Anisakis simplex (s.s.) larvae (Nematoda: Anisakidae) hidden in the mantle of European flying squid Todarodes sagittatus (Cephalopoda: Ommastrephidae) in NE Atlantic Ocean: Food safety implications International of Food Microbiology

Few reports exist upon the occurrence and localization of zoonotic anisakid nematodes in T. sagittatus , especially in the mantle of the squid. The occurrence and site of infection of larval anisakids in 98 T. sagittatus caught West off St. Kilda, NE Atlantic Ocean, were investigated. Squids were examined for anisakids using the UV-Press method. In total, 689 nematodes were detected in the viscera and mantle. According to morphology, all the larvae (L 3 ) were assigned to genus Anisakis . Diagnostic allozymes and mtDNA cox 2 sequence analysis permitted to genetically identify all larvae as Anisakis simplex (s.s.) (N = 100). Overall prevalence (P = 81%) and mean intensity (mI = 8.6) of infection with A. simplex are provided. Most of the larvae present in the mantle cavity were embedded in the stomach wall or attached in the outer layer of the stomach and caecum (49%). Over a third of squids (37%) hosted A. simplex (s.s.) larvae in the mantle. A novel schematized representation of larvae distribution in the mantle is provided, showing where they were mostly located. According to the results obtained, the risk of anisakiasis associated with consumption of raw or undercooked T. sagittatus should be considered.


Introduction
The flying squids belonging to the Family Ommastrephidae, are commercially valuable cephalopod species, occurring throughout the oceans. Members of this family have an important role in marine foodwebs, and they represent key trophic elements in marine communities (Clarke, 1996). Todarodes sagittatus (Lamarck, 1798) is a very abundant and widespread ommastrephid pelagic squid that lives over the continental slope and oceanic waters (Dunning and Wormuth, 1998).
Interest in cephalopods has increased considerably in the last decades, because they represent a valuable target for fisheries, retain a high market value, and constitute an important food resource for human consumption (Gestal et al., 2019). Species of the Ommastrephidae family are the most important commercial fishery among cephalopods . Commercial catches of Ommastrephidae are thought to be composed mainly of Illex coindetii, Illex argentinus, Todaropsis eblanae, Todarodes pacificus, Todarodes sagittatus, and even if reports of commercial catches requests data by species, most landings are still identified only to family level (Gestal et al., 2019;ICES, 2019). Todarodes sagittatus has been caught for centuries as a bycatch in southern European trawl fisheries and exploited in specific jigging fisheries of some northern countries, especially in Norway (Borges and Wallace, 1993). The total catch in 2017 was 1759 t. (FAO, 2020). The species is mostly exploited in the Mediterranean Sea by Italy (1005 t.) followed by Spain (230 t.), and in the North East Atlantic mainly by Spain (411 t.), United Kingdom (76 t.) and Norway (29 t.) (FAO, 2020). Todarodes sagittatus is currently considered an underexploited species .
According to the UN Food and Agriculture Organization reports (FAO, 2016(FAO, , 2018, the use of squid for human consumption is extensive worldwide. The main consumers and importers of squid are Spain, Italy and Japan (Gestal et al., 2019;Vieites et al., 2019). In European countries, cephalopods are most commonly consumed cooked, and squidbased products are commercialized fresh, refrigerated, frozen, canned, salted or dried (Gestal et al., 2019). In Japan, squids are a traditional and valuable food resource, consumed in a large quantity, mainly raw, as sashimi and sushi (Nagasawa and Moravec, 1995).
Despite an increasing commercial significance of cephalopods in the EU and not EU-markets, and the numerous European recommendations (EFSA, 2010) on the risk of parasites in fishery products issued in recent years, little is known about the presence of pathogens in cephalopods (Gestal et al., 2019). Identifying pathogens and the resulting diseases is one of the main requisites in 'assessment of health and welfare' of animals, as suggested in the Directive 2010/63/EU (Gestal et al., 2019). Cephalopods are intermediate, paratenic, or definitive hosts of a range of parasites with different life cycle strategies (Gestal et al., 2019). They occupy an ecological niche that makes them vulnerable to infection by specific groups of parasites, which are transmitted to the definitive host, namely fish, marine mammals, or birds (Gestal et al., 2019). Among the various parasites of cephalopods, ascaridoid nematodes, comprehending some potentially zoonotic species, have a remarkable importance.
The genus Anisakis includes so far nine species of heteroxenous parasites of marine organisms, with crustaceans as first intermediate hosts, fishes and squids as intermediate and/or paratenic hosts, and cetaceans as definitive hosts (reviewed by Mattiucci et al., 2018). In fish and squids, the third larval stage larva (L 3 ) of Anisakis spp. can reside encysted on the visceral organs (Abollo et al., 2001;Gestal et al., 2019), or into the fish flesh or squid mantle (Cipriani et al., 2019). A. simplex (s. s.) is the most frequently reported Anisakis species in the NE Atlantic, with its distribution comprising coastal and pelagic marine realm extending above the Gibraltar Strait until Northern tip of the Arctic (Mattiucci et al., 2017a).
Epidemiological studies of ascaridoid nematodes in T. sagittatus and other ommastrephid species are scanty and based on few squid specimens. So far, A. simplex (s.s.) was detected in T. sagittatus fished in Spanish NE Atlantic waters (Abollo et al., 2001;Mattiucci et al., 1997;Pascual et al., 1996), A. physeteris and A. pegreffii in T. sagittatus originating from southern Mediterranean (Ionian Sea) (Costa et al., 2012); A. pegreffii in T. sagittatus from Central Mediterranean Sea (Mattiucci and Nascetti, 2008) and in T. sagittatus angolensis fished off South African coast (Mattiucci et al., 1997); whereas, Anisakis type II larvae (presumably A. physeteris) in central Mediterranean Sea (off Sardinia) (Angelucci et al., 2011). Third stage larvae of anisakid nematodes in cephalopods are generally reported as free in the mantle cavity or "encapsulated in flat spirals in the sheath of connective tissue surrounding the mantle muscle, mesenteries, gonads and internal wall of the stomach" (Abollo et al., 2001). None of these Anisakis larval specimens were detected encysted inside the mantle tissue of T. sagittatus. More recent evidence revealed that L3 of Anisakis can penetrate the mantle muscular tissue of squids, as observed for A. pegreffii larvae in Illex argentinus (Cipriani et al., 2019). Being the mantle a conspicuous edible part of the host, consuming it raw, marinated or undercooked poses a potential human health risk, as demonstrated by recent cases of human anisakiasis associated with consumption of raw squid in Japan (Furuya et al., 2018;Ogata et al., 2015;Tamai and Kobayashi, 2015).
The aim of the present study was to investigate the occurrence, tissue distribution and species composition of larval anisakid nematodes in T. sagittatus caught off the St. Kilda islands, NE Atlantic Ocean, in order to: i) identify larval nematodes to species level using morphological and molecular methods; ii) determine parasitic infection levels; iii) define the anatomical infection sites in the squid host.

Squid sampling
In total, 98 specimens of T. sagittatus were obtained as bycatch during a commercial trawl fishing cruise aimed to fish blue whiting (Micromesistius poutassou) by trawling West off St. Kilda islands (N58 • 04 ′ W09 • 40 ′ ), in the NE Atlantic Ocean (FAO 27 area, Division VI a) in May/ June of 2018 and April 2019 (Fig. 1). The squid species was determined according to the diagnostic keys provided in Guerra (1992). The squids were inspected on-board the commercial fishery and research vessel MS Kings Bay (Institute of Marine Research cruise no. "Kings Bay" 2018843). All specimens were measured (mantle length) to the nearest 0.5 cm before inspection for parasitic nematodes. Total body weight was not reported because several squids lacked body parts such as tentacles or arms, that were damaged during catch.

Parasitological analysis
Squids were first examined for the presence of anisakids by plain visual inspection, followed by standard UV-press method procedures . Each specimen was cut along the ventral mid-line of the mantle, viscera removed.
Squids stomachs were emptied before pressing them, to avoid counting the larvae that could be free within the digestive tract (present in the stomach content). Mantle was visually inspected by naked eye for eventual visceral larvae accidentally left on the surface during manipulation. Subsequently, the mantle was positioned flat open in a triangle shape, in order to map the infection site of larvae into it. Viscera, mantle and stomach content of each squid were placed in separate plastic bags, flattened to 1-2 mm thick layers in a hydraulic press at 8 bar, frozen for ≥24 h, and subsequently checked for nematodes under a 366 nm UVlight source (dead nematodes fluoresce when irradiated by UV-light) (Karl and Leinemann, 1993;Karl and Levsen, 2011;Levsen et al., 2018;Pippy, 1970). Nematodes recovered from squids were counted, washed in saline solution, and stored at − 70 • C for further morphological and molecular identification.

Morphological and molecular identification of larval nematodes
The recovered nematodes were morphologically assigned to genus level using bright field microscopy and by following the diagnostic keys according to Berland (1961).
A subsample of 100 Anisakis spp. larvae randomly selected between the ones detected in viscera and mantle of all infected squids, were identified to species level using a multi-marker genotyping approach, consisting of three diagnostic allozymes loci (Mattiucci et al., 2014) and mitochondrial cytochrome c oxidase II (mtDNA cox2) gene sequence analyses. The diagnostic allozyme loci (Adk-2, Pep C-1 and Pep C-2) were analyzed according to established procedures (see Mattiucci et al., 2014) on 100 Anisakis spp. larvae. The total DNA was extracted from ≈2 mg of homogenized tissues from each specimen, using the DNeasy® Blood and Tissue Kit (QIAGEN® GmbH, Hilden, Germany). For sequencing the mitochondrial cytochrome C oxidase subunit II (cox2) gene, PCR amplification was performed using the primers 211F (5 ′ -TTTTCTAGT-TATATAGATTGRTTTYAT-3 ′ ) and 210R (5 ′ -CACCAACTCTTAAAATTA TC-3 ′ ) (Nadler and Hudspeth, 2000). Polymerase chain reaction (PCR) was carried out according to the procedures provided by Mattiucci et al. (2014). The sequences obtained at the mtDNA cox2 for the larval nematodes were compared with those already obtained for the same gene in our previous works and deposited in GenBank:

Epidemiological data and statistical analysis
Quantitative infection assessment focused on nematode prevalence and abundance in squids was carried out separately for viscera and mantle. The epidemiological parameters considered, calculated using Statistica 13.1, were: prevalence (P, %), mean abundance (mA), and mean intensity (mI) with standard deviation (±SD) and range of infection (min-max) (according to (Bush et al., 1997). Correlation between squid length and values of the parasites abundance in the viscera, mantle, and overall, were tested by means of a Spearman rank test using Statistica® 13.3.0 (TIBCO Software Inc., CA, USA).

Morphological and molecular identification
A total of 689 nematode larvae were collected in the viscera and mantle of the examined squids. Based on basic diagnostic morphological characters, all larval ascaridoid nematodes were recognized as Anisakis third-stage larvae (L3) showing larval Type I characters (sensu Berland, 1961).
According to the alleles observed at the diagnostic loci, i.e., Adk-2 105 ,

Epidemiology and site of infection
Anisakis simplex (s.s.) larvae were detected both in the mantle cavity encapsulated outside the internal organs, and inside the muscular mantle tissue of the squids (Figs. 2 and 3). Larvae positioned over visceral organs were coiled, mainly attached to outer surface of the stomach, mesenteries and alimentary tract (Fig. 2). No larvae were observed free in the stomach contents, thus recently acquired through diet.
Mantle-infecting larvae were mostly situated in the posterior half of the mantle adjacent to the squid hosts' digestive tract organs (especially, stomach) (Fig. 3). Some of the larvae were completely embedded in the muscular tissue, whereas few others were attached on the internal surface of mantle, but firmly adhering on the mantle tissue, only removable ripping the tissue with tweezers.
Data on infected squid number, prevalence (P), mean abundance (mA), and mean intensity (mI) of A. simplex (s.s.) larvae at different sites of infection (viscera and mantle) are given in Table 1. Concerning the larvae present in the viscera, the 54.4% of larvae were detected on the surface of stomach (seldom in the stomach wall), while the rest 45.6% of them were attached to viscera in the mantle cavity.

Discussion
In the present study, data on occurrence and distribution of A. simplex (s.s.) infecting T. sagittatus from West off St. Kilda islands (NE Atlantic Ocean) are presented. The findings represent the first molecular detection of the zoonotic nematode A. simplex (s.s.) in the tissue mantle of T. sagittatus.
The overall infection level of A. simplex (s.s.) in T. sagittatus in the present study (P = 81% mI = 8.58) were higher than previous findings of anisakid nematodes in the same host species originating from other localities of the NE Atlantic Ocean and from the Mediterranean Sea. Pascual et al. (1996) conducted an epidemiological study on a wide range of cephalopod species fished in several fishing grounds off Galicia (NW Spain), and reported the presence of A. simplex B (former name of A. simplex (s.s.)) in the visceral cavity of 22 out of 65 T. sagittatus specimens examined (P = 34%). In the same geographical area, Abollo et al. (2001) reported 34% prevalence and 7.55 mean intensity of A. simplex (s.s.) in 70 T. sagittatus (mean mantle length 333 mm). The authors inspected the squid mantle by using peptic digestion method, but they did not detect Anisakis larvae in this tissue. Regarding epidemiological data of nematodes in T. sagittatus from the Mediterranean Sea, only 4 out 60 specimens fished in the Ionian Sea and South of Sicily resulted infected with Hysterothylacium sp., A. pegreffii and A. physeteris (Costa et al., 2012). Squids were visually inspected, and no larvae were detected in the mantle. Further, Angelucci et al. (2011) reported the presence of Anisakis type II and Hysterothylacium sp. in a small batch of 5 T. sagittatus obtained from a catch off Sardinia (central Mediterranean Sea).
Some studies on the parasite load of ommastrephid squids from the North Pacific Ocean, most referring to Japanese waters, reported the presence of Anisakis spp. in Todarodes pacificus. In a really large sample size of 2153 T. pacificus, a 3.2% prevalence of A. simplex (s.l.) was observed by Takahara and Sakurai (2010). In the same squid host, Nagasawa and Moravec (1995) detected "A. simplex" larvae, without details on the epidemiology. Unfortunately, no genetic identification   was provided in support of these observations, and larval stages of the A. simplex (s.l.) complex cannot be identified on the basis of morphological traits. Several of the aforementioned studies were based on a visual approach methodology, and candling, for the detection of helminth parasite in squid. This methodology, as detailed by Levsen et al. (2005) for fish, can lead to an underestimation of the total burden of anisakid parasites, and consequently loss of information on the larvae present in certain organs/tissue of the host, mostly in the muscular tissue. The presence here reported of several larvae of A. simplex (s.s.) in the mantle of T. sagittatus, and the higher prevalence recorded, could be partially explained by the efficiency of UV-press method in detecting Anisakis larvae in the squid mantle. The efficiency of this method used to detect Anisakis spp. larvae in fish hosts (Gómez-Morales et al., 2018), and also in squids, as I. argentinus (Cipriani et al., 2019)  Besides this methodological aspect, the higher parasite burden of the squid examined in this study could be addressed to ecological drivers, such as the squid population of origin, their feeding habits and prey availability (discussed further), their migratory patterns, the year of capture (owing to the short life span of squids), or even large scale environmental changes occurring currently in NE Atlantic area (see Levsen et al., 2020). Considering that the geographically closest previous parasitological study on T. sagittatus in this ocean district was carried out in 2001 (Abollo et al., 2001), the overall scanty data on parasite in ommastrephids so far available, and the short life span of these voracious predators, it is difficult to propose any explanation supporting the higher parasite burden found in the present study with respect to the past surveys.
The mean length of the 98 specimens of T. sagittatus was 248 ± 46 mm (min 150 mm, max 395 mm) (Table 1). According to an age/size relationship of T. sagittatus proposed by Rosenberg et al. (1981), squid with a size ranging from 150 mm to 395 mm could have an age ranging from 220 to 365 days.
In host-parasite systems such as ommastrephid squid, the helminths usually survive the whole host-life cycle (Hochberg, 1990). Morsan et al. (1999) reported a similar trend, with Anisakis sp. larvae prevalence and abundance showing an increment with squid age. According to histopathological studies, a typical cell mediated immune response to anisakid nematodes was observed on cephalopod tissue (Ford, 1992;Gestal et al., 2019), resulting in an encapsulation of the parasite as immune strategy to avoid parasite migration (Gestal et al., 2019). Thus, the positive and statistically significant correlation (r = 0.394, p < 0.005) between T. sagittatus mantle length and A. simplex (s.s.) overall abundance (viscera and mantle) (Fig. 4) can be explained by a biological accumulation of larvae through diet during the life span of the squid host. Ecological and behavioral traits of the cephalopod life cycle (namely: deep range, bathymetry, mantle size, potential fecundity and vagility, feeding behavior) appear to be important determinants of parasite richness (González et al., 2003). Furthermore, larval accumulation seems to be facilitated by the longevity of Anisakis spp. larvae, which may stay alive for long periods or even over fish host's lifetime (Hemmingsen et al., 1993;Køie, 2001;Smith, 1984). Todarodes sagittatus is an opportunistic predator, in upper level in the trophic web of marine ecosystems, feeding mostly on pelagic and mesopelagic fish species of a certain size (Joy, 1990;Lordan et al., 2001;Piatkowski et al., 1998;Quetglas et al., 1999). A particularly important prey of T. sagittatus in NE Atlantic seems to be represented by myctophids Maurolicus muelleri, Micromesistius poutassou and Argentina sp. (Piatkowski et al., 1998;Lordan et al., 2001). Some observations of the stomach content of T. sagittatus here examined, showed the presence of fish scales and residuals probably traceable to small mesopelagic myctophids. Furthermore, T. sagittatus were sampled as bycatch of M. poutassou, and myctophids were also spotted in the same catch. These observation on squid feedings seems in accordance with findings reported by Lordan et al. (2001). Furthermore, the finding of A. simplex (s.s.) in some myctophid spp. and M. muelleri (Bao et al., paper in preparation), and a high prevalence and abundance of A. simplex (s.s.) in M. poutassou in the same fishing ground (personal observation; Levsen et al., 2018), suggest that both those myctophids species and M. potassou could represent a source of A. simplex (s.s.) infection for T. sagittatus.
The localization and distribution of A. simplex (s.s.) in T. sagittatus seems to be strictly linked to their foodborne transmission, as larvae are acquired through predation. Most of the larvae present in the mantle cavity were embedded in the stomach wall or attached in the outer layer of the stomach and caecum (49.3%), surrounded by a thin capsule (Fig. 2). It appears that the majority of larvae penetrate the stomach wall and then ensheath over the digestive tract or move into the portion of the mantle tissue directly in contact with these organs (Figs. 2, 3). In squids, the stomach can be extremely expandable, and it serves as a storage area for the initial digestion of prey items, while the caecum is a site of absorption (Gestal et al., 2019). Thus, all larvae acquired through diet tend probably to crawl out digesting matter and penetrate initially into the mantle cavity through these two organs. Furthermore, larvae seem to overcome limited migrations inside the squid host, remaining mostly (49.3%) "nearby" the site of absorption (stomach and caecum). The schematization of the distribution of larvae detected in the mantle proposed in Fig. 3 furthermore supports this finding, showing the most of muscle invading larvae located in the anterior half of the mantle, surrounding caecum and stomach.
Since larval L3 stage of A. simplex (s.s.) is the etiological agent of the zoonotic disease known as human anisakiasis (D'Amelio et al., 1999;Fumarola et al., 2009;Lim et al., 2015;Mattiucci et al., 2011Mattiucci et al., , 2013Mattiucci et al., , 2017bMattiucci et al., , 2018Umehara et al., 2007), the presence of larvae in T. sagittatus, above all in the edible mantle, might represent a risk if squid are consumed raw or poorly cooked. Even though ommastrephid squid catches are discontinuous, they assume a consistent importance in some countries. The use of squid for human consumption is extensive in Spain, Italy, and Japan, being these countries the main consumers and importers of these fish species (FAO, 2016(FAO, , 2018Gestal et al., 2019). Despite of this commercial interests, the presence of zoonotic anisakid nematodes in squids, and cephalopods in general, has been poorly investigated. The few epidemiological studies on anisakid nematodes in T. sagittatus report the presence of larvae in the visceral cavity, but the present study appears to be the first to report the presence of A. simplex (s.s.) in the mantle tissue of this squid species. The infection level of A. simplex in the mantle of T. sagittatus here reported, show that a third of caught squids (36.7%) had at least one larva in the mantle, with mean intensity 1.78, and with a single squid hosting 8 larvae. The most of mantle invading larvae were completely embedded in the muscular tissue, whereas few others were attached on the internal surface of mantle, but firmly attached to the mantle tissue. Thus, the risk of anisakiasis associated with consumption of raw or undercooked preparation should be considered. According to the larvae distribution schematized in Fig. 3, some specific portions of the squid mantle (anterior half, tissue surrounding caecum and stomach) could carry a higher number of parasites, while tentacles and other part of the edible tissue could have negligible risk of containing zoonotic agents. Applied rules to prevent zoonotic hazard by anisakid nematodes for commercialization, marketing and preparation of seafood, as recommended by EFSA (2010), should reduce the risk. Even if cephalopods in European countries are mostly consumed cooked, in Japan, indeed, a large quantity of squids are consumed fresh and raw, in sashimi and sushi preparations (Nagasawa and Moravec, 1995). In Japan the Anisakis risk is well known. The Japanese common squid (T. pacificus) is one of major fisheries resources (Nagasawa and Moravec, 1995), and its traditional consumption is considered one of the most frequent sources of anisakiasis cases in that country (Nagasawa, 1990). Several recent cases of human anisakiasis have been associated with consumption of raw squid in Japan (Furuya et al., 2018;Ogata et al., 2015;Tamai and Kobayashi, 2015).
Considering the current globalization of food preparations, and ongoing trend of consumption of raw or lightly processed seafood, getting common also in western countries, preventive measures to kill the parasite, as recommended by EFSA (2010), should be applied whenever T. sagittatus are to be consumed raw or lightly processed.

Declaration of competing interest
The authors declare that they have no conflicting interests