method that combines aspects of the alpha hull interpolation algorithm as well as the conditional interpolation approach. The results of our approach strongly indicate the existence of species-specific distribution patterns of Anisakis spp. within different climate zones and oceans that are in principle congruent with those of their respective final hosts. Our results support preceding studies that propose anisakid nematodes as useful biological indicators for their final host distribution and abundance as they closely follow the trophic relationships among their successive hosts. The modeling might although be helpful for predicting the likelihood of infection in order to reduce the risk of anisakiasis cases in a given area.

Background: So far, the frequency of Anisakis simplex-specific IgE antibodies has been determined by skin prick tests (SPTs) and the ImmunoCAP system. These commercial methods have good sensitivity, but their specificity is poor because they use complete parasite extracts. Our aim was to determine the frequency of sensitization to A. simplex using recombinant Ani s 1, Ani s 3, Ani s 5, Ani s 9 and Ani s 10 and to evaluate these allergens for diagnosis, comparing their performance with the commercial methods. Patients and Methods: We conducted a descriptive, cross-sectional validation study performed in an allergy outpatient hospital clinic. Patients without fish-related allergy (tolerant patients, n = 99), and A. simplex-allergic patients (n = 35) were studied by SPTs, ImmunoCAP assays and detection of specific IgE to A. simplex recombinant allergens by dot blotting. Results: SPTs and ImmunoCAP assays were positive in 18 and 17% of tolerant patients, respectively. All A. simplex-allergic patients had positive SPTs and ImmunoCAP assays. Specific IgE against at least one of the A. simplex recombinant allergens tested was detected in 15% of sera from tolerant patients and in 100% of sera from A. simplex-allergic patients. Detection of at least one A. simplex recombinant allergen by dot blotting and ImmunoCAP assay using complete extract showed a diagnostic sensitivity of 100% with both methods. However, the specificity of dot blotting with A. simplex recombinant allergens was higher compared with ImmunoCAP (84.85 vs. 82.83%). Conclusions: There are 15% of tolerant patients with specific IgE against important A. simplex allergens. The recombinant allergens studied here increase the specificity of A. simplex diagnosis while keeping the highest sensitivity. A. simplex recombinant allergens should be included with A. simplex allergy diagnostic tests to improve their specificity. Copyright [copy 2012 S. Karger AG, Basel

Anisakis simplex is a cosmopolitan parasitic nematode of marine organisms with a complex life cycle. Consuming fish infected with its larvae poses a health risk, as the parasites can penetrate gastrointestinal mucosa, damage stomach and intestinal walls, and trigger allergic reactions. The resulting disease is known as anisakiasis. The European Food Safety Authority (EFSA) classifies A. simplex as a biohazard. Most of its developmental stages (L3, L4, and adults) occur under anaerobic conditions, and larvae derive energy mainly from saccharides. However, the effect of glucose on L3 and L4 larvae—stages pathogenic to humans—has not been described. This study aimed to identify genes and pathways involved in glucose (10 mg/mL) response through transcriptomic and proteomic analyses of L3 and L4 larvae. Differentially expressed genes (DEGs), long non-coding RNAs (DELs), and differentially regulated proteins (DRPs) were identified. DEGs were involved in cuticle structure, lyase activity, and metabolic processes. Comparing L3 CTR and L4 CTR (control) to glucose-treated samples revealed 1,969 DEGs; 259 overlapped between L4 GLU vs. L3 GLU, (glucose-treated) with 11 showing reversed expression. Additionally, 84 DELs were identified in L3 GLU vs. control, 40 in L4 GLU vs. control, and 163 between glucose-treated L4 and L3. Larval and glucose-specific alternative splicing events were also analyzed. Proteomic analysis revealed 35 DRPs—5 more abundant in L4, 30 in L3. The data reveal that developmental stage exerts a more substantial influence than glucose exposure on gene and protein expression profiles. However, glucose still modulates several pathways related to translation, cytoskeletal remodeling, extracellular matrix (ECM) reorganization, and energy metabolism.

A multi-marker nuclear genotyping approach was performed on larval and adult specimens of Anisakis spp. (N = 689) collected from fish and cetaceans in allopatric and sympatric areas of the two species Anisakis pegreffii and Anisakis simplex (s. s.), in order to: (1) identify specimens belonging to the parental taxa by using nuclear markers (allozymes loci) and sequence analysis of a new diagnostic nuclear DNA locus (i.e. partial sequence of the EF1 α−1 nDNA region) and (2) recognize hybrid categories. According to the Bayesian clustering algorithms, based on those markers, most of the individuals (N = 678) were identified as the parental species [i.e. A. pegreffii or A. simplex (s. s.)], whereas a smaller portion (N = 11) were recognized as F1 hybrids. Discordant results were obtained when using the polymerase chain reaction–restriction fragment length polymorphisms (PCR–RFLPs) of the internal transcribed spacer (ITS) ribosomal DNA (rDNA) on the same specimens, which indicated the occurrence of a large number of ‘hybrids’ both in sympatry and allopatry. These findings raise the question of possible misidentification of specimens belonging to the two parental Anisakis and their hybrid categories derived from the application of that single marker (i.e. PCR–RFLPs analysis of the ITS of rDNA). Finally, Bayesian clustering, using allozymes and EF1 α−1 nDNA markers, has demonstrated that hybridization between A. pegreffii and A. simplex (s. s.) is a contemporary phenomenon in sympatric areas, while no introgressive hybridization takes place between the two species.

Anisakid nematodes are widespread marine parasites with complex life cycles involving invertebrates and fish as intermediate or transport hosts, and marine mammals as definitive hosts. Despite their ecological importance, and the zoonotic potential associated with the larval stages found in fish, recent data on anisakid species diversity in pinnipeds from Norwegian waters remain scarce. In this study, we investigated anisakid infections in two juvenile harbour seals ( Phoca vitulina ) stranded along the southern coast of Norway. Gastrointestinal nematodes were collected, morphologically classified to the genus level, and subsequently identified to species level through molecular analyses of mitochondrial (mtDNA cox2 ) and nuclear (rDNA ITS) markers. Five anisakid species were identified: Contracaecum osculatum sp. A (reported here for the first time in harbour seals), C. osculatum (sensu stricto), Phocanema decipiens (s.s.), P. krabbei , and Anisakis simplex (s.s.). The latter species was found in unexpectedly high abundance and in fully developed adult stages in one of the seals. Notably, these adult A. simplex (s.s.) exhibited large body size, in contrast with previous studies reporting either absence or minimal presence of adults in harbour seals. The underlying mechanisms promoting growth and reproductive development of A. simplex (s.s.) in this host species remain unclear, but may involve a combination of host-specific physiological traits, environmental factors, and parasite phenotypic plasticity. Gross pathological examination revealed multiple gastric and intestinal ulcers in the same seal, including seven crateriform lesions consistent with ulcerative gastritis and enteritis, associated with nematode attachment and feeding. These findings expand the current knowledge on anisakid diversity in P. vitulina and provide novel evidence of its role as a definitive host for A. simplex (s.s.) in Norwegian coastal waters. Furthermore, the results suggest that competitive interactions among anisakid species, combined with ecological and physiological host factors, may facilitate the development and maturation of A. simplex (s.s.) in harbour seals. Further studies are warranted to assess the frequency and health implications of such infections in wild pinniped populations.

Ascaridoid nematodes are widespread in marine fishes. Despite their major socioeconomic importance, mechanisms associated to the fish-borne zoonotic disease anisakiasis are still obscure. RNA-Seq and de-novo assembly were herein applied to RNA extracted from larvae and dissected pharynx of Hysterothylacium aduncum (HA), a non-pathogenic nematode. Assembled transcripts in HA were annotated and compared to the transcriptomes of the zoonotic species Anisakis simplex sensu stricto (AS) and Anisakis pegreffii (AP). Approximately 60,000,000 single-end reads were generated for HA, AS and AP. Transcripts in HA encoded for 30,254 putative peptides while AS and AP encoded for 20,574 and 20,840 putative peptides, respectively. Differential gene expression analyses yielded 471, 612 and 526 transcripts up regulated in the pharynx of HA, AS and AP. The transcriptomes of larvae and pharynx of HA were enriched in transcripts encoding collagen, peptidases, ribosomal proteins and in heat-shock motifs. Transcripts encoding proteolytic enzymes, anesthetics, inhibitors of primary hemostasis and virulence factors, anticoagulants and immunomodulatory peptides were up-regulated in AS and AP pharynx. This study represents the first transcriptomic characterization of a marine parasitic nematode commonly recovered in fish and probably of negligible concern for public health.

The species of Anisakis constitute one of the most widespread groups of ascaridoid nematodes in the marine ecosystem. Three closely related taxa are recognised in the A. simplex (s. l.) complex, i.e. A. pegreffii, A. simplex (s. s.) and A. berlandi. They are distributed in populations of their intermediate/paratenic (fish and squids) and definitive (cetaceans) hosts. A panel of seven microsatellite loci (Anisl 05784, Anisl 08059, Anisl 00875, Anisl 07132, Anisl 00314, Anisl 10535 and Anisl 00185), were developed and validated on a total of N = 943 specimens of A. pegreffii and A. simplex (s. s.), collected in fish and cetacean hosts from allopatric areas within the range of distribution of these parasite species. In addition, the locus Anisl 7, previously detected in those Anisakis spp., was investigated. The parasites were first identified by sequence analysis of the EF1 α-1 nDNA. The panel of the microsatellites loci here developed have allowed to: (i) detect diagnostic microsatellite loci between the two species; (ii) identify specimens of the two species A. pegreffii, A. simplex (s. s.) in a multi-marker nuclear genotyping approach; (iii) discover two sex-linked loci in both Anisakis species and (iv) estimate levels of genetic differentiation at both the inter- and intra-specific level.

Anisakis pegreffii and A. simplex (s.s.) are the two zoonotic anisakids infecting cetaceans as well as pelagic/demersal fish and squids. In European waters, A. pegreffii prevails in the Mediterranean Sea, while A. simplex (s.s.) in the NE Atlantic Ocean. Abiotic conditions likely play a significant role in shaping their geographical distribution. The Iberian Atlantic and Alboran Sea waters are sympatric areas of the two species. A total of 429 adults and L3 stage from both sympatric and allopatric areas were studied by a wide nuclear genotyping approach (including newly and previously found diagnostic single nucleotide polymorphisms (SNPs) at nuclear DNA (nDNA) and microsatellite DNA loci) and sequenced at mitochondrial DNA (mtDNA) cox 2. Admixture between the two species was detected in the sympatric areas studied by STRUCTURE Bayesian analysis; NEWHYBRIDS revealed different categories of hybridization between the two species, representing approximately 5%. A tendency for F1 female hybrids to interbreed with the parental species at the geographical distribution limits of both species was observed. This finding suggests that hybridization occurs when the two parental species significantly differ in abundance. Mitochondrial introgression of A. simplex (s.s.) in A. pegreffii from Mediterranean waters was also detected, likely as a result of past and/or paleo-introgression events. The high level of genetic differentiation between the two species and their backcrosses indicates that, despite current hybridization, reproductive isolation which maintains evolutionary boundaries between the two species, exists. Possible causes of hybridization phenomena are attempted, as well as their evolutionary and ecological implications, also considering a sea warming scenario in European waters.

Numerous specimens of the 3 sibling species of the Anisakis simplex species complex (A. pegreffii, A. simplex (senso stricto)), and A. simplex sp. C) recovered from cetacean species stranded within the known geographical ranges of these nematodes were studied morphologically and genetically. The genetic characterization was performed on diagnostic allozymes and sequences analysis of nuclear (internal transcribed spacer [ITS] of ribosomal [r]DNA) and mitochondrial (mitochondrial [mt]DNA cox2 and rrnS) genes. These markers showed (1) the occurrence of sympatry of the 2 sibling species A. pegreffii and A. simplex sp. C in the same individual host, the pilot whale, Globicephala melas Traill, from New Zealand waters; (2) the identification of specimens of A. pegreffii in the striped dolphin, Stenella coeruleoalba (Meyen), from the Mediterranean Sea; and (3) the presence of A. simplex (s.s.) in the pilot whale and the minke whale, Balaenoptera acutorostrata Lacépède, from the northeastern Atlantic waters. No F1 hybrids were detected among the 3 species using the nuclear markers. The phylogenetic inference, obtained by maximum parsimony (MP) analysis of separate nuclear (ITS rDNA region), combined mitochondrial (mtDNA cox2 and rrnS) sequences datasets, and by concatenated analysis obtained at both MP and Bayesian inference (BI) of the sequences datasets at the 3 studied genes, resulted in a similar topology. They were congruent in depicting the existence of the 3 species as distinct phylogenetic lineages, and the tree topologies support the finding that A. simplex (s.s.), A. pegreffii, and A. berlandi n. sp. (=A. simplex sp. C) represent a monophyletic group. The morphological and morphometric analyses revealed the presence of morphological features that differed among the 3 biological species. Morphological analysis using principal component analysis, and Procrustes analysis, combining morphological and genetic datasets, showed the specimens clustering into 3 well-defined groups. Nomenclatural designation and formal description are given for A. simplex species C: the name Anisakis berlandi n. sp. is proposed. Key morphological diagnostic traits are as follows between A. berlandi n. sp. and A. simplex (s.s.): ventriculus length, tail shape, tail length/total body length ratio, and left spicule length/total body length ratio; between A. berlandi n. sp. and A. pegreffii: ventriculus length and plectane 1 width/plectane 3 width ratio; and between A. simplex (s.s.) and A. pegreffii: ventriculus length, left and right spicule length/total body length ratios, and tail length/total body length ratio. Ecological data pertaining to the geographical ranges and host distribution of the 3 species are updated.

Anisakid nematodes are a globally distributed group of marine mammal parasites. Kogiid whales, including the pygmy sperm whale Kogia breviceps, host an assemblage of specific anisakid species. Currently, three species are known to be specific to kogiid hosts, i.e., Skrjabinisakis paggiae, S. brevispiculata, and the less studied Pseudoterranova ceticola. The aim of this study was to investigate the species diversity of anisakid nematodes sampled from a pygmy sperm whale stranded in 2013 at the edge of its distribution range in the Northeast Atlantic, specifically in the North of Scotland. Nematodes were assigned to genus level based on morphology and identified by sequence analysis of the mtDNA cox2 gene and the rDNA ITS region. The present finding represents the first observation of syntopic occurrence of adult stages of S. brevispiculata, S. paggiae, and P. ceticola in a pygmy sperm whale in the Northeast Atlantic, and represent the northernmost record of these species in this area. Skrjabinisakis brevispiculata was the most abundant species, accounting for 55% of the identified nematodes, predominantly in the adult stage. Anisakis simplex (s.s.) was also abundant, with most specimens in the preadult stage, followed by S. paggiae and P. ceticola. The pygmy sperm whale is rarely documented in Scottish waters, and its occurrence in the area could suggest expansion of its geographic range. The presence of S. brevispiculata, S. paggiae, and P. ceticola in this whale species in this region may indicate a shift in the whole host community involved in the life cycle of these parasites in northern waters. However, it is also plausible that these parasites were acquired while the whale was feeding in more southern regions, before migrating northbound.