INTRODUCTIONTop

Portugal has a long tradition of fishing and is the leading country in fish consumption in the European Union (EUMOFA 2016EUMOFA. 2016. The EU fish market. Accessed on 18/10/2016 at https://www.eumofa.eu/documents/20178/77960/The+EU+fish+market+-+2016+Edition.pdf), with 55.3 kg of sea food per capita per year in 2014. For this reason, zoonotic infections such as anisakiasis are a potential risk and a case of major public health concern in this country. Fish dishes, such as grilled (slightly cooked) fish, are one of the most popular food specialties in Portugal, not only among the local people but also among the many tourists who visit the country each year. Atlantic mackerels Scomber colias Gmelin, 1789 and Scomber scombrus L., 1758 are pelagic-neritic scombrids of great commercial interest that are consumed lightly grilled. According to the Portuguese Institution for Statistical Data, 29543 t of S. colias and 588 t of S. scombrus were landed in 2014 (DGRM 2015Direcção Geral de Recursos Naturais, Segurança e Serviços Marítimos (DGRM). 2015. Estatística da Pesca 2014. Instituto Nacional de Estatística, I.N.E.I.P., Lisboa.), with S. colias being one of the top four fish sold in the country.

Anisakid nematodes are the most abundant parasites of marine fishes worldwide (Mattiucci and Nascetti 2008Mattiucci M., 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. Adv. Parasitol. 66: 47-148.). They are parasites of zoonotic potential, having the ability to infect humans through the consumption of raw or lightly cooked fish, causing the emerging infection anisakiasis, and thus being a food safety concern (MacCarthy and Moore 2000MacCarthy J., Moore T.A. 2000. Emerging helminth zoonoses. Int. J. Parasitol. 30: 1351-1360.). Associated symptoms of anisakiasis include severe gastric or intestinal disease or, in the mild version, allergies (Audicana et al. 2002Audicana M.T., Ansotegui I.J., Fernández de Corres L. et al. 2002. Anisakis simplex: dangerous – dead and alive? Trends Parasitol. 18: 20-25., 2003Audicana M.T., del Pozo M.D., Iglesias R., et al. 2003. Anisakis simplex and Pseudoterranova decipiens. In: Miliotis M.D., Bier J.V. International Handbook of Foodborne Pathogens. CRC Press, pp. 613-636.). In Portugal, no reports of severe disease due to Anisakis infections have been recorded so far. However, in a survey conducted among the population in a fishery town located in the south of the country, it was found that 8% of the people were allergic to anisakids (Nunes et al. 2003Nunes C., Ladeira S., Mergulhão A. 2003. Allergy to Anisakis simplex in the Portuguese Population. Rev. Port. Imunoalergologia XI: 30-40.). In Spain, France, Italy, Germany, the Netherlands and Japan, severe episodes of anisakiasis have already been recorded (Arizono et al. 2012Arizono N., Yamada M., Tegoshi T., et al. 2012. Anisakis simplex sensu stricto and Anisakis pegreffii: biological characteristics and pathogenetic potential in Human Anisakiasis. Foodborne Pathog. Dis. 9: 517-521., Mattiucci et al. 2013Mattiucci S., Fazii P., De Rosa A., et al. 2013. Anisakiasis and gastroallergic reaction associated with Anisakis pegreffii infection, Italy. Emerg. Infect. Dis. 19: 496–499., Ubeira et al. 2000Ubeira F.M., Valinas B., Lorenzo S., et al. 2000. Anisaquiosis y alergia. Un estudio seroepidemiológico en la Comunidad Autónoma Gallega. Documentos Técnicos de Salud Pública, Serie B, 24. Ed. Conselleria de Sanidade e Servicios sociais (Xunta de Galicia, España).).

Both S. colias and S. scombrus are common hosts of anisakids in different geographic areas, particularly in Europe (Abollo et al. 2001Abollo E., Gestal C., Pascual S. 2001. Anisakis infestation in marine fish and cephalopods from Galician waters: an updated perspective. Parasitol. Res. 87: 492-499., Mattiuci and Nascetti 2008Mattiucci M., 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. Adv. Parasitol. 66: 47-148., Pontes et al. 2005Pontes T., D’Amelio S., Costa G., et al. 2005. Molecular characterization of larval Anisakid nematodes from marine fishes of Madeira by PCR-based approach, with evidence for a new species. J. Parasitol. 91: 1430-1434.). There are also several records from African countries (Abattouy et al. 2011Abattouy N., Valero A., Benajiba M.H., et al. 2011. Anisakis simplex s.l. parasitization in mackerel (Scomber japonicus) caught in the North of Morocco - Prevalence and analysis of risk factors. Int. J. Food Microbiol. 150: 136-139., Farjallah et al. 2008Farjallah S., Slimane B.B., Busi M., et al. 2008. Occurrence and molecular identification of Anisakis spp. from the North African coasts of Mediterranean Sea. Parasitol. Res. 102: 371-379., Kijewska et al. 2009Kijewska A., Dzido J., Shukhgalter O., et al. 2009. Anisakid parasites of fishes caught on the African shelf. J. Parasitol. 95: 639-645.). So far, no updated information is available for Atlantic mackerels fished off continental Portugal, and no attempt has been made to identify the anisakids isolated from S. scombrus to the species level (Rego et al. 1985Rego A.A., Carvalho-Varela M., Mendonça M.M., et al. 1985. Helmintofauna da sarda (Scomber scombrus L.) peixe da costa continental portuguesa. Mem. I. Oswaldo Cruz 80: 97-100.).

The main purpose of this study was to determine and compare the infection levels (prevalence and intensity) of anisakid nematodes in S. colias and S. scombrus, to correlate parasite and host data, and to evaluate whether the fish species can represent a danger when consumed raw or undercooked, taking into account (i) the infecting species and (ii) the recorded infection levels.

MATERIALS AND METHODSTop

Host sampling

Several samples of Atlantic chub mackerel, S. colias (n=40 in total), and Atlantic mackerel, S. scombrus (n=42 in total), were collected from October to December 2009 (n=19 and 21, respectively) and from January to June 2010 (n=21 for both species). The Atlantic mackerels were fished in the FAO area Atlantic Northeast 27, subarea Portuguese waters IX, by trawling fisheries operated by local boats. The fish were all purchased at the harbour fish market and then freshly dissected or frozen for subsequent parasitological analyses. Host identification was done according to Collette (1986)Collette B.B. 1986. Scombridae. In: Whitehead P.J.P., Bauchot M.-L., Hureau J.-C., et al. (eds) Fishes of the North-eastern Atlantic and the Mediterranean. vol 2. Unesco, United Kingdom, pp. 981-997., based on the external and internal features. Fish length was also evaluated [mean±standard deviation (range)]: 32.6±3.6 (25.4-39.2) cm for the Atlantic chub mackerel; and 31.9±2.3 (21.8-36.4) cm for the Atlantic mackerel (see Table 1). As some parasites might accumulate in older, i.e. larger fishes, and thus bias our sample, a set of hosts with similar lengths were used for the analysis. Also, fish weight (g), Fulton’s condition factor (K=weight/length³ g cm^–3), hepatosomatic index (HI=liver weight (g) / total weight (g) × 100), gonadosomatic index (GI=gonadal weight (g) / total weight (g) × 100) and sex ratio were recorded for each fish species (see Table 1).

Parasitological survey

During the parasitological survey for anisakids, the following infection sites were analysed under the stereo-microscope (magnification 30×): digestive tract, gonads, heart and muscle. The muscle portion examined was cut off from the ventral part of the fish, and also from the belly flaps, as this is considered one of the most infected muscular regions in the fish according to Mehrdana et al. (2014)Mehrdana F., Bahlool Q., Skov J., et al. 2014. Occurrence of zoonotic nematodes Pseudoterranova decipiens, Contracaecum osculatum and Anisakis simplex in cod (Gadus morhua) from the Baltic Sea. Vet. Parasitol. 205: 581-587.. The isolated worms were cleaned in saline solution (0.9% NaCl) and fixed and preserved in 70% ethanol. For morphological identification purposes, anisakids were cleared and mounted in glycerine, following Moravec (1998)Moravec F. 1998. Nematodes of freshwater fishes of the neotropical region. Academia Praha, Institute of Parasitology, Academy of Sciences of the Czech Republic, pp. 381-383.. Since around 700 worms were sampled from each fish species, a sub-sample of worms found around the viscera (and morphologically identified as Anisakis simplex s.l., type I) was identified to the species level using molecular tools. Moreover, around 19 anisakids were taken from about 19 specimens of each species of scombrids (i.e. one worm/fish at random). A minimum sample size of 15 worms was chosen as recommended for calculating reliable prevalence levels (Jovani and Tella 2006Jovani R., Tella J.L. 2006. Parasite prevalence and sample size: misconceptions and solutions. Trends Parasitol. 22: 214-218.). Specimens of Hysterothylacium, occasionally found in both fish hosts, were also characterized using the same molecular methods.

Molecular characterization of anisakids

This procedure follows closely the one described in Kuhn et al. (2011)Kuhn T., García-Màrquez J., Klimpel S. 2011. Adaptive Radiation within Marine Anisakid Nematodes: A Zoogeographical Modeling of Cosmopolitan, Zoonotic Parasites. PLoS ONE 6: e28642.. Eighteen and 20 anisakids (Anisakis sp. and Hysterothylacium sp.) from S. colias and S. scombrus, respectively, were analysed molecularly. DNA extraction was done for each larva, cut into pieces, using a genomic DNA extraction kit (peqGOLD MicroSpin Tissue, PEQLAB Biotechnology GmbH, Germany) and following the instructions of the manufacturer. The region of the rDNA included the ITS-1, 5.8S, ITS-2 and flanking sequences (=ITS+), and was amplified using the primers Forward TK1 (5’-GGC-AAA-AGT-CGT-AAC-AAG-GT-3’) and Reverse NC2 (5’-TTA-GTT-TCT-TTT-CCT-CCG-CT-3’). PCR reaction (50 µl) included 25 µl Master Mix (18.75 µl ddH₂O, 5 µl reaction buffer Y, 1 µl dNTP, 0.25 µl Taq-DNA-polymerase), 14 µl ddH₂O, 3 µl of each primer and 5 µl of genomic DNA. Each PCR reaction was performed in a PCR thermocycler (GeneTouch, Biozym scientific, Germany) under the following conditions: an initial denaturation at 95°C for 1 min, 40 cycles of 94°C for 45 sec (denaturation), 55°C for 45 sec (annealing), 72°C for 45 sec (extension), and a final extension at 72°C for 10 min. Samples with no DNA were included for negative control. PCR products were checked on 1% agarose gel that ran for 1 h 15 min at 80V provided by an electrophoresis power supply (EV 243, Consort, Belgium) inside an electrophoresis system (MSMIDI, MULTISUB, Biozym, Germany). The gel was then analysed with a BiodocAnalyse (Biometra, AnalytikJena, Germany) associated with a photographic camera (Canon EOS 1100D) and BioDoc Analyse software version 2.66.3.22 (Biometra, AnalytikJena, Germany). A GelRed 100 bp DNA ladder marker (peqGold Leadder Plus, PEQLAB Biotechologie GmbH, Germany) was used to estimate the size of the PCR products. The agarose gel products were afterwards purified with a Cycle-Pure Kit (peqGold, PEQLAB Biotechnology GmbH, Germany).

Twenty µl of each DNA sample and 2.5 µl of each primer per sample was sent to the laboratory (GATC Biotec AG, European Custom Sequencing Centre, Cologne, Germany) for sequencing. In total, 38 worms were sequenced. The species were identified by comparison with sequences previously deposited in GenBank, using the BLASTn algorithm and the BioEdit software version 7.1.3.0 (Hall 1999Hall T.A. 1999. BioEdit a user-friendly biology sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp. Ser. 41: 95-98.) to previously aligned sequences forward and reverse provided by the sequence laboratory.

Infection levels of anisakids

Prevalence and intensity [mean±SD (minimum-maximum)] were calculated for each identified taxon according to Bush et al. (1997)Bush A.O., Lafferty K.D., Lotz, J.M., et al. 1997. Parasitology meets ecology on its own terms: Margolis et al. revisited. J. Parasitol. 83: 575-583. and considering each of the two species of host.

Binary Pearson correlations among anisakid intensity and host total weight, total length, condition factor, hepatosomatic index and gonadosomatic index were conducted. Moreover, the anisakid intensities were analysed in smaller versus larger fish for host total weight, total length, condition factor, hepatosomatic index, gonadosomatic index and sex (male versus female), using the Mann-Whitney’s U test. In the later analysis, the two classes of fish were separated by the median value for each parameter. In the case of detecting significant differences, the anisakid intensities [mean±SD (minimum-maximum)] for the smaller and bigger hosts were determined. Non-parametric tests were chosen for the statistical analysis because the study of the normality using the Kolmogorov-Smirnov’s test for some variables, such as anisakid intensity, showed that they did not follow a normal distribution (needed in parametric tests), as for instance.

Occurrence of A. simplex s.s. and A. pegreffii was compared between the two species of host using the chi-squared test.

All statistical analyses were conducted in SPSS for Windows, version 23 (level of significance: P<0.05).

RESULTSTop

Anisakid infection levels

Both Atlantic mackerels recorded very high infection levels – a prevalence of anisakids of 85.0% and 83.3% for S. colias and S. scombrus, respectively. In total, 1312 anisakid worms were sampled from both fish, 737 from S. colias and 575 from S. scombrus. Mean intensity was higher for S. colias, with 21.7 worms/host, than for S. scombrus, with the 16.4 worms/host. These infection values were recorded for worms mainly found around the viscera, because the muscular tissue was found infected only once, with a single anisakid recorded in S. scombrus (prevalence of 2.4%) and none in S. colias (prevalence of 0%).

Binary correlations between anisakid intensity and host total weight, total length, condition factor, hepatosomatic index and gonadosomatic index were found to lack statistical significance.

The comparison of anisakid intensities among bigger and smaller hosts, separated by the median value, for host total weight, total length, condition factor, hepatosomatic index, gonadosomatic index and sex (males versus females) were in most cases non-significant, with probability values higher then 0.306, except for the gonadosomatic index, which was significant and recorded a probability value of 0.001. The intensities [mean±SD (range)] for the immature and mature hosts, according to the gonadosomatic index weight, were 6.0±8.6 (1-34) and 27.5±56.8 (2-245) worms per fish, respectively.

Anisakid molecular identification

Among the 36 Anisakis worms sequenced, several sequences were deposited in GenBank (accession numbers KF923929 and KF923930 for A. simplex (s.s.) and KF923927 for A. pegreffii from S. scombrus; and KF923928 for A. simplex (s.s.), and KF914636 and KF923926 for A. pegreffii from S. colias). The distribution for each Anisakis species for both fish hosts is given in Table 2. A. simplex (s.s.) was significantly more frequent in S. scombrus than in S. colias, whereas the opposite trend was reported for A. pegreffii (χ²=9.03, P<0.01).

Hysterothylacium aduncum specimens were also occasionally recorded, and two worms were sequenced, one worm per fish host species (GenBank accession numbers KF923932 from S. scombrus and KF923931 from S. colias, both showing 99% similarity to the H. aduncum sequences HM598666.1 and JQ934883.1 (Smrzlić et al. 2012Smrzlić V., Valić D., Kapetanović D., et al. 2012. Molecular characterisation of Anisakidae larvae from fish in Adriatic Sea. Parasitol. Res. 111: 2385-2391.)).

DISCUSSIONTop

In this study, infection levels for anisakids were evaluated considering two species of Atlantic mackerel, S. colias and S. scombrus, sampled at the continental Portuguese coast. The high infection levels (83%-85% prevalence) recorded for both fish are greater than the 10% reported for S. scombrus off continental Portugal by Rego et al. (1985)Rego A.A., Carvalho-Varela M., Mendonça M.M., et al. 1985. Helmintofauna da sarda (Scomber scombrus L.) peixe da costa continental portuguesa. Mem. I. Oswaldo Cruz 80: 97-100., and the 69.5% reported for S. colias off the Madeira Islands, Portugal, by Costa et al. (2003)Costa G., Pontes T., Mattiucci S., et al. 2003. The occurrence and infection dynamics of Anisakis larvae in the black-scabbard fish, Aphanopus carbo, chub mackerel, Scomber japonicus, and oceanic horse mackerel, Trachurus picturatus from Madeira, Portugal. J. Helminthol. 77: 163-166.. However, they are lower than the 100% reported for S. colias off the Azores Islands, Portugal, by Shukhgalter (2004)Shukhgalter O.A. 2004. The parasite fauna of the chub mackerel (Scombridae: Scomber japonicus Houttuyn, 1782) in the central-eastern Atlantic (Atlantic coast of Northern Africa and the Azores Archipelago banks). Parazitol. 38: 160-170.. High prevalence values were also recorded in other geographic regions: 87% for S. colias in El Rincon, Argentina (Cremonte and Sardella 1997Cremonte F., Sardella N.H. 1997. The parasito fauna of Scomber japonicus Houttuyn, 1782 (Pisces: Scombridae) in two zones of the Argentine Sea. Fish. Res. 31: 1-9.) and 92% for S. scombrus and 100% for S. colias in the Adriatic Sea off Croatia (Mladineo 2003Mladineo I. 2003. Anisakis simplex in the Adriatic Sea. Periodicum. Biologorum 105: 389-392., Mladineo and Poljak 2014Mladineo I., Poljak V. 2014. Ecology and Genetic Structure of Zoonotic Anisakis spp. from Adriatic. Appl. Environ. Microb. 80: 1281-1290.). In contrast, in Moroccan waters, prevalence of anisakids in S. colias varied from 57% to 67.9% (Abattouy et al. 2011Abattouy N., Valero A., Benajiba M.H., et al. 2011. Anisakis simplex s.l. parasitization in mackerel (Scomber japonicus) caught in the North of Morocco - Prevalence and analysis of risk factors. Int. J. Food Microbiol. 150: 136-139.). High prevalence values were also recorded for anisakids of other fish species from the Portuguese mainland, such as the blue whiting (Micromesistius poutassou, 77.7%), the black scabbardfish (Aphanopus carbo, 100%) (Cruz et al. 2007Cruz C., Barbosa C., Saraiva A. 2007. Distribution of larval anisakids in blue whiting off Portuguese fish market. Helminthol. 44: 21-24., 2009Cruz C., Saraiva A., Santos M.J., et al. 2009. Parasitic infection levels by Anisakis spp. larvae (Nematoda: Anisakidae) in black scabbardfish Aphanopus carbo (Osteichthyes: Trichiuridae) from Portuguese waters. Sci. Mar. 73S2: 115-120.) and the blackspot seabream (Pagellus bogaraveo, 100%) (Hermida et al. 2012Hermida M., Mota R., Pacheco C.C., et al. 2012. Infection levels and diversity of anisakid nematodes in blackspot seabream, Pagellus bogaraveo, from Portuguese waters. Parasitol. Res. 110: 1919-1928.). Moreover, Costa et al. (2004)Costa G., Madeira A., Pontes T. et al. 2004. Anisakid nematodes of the blackspot seabream, Pagellus bogaraveo, from Madeiran waters, Portugal. Acta Parasitologica. 49: 156-161. also reported a high prevalence (89.6%) for the blackspot seabream off Madeira Islands, Portugal.

In our samples, not only the overall prevalence but also the mean intensity of anisakids was high, with 21.7 worms per host for S. colias and 16.4 worms per host for S. scombrus. These values were similar to the ones recorded from the Azores Islands (Hyeres Bank), with 17.1 worms per host for S. colias (Shukhgalter 2004Shukhgalter O.A. 2004. The parasite fauna of the chub mackerel (Scombridae: Scomber japonicus Houttuyn, 1782) in the central-eastern Atlantic (Atlantic coast of Northern Africa and the Azores Archipelago banks). Parazitol. 38: 160-170.).

The correlation analysis of anisakid intensity and host total weight, total length, condition factor, hepatosomatic index and gonadosomatic index for S. colias and S. scombrus was found not to be significant. Moreover, the comparison analysis of anisakid intensity among smaller and bigger fish according to host total weight, total length, condition factor, hepatosomatic index, gonadosomatic index and sex (males versus females) showed mostly the same behaviour: the results were non-significant, except for the gonadosomatic index in S. scombrus. In the latter, more mature fish, with higher index values, recorded significantly higher intensities of anisakids than fish with lighter gonads, with mean values of 6.0 and 27.5, respectively. These findings can be interpreted as a conspicuous high preference for the first intermediate host by the more mature hosts. The lack of significant values for correlation between intensity of anisakids and host data was also recorded by Mladineo and Poljak (2014)Mladineo I., Poljak V. 2014. Ecology and Genetic Structure of Zoonotic Anisakis spp. from Adriatic. Appl. Environ. Microb. 80: 1281-1290. for S. colias and Sardina pilchardus in the Adriatic Sea, and by Costa et al. (2004)Costa G., Madeira A., Pontes T. et al. 2004. Anisakid nematodes of the blackspot seabream, Pagellus bogaraveo, from Madeiran waters, Portugal. Acta Parasitologica. 49: 156-161. for Pagellus bogaraveo off Madeira Island. By contrast, Cruz et al. (2009)Cruz C., Saraiva A., Santos M.J., et al. 2009. Parasitic infection levels by Anisakis spp. larvae (Nematoda: Anisakidae) in black scabbardfish Aphanopus carbo (Osteichthyes: Trichiuridae) from Portuguese waters. Sci. Mar. 73S2: 115-120. recorded a significant increase in anisakid intensity according to the host length for Aphanopus carbo off Sesimbra (mainland) and Madeira Islands, Portugal. Additionally, Chou et al. (2011)Chou Y.-Y., Wang C.-S., Chen H.-G., et al. 2011. Parasitism between Anisakis simplex (Nematoda: Anisakidae) third-stage larvae and the spotted mackerel Scomber australasicus with regard to the application of stock identification. Vet. Parasitol. 177: 324-331. found significantly higher intensity of Anisakis larvae in larger and older spotted mackerel (Scomber australasicus) off the Taiwanese coast. Furthermore, Mladineo and Poljak (2014)Mladineo I., Poljak V. 2014. Ecology and Genetic Structure of Zoonotic Anisakis spp. from Adriatic. Appl. Environ. Microb. 80: 1281-1290. recorded significant values for host length and abundance of Anisakis from anchovies (Engraulis encrasicolus), European hake (Merluccius merluccius) and whiting (Merlangius merlangus) in the Adriatic Sea. The correlation significance between either intensity or abundance of Anisakis and host length, seems to be dependent on the fish species, and for S. colias the present work is at least the second time that it has been measured (Mladineo and Poljak 2014Mladineo I., Poljak V. 2014. Ecology and Genetic Structure of Zoonotic Anisakis spp. from Adriatic. Appl. Environ. Microb. 80: 1281-1290.), but with no significance.

In our study, the edible portion of the fishes, the muscle, recorded low or absent infection values, with 2.4% for S. scombrus and 0% for S. colias. Nevertheless, a potential risk of infection should not be discarded, because anisakid larvae can migrate from the viscera to the muscle after death of the host (Smith and Wootten 1975Smith J. W., Wootten R. 1975. Experimental studies on the migration of Anisakis sp. larvae (Nematoda: Ascaridida) into the flesh of herring, Clupea harengus L. Int. J. Parasitol. 5: 133-136., Hauck 1977Hauck A. K. 1977. Occurrence and survival of the larval nematode Anisakis sp. in the flesh of fresh, frozen, brined, and smoked Pacific Herring, Clupea harengus pallasi. J. Parasitol. 63: 515-519.). These results suggest that fish caught from Portuguese northern Atlantic waters are highly susceptible to carry infections with anisakid nematodes and thus represent a potential human health problem. Taking into account these infection values, we advise that Atlantic mackerels caught off Portugal be ingested with care, and only after being properly frozen or cooked. The European Union (2011)European Union (E.U.). 9 December 2011. Regulation no 1276/2011 of the European Parliament and of the Council amending annex III to regulation (EC) no 853/2004 of the European Parliament and of the Council as regards the treatment to kill viable parasites in fishery products for human consumption. Eur. Lex 54: 39. recommends that for the safe consumption of fishery products, raw or undercooked, a previous period of freezing and storage at a core temperature of –20°C or below for not less than 24 h, or of –35°C or below for not less than 15 hours should be applied. In addition, the recommendation of fish evisceration and low temperatures on board just after the catch should be followed more closely. These procedures will avoid as much as possible the migration of the worm to the muscle after the host dies.

The present study identifies for the first time the anisakid species found in mainland Portuguese Atlantic mackerels, S. scombrus, and recognizes the presence of mixed infections with both A. simplex s.s. and A. pegreffii in the two scombrid fish species. For S. colias off Madeira Island, Pontes et al. (2005)Pontes T., D’Amelio S., Costa G., et al. 2005. Molecular characterization of larval Anisakid nematodes from marine fishes of Madeira by PCR-based approach, with evidence for a new species. J. Parasitol. 91: 1430-1434. recorded six different Anisakis species for S. colias, namely A. simplex s.s., A. pegreffii, A. nascetti, A. typica, A. ziphidarum and A. physeteris. The detection of A. simplex (s. s.) and A. pegreffii mixed infections have also been recorded for S. scombrus in Galicia from the northwestern coast of Spain (Abollo et al. 2001Abollo E., Gestal C., Pascual S. 2001. Anisakis infestation in marine fish and cephalopods from Galician waters: an updated perspective. Parasitol. Res. 87: 492-499.), and for S. colias and S. scombrus in the Alboran Sea off the southern coast of Spain (Abollo et al. 2003Abollo E., Paggi L., Pascual S., et al. 2003. Occurrence of recombinant genotypes of Anisakis simplex s.s. and Anisakis pegreffii (Nematoda: Anisakidae) in an area of sympatry. Infect. Genet. Evol. 3: 175-181.). These findings corroborate the statement that the Portuguese and Spanish coasts are a sympatric area for both parasite species (Abollo et al. 2001Abollo E., Gestal C., Pascual S. 2001. Anisakis infestation in marine fish and cephalopods from Galician waters: an updated perspective. Parasitol. Res. 87: 492-499., Marques et al. 2006Marques J.M., Cabral H.N., Busi M., et al. 2006. Molecular identification of Anisakis species from Pleuronectiformes off the Portuguese coast. J. Helminthol. 80: 47-51.).

Taking into account the distribution of the different Anisakis species in our fish sample, we further hypothesize that this pattern may be related to differences in the anisakid life cycle hosts, since the two fish mainly occur at different depths. A. pegreffii larvae are probably found in deeper waters, as are their main first host, than A. simplex (s.s.) larvae in less deeper waters, because A. pegreffii larvae were more frequent in S. colias (a deeper fish living at 50-300 m) than in S. scombrus (living at 0-200 m depth). However, this subject is controversial. According to Mattiucci et al. (1997)Mattiucci M., Nascetti G., Gianchi R., et al. 1997. Genetic and ecological data on the Anisakis simplex complex, with evidence for a new species (Nematoda, Ascaridoidea, Anisakidae). J. Parasitol. 83: 401-406., A. simplex (s.s.) is found mainly in benthic or demersal fishes, while A. pegreffii is found mainly in pelagic fishes, based on the marine mammal final host distribution. Abollo et al. (2001)Abollo E., Gestal C., Pascual S. 2001. Anisakis infestation in marine fish and cephalopods from Galician waters: an updated perspective. Parasitol. Res. 87: 492-499. confirmed the occurrence of A. simplex (s.s.) in benthic and demersal fishes but could not confirm the occurrence of A. pegreffii in pelagic fishes, having analysed a range of different fishes and cephalopods. Kuhn et al. (2013)Kuhn T., Hailer F., Palm H.W., et al. 2013. Global assessment of molecularly identified Anisakis Dujardin, 1845 (Nematoda: Anisakidae) in their teleost intermediate hosts. Folia Parasitol. 60: 123-134. found that both Anisakis species have similar proportions of fish hosts that live in pelagic, benthopelagic and demersal environments. However, after a second analysis of this author’s data, we may see that A. pegreffii occurs in a higher number of reef-associated fish species than A. simplex (s.s.), thus supporting our hypothesis. Also, the high abundance of A. pegreffii in Trachurus trachurus (Abattouy et al. 2013Abattouy N., Lopez A.V., Maldonado J.L., et al. 2013. Epidemiology and molecular identification of Anisakis pegreffii (Nematoda: Anisakidae) in the horse mackerel Trachurus trachurus from northern Morocco. J. Helminthol. 88: 257-263.), a benthopelagic fish reaching depths of 0 to 1050 m (Lloris and Moreno 1995Lloris D., Moreno T. 1995. Distribution model and association in three pelagic congeneric species (Trachurus spp.) present in the Mediterranean Iberic Sea. Sci. Mar. 59: 399-403.), supports our hypotheses. The geographic distribution of a parasite will be the conjunction of the distributions of its different hosts belonging to its life cycle. And a parasite with high host specificity will have a smaller distribution, probably limited to that of its hosts, than a non-specific parasite, which is expected to have a broad distribution.

We are aware that further data on the distribution within the Anisakis simplex complex at the invertebrate host level is needed in order to prove our hypothesis. Smith (1983)Smith J. 1983. Anisakis simplex: Morphology and morphometry of larvae from euphausiids and fish, and a review of the life-history and ecology. J. Helminthol. 57: 205-224. proposed that euphausiids were the major intermediate host of A. simplex s.l., in the northeast Atlantic and northern North Sea, and perhaps universally. Recently, Gregori et al. (2015)Gregori M., Roura A., Abollo E., et al. 2015. Anisakis simplex complex (Nematoda: Anisakidae) in zooplankton communities from temperate NE Atlantic waters. J. Nat. Hist. 49: 13-14. detected the occurrence of larvae of A. simplex (s.s.) and A. pegreffii in the euphausiid Nyctiphanes couchii, justifying the co-occurrence of both species in the same fish species, and its sympatry in the northeast Atlantic. However, other invertebrate species were also indicated as host candidates for Anisakis species, such as copepods (eg. Acartia tonsa, and Oitona similis) (Koie 2001Koie M. 2001. Experimental infections of copepods and sticklebacks Gasterosteus aculeatus with small ensheated and large third-stage larvae of Anisakis simplex (Nematoda, Ascaridoidea, Anisakidae). Parasitol. Res. 87: 32-36.). The low infection levels recorded for these first intermediate hosts to date in the ocean do not allow us to fully understand what species are more important for the life cycle of each Anisakis species, but further research studies will certainly solve this problem in the short term.

According to earlier studies, the two recorded Anisakis species have a different potential zoonotic level in humans: A. simplex (s.s.) is more prone to migrate into the muscle than the other species, and also withstands better the gut juices of the human stomach, with high survival rates (Arizono et al. 2012Arizono N., Yamada M., Tegoshi T., et al. 2012. Anisakis simplex sensu stricto and Anisakis pegreffii: biological characteristics and pathogenetic potential in Human Anisakiasis. Foodborne Pathog. Dis. 9: 517-521.). These differences in parasite behaviour explain why more human infections have been reported due to A. simplex (s.s.) than to A. pegreffii in Japan, where both species co-occur and the ingestion of raw fish is particularly common (Umehara et al. 2007Umehara A., Kawakami Y., Araki J., et al. 2007. Molecular identification of the etiological agent of the human anisakiasis in Japan. Parasitol Int. 56: 211-215.). Moreover, the average number of Anisakis muscle larvae in Scomber japonicus can be 12 times higher for A. simplex s.s. than for A. pegreffii, (Suzuki et al. 2010Suzuki J., Murata R., Hosaka M., et al. 2010. Risk factors for human Anisakis infection and association between the geographic origins of Scomber japonicus and anisakid nematodes. Int. J. Food Microbiol. 137: 88-93.). Taking into account that our Atlantic mackerels have significantly different infections with these two Anisakis species and likewise a similar anisakid load, we may conclude that it is less safe to eat S. scombrus, since it harbours more A. simplex (s.s.) than S. colias, unless it is appropriately frozen or well cooked.

ACKNOWLEDGEMENTSTop

The authors would like to acknowledge all the helpful suggestions provided by one of the anonymous reviewers. This research was partially supported by the Research Line INSEAFOOD Innovation and valorization of seafood products: meeting local challenges and opportunities, within the R&D Institution CIIMAR (Interdisciplinary Centre of Marine and Environmental Research) funded by the Northern Regional Operational Programme (NORTE2020) through the European Regional Development Fund (ERDF). It was also supported by FCT grants for M.J.S. (Sabbatical leave, nº BSAB-1239/2012), A.S. and R.C. (CIIMAR/C2008/BII-11/2010). We also thank Ana Silva for helping with the anisakid sampling.

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