Anisakid Nematodes and Potential Risk of Human Anisakiasis through the Consumption of Hake, Merluccius spp., Sold Fresh in Spanish Supermarkets

Nematode parasite species belonging to the Anisakis simplex complex are the most important cause of human anisakiasis through the consumption of (mainly) undercooked, previously not frozen, or conveniently treated fish. In Spain, the consumption of hake has been recognized as an important source of this parasitosis. With the aim of shedding light on the risk factors that can influence the potential risk of human anisakiasis in Spain through the consumption of fresh hake sold by nationwide supermarket chains, a total of 536 small hake specimens belonging to the species Merluccius bilinearis caught off the Northeast American coasts and Merluccius merluccius caught in the Northeast Atlantic and Mediterranean waters was analysed. Anisakids morphologically identified as Anisakis type I were found as the most prevalent and the most abundant species and were considered the main potential cause of human anisakiasis. Intrinsic and extrinsic factors concerning the hake, such as its origin and season of capture, its size, as well as the days passed between its capture and consumption, should be taken into account to avoid this human parasitosis. It is essential that consumers have access to fish label information which should include, as regulated by the European Commission, traceability data.


Introduction
Human anisakiasis, the most frequent form of human anisakidosis, has been recognized as an emerging food-borne disease worldwide, especially in Japan and in some Western European countries such as Spain and Italy, where fish consumption is high and consumers are able to purchase fresh and uneviscerated fish (in some of which Anisakidae larvae are very common) [1,2].
Spain seems to have the highest incidence in Europe [3], with about 8000 annual cases estimated in recent years, although the anisakiasis burden in the human population is unknown due to of the scarcity of epidemiological data [4], with the traditional consumption of raw anchovies, Engraulis encrasicolus, in vinegar posing the main source of risk [5], although the consumption of other fish species has also been reported multiple times as the cause of human anisakiasis in Spain.
Anisakis spp. larvae can be found parasitizing a wide range of marine teleost species inhabiting the Atlantic and the Mediterranean, as well as the Pacific to the Antarctic area, affecting overall fish product quality [6].
reported due to its consumption such as the three recently reported cases in Barcelona [24]. Previous studies have reported high levels of Anisakis larvae in the European hake, mainly from the North East Atlantic [22]. Moreover, the two most often reported Anisakis species in M. merluccius originating from the North East Atlantic and the Mediterranean are A. simplex (s.s.) and A. pegreffii, respectively, both belonging to the morphotype I [2,22,[25][26][27]. In M. bilinearis, the scarce studies carried out have reported Anisakis type I larvae, some of them identified molecularly mainly as A. simplex (s.s.) but also, surprisingly, as the hybrid genotype A. simplex (s.s.) × A. pegreffii also [12,28].
Epidemiological studies carried out to determine which commercially available fish species are most parasitized and which fishing grounds have the highest rates of parasitization are of great interest [29]. Although there are many reports on the population dynamics of anisakid nematodes, mainly Anisakis spp. In the Mediterranean and in the Atlantic, an updated assessment of infection levels in commercially available fish species, such as Merluccius spp. In this case, is crucial to assess the human anisakidosis risk [30].
Consequently, the main objectives of this study are: (1) to assess the risk of human anisakidosis, mainly anisakiasis, through the consumption of M. merluccius and M. bilinearis sold by nationwide Spanish supermarket chains, by means of the analysis of the potential influence of intrinsic and extrinsic factors of these fish species on the presence and the worm burden of anisakid larvae; (2) to inform consumers on the importance of the information on fish crate labels to prevent and diminish the human anisakidosis risk; and (3) to inform the institutions dealing with the fish trade and food safety admitted to the market about the parameters of infestation of commercially sold fish from the fishing areas included in the study. Moreover, since the most common anisakid species reported in hake cannot be easily distinguished visually by consumers, fishermen, and handlers, only morphological and not molecular identification has been carried out, differentiating the larvae found at genus level and at morphotype level (types I and II) in the case of Anisakis. Our study hypotheses are that: geographic origin, size (weight and length) and seasons of capture may influence the prevalence and the abundance of anisakid larvae in hakes; and that the days passed since capture may influence migration of larvae from viscera to the flesh in previously parasitized fish. If these hypotheses are verified, consumers would have additional criteria at their disposal when choosing fish, considering the information on fish crate labels. The European Union makes provision of this information at the selling point obligatory, with the aim to complement prevention measures and ultimately diminish the consumer risk of parasitization.

Results
The three populations of hake were found parasitized by Ascaridoidea nematode larvae, with the Atlantic population of the European hake being the most parasitized (87.8%), followed by the silver hake (65.7%) and the Mediterranean population of the European hake (26.0%) (Table 1). However, the Mediterranean European hake presented a higher abundance than the silver hake, although the parasite load of the Atlantic European hake was much higher than that of the other two (Table 2).
Three clearly different Ascaridoidea morphotypes were found in the three hake populations, which were morphologically classified as family Anisakidae (Anisakis type I larvae and Contracaecum spp.) and family Raphidascaridae (Hysterothylacium spp.). Anisakis type I was the most prevalent and abundant morphotype in the three populations, with the two other morphotypes being much rarer in terms of prevalence and abundance (Tables 1 and 2). Additionally, two European Mediterranean hakes were found parasitized by larvae of Anisakis type II, one of which carried two larvae in the viscera and the other one larva in the flesh.  Parasitization by Anisakis type I larvae was also higher in the Atlantic population of the European hake than in the two other populations (Table 1), with this difference being statistically significant (χ 2 = 165.752; p < 0.0001). The Odds Ratio (OR) was 22.82 (12.98-40.12 95% CI) with respect to the Mediterranean population and 4.48 (2.58-7.76) with respect to the silver hake population. Moreover, the OR of the Northwest American population with respect to the Mediterranean was 5.10 (3.24-8.01).
Likewise, the mean abundance of Anisakis type I larvae was also higher in the Northeast Atlantic population than in the two other populations (Table 2), also with statistically significant differences: U = 6638.0; p < 0.0001 when compared to the silver hake population; and U = 5560.0; p < 0.0001 when compared to the Mediterranean population. However, surprisingly, as was appreciated in the total of Ascaridoidea larvae, the mean abundance of the Mediterranean hake population was higher than that of the silver hake population (U = 11333.5; p < 0.0001).
A higher presence of Anisakis type I larvae in the viscera than in the flesh was found in the three populations of hake analysed (Table 3), and with statistically significant differences in the Atlantic European hake (χ 2 = 4.704; p = 0.044) (OR = 1.67; 1.04-2.67) and in the silver hake populations (χ 2 = 165.752; p < 0.0001) (OR = 2.87; 1.83-4.515). When comparing the abundance between viscera and flesh, the highest larvae burden was found in the viscera in the Mediterranean (Z = 4.462; p < 0.0001) and in the silver hake (Z = 5.941; p < 0.0001) populations. However, in the Atlantic European hake population the mean abundance was higher in the flesh than in the viscera (Table 4), also containing a significant statistical difference (Z = 2.190; p = 0.029).
SE, standard error.

Influence of Intrinsic and Extrinsic Factors
Concerning the season of capture, the prevalence and the mean abundance of Anisakis type I larvae were, in the three hake populations, higher in the specimens caught in autumnwinter than those caught in spring-summer (Tables 3 and 4). However, only the differences of mean abundance of the Atlantic European hake (U = 1692.5; p < 0.0001) and the silver hake (U = 2705.0; p = 0.029) showed statistically significant differences.
Considering the two groups of weight and length in which each of the three populations of hake were divided (see Material and Methods section), Anisakis type I parasitization was always higher in the group of a larger host size. However, there were statistically significant differences concerning the prevalence in M. merluccius only, both in the Atlantic hake and in the Mediterranean hake populations. Concerning the mean abundance, the statistically significant differences were found in the three populations of hake (Tables 5 and 6). Table 5. Statistically significant differences found between weight groups and the prevalence and mean abundance of Anisakis type I of the three populations of hake (Merluccius spp.) analysed.  The higher helminth burden in the weight and length groups of the three populations of hake was confirmed when studying the influence of quantitative host size, with positive correlations related to length and weight and all of them statistically significant (Table 7). Moreover, the proportion of Anisakis type I larvae in the flesh related to the fish size of the three populations of hake analysed showed statistically significant negative correlations (Table 8). Regarding the presence of parasites in the flesh and the number of days passed between the catch and the analysis of the specimens, the results of the Binary Logistic Regression show that the prevalence of Anisakis type I increases with the days passed after the catch of the hake in the Atlantic hake (χ 2 = 15.304; p = 0.004) and in the Mediterranean hake (χ 2 = 23.849; p < 0.0001) populations. However, in the case of the silver hake population this positive relation was observed considering the spring-summer subpopulation (χ 2 = 8.342; p = 0.015) only.

Global Parasitization
The results obtained in this regional study from València can easily be extrapolated to the whole of Spain, as the supermarket chains surveyed are nationwide. Although they tend to sell fish from local and nearby fishing grounds, from the Atlantic or the Mediterranean, depending on their geographical location in the Iberian Peninsula, other fish stocks are usually also offered at the selling points originating from almost the same fishery areas.
The very high global prevalence of Hysterothylacium and Anisakis type I larvae in the samples of European Atlantic hake and silver hake, as well as the low prevalence in the sample of European Mediterranean hake stands out. These results are in agreement with the most of the previous studies carried out on samples of the European Atlantic hake [12,22,25,27,[31][32][33][34][35][36], the silver hake [37], and the European Mediterranean hake [12,25,33,[38][39][40]. In this last case, the studies carried out in the same Mediterranean subdivision, FAO 37.1.1-Balearic, were taken into account, with the exception of the study carried out by Valero et al. [26], which found a higher prevalence of parasitization in this area. However, the higher mean abundance of Anisakis type I found in the Mediterranean sample compared to the silver hake sample stands out.
This higher parasitization of M. merluccius in the fishery grounds of the Atlantic Ocean compared with Balearic division has also been reported in previous studies [12,25], as well as in other fish species too. This variation of the parasitization parameters in both geographical locations could be the result of various factors [26,41] such as the infection level of marine mammals, the definitive hosts of anisakids; the abundance of intermediate and paratenic hosts; and the role of each fish stock in the trophic chain (especially in the case of hake).
The presence of larvae belonging to the genera Hysterothylacium and Contracaecum was reported previously by other authors in some of the three hake populations analysed [12,28,37]. However, as far as we are aware, the report of Contracaecum in M. merluccius, both in European and Mediterranean hakes, as well as Hysterothylacium in M. bilinearis, are new records for this fish species.
In an analysis made on a smaller part of the samples analysed in the present research, the molecular analysis of some anisakid larvae was carried out [28] as previously described by Madrid et al. [1], identifying the following Anisakis species: A. simplex (s.s.), A. pegreffii and the hybrid genotype of both species, in the viscera and the flesh of the European Atlantic hake; A. simplex (s.s.) and the above-mentioned hybrid genotype in the viscera and the flesh of the silver hake, and A. pegreffii in the flesh of the European Mediterranean hake, although only one larva could be analysed from this population. On the other hand, the three larvae identified as Anisakis type II in two hakes originating from the Balearic division, could belong to the species A. physeteris according to the results of previous studies carried out in this fishing ground [25,26,[38][39][40].

Parasitization in the Viscera and the Flesh
In the three populations of hake analysed, the prevalence of Anisakis type I larvae was higher in the viscera than in the flesh. However, in the case of mean abundance, the European Atlantic hake population did not follow the usual pattern, with the larvae burden being higher in the flesh than in the viscera, reaching a total of 31.2 larvae per fish flesh.
The lowest value of parasitization observed in the European Mediterranean hake might be due to the fact that A. pegreffii is the most common Anisakis type I species in the Balearic division, a species that has been recognized to present a lower capacity of migration to the flesh than other Anisakis species, such as A. simplex (s.s.) [27,40,42,43].
Although almost all parameters of parasitization were higher in the viscera than in the flesh, with the above-mentioned exception, the high indices of parasitization in the edible fish part implies a real risk for consumers who eat undercooked or not conveniently processed fish. Additionally, this risk may increase with the post-mortem migration of Anisakis larvae from the viscera to the flesh, as will be discussed below.

Influence of the of Fish Catching Season on Anisakis Parasitization
The analysis of the fish catching season on larvae parasitization, mainly on Anisakis larvae showed a similar pattern in the three populations of hakes, with the autumn-winter period reaching the highest parasitization parameters, prevalence and mean abundance, compared to the spring-summer period, with or without statistically significant differences. However, these results are not in accordance with those reported in previous studies in this and other fish species previously analysed, which reported mainly, the highest parasitization parameters during the spring season [12,18,22,36,44,45]. Only a few studies reported a higher parasitation in fish caught during the autumn season, such as in the silver hake [1,12].
The reason for seasonal fluctuations in the population dynamics of Anisakis spp. Could be associated with the seasonal fluctuations of biotic and abiotic environmental conditions that indirectly influence the migration of aquatic mammals (final hosts), the amounts of parasite eggs shed, and the availability of zooplankton (intermediate Anisakis hosts) [30,34,44,[46][47][48].
Moreover, these differences are probably related to the seasonal patterns of migration of the fish species [12,48]. Nevertheless, in the opinion of other authors [49], seasonality in prevalence and intensity of Anisakis might not be expected as parasite eggs are shed by the final hosts throughout the year and, similarly, parasitation in crustaceans is non-seasonal.

Influence of the Fish Size on Anisakis Parasitization
Fish size, length and weight, influenced the Anisakis larvae parasitization parameters of the three hake population analysed, with the only exception being the prevalence in the silver hake. This positive relation between the size (age) of the fish and the increase of parasitization has been reported particularly in the case of hake analysed in various fishing grounds of the Atlantic Ocean and the Mediterranean Sea [12,22,[25][26][27][28]30,33,34,36,38,40], although this is not a universal phenomenon in fish, and this correlation might even be negative [22].
This positive correlation between the size of the hake an the increase of Anisakis larvae parasitation may be attributed to various factors [2,8,12,22,25,26,30,33,40]: (a) the cumulative parasitization of larvae along time and the higher likelihood of infection; (b)the reinfection over time through the diet of the hake and the fish spent feeding on its prey; (c) the change in the diet of the hake; piscivorous species such as adult hakes are usually more heavily infected with anisakid larvae compared to strict plankton fish feeders, e.g., sardines and anchovies; hake is a top predator that occupies different trophic levels during its life, and its diet shifts from euphausiids (<16 cm), to fishes consumed by larger individuals.

Migration of Anisakis Larvae to the Fish Flesh
In the three populations of hake analysed, the number of days that passed between fishing and the analysis of fish (which corresponds to the theoretical day of consumption) influenced the presence of larvae in the flesh. Other authors have also reported in various populations of hake analysed that Anisakis spp. Larvae are able to migrate from the visceral organs to the flesh after the death of the host, suggesting that this movement may be facilitated by the cold storage or processing of uneviscerated fish [12,22,28,31,46,49]. The days between the catch and the sale influenced the A. simplex complex presence in the flesh of parasitized fish that mainly feed on euphausiids, such as the blue whiting tend to accumulate anisakid larvae in their body cavity and viscera, although larvae are more abundant in the hypoaxial musculature of piscivorous fish such as hake and cod [18,28,50,51]. Moreover, the accumulation of larvae in the viscera seems to favour their migration to the flesh. Some authors suggest that factors such as temperature [4,52] or pH [53] play an important role in the post-mortem motility of larvae in the host. However, other experiments suggest that keeping the fish at low temperature could reduce larval migration [43].
Additionally, a negative correlation between the proportion of larvae in the flesh and the fish size was also observed in the three hake populations. Cruz et al. [51] put forward another hypothesis concerning the distribution of Anisakis larvae between the flesh and the viscera, proposing that the percentage of larvae in the flesh should be inversely related to the fish size as the migratory distance to the flesh increases with fish size. Intra-vitam migration of Anisakis larvae into the flesh is recognizable by the high number of melanized parasitic capsules in the flesh. It seems likely that the proximity of the fish gut and visceral cavity to the antero-ventral musculature facilitates the migration of worms toward this part of the fish but other factors (e.g., related to the immunological response of the fish, condition, density-dependent mechanisms and aggregating behavior) may also contribute to this pattern [22].
Conversely, some authors argue that fish size cannot be used as a reliable predictor of post-mortem larvae migration [1,44], suggesting that larval distribution might be governed by the conditions encountered within host tissues but not by distance.

Assessment of Human Anisakiasis Risk through the Consumption of Hake
The populations of European Atlantic hake and the silver hake can be classified as "high-risk" fish species for human anisakiasis, as previously proposed [12]. Only the European Mediterranean hake, due to its lower parasitization parameters, mainly flesh parasitization, and considering that the sample was caught from the Balearic division, can be classified as intermediate-risk fish species.
Considering the prevalence and abundance registered, all of them could represent a risk of infection when consumed raw, marinated, or poorly cooked, if not being adequately frozen beforehand. Related to this issue, although the disease may appear in different forms, it is necessary to consider the anisakiasis allergy risk due to the allergenic potential of dead larvae, as the presence of thermostable parasite allergens has actually been proved [54].
In general, the proposed human anisakiasis risk through hake consumption is in accordance with other results previously reported, specifically, that carried out by [34] in hake caught in some Northeast Atlantic fishing grounds, classifying the samples from the Grand Sole and Galician coast as FPR Poor Standard (that means very high-risk), and the sample from the Portugal coast as FPR Fair Standard (that means high-risk) of human anisakiasis.
Although anchovies have been recognized as the main fish species causing human anisakiasis, at least in Spain [4], other fish species, such as hake, have also been implicated in human parasitization (e.g.,: 9 cases of 96 gastrointestinal cases, reported in Madrid, after the consumption of undercooked hake [55]; a case of acute anisakiasis, also reported in Madrid, after the ingestion of fried hake and fish ova [56]; and three cases of gastric anisakiasis, reported in Barcelona, caused by the consumption of undercooked hake [24]).
This high-risk of hake has an important impact both on public health and in the fisheries economy, considering that this fish species is the most important demersal fish resource caught off temperate Western Europe, highly priced and mostly sold fresh in European markets [34]. On the other hand, the finding of the other two nematode larvae found parasitizing the three hake populations (i.e., the anisakid Contracaecum spp. and the Ascaridoidea Hysterothylacium, spp.), although the prevalence observed is relatively low (2-10%), cannot be overlooked, not due to their unclear zoonotic potential with only few human cases reported [57][58][59][60][61] if not mainly due to their deleterious effect at commercial level. Moreover, these two nematode larvae resembling Anisakis larvae to a non-expert, pose additional issues associated to the quality of the product. Consequently, consumers should be warned about its presence, especially in the viscera as the consumer's naked eye is not able to differentiate between these genera and Anisakis or other Ascaridoidea nematodes [12,45].

Global Preventive Measures and Recommendations to Consumers to Prevent the Human Anisakiasis Risk
To prevent human anisakiasis, the most effective measure is, according to the recommendations of the Commission Regulation (EU) No1276/2011 [62] and the Spanish Royal Decree 1420/2006 [63], cooking the piece until a core temperature of 60-70 • C for 5-10 min has been reached, or freezing fish at −20 • C at least for 24 h., at −35 • C for at least 15 h, in case of fish meant to be consumed raw or undercooked. Nevertheless, domestic freezers do not always provide a low homogenous temperature and are, therefore, not able to inactivate larvae. The freezing tolerance of these nematodes constitutes a safety risk when domestic freezers operating at low cooling capacity are used for the inactivation of these larvae, as temperatures are not sufficiently low or homogenous [12,45].
Moreover, the increase of the transmission risk through a prolonged time lag between the catch and the consumption of hake suggest the immediate evisceration after capture recommendable as an effective measure to prevent parasite presence [12].
Additionally, consumers must be warned about the presence and the potential effect of the parasite, thus preventing the possible confusion with Anisakis spp. larvae [12,45].

Samples and Parasitological Procedures
A total of 536 hake specimens (Table 9), belonging to the species M. bilinearis and M. merluccius, were purchased in branches of various nationwide Spanish supermarket chains located in the city of València and its metropolitan area. Hake specimens, fresh and uneviscerated, were chosen at random. All available information about the acquired fish specimens was obtained from the fish crates at the selling point. This information consisted mainly of: scientific and common names; date of capture; and FAO (Food and Agriculture Organization of the United Nations) major fishing area, subarea and division.  All specimens were refrigerated at 4 • C in the laboratory until dissection. Each hake specimen was weighed and the total length was measured. Specimens were classified into two groups according to weight (W1 and W2) and length (L1 and L2). However, different groups of weight and length were established considering that two species of Merluccius were analysed and, as above-mentioned, Atlantic and Mediterranean M. merluccius populations have to be considered as separated stocks. Moreover, specimens were also classified according to the season of capture in two periods: autumn-winter and spring-summer (Table 9). Additionally, the days (between 1-8 days) passed between the fishing date and the analysis (the hypothetical date of consumption) were also recorded.
As described by [12], the viscera and flesh of each hake were placed in two different Petri dishes and examined separately for the presence of nematode larvae. The viscera were dissected under a stereoscopic microscope and the flesh, after a previous visual inspection, underwent artificial enzymatic digestion [64]. The resulting product from the digestion was also examined under a stereoscopic microscope. Identification of each larva found was based on the morphological characteristics described in the literature [65,66]. The main characteristics considered for these classifications were the position of the excretory pore, the arrangement and separation of the digestive tract into the oesophagus, ventricle and the presence/absence of structures such as intestinal caeca and oesophageal appendix, as well as the shape of the tail. In the case of Anisakis, the differentiation between two groups of larvae (Anisakis type I and type II) was also carried out. As above-mentioned, molecular diagnosis of each specimen at specific level was not carried out, as the main objective of the study is to inform consumers about the presence of nematode larvae and how to minimize the risk of infection.

Statistical Analysis
The number of parasitized hosts, prevalence (frequency of recording), mean abundance and range of parasitization were analysed according to Bush et al. [67] for the total of nematode Ascaridoidea larvae found, as well as for each of the genera identified. Moreover, these parameters were also calculated for the parasitization site (viscera and flesh) and the season of capture.
The potential risk of human anisakiasis was assessed through the analysis of the influence of intrinsic and extrinsic factors on the prevalence and abundance of larvae identified as Anisakis type I. The complete statistical analysis comprised the comparison of prevalence (χ 2 test) and abundances (Mann-Whitney-U-test, in the case of season of capture, weight and length groups, and Wilcoxon-Z-test, in the case of sites of parasitization). The possible influence of the number of days after capture (which corresponds to the theoretical day of consumption) on the presence of larvae in the flesh was also analysed by Binary Logistic Regression (BLR), considering parasitized hosts only. Furthermore, the Spearman's rank correlation coefficient (Rho) was also applied to analyse the potential influence of quantitative intrinsic factors (weight and length) on the Anisakis type I burden, as well as their influence on the proportion of larvae in the flesh.
Statistical significance was established at p < 0.05. Statistical analyses were carried out using the IBM SPSS 26.0 for Windows (International Business Machines Corporation, Armonk, New York, NY, USA) and StatView 5.0 (Statistical Analysis System Institute Inc., Cary, NC, USA) software packages.

Conclusions
The following recommendations should be considered by consumers to avoid human anisakiasis through the consumption of hake, Merluccius spp., in any of its forms: cooking the piece until a core temperature of 60-70 • C for 5-10 min has been reached, or freezing fish at −20 • C at least for 24 h, at −35 • C for at least 15 h, in case of fish meant to be consumed raw or undercooked, and above all of the fish ought to be consumed fresh, avoiding prolonged refrigeration, and it should be eviscerated beforehand, preferably straight after the catch.
Consumers should be informed about the most important variables likely to increase the risk of human anisakiasis (i.e., the traceability data concerning the place and the date of capture), preservation method, as well as the scientific and the common name of the fish, the place of capture (FAO area and division) and the day of capture, available at the fish selling point and included on the crate of the fish, as regulated by the European Commission [68], that should be easily visible for the consumers.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.

Conflicts of Interest:
The authors declare no conflict of interest.