Ecology and Genetic Structure of Zoonotic Anisakis spp. from AdriaticCommercial Fish Species

Ivona Mladineo,a Vedran Poljakb

Institute of Oceanography and Fisheries, Laboratory of Aquaculture, Split, Croatiaa; Health Ecology Department, Croatian National Institute of Public Health, Zagreb,Croatiab

Consumption of raw or thermally inadequately treated fishery products represents a public health risk, with the possibility ofpropagation of live Anisakis larvae, the causative agent of the zoonotic disease anisakidosis, or anisakiasis. We investigated thepopulation dynamics of Anisakis spp. in commercially important fish—anchovies (Engraulis encrasicolus), sardines (Sardinapilchardus), European hake (Merluccius merluccius), whiting (Merlangius merlangus), chub mackerel (Scomber japonicus), andAtlantic bluefin tuna (Thunnus thynnus)— captured in the main Adriatic Sea fishing ground. We observed a significant differ-ence in the numbers of parasite larvae (1 to 32) in individual hosts and between species, with most fish showing high or very highAnisakis population indices. Phylogenetic analysis confirmed that commercial fish in the Adriatic Sea are parasitized by Anisakispegreffii (95.95%) and Anisakis simplex sensu stricto (4.05%). The genetic structure of A. pegreffii in demersal, pelagic, and toppredator hosts was unstructured, and the highest frequency of haplotype sharing (n � 10) was between demersal and pelagicfish.

Global migration and foodstuff trade, climate change, andnovel trends in human eating habits characterized by an ele-

vated demand for raw food are today considered major reasonswhy reports of food-borne infections, especially zoonotic parasi-tosis, have increased in the last decade. Therefore, the preventionof and protection against zoonotic parasites in fishery productsdestined for human consumption have recently become a priority(1). Consequently, health authorities and the fishing industryhave become aware of the significance of the nematode genusAnisakis Dujardin, 1845, whose members affect both humanhealth and the commercial value of fish products. Although it isconsidered one of the most significant emerging food-borne zoo-noses, anisakiasis in many countries is still misdiagnosed or un-derestimated (2).

The genus Anisakis comprises parasites with a complex lifecycle (3) and a generalist character that enables it to have a world-wide distribution. Its infective third-stage (L3) larvae are com-monly found in the viscera and musculature of many teleost spe-cies (4) and migrate postmortem from the abdominal cavity intothe fish flesh. Larvae penetrating deeply into the fillets are difficultto detect by visual inspection (5), and a hazard analysis criticalcontrol point (HACCP) program must be implemented to reducethe risk (6). Risk management measures for mitigation of anisaki-dosis have been regulated by the European Community (EC) andthe U.S. Food and Drug Administration (7, 8).

Consumption of raw or thermally inadequately treated fisheryproducts represents a public health risk with the possibility ofpropagation of live Anisakis larvae, the causative agent of the zoo-notic disease anisakidosis, or anisakiasis (9, 10). Thermally unpro-cessed or lightly processed traditional seafood has the highest riskof Anisakis propagation: Japanese sushi and sashimi (9), tuna orsparid carpaccio, marinated salted sardines, or pickled anchoviesin the Mediterranean (11, 12); smoked or fermented herrings(maatjes) in The Netherlands; (5) dry, cured salmon (gravlax) inNorway; raw salmon (lomi lomi) in Hawaii; or ceviche in SouthAmerica (13). In humans, larvae penetrate the mucosa of thestomach, small intestine, or colon, causing eosinophilic granu-

loma formation with severe symptoms of disease (for a review, seereferences 10, 14, and 15). The other clinical form is triggered byrepeated consumption of thermally processed seafood that con-tains the whole nematode or its molecular traces, inducing hyper-sensitivity in the consumer and an array of allergic symptoms:acute urticaria and angioedema, bronchospasm, fatal respiratoryarrest, and anaphylaxis (16, 17). Rheumatologic diseases and dis-orders like asthma, gingivostomatitis, conjunctivitis, and contactdermatitis have also been related to occupational exposure to al-lergens in fishmongers, fishermen, or employees of the fish-pro-cessing industry (18).

Although there are many reports on the population dynamicsof Anisakis spp. in the Mediterranean (19–22) and the Adriatic Sea(23–25), an updated assessment of infection levels in commercialfish species is important for the implementation of risk analysis. Incoastal parts of the Adriatic, traditional home-made, thermallyunprocessed fish, mostly pickled, marinated, or salted anchoviesand sardines, are particularly well-kown ethnic dishes that need tobe considered in relation to Anisakis infection in humans, and alsobecause their elevated consumption correlates with the touristseason in the Adriatic. Additionally, Adriatic fish species marketedfresh, frozen, salted, or marinated are also exported to the EC, andAnisakis infection is frequently the reason for rejection at the bor-der (26). Therefore, we investigated the population dynamics ofAnisakis spp. in commercially important fish—anchovies (En-graulis encrasicolus), sardines (Sardina pilchardus), European hake(Merluccius merluccius), whiting (Merlangius merlangus), chub

Received 4 November 2013 Accepted 2 December 2013

Published ahead of print 6 December 2013

Address correspondence to Ivona Mladineo, mladineo@izor.hr.

Supplemental material for this article may be found at http://dx.doi.org/10.1128/AEM.03561-13.

Copyright © 2014, American Society for Microbiology. All Rights Reserved.

doi:10.1128/AEM.03561-13

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mackerel (Scomber japonicus), and Atlantic bluefin tuna (Thunnusthynnus)— captured in the main Adriatic fishing ground (GSA17). Molecular identification of isolated larvae was inferred fromthe mitochondrial DNA (mtDNA) cytochrome oxidase subunit 2(cox2) locus, and a population genetic study was performed tobetter understand the parasite’s ecological and phylogenetic traits.

MATERIALS AND METHODSFish sampling. The sample consisted of 120 individuals of each commer-cially important species—anchovies (E. encrasicolus), sardines (S. pilchar-dus), European hake (M. merluccius), whiting (M. merlangus), and chubmackerel (S. japonicus) (n � 600)— caught in the wild and 120 bluefintuna designated for cage culture. The fish (except bluefin tuna) were cap-tured in Croatian fishing area C (43°15=N, 15°10=E; 43°15=N, 16°45=E;42°10=N, 15°05=E; 42°10=N, 16°45=E), Adriatic region (GSA 17), sampledevery month from September 2009 to September 2010. The fish werebrought to the laboratory on ice and examined immediately (36 to 48 hpostcapture), and the total fork length (cm) and total weight (g) weremeasured. The temperature of the fish upon arrival at the laboratory was3°C (�1°C), measured with a temperature probe. The surface of the vis-ceral mass was examined under a stereomicroscope (Olympus; SZX10)for the presence of anisakid larvae. Larvae were isolated and morpholog-ically identified as Anisakis spp. based on mucron and boring tooth pres-ence and the appearance of an esophagus and ventriculus (27). The site ofparasitation was noted. The abdominal cavity was checked for the pres-ence of nematodes by visual observation, and the pressing-UV method (6)was used to investigate the presence of larval anisakids in the flesh. Theprevalence (P) (percent), mean intensity (I), and mean abundance (A) infish visceral organs and the intensity in fillets (J) were calculated accordingto the method of Bush et al. (28). Wild bluefin tuna (T. thynnus) was caughtduring June 2008 in the central Adriatic waters around the Island of Jabuka(43°5=67�N, 15°28=63�E). The fish (n � 120) were transported to the farm,and 30 fish were sampled every 3 months from summer 2008 until spring2009. The same procedure as for the wild-fish analysis was used for bluefintuna, except for fillet inspection. Isolated Anisakis L3 larvae (n � 77) from fishfillets and visceral cavities were morphologically identified as type I larvae,washed in phosphate-buffered saline solution, and fixed in ethyl alcohol formolecular identification.

Statistical analysis. The data obtained were tested for normality (An-derson-Darling test) and, when necessary, log transformed. QuantitativeParasitology 3.0 software (29) was used to calculate Sterne’s exact 95%confidence limits for prevalence, bootstrap 95% confidence limits (2,000bootstrap replications) for mean abundances, mean intensity, variance tomean ratio, and the exponent of the negative binomial (k). Analysis ofvariance (ANOVA) (Statistica 7.0; StatSoft, Inc.) was used to test the dif-ferences in abundance between the sexes and sampling seasons, apply-ing the general linear model (GLM) procedure for fixed effect of theinvestigated factors with the accepted type I error of 5%. The linearregression model and Pearson’s correlation coefficient were used todetermine the relationship between fish length and abundance. Simi-larities in parasite seasonal abundance were analyzed with PER-MANOVA (Primer 6; Primer-E Ltd.) based on the Bray-Curtis simi-larity index.

Molecular identification of anisakid larvae. For phylogenetic analy-sis, genomic DNA was isolated, amplified, purified, and sequenced at themitochondrial cox2 locus (�645 bp), as described previously (30), fromanisakid larvae isolated from the fillets and visceral cavities of 6 fish species(n � 77). The sequences were aligned with other anisakid sequencesstored in GenBank (http://www.ncbi.nlm.nih.gov/GenBank/GenbankSearch.html)—Anisakis simplex sensu stricto (DQ116426), A. pegreffii(DQ116428), A. simplex C (DQ116429), A. typica (DQ116427), A.ziphidarum (DQ116430), A. physeteris (DQ116432), A. brevispiculata(DQ116433), A. paggiae (DQ116434), and A. nascettii (DQ116431) (asreported in reference 31)— by Clustal X (32) implemented in MEGA 5.05

software, using default parameters, and further verified by GBlocks (http://molevol.cmima.csic.es/castresana/Gblocks.html).

Genetic diversity and population structure of A. pegreffii. Moleculardiversity was analyzed using Dnasp 5.0 (33) and Arlequin 3.5 (34), and thenumber of haplotypes (H) and polymorphic sites (S), haplotype diversity(h), nucleotide diversity (p), and the average numbers of pairwise nucle-otide differences (k) were estimated. Pairwise and overall distances amonghaplotype sequences were calculated in MEGA 5 (35). A substitutionmodel for both genes and the gamma distribution shape parameter for therate of heterogeneity among sites were determined using Modeltest 3.07(36), based on the hierarchical likelihood ratio tests (hLRTs). For cox2data, the TrN model (37) of evolution with the gamma shape parameter(G � 0.01) was selected for the analysis of molecular variances (AMOVA)and phylogenetic analysis. Populations were constructed based on hostfeeding habits, not fish species, because only a small number of sequenceswere available for each species. Thus, the pelagic population consisted ofnematode sequences from pelagic hosts (E. encrasicolus, S. japonicus, S.pilchardus, and Illex coindeti [obtained from our previous research {30}]),the demersal population consisted of sequences from two demersal hosts(M. merluccius and M. merlangus), and the top predator population con-sisted of one cage-farmed host (T. thynnus). For evaluation of hypothe-sized patterns of the spatial genetic structure, a hierarchical AMOVA wasused to partition variance components attributable to population vari-ance and to individuals within the populations, where 10,000 permuta-tions were performed to test the significance of pairwise populationcomparison. Pairwise genetic differentiation between populations was es-timated using the fixation index (FST), and statistical significances weretested with 10,000 permutations. Both AMOVA and FST calculations wereperformed in Arlequin 3.5 (http://cmpg.unibe.ch/software/arlequin35/).The null hypothesis of population panmixia was also tested in Arlequin3.5 using an exact test of the differentiation of haplotypes among popula-tions.

Tajima’s D and Fu’s Fs statistics were calculated to verify the nullhypothesis of selectivity neutrality inferred by mtDNA sequences, whichwould be expected with population expansion.

Mismatch distributions (38) were constructed using Arlequin 3.5. Theshapes of the mismatch distributions were used to deduce whether a pop-ulation had undergone sudden population expansion. The fit between theobserved and expected distributions was tested using the Harpendingraggedness index (HRI) and sum of squared deviations (SSD) for theestimated stepwise expansion models (39), implemented in Arlequin 3.5.Significance was assessed for the parameters with permutation tests underthe null hypothesis that sudden population expansion cannot be rejected.Graphic presentation of genetic diversity and population structure wasinferred by activating the R statistics (R.2.15.2) command in Arlequin 3.5.

Phylogenetic analysis. Bayesian inference (BI) analysis (40) was per-formed on the 579-bp sequences using MrBayes v3.1.2 (41) and the Ta-mura-Nei with gamma distribution (TrN�G) model. Four incrementallyheated Markov chains were run for 2,000,000 generations, with samplingevery 100 generations, and 5,000 samples were discarded. Default valueswere used for Markov chain Monte Carlo (MCMC) parameters. A 50%majority rule consensus tree was constructed from the tree output filesproduced in the Bayesian inference analysis and visualized using FigTree(http://tree.bio.ed.ac.uk/software/figtree/). For tree rooting, Pseudoterra-nova decipiens sensu stricto (AF179920) was used. A median-joining net-work (42) was used to illustrate cox2 haplotype distribution among hostgroups (NETWORK v 4.5.1.6, available at http://www.fluxes-engineering.com/sharenet.htm).

Nucleotide sequence accession numbers. The GenBank accessionnumbers of the sequences obtained in this study are as follows: T. thynnus,KC479822 to KC479852; E. encrasicolus KC479853 to KC479860; M. mer-luccius, KC479861 to KC479870; S. pilchardus, KC479871 to KC479874;M. merlangus, KC479875 to KC479883; S. japonicus, KC479884 toKC479890.

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RESULTSParasite population dynamics. Anisakid larvae were isolated inall sampled species from visceral surfaces of the gastrointestinaltract and from muscle fillets, except in sardines (Table 1), andwere morphologically identified as type I larvae. Bluefin tuna fil-lets have not been investigated because of their high commercialvalue. The larval ecological parameters of infection are shown inTable 2. The observed parasite frequency distributions in fish spe-cies showed a negative binomial distribution. Significant differ-ences were found in abundance values through sampling seasons,except for the spring-autumn pair (pairwise t test; P � 0.103).Significant correlation was found between host length and Anisa-kis abundance in anchovies (Pearson’s r � 0.45), hake (Pearson’sr � 0.64), and whiting (Pearson’s r � 0.58), in contrast to chubmackerel (Pearson’s r � 0.33) and sardines (Pearson’s r � 0.17).No statistical difference was observed between abundances of ani-sakids in male and female fish in anchovies, sardines, chub mack-erel, and tuna (bootstrap t test; P � 0.05), in contrast to hake(bootstrap t test; t � 5.488; P � 0.05) and whiting (bootstrap t test;t � 5.684; P � 0.05), where females were more parasitized. In hakeand whiting, the females were significantly longer than the males(Fisher’s exact test; P � 0.05).

Genetic diversity of the cox2 gene fragments. Unambiguoussequences for the mitochondrial cox2 genes of three A. pegreffiipopulations from the Adriatic fishing area GSA 17 were obtainedand deposited in GenBank. The length of cox2 in the analysis was597 bp (n � 74). In total, 59 variable sites were observed and 36haplotypes were detected for the cox2 fragment. The sequence

divergence (Tamura-Nei distance) among cox2 haplotypes rangedfrom 0.2% to 3.5%, with an average of 0.7%. Among 59 polymor-phic sites observed in the cox2 fragment, 23 were singleton vari-able sites and 36 were parsimony informative. Among 36 haplo-types defined, most (58.3%) were unique and were represented bya single individual (Fig. 1A). Only three haplotypes were sharedamong hosts that came from all three different feeding niches (H6,H12, and H16), and H6 was the most abundant haplotype presentin all feeding habitats of the Adriatic Sea host populations. Toppredators and the pelagic fish population shared haplotypes H6,H12, H13, and H16; top predators and demersal fish shared H6,H9, H12, and H16, while pelagic and demersal fish, surprisingly,shared the highest number of haplotypes (H6, H12, H13, and H23to H29). The highest Anisakis haplotype frequencies were ob-served for H6 in the top predator (0.138) and pelagic (0.148) pop-ulations and for H12 in the pelagic host population (0.185). Thegenetic diversity indices for each population are summarized inTable S1 in the supplemental material, giving an overall haplotypediversity (h) of 0.957 � 0.011 and nucleotide diversity () of0.00723 � 0.0011, indicating high haplotype and low nucleotidediversity. Also, cox2 displayed a high average number of nucleo-tide differences (k) (4.310 � 1.34519) (Fig. 1B and C).

Population genetic structure. The genetic differentiationamong three Anisakis populations was assessed using FST pairwisecomparison. Global FST values were very low (see Table S2 in thesupplemental material), showing no significant genetic structurein the investigated feeding habitats [FST(cox2) � 0.005366; P �0.77126). The same pattern of genetic structure was confirmed by

TABLE 1 Numbers of isolated anisakid larvae (L3) and their parasitation sites in commercial Adriatic Sea fish

Host species No. of larvae

% parasitation at indicated sitea

No. of molecularlyidentified larvaeL S I G F

Anchovy (E. encrasicolus) 827 0 0 99.4 0 0.6 8Sardine (S. pilchardus) 26 0 0 100 0 0 4Hake (M. merluccius) 824 64.53 0 20.87 13.51 1.09 10Whiting (Gadus merlangus) 343 63.72 0 22.29 12.83 1.16 9Chub mackerel (S. japonicus) 1,257 9.07 20.6 66.86 2.12 1.35 7Bluefin tuna (T. thynnus) 202 0 0 100 0 0 31

Total 3,479 77a L, liver; S, stomach; I, intestine; G, gonads; F, musculature.

TABLE 2 Larval ecological parameters of infection of Anisakis sp. parasites isolated from six fish species over a 1-year period

Fish species

Mean hostlength � SE(mm)

Mean host wt �SE (g)

Prevalence(CI)a (%) Mean I (CI), rangeb Mean A (CI)c

Mean J(range)d v/xe k Dg

E. encrasicolus 142.29 � 7.74 17.73 � 0.3 81.7 (73.81–87.76) 8.44 (7.26–9.57), 1–23 6.89 (5.83–8.07) 1.25 (1–2) 5.84 0.889f 0.507S. pilchardus 149.53 � 12.95 29.8 � 0.41 3.3 (1.15–8.21) 1.25 (1.18–2.12), 1–5 0.04 (0.01–0.09) 1.37 0.227f 0.890M. merluccius 342.92 � 39.71 322.12 � 6.54 70.8 (62,12–78,41) 9.69 (8,39–11,25), 1–32 6.87 (5,57–8,20) 1.33 (1–2) 7.80 0.566 0.567M. merlangus 279.54 � 18.23 140.3 � 1.68 65.8 (56.69–73.80) 4,34 (2.32–3.47), 1–13 2,86 (2.32–3.47) 1 (1–1) 3.59 0.790 0.580S. japonicus 300.725 � 8.81 222.63 � 1.62 100 (96.87–100) 10,48 (9.80–11.28), 1–22 10.48 (9.8–11.28) 2.13 (1–4) 1.60 0.211T. thynnus 904.66 � 99.37 1643 � 49.1 23.3 (16.59–31.66) 6.5 (5.0–10.00), 1–6 1.78 (1.15–2.67) 10.06 0.091 0.851a Total mean prevalence with Stern’s exact 95% confidence limits (CI).b I, intensity.c A, abundance, with bootstrap 95% CI.d J, intensity in fillets.e v/x, variance-to-mean ratio.f The exponent of the negative binomial (k) showed no statistical difference between observed and expected frequencies at P � 0.05.g D, discrepancy index (D � 0 to 1).

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FIG 1 Graphical representation of genetic diversity and population structures of three A. pegreffii populations isolated from hosts from different feeding habitatsinferred from the mtDNA cox2 locus. (A) Frequencies of haplotypes in top predator, demersal, and pelagic fish. (B) Molecular diversity indices: �k, averagenumber of nucleotide differences; �H, haplotype diversity; �S, number of polymorphic sites; �, nucleotide diversity. (C) Haplotype distance matrix inferred fromthe number of pairwise differences (diff.). (D to F) Observed and expected mismatch distributions with 90 to 99% confidence intervals for A. pegreffii populationsparasitizing pelagic (D), demersal (E), and top predator (F) fish.

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AMOVA, which attributed 0.54% of the genetic variation tovariability among populations and 100% variation within popu-lations (see Table S3 in the supplemental material). Over all sam-ples, the nondifferentiation exact P values were not significant(P � 0.83906 � 0.01109), not rejecting the idea that the popula-tion of A. pegreffii among hosts that feed in three different envi-ronmental habitats is panmictic.

Phylogenetic and network analyses. The topology of treesbuilt from the cox2 locus using BI (Fig. 2) clustered most of theAdriatic taxa in a younger clade, along with the A. pegreffii refer-ence sequence, which in part branched in well-supported groupsthat formed small, relatively short-branched clades. In the tree’sother segment, polytomy was observed in the lower part withinmostly bluefin tuna and a few other fish taxa. No structuring basedon habitat use (feeding ground) or fish species was detected. Mostof the isolated larvae (n � 74; 95.95%) with no ambiguity be-longed to A. pegreffii, while only three samples (4.05%) clusteredwith the A. simplex sensu stricto isolate. Adriatic isolates of A. sim-plex sensu stricto were isolated from bluefin tuna (n � 2) and hake(n � 1). The haplotype network showed a star-like phylogeny,with most of the unique haplotypes closely related to the commoncentral haplotype (H6) (Fig. 3), composed of Anisakis parasitehosts from all three examined habitats. If A. pegreffii underwentexpansion, this central common haplotype was probably the an-cestral one, from which further stepwise nucleotide substitutionscould explain all other unique haplotypes.

Demographic patterns. The demographic history of A. pegref-fii was investigated using mismatch distribution. The goodness-of-fit test showed that no mismatch distributions for sample lo-calities deviated significantly (P � 0.05) from predicted valuesunder the sudden-expansion model of Harpending (see Table S4in the supplemental material). Furthermore, the HRI values werelow for all Anisakis populations from different habitats, indicatinga significant fit between the observed and expected distributions,thereby providing further evidence for population expansion inthe past (Fig. 1D, E, and F). The sum of squared deviation alsosupported a nondeparture from the null hypothesis of populationexpansion. The � values for all cases confirmed the scenario ofpopulation expansion; the initial � values were always muchsmaller than the final � values (see Table S4 in the supplementalmaterial). Only slight departure from the age expansion parame-ter (�) between populations from different habitats suggested thatthe population expansion may date back to a recent historicalperiod.

The results of Tajima’s D test and Fu’s Fs test are presented inTable S4 in the supplemental material. Tajima’s D values werenegative for each population and the populations overall, indicat-ing an excess of rare nucleotide site variants compared to the ex-pectation under a neutral model of evolution, with statisticallysignificant differentiation from the model of neutral evolution.Fu’s Fs test, which is based on the distribution of haplotypes, alsoshowed negative values for each population and the populationsoverall, indicating an excess of rare haplotypes over what would beexpected under neutrality. Following these tests, the hypothesis ofneutral evolution was rejected for all populations.

DISCUSSIONEcological factors of Ansakis-fish interaction. In three commer-cially important pelagic fish species (anchovies, chub mackerel,and sardines), two demersal fish species (hake and whiting), and

one cage-farmed fish species (bluefin tuna) we investigated, pre-vious studies have also identified Anisakis spp. in the Adriatic (23,25, 43, 44). However, no published records exist concerning Ani-sakis infection in sardines and whiting in the east Adriatic, while toour knowledge, Anisakis sp. infections in sardines have been re-corded only in the eastern Mediterranean (45–47).

We observed a significant difference in the numbers of parasitelarvae (1 to 32) in individual hosts and between species, as de-scribed previously (2, 23), which underscores the necessity tomonitor anisakid infections over a longer time span in order todevelop an adequate risk assessment analysis for commerciallyimportant fish. Interestingly, the aggregation of the parasite varieswith the host species. In anchovies and sardines, it was observedthat Anisakis spp. have an aggregated, left-biased binominal dis-tribution very common among parasites (48). In contrast, thenormal distribution of aggregating larvae was observed in chubmackerel, possibly associated with the limited life span of the par-asite and the robustness of the host immune response at a specificlength/age (49). We also confirmed the tendency of Anisakis larvaetoward a particular organ in a specific host (2, 43, 44, 49), whichhas been related to the availability of nutrients to the parasite (2),although firm evidence has never been provided. We argue that ananatomical relationship between the point of anisakid penetrationthrough the gastrointestinal tract (related to intrinsic host factors,such as gut peristalsis or the quantity of its content) and the loca-tion of the visceral organ that is close to that point governs thepredilection site in the host.

Most commercially important fish species in this study showedhigh or very high population index values for the parasite, whilelower values were observed in farmed bluefin tuna and sardines.Compared to other studies, very similar patterns exist in general,and the variation might be attributed to the initial sampling effort(sample size and period) and/or the sampling ground (ecologicaland oceanographic characteristics). Similarly, Abattouy et al. (50)concluded that the potential risk factors of Anisakis in fish areinfluenced by the fishing season and fishing grounds and the sex,weight, and length (age) of the fish, for example, previously ob-served higher values for anchovies (100%) and hake (88%) andlower values for chub mackerel (54%) (43). In the Aegean Sea(47), prevalences in hake (66.6%) and chub mackerel (75%) weresimilar to our findings, while the prevalences in sardines (5.5%)and anchovies (3.9) were much lower, probably due to the smallersample size (36 and 77, respectively) and different fishinggrounds. Data on the infection of mackerel in the northeast At-lantic Ocean (50) and in reared bluefin tuna (25%) (44, 51) arecongruent with this study. We observed oscillations in the sea-sonal pattern of infection (significant through all sampling sea-sons, except for spring-autumn) in all examined species exceptcage-farmed tuna. Temporal variations in the intensity of parasiticinfections are common in marine ecosystems due to seasonalchanges affecting the physiology of the host (e.g., the intensity offeeding and immune function) (52). The highest level of infectionwas observed in the warm part of the year, which in the literaturehas been described as typical for Anisakis spp. (50, 53). Likewise,Gutiérrez-Galindo et al. (53) reported the highest intensity inmackerel from the Spanish Mediterranean coast in the summerseason. The reasons for seasonal fluctuations in the populationdynamics of Anisakis spp. are associated with the seasonal fluctu-ation of biotic and abiotic environmental conditions that indi-rectly influence the migration of aquatic mammals (final hosts),

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FIG 2 Rooted phylogenetic tree inferred by Bayesian analysis of mtDNA cox2 locus fragments from Anisakis spp., with posterior probabilities shown in differentcolors (thickest [red] line, 0.9 to 1; thick [orange] line, 0.8 to 0.9; thin [coral] line, 0.7 to 0.8; thinnest [yellow] line, 0.6 to 0.7). A. pegreffii isolated from fish speciesin the Adriatic Sea is represented by 74 isolates forming a sister clade with A. pegreffii (DQ116428), while 3 isolates (M. merluccius 1, T. thynnus 3, and T. thynnus4) branched from A. simplex sensu stricto (DQ116426), apart from the A. pegreffii group. (Inset) Magnification of part of the tree with clades composed of A.simplex sensu stricto and A. pegreffii taxa.

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the amounts of parasite eggs laid, and zooplankton (intermediateAnisakis hosts) availability (51). Regular seasonal fluctuations oftemperature and salinity, the impact of open and deeper waters,and various topographic and hydrographical factors in the fishingarea of the eastern Adriatic control the concentrations of chloro-phyll a. In turn, phytoplankton has a decisive influence on thedistribution and abundance of zooplankton, the first link in theAnisakis life cycle and the main nutritive base for fish. We ob-served that the host sex had no statistically significant effect on thedegree of infection in most studied species, except in hake andwhiting, where females were more infected, probably related tothe faster growth of females in these demersal species (54).Šimková et al. (55) related the greater infection of the gill mono-genean parasites in female fish to the greater investment of femalesin reproduction, which produces larger gametes than in males.The authors observed a positive correlation between parasite in-fection and gonad weight and the gonadosomatic index, althoughthis does not fully explain the higher affinity of the parasite forfemale hosts. In contrast to our study, Mladineo et al. (25) ob-served a correlation between sex and anisakid infection in a sam-ple of 4,600 anchovies, which highlights the importance of theeffect of sample size in parasitic studies.

Positive correlation between the length of the host and thenumber of larvae was observed in all captured species and wasattributed to the cumulative effect of repeated parasite infections,acquired over a longer lifetime for larger (older) fish and constantreinfection through the diet. Bioaccumulation of anisakids thatleads to the accumulation of hundreds of parasites in the paratenichost has been described in many studies (21, 23, 30). Mladineo(43) reported a negative correlation for anchovies and a significantpositive correlation for hake, while in a more recent work (25), thesame author reported a high positive correlation for anchovies.Rello et al. (21) also reported a positive correlation between fourgroups of anchovies of different lengths and the prevalence of theparasite, and Abattouy et al. (50) recorded a high correlation be-tween parasitic infection and age (length and weight) in S. japoni-cus from Moroccan coast waters. The only negative correlation inour study was found between the length of the cage-farmed tunaand the abundance of nematodes, indicating that larger individu-

als were less infected, in contrast to other examined hosts. Thispattern has also been described in other parasite species isolatedfrom reared tuna (51), which is very interesting, because the fish incages are intensively fed the same bait fish they feed upon in thewild, which are significant paratenic hosts for anisakids. This de-crease in anisakids (and other parasite species) in tuna has beenattributed to the strengthened and stable immunity of properlyfarmed fish. Similarly, low correlation between the length ofmackerel and Anisakis numbers was previously recorded in agroup of larger fish (�500 mm), where increasing the length of thehosts decreased parasite intensity. As previously argued, this couldbe attributed to the immune response of the larger host specimens(49) and limited larval lifetime (2); however, it can have importantimplications in the risk assessment analysis of categories of largefish. In the case of tuna, this would imply that larger specimensdestined for the Japanese sushi and sashimi market are less risky interms of human anisakidosis than smaller fish, which are usu-ally sold in local markets. Migration of Anisakis sp. larvae inmuscle tissue was observed in all sampled host species excepttuna and sardines, with an intensity range from 1.00 (whiting)to 2.13 (chub mackerel) larvae. We argue that these numbersmight be underestimated, because the visual method we em-ployed, although a reference method of the EC (6), has loweraccuracy and sensitivity than molecular methods. Such migrat-ing larvae in fish fillets are the main microbiological (parasitic)hazards in fishery products and thermally untreated dishesmade from fish (2), as previously described (21, 49). BecauseAnisakis prevalence and intensity present the first risk factorsfor human infection (2), we can conclude that there is a highlevel of parasite hazard in raw materials originating from ourstudied species. Moreover, the existence of A. simplex sensustricto in two Adriatic hosts further aggravates the risk for con-sumers, as the species has a 12 times greater ability to penetrateinto the fillets than A. pegreffii (56). However, such a risk is ulti-mately negligible, given the very low prevalence and abundance ofA. simplex sensu stricto observed in the Adriatic Sea.

Phylogeny and genetic structure of Ansakis in the Adriatic.Phylogenetic analysis confirmed that commercial fish in the Adri-atic Sea are parasitized by A. pegreffii (95.95%) and A. simplexsensu stricto (4.05%; bluefin tuna and hake), both belonging to theA. simplex complex (57). Previous studies in the Adriatic foundonly A. pegreffii infection in paratenic hosts (23–25, 30), and ourevidence of sympatry of A. simplex in fish is the first recorded forthe Adriatic Sea. We argue against the possibility that A. simplexsensu stricto isolated from bluefin is in fact “imported” throughtuna feed (North Sea herring) and not an actual Adriatic resident.First, the technical management of the imported bait fish is rigor-ous: herrings are caught in the North Sea and frozen below 20°Cfor a period of over 30 days, shipped, and stored deep frozen inorder to maintain feed quality. Second, A. simplex sensu stricto wasalso isolated from hake native to the Adriatic, offering furtherevidence that the propagation of the species in the Adriatic isachievable. Previously, sympatry of A. simplex sensu stricto and A.pegreffii has been recorded in Mediterranean fish (4), while in theAdriatic, A. simplex sensu stricto was observed, in addition to an-other anisakid species, A. physeteris, in the final cetacean hosts(58). The authors isolated A. physeteris and A. simplex sensu strictofrom Cuvier’s beaked whale (Ziphius cavirostris), spinner dolphin(Stenella coeruleoalba), and bottlenose dolphins (Tursiops trunca-tus), with an incidence of 5% in syntopy with A. pegreffii (95%).

FIG 3 Haplotype network showing a star-like phylogeny, with most of theunique haplotypes closely related to the common central haplotype (H6). Thesizes of the circles match the numbers of sequences belonging to the specifichaplotypes. The circle colors represent the three A. pegreffii populations: black,demersal; white, pelagic; gray, top predator. The smallest dark-gray polygonalnodes represent hypothetical haplotypes that were required for the establish-ment of the existing (sampled) haplotypes.

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This strongly supports the presence of anisakid species otherthan the typical A. pegreffii in Adriatic fish, although in a muchsmaller quantity that makes them almost rare. Cuvier’s beakedwhale (Z. cavirostris) and spinner dolphin (S. coeruleoalba) arehighly migratory species whose presence in the open middleAdriatic is occasionally recorded, in contrast to the constantlypresent bottlenose dolphin (T. truncatus) (59), implying dis-semination of more than one anisakid species from other seas.Based on a very low incidence of these other anisakid species inAdriatic paratenic hosts, it is still questionable if their coloni-zation and propagation in the Adriatic is successful or is limitedto only occasional events.

Analyzing the population genetic structure of A. pegreffii inthree groups of paratenic hosts divided by their feeding grounds(demersal, pelagic, and top predator), we evidenced the existenceof genetically unstructured populations. Mes (60) suggested thatparasite ecological characteristics, such as a wide distributionrange, the fragmented nature of the habitat, and/or a low ex-pected rate of long-distance dispersal and hermaphroditic re-production type, contribute to genetic structuring of parasitepopulations. In contrast, A. pegreffii is a generalist, cosmopol-itan species with separate sexes, which enables the observedhigh gene flow and consequent parasite panmixia in the Adri-atic. This is further confirmed by negligible FST values used todifferentiate potential populations. Comparing the haplotypeand nucleotide diversity over three anisakid populations, sim-ilar values were observed for each of them, further suggestingthe existence of a single population that circulates betweendifferent levels of the marine food chain in the Adriatic. Acombination of high haplotype diversity and low nucleotidediversity is a signature of rapid demographic expansion from asmall effective population size (61), which was also supportedby Tajima’s D test and the Fu test. The negative value of thesetests suggests bias toward rare alleles, typical of recent popula-tion expansion and recent colonization events in specific areas.The observed haplotype divergence (cox2, 0.2% to 3.5%; average,0.7%) is mostly in accordance with previous data for anisakid species(4). Mattiucci and Nascetti (4) concluded that such findings confirmthat the complex life cycle of these nematodes does not limit geneexchange but enhances it through the high mobility of the differenthosts, such as fish and marine mammals.

Three Adriatic haplotypes included anisakids from all feedinggrounds (H6, H12, and H16), which can contribute to our knowl-edge of parasite ecology. The highest frequency of haplotype shar-ing (n � 10) was found between demersal and pelagic fish; whitingand the European hake are demersal, predatory carnivorous spe-cies whose main diet consist of small pelagic fish, especially ancho-vies and sardines (54), supporting our data on shared haplotypes.Thus, both demersal fish species can be considered secondary Ani-sakis paratenic hosts. Haplotypes are also shared between the toppredator population and the demersal/pelagic population withsimilar frequencies (n � 4 and 5, respectively). During cage rear-ing, bluefin tuna is mostly fed small pelagic fish and cephalopodsand rarely any demersal species (62). This suggests that part of thetuna infection with A. pegreffii originates from its wild juvenilephase prior to rearing (age category, �1 year in 2008). We col-lected the last reared tuna samples in 2009, which implies that A.pegreffii larvae survived in the bluefin for at least 1 year, and prob-ably even longer, enabling a first robust estimate of larval survivalwithin a secondary paratenic host. The second group of A. pegreffii

haplotypes isolated from the reared bluefin is shared by small pe-lagic fish, as expected, given the current feeding regime of thebluefin in aquaculture (62).

In conclusion, the risk management measures for Anisakis in sea-food need to be adapted to each commercial fish species. In this way,all ecological and phylogenetic traits of the host and parasite found ina specific fishing ground will be encompassed, resulting in satisfac-tory control and mitigation conditions.

ACKNOWLEDGMENTS

This work was supported by the Croatian Ministry of Science, Educationand Sport (MZOS), grant no. 001-0000000-3633.

We are thankful to Relja Beck from the Croatian Veterinary Institute,Zagreb, Croatia, for technical help with amplification of Anisakis sp.samples.

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