Molecular identification of Anisakis and Hysterothylacium larvae in commercial cephalopods from the Spanish Mediterranean coast

Veterinary Parasitology 220 (2016) 47–53

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Molecular identification of Anisakis and Hysterothylacium larvae in commercial cephalopods from the Spanish Mediterranean coast Gabriela Picó-Durán a,b , Lorena Pulleiro-Potel a,b , Elvira Abollo c , Santiago Pascual d , ˜ a,b,∗ Pilar Munoz a

Dpto. Sanidad Animal, Universidad de Murcia, 30100 Murcia, Spain Campus de Excelencia Internacional Regional “Campus Mare Nostrum”, Spain c Centro Tecnológico del Mar, Eduardo Cabello s/n, 36208 Vigo, Spain d Ecobiomar, Instituto de Investigaciones Marinas de Vigo. CSIC, Eduardo Cabello 6, 36208 Vigo, Spain b

a r t i c l e

i n f o

Article history: Received 24 September 2015 Received in revised form 18 February 2016 Accepted 19 February 2016 Keywords: Nematodes Anisakis Hybrid Hysterothylacium Cephalopods Mediterranean sea

a b s t r a c t This study aims to investigate the occurrence of nematode larvae in commercial cephalopods in the Western Mediterranean Sea. A total of 202 animals comprising 123 broadtail shortfin squid (Illex coindetii), 34 European squid (Loligo vulgaris) and 45 common octopus (Octopus vulgaris) were examined using enzymatic digestion. A total of 31 larvae were isolated (prevalence: 14.6%) and identified using molecular analyses which included PCR and sequencing of the ITS (ITS1-5.8S rDNA-ITS2) region. Phylogenetic tree inferred from ITS sequences yielded supported relationships for Anisakis (P: 12.2%) and Hysterothylacium species (P: 4.1%). All parasites were found parasitizing I. coindetii and, as expected, A. pegreffii presented the highest prevalence (11.4%). A. physeteris was also found with a lower prevalence (1.6%) but confirming the role of the broadtail shortfin squid as paratenic host and, its potential host for anisakidosis transmission. A hybrid larva between Anisakis simplex and A. pegreffi was also identified. All Anisakis larvae were found within the visceral cavity; in contrast most of the Hysterothylacium larvae were isolated from the mantle. A significant correlation was found between total nematode prevalence and depth, explained by the presence of larger broadtail shortfin squids inhabiting deeper depths. Therefore, the results obtained in the present study improve the knowledge of the occurrence of Anisakis and Hysterothylacium species in the I. coindetii from the Spanish Mediterranean Sea highlighting the importance of considering I. coindetii as a potential hazard for humans if it is consumed raw or not well cooked and the need of further research in other cephalopods. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Anisakidosis, produced by nematodes of the family Anisakidae or Raphidsacaridae, which are commonly referred to as anisakids, has acquired a high social relevance for causing digestive disorders or initiating hypersensitivity states or allergies (Audicana and Kennedy 2008; Mattiucci et al., 2013). These nematodes comprise a parasitic group widely distributed at geographical level, displaying a complex life cycle in aquatic ecosystem that involves various hosts at different levels in the food-web (Køie, 1993, 2001). Cephalopods and fishes are paratenic hosts for the anisakid larvae, while adult’s parasites are found in marine mammals, marine birds or fish.

∗ Corresponding author at: Departamento de Sanidad Animal, Facultad de Veterinaria, Universidad de Murcia, 30100, Murcia, Spain. ˜ E-mail address: [email protected] (P. Munoz). http://dx.doi.org/10.1016/j.vetpar.2016.02.020 0304-4017/© 2016 Elsevier B.V. All rights reserved.

Humans can become part of the cycle as accidental hosts by consuming raw or lightly cooked fish and cephalopods contaminated with third-stage larvae (Audicana and Kennedy, 2008). The genera Anisakis and Pseudoterranova are described as primarily responsible (Klimpel and Palm, 2011) whereas Hysterothylacium and Contracaecum are believed to be minor (Valero et al., 2003). According to the scientific opinion of the European Food Safety Authority (EFSA, 2010) protection and prevention are priorities in zoosanitary parasite control of fishery products for human consumption (Brogli and Kapel, 2011). In fact, EFSA (2010) recommended that research should be continued in parasites of public health importance in fishery products, regarding prevalence, intensity, anatomical location, as well as geographical and seasonal distribution. The present study has investigated the occurrence and taxonomic identification of Anisakis and Hysterothylacium larvae in three commercially-important cephalopod species in the Spanish Mediterranean coast. Remarkably, cephalopods are of increasing

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economic importance which is manifested by the rise in their global landings over recent decades (International Council for the Exploration of the Sea [ICES], 2012). They constitute a large portion of the species fished in the Spanish Mediterranean Sea, with annual landings of approximately 7501 tons between 2004 and 2012 according to the FAO, being one of the main compounding of the Mediterranean diet (Illescas et al., 2007; Osanz Mur, 2001). 2. Materials and methods 2.1. Sample collection The sampling plan was carried out within the framework of the Mediterranean International Trawl Survey (MEDITS financed by DG MARE and UE members Council Regulation (EC) N◦ 199/2008) by the Spanish Institute of Oceanography between May and June 2013. Trawlings were conducted in 91 different geographical sectors (Fig. 1 and Supplementary material) which were stratified into three zones based on the differences in seabed topography and water currents according to Lobo et al. (2014). Zone 1 (fishing hauls 1–14) is characterised by a very narrow continental shelf (from a few km to >20 km) and a steep slope to the shelf edge close to the coast. Zone 2 (fishing hauls 15–35) is characterised by a medium width shelf (from a few to >40 km). Zone 3 (fishing hauls 36–91) is characterised by a wide shelf (from a few to >80 km), by the presence of the Columbretes Islands which have been protected as a marine reserve since 1990 and an important current cetaceans passing between the island of Ibiza and the Iberian Peninsula. Trawlings were conducted with a bottom trawl (model GOC73) with a 4 m vertical opening and a 20 mm cod end mesh size. Depth was recorded by means of a CTD SBE-37 probe located in the mouth of the gear and ranged between 36.25 and 526 m. Further information on the sampling design and on the characteristics of the gear is available in the MEDITS-Handbook (2012). A total of 202 cephalopods specimens belonging to three different species (123 broadtail shortfin squid (Illex coindetii), 34 European squid (Linaria vulgaris) and 45 common octopus (Octopus vulgaris)) were randomly collected. Each specimen was identified at species level, sexed, weighted at the nearest gram, and the dorsal mantle length (DML) measured from the tip of the mantle to the midpoint between the eyes at the nearest millimeter. Body condition score (K) was calculated following Fulton’s index as K = BW/DML3 × 100 (Ricker, 1975) were BW was body weight. Maturity stage was determined according to the three-stage maturity scale described by Sánchez and Obarti (1993) which includes (I) immature (ovary whitish, very small and with no signs of granulation in females; spermatophoric organ transparent or whitish in males), (II) maturing (ovary yellowish with a granular structure; spermatophoric organ with white streaks of sperm) and (III) mature (ovary very large with plenty of eggs; spermatophoric sac with spermatophores). Then, cephalopods were immediately frozen on board and stored at −20 ◦ C. Samples were transferred in frozen conditions to laboratory and stored at −20 ◦ C until examination. 2.2. Parasite isolation and enzymatic digestion Once thawed, cephalopods were visually examined for the presence of nematodes. The viscera and muscle were digested separately in order to establish whether nematodes were present at the muscular level. The enzymatic digestion method used was based on the Codex Alimentarius Commission (CODEX, 2004) and the EU Regulation (EC) No 2075/2005 (EU, 2016). Digestion was carried out in a freshly prepared solution of 0.5% (w/v) pepsin (10000 FIP-U/g) and 0.063 M hydrochloric acid in distilled water in a ratio 1:10. The mixture was heated at 37◦C and

continuously stirred for 2, 4 or 24 h depending on whether a specimen of I. coindetii, L. vulgaris or O. vulgaris respectively was being digested. Digested tissue was poured through a sieve with a mesh size of 400 ␮m and flushed carefully with tap water. When the flesh was not dissolved completely, the solution was filtered through a sieve and washed with tap water. Then the remaining flesh was quantitatively replaced in the beaker with a proportional amount of solution and stirred under the same conditions until there were no large pieces left (CODEX, 2004). The presence of nematode larvae was evaluated observing the retained part by sieve on a Petri dish under stereomicroscope. All nematodes found were removed and preserved in ethanol 70% for molecular diagnosis. 2.3. Molecular identification Genomic DNA purification was performed employing NucleoSpin Tissue Kit (Macherey-Nagel, Easton, PA), according to manufacturer’s instructions. DNA quality and quantity was checked in a spectrophotometer Nanodrop® ND-2000 (Thermo Scientific). The entire ITS region (ITS-1, 5.8S rDNA gene and ITS-2) was amplified using the forward primer NC5 (5 -GTA GGT GAA CCT GCG GAA GGA TCA TT-3 ) and reverse primer NC2 (5 -TTA GTT TCT TTT CCT CCG CT-3 ) (Zhu et al., 2000). The primer pair amplified an approximately 950 bp product. PCR reactions were performed in a total volume of 25 ml containing 1 ␮l of genomic DNA (100 ng), PCR buffer at 1x concentration, 0.3 ␮M primers, 0.2 mM nucleotides and 0.625 U Taq DNA polymerase (Roche Applied Science). PCRs were carried out in a GeneAmp PCR System 9700 (Applied Biosystems). The cycling protocol was 10 min at 94 ◦ C, 35 cycles of 30 s at 94 ◦ C, 30 s at 55 ◦ C, and 1 min 15 s at 72 ◦ C, followed by 7 min at 72 ◦ C. A negative control (no DNA) was included in all PCR amplifications. The PCR products were separated on a 1% agarose gel in Trisacetate EDTA buffer, stained with Red Safe and scanned in a GelDoc XR documentation system (Bio-Rad Laboratories). PCR products were cleaned for sequencing using ExoSap-It reagent (GE, Healthcare, NJ, USA) for 15 min at 37 ◦ C, followed by inactivation for 15 min at 80 ◦ C. Sequencing was performed in a specialised service (Secugen, Madrid). Chromatograms were analysed using ChromasPro v.1.41 Technelysium Pty Ltd., All generated sequences were searched for identity using BLAST (Basic Local Alignment Search Tool) through web servers of the National Center for Biotechnology Information (USA). Multiple aligments based on ITS region of nematode parasites were performed using the sequences obtained in this study and others available from GenBank. Aligments were performed using Clustal W (Thompson et al., 1994) included in MEGA6 (Tamura et al., 2013). Maximum parsimony tree was constructed using MEGA6 software and reliability of the inferred tree was tested by 2000 bootstrap replications. The analyses of the ITS1-5.8SITS2 region included the sequences obtained in this study (n = 31) and the following sequences deposited in the GenBank: A. pegreffii (KJ011495, KF923927), A. simplex s.s. (KC663498, KF512906, KF953967, KF953969), A. physeteris (JQ912693, JN005754), hybrid beteween A. simplex and A. pegreffii (KF032056), Hysterothylacium sp. (JQ798963), H. deardorffoverstreetorum (JF30201) and H. tetrapteri (KF601901). The sequence of Ascaridia columbae (KF147909) was used as outgroup. 2.4. Statistical analysis Quantitative parasite descriptors such as prevalence (P), mean intensity (MI), and mean abundance (MA) were calculated according to Bush et al. (1997). Odds ratio and 95% confidence interval were obtained. Normality of the data was tested using a SHAPIROtest. Pearson’s correlation coefficient (r) was applied to determinate the degree of association between prevalence and biological

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Fig. 1. Map of the studied area (West Mediterranean) showing the geographical location of each zone (1–3) and the distribution of the sampled fishing haul.

(weight, length, body condition) or environmental (depth) variables. Associations between total nematode prevalence and the non-parametric variables (sex, maturity and geographical sector of capture) were explored using the correlation coefficient (r) with the ANOVA’s test (Kruskal-Wallis). According to geographical sector stratification, associations between total nematode prevalence as well as different nematode species prevalences and geographical sector of capture were calculated. Statistical significance was established at a p < 0.05. All statistical analyses were performed using RStudio statistical package (R Development Core Team, 2010). 3. Results Nematode prevalence in I. coindetii was 14.6% while no presence of nematode larvae neither in L. vulgaris nor O. vulgaris (Table 1). Table 2 shows P, MI, and MA of Anisakis and Hysterothylacium larvae found either in muscle or viscera of I. coindetii. In total, 37 (10 in the musculature, 27 in viscera) nematode specimens were recovered. Of them, 31 larvae were identified using molecular methods. Blast results showed that 21 nematodes belonged to A. pegreffii with identity values of 100%, 1 larvae was identified as a hybrid between A. simplex and A. pegreffii (identity value = 100%), 3 larvae as A. physeteris (identity value = 100%), and 6 larvae as Hysterothylacium sp. with identity values of 95% with Hysterothylacium sp. (JQ798963, HE610414) and Hysterothylacium deardorffoverstreetorum (JF30199, JF30200, JF30201, JF30202, JF30203, JF30204). ITS tree, constructed with a total of 740 sites and 44 sequences, showed that 21 sequences identified as belonging to A. pegreffii were grouped in the same clade as other known A. pegreffii

sequences with bootstrap values of 86% and three sequences were place together with A. physeteris sequences with bootstrap values of 100%. The sequence identified as belonging to recombinant genotype was grouped with other hybrid sequence between A. simplex and A. pegreffii and both were grouped closer to A. simplex sequences with bootstrap value of 76%. Six sequences identified as Hysterothylacium sp. were placed together with bootstrap values of 100% and they were clustered in the same clade that other sequences of the genus Hysterothylacium (bootstrap value of 100%) (Fig. 2). A significant correlation (Table 3) was found between total nematode prevalence and weight (p-value 0.00017), length (pvalue 0.0059) and depth (p-value 0.0091). Also, a significant correlation was observed between Anisakis spp. and A. pegreffii prevalence and weight, length and depth. In contrast, a significant correlation was found between the hybrid A. simplex and A. pegreffii prevalence and body condition score (p-value 0.049). No significant correlation between overall nematode prevalence and host sex or host maturity stage was observed. Although no significant correlation was observed at the 5% level between geographical sector of capture and Anisakis sp., A. pegreffi or overall anisakid prevalence, these prevalences were higher in Zone 3 of the study area than the other zones (Supplementary material). 4. Discussion Nematodes species isolated from I. coindetii summed up a total of 3 species belonging to two different genera. Anisakis spp. has already been reported from cephalopods inhabiting the Northwestern Atlantic (Abollo et al., 1998, 2001; González et al., 2003;

Table 1 Data on cephalopod species from West Mediterranean Sea sampled for nematode larvae. Species

No

DML ± SE (cm)

BW ± SE (g)

Fishing haul

Anisakid

Prevalence (CI 95%)

Illex coindetti Loligo vulgaris Octopus vulgaris

123 34 45

169.1 ± 24.53 155.2 ± 45.79 87.42 ± 20.95

137.5 ± 48.85 136.1 ± 101.34 550 ± 339.84

1,3–8, 10–16, 20−91 1, 7, 16, 18, 26, 32, 33, 44, 45, 74, 78, 79 1−4, 7–12, 17, 19, 27, 33, 40, 41, 43, 44, 46–48, 52, 59, 79, 88, 89

18/123 0/34 0/45

14.6 (0.091–0.224) 0 0

No = number of specimens sampled, DML = dorsal mantle length, SE = standard error, BW = body weight, CI = confidence interval.

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Fig. 2. Maximum parsimony tree of the different sequences obtained in this study (codes in italics) and others available from GenBank, showing the taxonomic position of the nematode larvae that were found infecting Illex coindettii from Mediterranean waters. The numbers above the branches are the parsimony bootstrap support value.

Mattiucci et al., 1997; Pascual et al., 1995, 1996, 1999) and Mediterranean waters (Angelucci et al., 2011; Farjallah et al., 2008; Gestal et al., 1999; Petric´ et al., 2011; Serracca et al., 2013). In fact, the most widespread species in the Mediterranean Sea is A. pegreffii (Abollo et al., 2003; Cavallero et al., 2012; Mattiucci et al., 2001; Mattiucci and Nascetti, 2006), showing a prevalence of 11.4% in our study. Nevertheless, most studies are based on fish, while only few studies have been focus in I. coindetti. Petric´ et al. (2011) recorded a

higher prevalence (P = 30.5%) while Serracca et al. (2013) reported its absence in this cephalopod. These differences may be due to heterogeneity in sample size and size of the squid, that seem to be the most important categorical predictors of parasite recruitment in squid populations (Pascual et al., 2005; Petric´ et al., 2011). Anisakis simplex sensu stricto is known to have a worldwide distribution mainly with benthic or demersal life cycle (Mattiucci et al., 1997; Abollo et al., 2001), although few records are from Mediter-

26/123 0.211 (0.145–0.296) 5/123 0.041 (0.015–0.097) 31/123 0.252(0.18–0.34) 1.62(1–5) 1.25 (1–2) 1.72 (1–6) 4/123 3.3 (0.01–0.086) 18/123 14.6 (0.091–0.224) Total

Hysterothylacium sp.

0 A. simplex x A. pegreffii

0 A. physeteris

CI = confidence interval.

1 (1–1) 1.25 (1–2) 1.2 (1–2)

1 (1–1) 0 1 (1–1)

1.5 (1–2) 0 1.5 (1–2)

1.5 (1.4) 0 1.5 (1–4) A. pegreffii

0

4/123 3.3 (0.01–0.086)

16/123 13 (0.078–0.205)

5/123 0.041 (0.015–0.097)

0

0

0

25/123 0.203 (0.138–0.287) 21/123 0.171 (0.111–0.251) 3/123 0.024 (0.006–0075) 1/123 0.008 (0.0004–0.051) 1/123 0.008 (0.0004–0.051) 0

Muscle Total

25/123 0.203 (0.138–0.287) 21/123 0.171 (0.111–0.251) 3/123 0.024 (0.006–0075) 1/123 0.008 (0.0004–0.051) 6/123 0.049 (0.0199–0.108) 1.8 (1–5) 0 1.8 (1–5)

15/123 12.2 (0.072–0.196) 14/123 11.4 (0.066–0.187) 2/123 1.6 (0.003–0.063) 1/123 0.8 (0.0004–0.051) 1/123 0.8 (0.0004–0.051) 0

15/123 12.2 (0.072–0.196) 14/123 11.4 (0.066–0.187) 2/123 1.6 (0.003–0.063) 1/123 0.8 (0.0004–0.051) 5/123 4.1 (0.015–0.097) Anisakis sp.

Mean abundance (CI)

Viscera Muscle Total

Mean intensity (range)

Viscera Muscle Total

Prevalence (%) CI Species

Table 2 Prevalence, mean intensity and mean abundance of larvae of Anisakis spp. (A. pegreffii, A. physeteris, hybrid between A. simplex x A. pegreffii) and Hysterothylacium sp. in the sampled IIlex coindetii.

Viscera

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ranean Sea. Gestal et al. (1999) reported a prevalence of 4.8% in I. coindetti (N = 42), while Angelucci et al. (2011) observed 50% (N = 4) and Petric´ et al. (2011) 30.5% (N = 439). Although we did not find this nematode species in I. coindetti, their absence cannot be confirmed in the studied areas due to the finding of a hybrid larva between A. simplex and A. pegreffii seems to indicate their presence in the surrounding waters. Abollo et al. (2003) was the first one to report the presence of a hybrid of A. simplex and A. pegreffii, suggesting Gibraltar Strait waters as a hybrid zone. Subsequent reports of heterozygote larval individuals were performed in the Mediterranean Sea (Cavallero et al., 2012; Chaligiannis et al., 2012; Farjallah et al., 2008; Abattouy 2012), Gilbraltar Strait (Marqués et al., 2006; Martin-Sanchez et al., 2005; Sequeira et al., 2010; Abattouy 2012), Japanese waters (Quiazon et al., 2008; Suzuki et al., 2010) and the Baltic Sea (Kuhn et al., 2013). Recently, Cavallero et al. (2014) reported the first record of adult hybrids in the definitive host Stenella coeruleoalba. The occurrence of A. physeteris has been reported in different Mediterranean fish but with low prevalences (Mattiucci et al., 2004; Valero et al., 2006; Farjallah et al., 2008) while a recent study has detected higher prevalence (33%) in Alepocephalus rostratus from Balearic Sea (NW Mediterranea). Farjallah et al. (2008) showed that A. pegreffii and, especially, A. physeteris are detected infecting mainly demersal fish rather than pelagic or benthic ones. Considering that I. coindetii is a demersal species, our study confirms the role of the broadtail shortfin squid as a paratecnic host of A. physeteris, as proposed by Mattiucci et al. (2001). They argued that the low prevalence of A. physeteris may point to cephalopods, rather than fish, as their main intermediate host. Our study showed a low but non-negligible prevalence of 1.6%, refuting the claims by Petric´ et al. (2011) against such hypothesis. Ceriola et al. (2006) described peak concentrations of I. coindetii in the Mediterranean Sea between 100 and 200m, but this distribution pattern differs depending on their life cycle phase, inhabiting a wide range of bottom depth from 50 to 200 m (Ceriola et al., 2006; Guerra, 1992). The specimens sampled in this study were caught in a range between 36.25 and 526 m depth, which is in concordance with previous data (González and Sánchez, 2002; MEDITS, 2012). The statistical correlation between depth and nematode prevalence can be explained by the presence of largest broadtail shortfin squids inhabiting deeper depths. In fact this statement was clear when a Pearson test was used to test the correlation between depth and weight (P = 0.001466, r = 0.284). Similarly, nematode prevalence was found to be positively correlated with I. coindetii mantle length and weight. It can be attributed to the cumulative effect of repeated parasite infections, acquired over a longer lifetime for larger (older) specimens through the diet (Adroher et al., 1996; Mattiucci et al., 2004), and also to the change of diet during life cycle (Valero et al., 2006). The broadtail shortfin squids’ juveniles usually prey on crustaceans and switch to fish and cephalopods as they grow (Boyle and Rodhouse, 2005), suggesting larger broadtail shortfin squids can harbour more nematodes. Compared with Petric´ et al. (2011) our infected broadtail shortfin squids presented higher size. However, our greatest number of larvae (6) was half from those recorded by them. All this indicates that infection in I. coindetii was detected at a later age in our study but not necessarily presenting a greater number of larvae. Even though this may suggest a lower risk of infection in I. coindetii from the Spanish Mediterranean coasts, our sample size might had not been big enough to confirm such a comparison with the specimens from the Adriatic Sea (Petric´ et al., 2011). Differences in values of infection in relation to sampling areas may be related to differences in habitat structure (Sousa and Grosholz, 1991) and bottom type (Smith, 1983); species biodiversity and biomass of neighbouring intermediate (mainly euphausiids) and paratenic hosts; trophic ecology; and climatic variables (wind speed and direction) which affect seasonal hydro-

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Table 3 Correlation coefficients between nematode prevalence in IIlex coindetii and biological and environmental variables. Species

Weight r (p)

Length r (p)

K r (p)

Sex r (p)

Madurity r (p)

Depth r (p)

Sector r (p)

Anisakis sp. A. pegreffii A. physeteris A. simplex x A. pegreffii Hysterothylacium sp. Total

0.282 (0.0016)a 0.305 (0.0006)a 0.049 (0.592) −0.053 (0.558) 0.177 (0.0497)a 0.332 (0.00017)a

0.235 (0.009)a 0.269 (0.0026)a 0.073 (0.423) −0.097 (0.286) 0.0999 (0.272) 0.247 (0.0059)a

0.021 (0.8201) −0.0289 (0.751) −0.049 (0.588) 0.178 (0.049)a 0.1402 (0.1219) 0.099 (0.275)

0.01473 (0.227) 0.01361 (0.2457) 0.001734 (0.678) 0.0008592 (0.77) 0.00255 (0.6145) 0.00472 (0.4932)

0.0038 (0.5387) 0.00314 (0.576) 0.001397 (0.709) 0.000692 (0.7929) 0.000311 (0.8603) 0.001141 (0.736)

0.268 (0.003)a 0.221 (0.014)a 0.069 (0.444) 0.195 (0.031)a 0.0684 (0.4524) 0.234 (0.0091)a

0.0287 (0.2384) 0.0451 (0.1048) 0.02377 (0.3047) 0.01196 (0.5498) 0.0202 (0.0904) 0.0256 (0.2781)

r = correlation coefficient, K = Body condition score, p = P-value, a correlation is significant at the 0.05 level.

graphic patterns (Pascual et al., 1999) as well as the occurrence of their preferential definitive hosts (Osanz Mur, 2001). In the case of A. pegreffii, the definitive host includes the bottlenose dolphin (Tursiops truncattus) as its main definitive host and common dolphin (Delphinus delphis) and striped dolphin (Stenella coeruleoalba) to a lesser extent (Mattiucci and Nascetti, 2008). As for A. physeteris, its main definitive host is the sperm whale (Physeter catodon), but they have been also described in the beaked whale (Ziphius cavirostris) (Mattiucci et al., 2001). The absence of significant differences in cetacean abundances reported among the three studies zones (Gómez de Segura et al., 2006), would explain the lack of relation between geographical zone of capture and nematode prevalence. Nevertheless, the lower parasite recruitment took place in broadtail shortfin squid populations at zone 1 which is associated with the oceanographic conditions of the Alboran-Oran front. It appears that instability in water masses caused by physical perturbations in this zone (i.e. water mass convergence and turbulent mixing) is associated with instability of trophic interactions over time, which in turn leads to a paucity of parasite communities in that area. This result also reinforces recent evidence provided by Gregori et al. (2015) supporting the hypothesis of Pascual and González (2007) on the relationship between parasite recruitment and oceanographic regime associated with major current systems of the World’s oceans. No nematodes were detected in the two other cephalopod species sampled in the present study, L. vulgaris and O. vulgaris. Gestal et al. (1999) and de la Torre Molina et al. (2000) reported the absence of nematodes in specimens of these two cephalopods caught in Mediterranean Sea, while Angelucci et al. (2011) reported Hysterothylacium sp. in O. vulgaris but not in L. vulgaris in the waters off Sardinia. Therefore, further research on these species is required in order to clarify the role of these cephalopods as paratenic hosts. Overall, results obtained in the present work improve the picture of the occurrence of Anisakis and Hysterothylacium species in the I. coindetii from the Spanish Mediterranean Sea, where is one of the most important cephalopods species caught by trawlers (Ceriola et al., 2007). As host for anisakid transmission, this squid species should be considered a potential alimentary hazard if it is consumed raw or not well cooked. For this reason, the presence of these nematode larvae especially in the muscle is a public health concern. As Cavallero et al. (2012) stated, anisakidosis is rarely caused by Contracaecum sp. and Hysterothylacium sp. but the possibility of causing allergic syndromes needs to be clarified. In the present study, the prevalence of Hysterothylacium sp. observed was slightly higher (P = 4.1%) than the one recorded by Serracca et al. (2013) (P = 1.6%) but, our cephalopod sample size was significantly higher. In contrast, members of Anisakis genus are of great concern been described as the most frequent cause of human anisakidosis in the Mediterranean Sea (Amelio et al., 2000; Fumarola et al., 2009; Serracca et al., 2013). Moreover, anisakidosis caused by A. physeteris has been reported in Spain (Clavel et al., 1993), confirming it as a human pathogen as well (Cavallero et al., 2012). Nevertheless, Anisakis spp. pathogenicity differs among the different species. A. simplex (s.s.) is more invasive and more tolerant of the acidity of

human stomachs than A. pegreffii (Jeon and Kim, 2015) which has similar pathogenicity to that of A. physeteris (Romero et al., 2014). The three species may be implicated in human anisakiasis, as they are capable of attaching to and penetrating the gastrointestinal wall of animals (Romero et al., 2014). Although the infection levels detected were low, mainly intensity, our study suggests that the implications of cephalopod consumption to human health must be taken into consideration. Acknowledgements The authors wish to thank the Spanish Institute of Oceanography, to all participants, in the cruise MEDIT GSA06 on board R/V “Cornide de Saavedra” as well as to Antonio Esteban, regional coordinator of the MEDIT program, for all help and support provided during sampling. J.M. Antonio Durán and Mariana Cueto (IIM-CSIC) were also thanks for their technical assistance. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.vetpar.2016.02. 020. References Abattouy, N., 2012. Prevalencia Y Factores De Riesgo De La Anisakiosis En El Norte De Marruecos (PhD Dissertation). Universidad De Granada, Granada, Retrieved from http://digibug.ugr.es/bitstream/10481/21040/1/20762173.pdf (accessed on 24/09/2015). Abollo, E., Gestal, C., López, A., González, A.F., Pascual, S., 1998. Squid as trophic bridges for parasite flow within marine ecosystems: the case of Anisakis simplex (Nematoda: anisakidae), or when the wrong way can be right. Afr. J. Mar. Sci. 20, 223–232. Abollo, E., Gestal, C., Pascual, S., 2001. Anisakis infestation in marine fish and cephalopods in Galician waters: an update perspective. Parasitol. Res. 87, 492–499. Abollo, E., Paggi, L., Pascual, S., DAmelio, S., 2003. Occurrence of recombinant genotypes of Anisakis simplex s.s. and Anisakis pegreffii (Nematoda: anisakidae) in an area of sympatry. Infect. Genet. Evol. 3, 175–181. Adroher, F.J., Valero, A., Ruiz-Valero, J., Iglesias, L., 1996. Larval anisakids (Nematoda: Ascaridoidea) in horse mackerel (Trachurus trachurus) from the fish market in Granada (Spain). Parasitol. Res. 82, 253–256. Angelucci, G., Meloni, M., Merella, P., Sardu, F., Madeddu, S., Marrosu, R., Salati, F., 2011. Prevalence of Anisakis spp. and Hysterothylacium spp. larvae in teleosts and cephalopods sampled from waters off Sardinia. J. Food Prot. 74 (10), 1769–1775. Audicana, M.T., Kennedy, M.W., 2008. Anisakis simplex: from obscure infectious worm to inducer of immune hypersensitivity. Clin. Microbiol. Rev. 21, 360–379. Boyle, P., Rodhouse, P., 2005. Cephalopods: Ecology and Fisheries. Blackwell Publishing, London, pp. 464. Brogli, A., Kapel, C., 2011. Changing dietary habits in a changing world: emerging drivers for the transmission of foodborne parasitic zoonoses. Vet. Parasitol. 182, 2–13. Bush, A.O., Lafferty, K.D., Lotz, J.M., Shostak, A.W., 1997. Parasitology meets ecology on its own terms: Margolis et al. revisited. J. Parasitol. 83, 575–583. Codex Alimentarius Commission, 2004. Standard for salted Atlantic herring and salted sprat. Codex Stan 244–2004, 1–8. D’Amelio, S., Mathiopoulos, K.D., Santos, C.P., Pugachev, O.N., Webb, S.C., Picanc¸o, M., Paggi, L., 2000. Genetic markers in ribosomal DNA for the identification of members of the genus Anisakis (Nematoda: Ascaridoidea) defined by polymerase chain

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