Anisakid and Raphidascaridid parasites in Trachurus trachurus: infection drivers and possible effects on the host’s condition

This study investigated the distribution of nematode larvae of Anisakidae and Raphidascarididae (genera Anisakis and Hysterothylacium) in Trachurus trachurus (Linnaeus, 1758) in the Ligurian and central-northern Tyrrhenian Seas. The relationship between the number of parasites and the length and weight parameters of the fish was assessed, and the possible effect of the parasites on the condition factor was evaluated. A total of 190 T. trachurus specimens were collected in July 2019. Parasites were found in 70 individuals. A total of 161 visible larvae were collected in the viscera. Morphological analysis revealed the presence of Anisakis spp. in 55 fish and Hysterothylacium spp. in 15 fish, while 5 fish showed coinfection with both genera. The specimens subjected to PCR (n = 67) showed that 85% of the Anisakis larvae analyzed belonged to the species A. pegreffii, while the remaining 15% belonged to hybrids of A. pegreffii-A. simplex (s.s.). A total of 58% (n = 7) of the Hysterothylacium larvae analyzed belonged to the species H. fabri, while 42% belonged to the species H. aduncum. Our results support the hypothesis that infection with these parasites does not affect the condition of the fish host analyzed, and that body size and depth are major drivers in determining infection levels with Anisakid and Raphidascaridid nematodes.


Introduction
Anisakidosis is a fish-borne zoonosis following ingestion of the third larval stage of nematodes of the family Anisakidae. Within the Anisakis simplex Rudolphi, 1809 complex, the species A. simplex (s.s.) and A. pegreffii Campana-Rouget andBiocca, 1955 (Mattiucci et al. 2014) are recognized as the main causative agents of anisakiasis, a condition related to the consumption of raw, marinated, or undercooked fish filets infected by the third-stage larvae of these parasites. In Mediterranean waters, the species A. pegreffii is dominant and is also the main etiological agent of anisakiasis and is distributed in numerous paratenic and definitive hosts (Mattiucci and D'Amelio 2014;Mattiucci et al. 2019).
Species of the genus Hysterothylacium Ward and Magath, 1917, formerly belonging to the Anisakidae and currently assigned to the family Raphidascarididae, are common parasites in different marine and freshwater fish species (Bezerra et al. 2020). H. aduncum Rudolphi, 1802 andH. fabri Rudolphi, 1819 are the most frequently reported species in teleost fish from the Mediterranean region (Roca-Geronès et al. 2018;Tedesco et al. 2018). Evidence of the direct consequences of Hysterothylacium infection on fish health is limited: parasites of this genus are considered only mildly pathogenic for adult fish (Ishikura et al. 1993;Yagi et al. 1996;Valero et al. 2003;Cavallero et al. 2012); however, mortality episodes in larval and juvenile fish have been reported 1 3 (Bristow 1990;Balbuena et al. 2000). Although generally not listed among fish-borne zoonotic agents, preliminary evidence on the allergenic potential of Hysterothylacium species (Fernández-Caldas et al. 1998;Valero et al. 2003) suggests their importance in relation to food safety and human health.
Monitoring the occurrence of Anisakis and Hysterothylacium in wild fish for human consumption is therefore necessary, particularly regarding selected species (Debenedetti et al. 2019) considered at higher risk of infection. Furthermore, the high parasite load reported in susceptible fish species (Manfredi et al. 2000;Angelucci et al. 2011) highlights the need to investigate the effects of parasites on the host's condition.
Among Mediterranean fish species at high risk of Anisakids and Raphidascaridids, the Atlantic horse mackerel Trachurus trachurus Linnaeus, 1758 (Trachuridae, Carangidae) is a gregarious bentho-pelagic species, widely distributed throughout the Mediterranean Sea including the Black Sea (Bini 1967) and eastern Atlantic from Iceland to Senegal , and supports large fisheries (Abaunza et al. 2003), both as target and by-catch species. This species feeds on small fish and planktonic crustaceans and may become infected by both Anisakis and Hysterothylacium larvae by consuming euphausiids, which are intermediate hosts of these nematodes (Smith 1983;Adroher et al. 1996).
In the present study, we surveyed the occurrence and distribution of Anisakis spp. and Hysterothylacium spp. in the Atlantic horse mackerel, T. trachurus, caught in the FAO-GFCM Geographic Sub-area 9 (GSA9), Ligurian Sea and central-northern Tyrrhenian Sea, investigating the effect of infection on the host's condition and the influence of different biological (total length, total weight, sex) and environmental (depth) variables.

Study area
Atlantic horse mackerel specimens were sampled in July 2019 in the Ligurian and central-northern Tyrrhenian Seas (FAO-GFCM Geographic Sub-area 9) (Fig. 1) by trawling at depths ranging from 18 to 330 m during the implementation of the EU-funded Mediterranean international trawl survey (MEDITS project, Spedicato et al. 2019). After capture, samples were frozen immediately on board and transported to the Centro Interuniversitario di Biologia Marina "A. Bacci" (CIBM) labs for the analysis.

Fish samples and parasitological examination
For each specimen, total length (TL, to 0.5 cm below) from the tip of the snout to the end of the tail and total weight (TW, g) (weighing scale precision 0.1 g) were recorded. Sex was determined through macroscopical examination of gonads.
Length-weight relationship was analyzed by means of the power equation W = aTL b , where W is the total weight and TL is the total length. The Le Cren (1951) relative condition factor (Kn), expressing the condition of a fish in numerical terms, was calculated from the observed total weight and theoretical weight (EW, g) estimated from "a" and "b" parameters of the length-weight relation. For the parasitological examination, the abdominal cavity was examined by visual inspection, while the internal organs were observed under a stereomicroscope (magnification 8-× 35) for the presence of third-stage larvae (L3) of Anisakid and Raphidascaridid nematodes.

Morphological analyses
All collected larvae were identified at the genus level according to their general morphology (Hartwich 2009;Gibbons 2010), through observation under light microscopy. The prevalence, mean intensity (MI), and mean abundance (MA) values of larvae belonging to each genus were calculated according to Bush et al. (1997).

Molecular analyses
Genomic DNA was extracted from the central part of the larvae body by the PureLink® Genomic DNA Kit (Life Technologies, Carlsbad, CA) following the manufacturer's instructions. Amplification of the complete ITS rDNA region was performed with primers NC5_f (5′-GTA GGT GAA CCT GCG GAA GGA TCA TT-3′) and NC2_r (5′-TTA GTT TCT TCC TCC GCT -3′) (Zhu et al. 1998). The PCR products were electrophoresed on 1% agarose gel stained with SYBR Safe DNA Gel Stain (Thermo Fisher Scientific, Carlsbad, CA) in 0.5X TBE. For the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), 10 µl of the PCR product were digested with 1.5 µl of restriction enzymes HinfI, HaeIII, and AluI (D'Amelio et al. 2000;Tedesco et al. 2018), in a volume of 20 µl at 37 °C for 90 min (Abollo et al. 2003). The restriction fragments were separated in 3% agarose gel stained with SYBR Safe DNA Gel Stain in 0.5X TBE. Sequenced A. pegreffii and A. simplex (s.s.) were used as positive controls in every reaction. After the electrophoresis, some specimens showed hybrid restriction patterns; therefore, in order to exclude the possibility of incomplete digestion, they were digested for longer time (240 min).
Sequenced A. pegreffii and A. simplex (s.s.) were used as positive controls (K +) in every reaction.

Data analysis
The Chi-square test (significance level 0.05) was performed to assess possible significant differences in the prevalence of Anisakid and Raphidascaridid parasites between male and female fish and also to test the relationship between the prevalence of nematode parasites and depth.
The analysis was performed using the JMP statistical package (SAS, Jmp 2007).
Regarding the length-weight relationship, Student's t-test was applied to test allometric growth ("b" = 3) (Pauly 1984) and differences between sexes.
Data exploration was performed to check correlation among variables (TL, TW, sex, Kn, and number of parasites), and a graphic output was produced (pairplot); the relationship between the number of parasites and biological parameters (TL, TW, sex) was tested by ANOVA. The possible effect of parasites on the condition factor was evaluated by Student's t-test.
The prevalence of single or multiple infections of nematodes larvae with a 95% confidence level, based on the results of microscopic analysis, was calculated for the whole sample. The prevalence of Anisakids and Raphidascaridids in both fish sexes was subjected to statistical analysis, using the Chisquare test, and was considered significant at P < 0.05.

Results
A total of 190 specimens of T. trachurus were collected during the MEDITS survey in July 2019, of which 107 were female and 83 were male. Body size ranged from 10.0 to 31.0 cm TL in females and from 10.0 to 31.5 cm TL in males.
Parasites were found in 70 individuals: 30 males and 40 females; prevalence, mean intensity, and mean abundance of parasites recorded for all specimens are reported in Table 1.
A total of 161 visible larvae were collected in the viscera. Morphological analysis revealed the presence of 129 (28%) Anisakis spp. larvae in 55 fish: 21 males and 34 females and 31 larvae (10.5%) Hysterothylacium spp. in 15 fish: 9 males and 6 females, while 5 fish showed coinfection with both.
All the values are reported in Table 2. Statistical analysis showed that the fish were more significantly infected with Anisakis larvae than with Hysterothylacium spp. (p = 0.032); however, no statistically significant differences in infection values were observed between sexes.
Length-weight relationship was calculated by sex and the results are shown in Fig. 2a and b and Table 3. The "b" parameter differed significantly for each sex: females showed positive allometric growth, while males showed isometric growth. Statistically significant differences between sexes were not detected (t-value 0.498; p > 0.05).
The condition factor (Kn) ranged from 0.80 to 1.98: the minimum values were 0.81 and 0.80 for males and females, respectively, while the maximum values were 1.98 for males and 1.27 for females. No statistically significant differences in the condition factor emerged between males and females (t = 0.190; p > 0.05) and between parasitized and non-parasitized individuals (t = 0.986; p = 0.325). Variations in the condition factor in relation to total length are shown in Table 3. A preliminary data exploration highlighted a relationship between total length and total weight with the number of parasites (Pearson correlation coefficient (PCC) = 0.5); there was also a correlation between total length and total weight (PCC = 0.9).
A significant and positive correlation was found between the number of parasites and total length (t = 7.532; p < 0.05) and total weight (t = 8.786; p < 0.05), while sex was not significantly correlated (t = 0.925; p > 0.05).
With regard to molecular analyses, all the specimens subjected to PCR (n = 67) were successfully amplified, showing bands of ~ 1000 bp. The PCR-RFLP showed that 85% (n = 47) of the Anisakis larvae analyzed belonged to the species A. pegreffii, while in the remaining 15% (n = 8), hybrids of A. pegreffii-A. simplex (s.s.) were detected (Fig. 3a).
A total of 58% (n = 7) of the Hysterothylacium larvae analyzed belonged to the species H. fabri, while 42% (n = 5) belonged to the species H. aduncum (Fig. 3b and  c). For confirmation, the hybrids were re-digested with the same enzymes for 240 min.

Discussion
The present study provides information on the distribution of third-stage larvae of A pegreffii, H. aduncum, and H. fabri in T. trachurus from the Ligurian and Tyrrhenian Seas (western Mediterranean), correlating the infection data with the biological and biometric features of the hosts.
Concerning the molecular analysis, the PCR-RFLP identified A. pegreffii and hybrids A pegreffii-A. simplex and H. fabri and H. aduncum. The hybrid A. pegreffii-A. simplex (s.s.) has been described in T. trachurus from the Cantabrian Sea (Abollo et al. 2003) and from the coasts off Sardinia .
The results are in agreement with the evidence that A. pegreffii is the dominant Anisakis species in the Mediterranean Sea, as highlighted by Mattiucci et al. (2018). The occurrence of hybrids in the Mediterranean Sea, detected through PCR-RFLP of the ITS region of rDNA and other molecular markers, has also been reported by numerous studies in other fish species and in marine mammals (Abollo et al. 2003;Meloni et al. 2011;Cavallero et al. 2012;. The reason for the spread of hybrid genotypes in the Mediterranean is still unclear ). In the northeastern Atlantic and in the western Mediterranean, A. simplex (s.s.) and A. pegreffii are known to occur in sympatry (Abollo et al. 2001; and may undergo interspecific hybridization. However it is unclear whether this phenomenon results in a higher or lower fitness of hybrids compared to parental species and therefore in a higher or lower infectivity or the possibility of parasitizing different host species. Future investigations considering multiple molecular markers (Mattiucci et al. 2018) may shed further light on these aspects.
With respect to the biological and biometric features of the T. trachurus examined, the "b" values for females (b = 3.145) showed positive allometric growth, with the growth in length proportionally bigger than the growth in weight. For males (b = 2.937) and the total sample, isometric growth was recorded. Similar results have been found in other areas of the western (Gancitano et al. 2011;Ligas et al. 2012;Spedicato et al. 2012) and eastern Carbonara et al. 2012;Santojanni et al. 2013) Italian Seas.
Le Cren's condition factor was applied to assess the fish welfare linked with the length-weight relationship, which can be influenced by parasites (Dias et al. 2015;Silva et al. 2013;Santos et al. 2013) as well as factors such as gonad maturation and feeding (Verani et al. 1997).
A strong correlation (P < 0.0001) was found between the number of nematode parasites in the viscera and the body size. However, our results suggest that Anisakid and Raphidascaridid parasites do not influence the state of health of the horse mackerel in terms of body condition. This result is in accordance with the results of a previous study (Ichalal et al. 2015) which failed to detect a negative impact of A. simplex and H. aduncum on the condition of T. trachurus based on the analysis of Fulton's condition index. In fact, very few studies have explored the effect of Anisakis infection on the body condition of fish and with contrasting results (Podolska and Horbowy 2003;Lagrue and Poulin 2015).
In our study, a trend in Kn value was observed in relation to the length, but it did not differ from 1. Kn increased between 16.5 and 18.0 cm TL and then decreased. A similar trend was recorded by Alegria-Hernandez (1994) and Šantić et al. (2011), which is linked to the development and maturation of the gonads: after length at first maturity, 18.8 cm in GSA9, (MEDISEH 2013), Kn values decrease due to the high energy demand required by reproduction. Differences in the maturity stage of the fish could therefore mask the effect of parasitic infections on the body condition of the fish host and result in the contrasting evidence found in the literature.
With respect to body size, we found a positive correlation between fish size and the prevalence of Anisakis, in accordance with the results of several parasitological surveys on different fish species (Mattiucci et al. 2018 and references therein), suggesting that fish size could be a good predictor of infection with Anisakis spp. and of the associated risk of anisakiasis in humans (Madrid et al. 2016). However, other research failed to detect any relationship between fish length and the number of Anisakis larvae in the edible parts of fish (Karl et al. 2011).
In addition, the time after capture and storage temperature can play an important role in defining the distribution of Anisakis larvae in fish filets (Cipriani et al. 2016). The relationship between fish size and the zoonotic potential of Anisakis in the fish host is therefore not always obvious.
Fish age, which is positively correlated to body size, is one of the main factors to be considered in the analysis of infection levels in long-lived parasites, such as Anisakis spp. (Abaunza et al. 1995). Higher infection levels in older and larger fish are the result of a bioaccumulation of parasites throughout the fish's life span and, possibly, of ontogenetic dietary shifts. Furthermore, larger fish feed at a higher rate with a variety of potential intermediate/paratenic hosts, thus favoring higher parasitization levels (Abattouy et al. 2011). In fact, larger fish tend to occupy higher levels in the food chain with the increased possibility of ingesting intermediate/paratenic hosts parasitized with Anisakids (Strømnes and Andersen 2000).
In the present study, the sampled fish were stratified according to the depth of capture (< 250 m and > 250 m). Our results showed a statistically significant correlation values (P < 0.0001) between depth and prevalence of nematode parasites, which are more prevalent in fish from deeper waters (> 250 m). Such a correlation could be explained by the presence of larger (thus more parasitized) fish at a greater depth. However, this variable appears to be a main risk factor for Anisakid and Raphidascaridid infection in commercially important marine fish, as previously reported in a variety of teleost species (e.g., Sardina pilchardus, Engraulis encrasicolus, Phycis blennoides) independently of fish size (Pulleiro-Potel et al. 2015). Specific oceanographic and ecological factors, such as temperature, oceanic currents, depth, salinity, and primary production, have been identified as the main variables affecting the distribution of Anisakis spp. (Højgaard 1998;Kuhn et al. 2016).

Conclusion
In conclusion, our study provides information on the infection pattern of Anisakis and Hysterothylacium larvae in T. trachurus from the Ligurian and Tyrrhenian Seas, and the occurrence of the species A. pegreffii together with A. pegreffii/A. simplex (s.s.) hybrids, and H. aduncum and H. fabri, identified by molecular methods. Our results also support the hypothesis that infection with these parasites does not affect the condition of the fish host analyzed, and that body size and depth are major drivers in determining infection levels with Anisakid and Raphidascaridid nematodes. Funding Open access funding provided by Università di Pisa within the CRUI-CARE Agreement.

Data availability Not applicable.
Code availability Not applicable.

Declarations
Ethics approval Not applicable.

Conflict of interest
The authors declare no competing interests.
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