Skip to main content Accessibility help

No more time to stay ‘single’ in the detection of Anisakis pegreffii, A. simplex (s. s.) and hybridization events between them: a multi-marker nuclear genotyping approach

  • S. MATTIUCCI (a1), V. ACERRA (a1) (a2), M. PAOLETTI (a1) (a2), P. CIPRIANI (a1) (a2), A. LEVSEN (a3), S. C. WEBB (a4), D. CANESTRELLI (a2) and G. NASCETTI (a2)...


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.

  • View HTML
    • Send article to Kindle

      To send this article to your Kindle, first ensure is added to your Approved Personal Document E-mail List under your Personal Document Settings on the Manage Your Content and Devices page of your Amazon account. Then enter the ‘name’ part of your Kindle email address below. Find out more about sending to your Kindle. Find out more about sending to your Kindle.

      Note you can select to send to either the or variations. ‘’ emails are free but can only be sent to your device when it is connected to wi-fi. ‘’ emails can be delivered even when you are not connected to wi-fi, but note that service fees apply.

      Find out more about the Kindle Personal Document Service.

      No more time to stay ‘single’ in the detection of Anisakis pegreffii, A. simplex (s. s.) and hybridization events between them: a multi-marker nuclear genotyping approach
      Available formats

      Send article to Dropbox

      To send this article to your Dropbox account, please select one or more formats and confirm that you agree to abide by our usage policies. If this is the first time you use this feature, you will be asked to authorise Cambridge Core to connect with your <service> account. Find out more about sending content to Dropbox.

      No more time to stay ‘single’ in the detection of Anisakis pegreffii, A. simplex (s. s.) and hybridization events between them: a multi-marker nuclear genotyping approach
      Available formats

      Send article to Google Drive

      To send this article to your Google Drive account, please select one or more formats and confirm that you agree to abide by our usage policies. If this is the first time you use this feature, you will be asked to authorise Cambridge Core to connect with your <service> account. Find out more about sending content to Google Drive.

      No more time to stay ‘single’ in the detection of Anisakis pegreffii, A. simplex (s. s.) and hybridization events between them: a multi-marker nuclear genotyping approach
      Available formats


This is an Open Access article, distributed under the terms of the Creative Commons Attribution licence (, which permits unrestricted re-use, distribution, and reproduction in any medium, provided the original work is properly cited.

Corresponding author

*Corresponding author: Department of Public Health and Infectious Diseases, Section of Parasitology, Sapienza-University of Rome, P.le Aldo Moro, 5 00185 Rome, Italy. E-mail:


Hide All
Abollo, E., Gestal, C. and Pascual, S. (2001). Anisakis infestation in marine fish and cephalopods from Galician waters: an updated perspective. Parasitology Research 87, 492499.
Abollo, E., Paggi, L., Pascual, S. and D'Amelio, S. (2003). Occurrence of recombinant genotypes of Anisakis simplex s.s. and Anisakis pegreffii (Nematoda: Anisakidae) in an area of sympatry. Infection, Genetics and Evolution 3, 175181.
Agatsuma, T., Arakawa, Y., Iwagami, M., Honzako, Y., Cahyaningsih, U., Kang, S. and Hong, S. (2000). Molecular evidence of natural hybridization between Fasciola hepatica and F. gigantica. Parasitology International 49, 231238.
Anderson, E. C. and Thompson, E. A. (2002). A model-based method for identifying species hybrids using multilocus genetic data. Genetics 160, 12171229.
Anderson, T. J. C. (2001). The dangers of using single locus markers in parasite epidemiology: Ascaris as a case study. Trends in Parasitology 17, 183188.
Avise, J. C. and Saunders, N. C. (1984). Hybridization and introgression among species of sunfish (Lepomis): analysis by mitochondrial DNA and allozyme markers. Genetics 108, 237255.
Avise, J. C. and Wollenberg, K. (1997). Phylogenetics and the origin of species. Proceedings of the National Academy of Science 94, 77487755.
Biocca, E., Nascetti, G., Iori, A., Costantini, R. and Bullini, L. (1978). Descrizione di Parascaris univalens, parassita degli equini, e suo differenziamento da Parascaris equorum. Accademia Nazionale Lincei Rendiconti Classe Scienze Fisiche Matematiche Naturali Serie 65, 133141.
Bullini, L., Nascetti, G., Ciafrè, S., Rumore, F. and Biocca, E. (1978). Ricerche cariologiche ed elettroforetiche su Parascaris univalens e Parascaris equorum. Accademia Nazionale Lincei Rendiconti Classe Scienze Fisiche Matematiche Naturali Serie 65, 151156.
Cavallero, S., Ligas, A., Bruschi, F. and D'Amelio, S. (2012). Molecular identification of Anisakis spp. from fishes collected in the Tyrrhenian Sea (NW Mediterranean). Veterinary Parasitology 187, 563566.
Cavallero, S., Costa, A., Caracappa, S., Gambetta, B. and D'Amelio, S. (2014). Putative hybrids between two Anisakis cryptic species: molecular genotyping using high resolution melting. Experimental Parasitology 146, 8793.
Chaligiannis, I., Lalle, M., Pozio, E. and Sotiraki, S. (2012). Anisakidae infection in fish of the Aegean Sea. Veterinary Parasitology 184, 362366.
Chilton, N. B., Beveridge, I., Hoste, H. and Gasser, R. B. (1997). Evidence for hybridisation between Paramacropostrongylus iugalis and P. typicus (Nematoda: Strongyloidea) in grey kangaroos, Macropus fuliginosus and M. giganteus, in a zone of sympatry in eastern Australia. International Journal for Parasitology 27, 475482.
Chou, Y. Y., Wang, C. S., Chen, H. G., Chen, H. Y., Chen, S. N. and Shih, H. H. (2011). Parasitism between Anisakis simplex (Nematoda: Anisakidae) third-stage larvae and the spotted mackerel Scomber australasicus with regard to the application of stock identification. Veterinary Parasitology 177, 324331.
Cipriani, P., Smaldone, G., Acerra, V., D'Angelo, L., Anastasio, A., Bellisario, B., Palma, G., Nascetti, G. and Mattiucci, S. (2015). Genetic identification and distribution of the parasitic larvae of Anisakis pegreffii and Anisakis simplex (s. s.) in European hake Merluccius merluccius from the Tyrrhenian Sea and Spanish Atlantic coast: implications for food safety. International Journal of Food Microbiology 198, 18.
Criscione, C. D., Poulin, R. and Blouin, M. S. (2005). Molecular ecology of parasites: elucidating ecological and microevolutionary processes. Molecular Ecology 14, 22472257.
Criscione, C. D., Anderson, J. D., Sudimack, D., Peng, W., Jha, B., Williams-Blangero, S. and Anderson, T. J. C. (2007). Disentangling hybridization and host colonization in parasitic roundworms of humans and pigs. Proceedings of the Royal Society B 274, 26692677.
D'Amelio, S., Mathiopoulos, K. D., Brandonisio, O., Lucarelli, G., Doronzo, F. and Paggi, L. (1999). Diagnosis of a case of gastric anisakidosis by PCR-based restriction fragment length polymorphism analysis. Parassitologia 41, 591593.
D'Amelio, S., Mathiopoulos, K. D., Santos, C. P., Pugachev, O. N., Webb, S. C., Picanço, M. and Paggi, L. (2000). Genetic markers in ribosomal DNA for the identification of members of the genus Anisakis (Nematoda: Ascaridoidea) defined by polymerase chain reaction-based restriction fragment length polymorphism. International Journal for Parasitology 30, 223226.
Detwiler, J. T. and Criscione, C. D. (2010). An infectious topic in reticulate evolution: introgression and hybridization in animal parasites. Genes 1, 102123.
Dunams-Morel, D. B., Reichard, M. V., Torretti, L., Zarlenga, D. S. and Rosenthal, B. M. (2012). Discernible but limited introgression has occurred where Trichinella nativa and the T6 genotype occur in sympatry. Infection Genetics and Evolution 12, 530538.
Dybdahl, M. F., Jokela, J., Delph, L. F., Koskella, B. and Lively, C. M. (2008). Hybrid fitness in a locally adapted parasite. The American Naturalist 172, 772782.
Elder, J. F. and Turner, B. J. (1995). Concerted evolution of repetitive DNA sequences in eukaryotes. The Quarterly Review of Biology 70, 297320.
Evanno, G., Regnaut, S. and Goudet, J. (2005). Detecting the number of clusters of individuals using the software STRUCTURE: a simulation study. Molecular Ecology 14, 26112620.
Farjallah, S., Slimane, B. B., Busi, M., Paggi, L., Amor, N., Blel, H., Said, K. and D’Amelio, S. (2008). Occurrence and molecular identification of Anisakis spp. from the North African coasts of Mediterranean Sea. Parasitology Research 102, 371379.
Fritz, R. S., Moulia, C. and Newcombe, G. (1999). Resistance of hybrid plants and animals to herbivores, pathogens, and parasites. Annual Review of Ecology, Evolution, and Systematics 30, 565591.
Ganley, A. R. D. and Kobayashi, T. (2007). Highly efficient concerted evolution in the ribosomal DNA repeats: total rDNA repeat variation revealed by whole-genome shotgun sequence data. Genome Research 17, 184191.
Gilabert, A. and Wasmuth, J. D. (2013). Unravelling parasitic nematode natural history using population genetics. Trends in Parasitology 29, 438448.
Goday, C. and Pimpinelli, S. (1986). Cytological analysis of chromosomes in the two species Parascaris univalens and P. equorum. Chromosoma 94, 110.
Larkin, M. A., Blackshields, G., Brown, N. P., Chenna, R., McGettigan, P. A., McWilliam, H., Valentin, F., Wallace, I. M., Wilm, A., Lopez, R., Thompson, J. D., Gibson, T. J. and Higgins, D. G. (2007). Clustal W and Clustal X version 2.0. Bioinformatics 23, 29472948.
Lee, M. H., Cheon, D. and Choi, C. (2009). Molecular genotyping of Anisakis species from Korean sea fish by polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP). Food Control 20, 623626.
Levsen, A. and Karl, H. (2014). Anisakis simplex (s. l.) in grey gurnard (Eutrigla gurnardus) from the North Sea: food safety considerations in relation to fishing ground and distribution in the flesh. Food Control 36, 1519.
Levsen, A. and Lunestad, B. T. (2010). Anisakis simplex third stage larvae in Norwegian spring spawning herring (Clupea harengus L.), with emphasis on larval distribution in the flesh. Veterinary Parasitology 17, 247253.
Martìn-Sànchez, J., Artacho-Reinoso, M. E., Dìaz-Gavilàn, M. and Valero-Lòpez, A. (2005). Structure of Anisakis simplex s. l. populations in a region sympatric for A. pegreffii and A. simplex s.s. Absence of reproductive isolation between both species. Molecular and Biochemical Parasitology 141, 155162.
Mattiucci, S. and Nascetti, G. (2008). Advances and trends in the molecular systematics of anisakid nematodes, with implications for their evolutionary ecology and host-parasite co-evolutionary processes. Advances in Parasitology 66, 47148.
Mattiucci, S., Nascetti, G., Cianchi, R., Paggi, L., Arduino, P., Margolis, L., Brattey, J., Webb, S. C., D'Amelio, S., Orecchia, P. and Bullini, L. (1997). Genetic and ecological data on the Anisakis simplex complex with evidence for a new species (Nematoda, Ascaridoidea, Anisakidae). Journal of Parasitology 83, 401416.
Mattiucci, S., Abaunza, P., Ramadori, L. and Nascetti, G. (2004). Genetic identification of Anisakis larvae in European hake from Atlantic and Mediterranean waters for stock recognition. Journal of Fish Biology 65, 495510.
Mattiucci, S., Cimmaruta, R., Cipriani, P., Abaunza, P., Bellisario, B. and Nascetti, G. (2015). Integrating Anisakis spp. parasites data and host genetic structure in the frame of a holistic approach for stock identification of selected Mediterranean Sea fish species. Parasitology 142, 90108.
Mattiucci, S., Farina, V., Campbell, N., Mackenzie, K., Ramos, P., Pinto, A. L., Abaunza, P. and Nascetti, G. (2008). Anisakis spp. larvae (Nematoda: Anisakidae) from Atlantic horse mackerel: their genetic identification and use as biological tags for host stock identification. Fisheries Research 89, 146171.
Mattiucci, S., Paoletti, M., Borrini, F., Palumbo, M., Palmieri, R. M., Gomes, V., Casati, A. and Nascetti, G. (2011). First molecular identification of the zoonotic parasite Anisakis pegreffii (Nematoda: Anisakidae) in a paraffin-embedded granuloma taken from a case of human intestinal anisakiasis in Italy. BMC Infectious Diseases 11, 82.
Mattiucci, S., Fazii, P., De Rosa, A., Paoletti, M., Salomone Megna, A., Glielmo, A., De Angelis, M., Costa, A., Meucci, C., Calvaruso, V., Sorrentini, I., Palma, G., Bruschi, F. and Nascetti, G. (2013). Anisakiasis and Gastroallergic reactions associated with Anisakis pegreffii infection, Italy. Emerging Infectious Diseases 19, 496499.
Mattiucci, S., Cipriani, P., Webb, S. C., Paoletti, M., Marcer, F., Bellisario, B., Gibson, D. I. and Nascetti, G. (2014). Genetic and morphological approaches distinguish the three sibling species of the Anisakis simplex species complex, with a species designation as Anisakis berlandi n. sp. for A. simplex sp. C (Nematoda: Anisakidae). Journal of Parasitology 100, 199214.
Mayr, E. and Ashlock, P. D. (1969). Principles of Systematic Zoology. Mc-Graw-Hill, New York, 145166.
Meloni, M., Angelucci, G., Merella, P., Siddi, R., Deiana, C., Orrù, G. and Salati, F. (2011). Molecular characterization of Anisakis larvae from fish caught off Sardinia. Journal of Parasitology 97, 908914.
Molina-Fernández, D., Malagón, D., Gómez-Mateos, M., Benítez, R., Martín-Sánchez, J., and Adroher, F. J. (2015). Fishing area and fish size as risk factors of Anisakis infection in sardines (Sardina pilchardus) from Iberian waters, southwestern Europe. International Journal of Food Microbiology 203, 2734.
Nadler, S. A. and Pérez-Ponce De León, G. (2011). Integrating molecular and morphological approaches for characterizing parasite cryptic species: implications for parasitology. Parasitology 138, 16881709.
Nascetti, G., Paggi, L., Orecchia, P., Smith, J. W., Mattiucci, S. and Bullini, L. (1986). Electrophoretic studies on the Anisakis simplex complex (Ascaridida: Anisakidae) from the Mediterranean and North-East Atlantic. International Journal for Parasitology 16, 633640.
Okamoto, M., Nakao, M., Blair, D., Anantaphruti, M. T., Waikagul, J. and Ito, A. (2010). Evidence of hybridization between Taenia saginata and Taenia asiatica. Parasitology International 59, 7074.
Paggi, L., Nascetti, G., Cianchi, R., Orecchia, P., Mattiucci, S., D'Amelio, S., Berland, B., Brattey, J., Smith, J. W. and Bullini, L. (1991). Genetic evidence for three species within Pseudoterranova decipiens (Nematoda, Ascaridida, Ascaridoidea) in the North Atlantic and Norwegian and Barents Seas. International Journal for Parasitology 21, 195212.
Pekmezci, G. Z., Onuk, E. E., Bolukbas, C. S., Yardimci, B., Gurler, A. T., Acici, M. and Umur, S. (2014). Molecular identification of Anisakis species (Nematoda: Anisakidae) from marine fishes collected in Turkish waters. Veterinary Parasitology 201, 8294.
Pérez-Ponce de León, G., and Nadler, S. A. (2010). What we don't recognize can hurt us: a plea for awareness about cryptic species. Journal of Parasitology 96, 453464.
Pontes, T., D'Amelio, S., Costa, G. and Paggi, L. (2005). Molecular characterization of larval anisakid nematodes from marine fishes of Madeira by a PCR-based approach, with evidence for a new species. Journal of Parasitology 91, 14301434.
Pritchard, J. K., Stephens, M. and Donnelly, P. (2000). Inference of population structure using multilocus genotype data. Genetics 155, 945959.
Quiazon, K. M., Yoshinaga, T. and Ogawa, K. (2011). Distribution of Anisakis species larvae from fishes of the Japanese waters. Parasitology International 60, 223226.
Rice, W. R. (1989). Analyzing tables of statistical tests. Evolution 43, 223225.
Rousset, F. (2008). GENEPOP'007: a complete re-implementation of the GENEPOP software for Windows and Linux. Molecular Ecology Resources 8, 103106.
Steinauer, M. L., Hanelt, B., Mwangi, I. N., Maina, G. M., Agola, L. E., Kinuthia, J. M., Mutuku, M. W., Mungai, B. N., Wilson, W. D., Mkoji, G. M. and Loker, E. S. (2008). Introgressive hybridization of human and rodent schistosome parasites in western Kenya. Molecular Ecology 17, 50625074.
Steinauer, M. L., Blouin, M. S. and Criscione, C. D. (2010). Applying evolutionary genetics to schistosome epidemiology. Infection, Genetics and Evolution 10, 433443.
Suzuki, J., Murata, R., Hosaka, M. and Araki, J. (2010). Risk factors for human Anisakis infection and association between the geographic origins of Scomber japonicus and anisakid nematodes. International Journal of Food Microbiology 137, 8893.
Théron, A. (1989). Hybrids between Schistosoma mansoni and S. rodhaini: characterization by cercarial emergence rhythms. Parasitology 99, 225228.
Tintore, J., La Violette, P. E., Blade, I. and Cruzado, G. (1988). A study of intense density front in the eastern Alboran Sea: the Almeria-Oran front. Journal of Physical Oceanography 18, 13841397.
Umehara, A., Kawakami, Y., Matsui, T., Araki, J. and Uchida, A. (2006). Molecular identification of Anisakis simplex sensu stricto and Anisakis pegreffii (Nematoda: Anisakidae) from fish and cetaceans in Japanese waters. Parasitology International 55, 267271.
Umehara, A., Kawakami, Y., Araki, J. and Uchida, A. (2007). Molecular identification of the etiological agent of the human anisakiasis in Japan. Parasitology International 56, 211215.
Valentini, A., Mattiucci, S., Bondanelli, P., Webb, S. C., Mignucci-Giannone, A., Colon-Llavina, M. M. and Nascetti, G. (2006). Genetic relationships among Anisakis species (Nematoda: Anisakidae) inferred from mitochondrial cox2 sequences and comparison with allozyme data. Journal of Parasitology 92, 156166.
Volf, P., Benkova, I., Myskova, J., Sadlova, J., Campino, L. and Ravel, C. (2007). Increased transmission potential of Leishmania major/Leishmania infantum hybrids. International Journal for Parasitology 37, 589593.
Zhu, X. Q., D'Amelio, S., Paggi, L. and Gasser, R. B. (2000). Assessing sequence variation in the internal transcribed spacer of ribosomal DNA within and among members of the Contracaecum osculatum complex (Nematoda: Ascaridoidea: Anisakidae). Parasitology Research 86, 677683.


Related content

Powered by UNSILO
Type Description Title
Supplementary materials

Mattiucci supplementary material
Figure S1

 Unknown (428 KB)
428 KB

No more time to stay ‘single’ in the detection of Anisakis pegreffii, A. simplex (s. s.) and hybridization events between them: a multi-marker nuclear genotyping approach

  • S. MATTIUCCI (a1), V. ACERRA (a1) (a2), M. PAOLETTI (a1) (a2), P. CIPRIANI (a1) (a2), A. LEVSEN (a3), S. C. WEBB (a4), D. CANESTRELLI (a2) and G. NASCETTI (a2)...


Altmetric attention score

Full text views

Total number of HTML views: 0
Total number of PDF views: 0 *
Loading metrics...

Abstract views

Total abstract views: 0 *
Loading metrics...

* Views captured on Cambridge Core between <date>. This data will be updated every 24 hours.

Usage data cannot currently be displayed.