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

Abstract

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.

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Vol.:(0123456789)
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https://doi.org/10.1007/s00436-021-07200-0
FISH PARASITOLOGY - ORIGINAL PAPER
Anisakid andRaphidascaridid parasites inTrachurus trachurus:
infection drivers andpossible effects onthehost’s condition
FabioMacchioni1 · PerlaTedesco2· VanessaCocca1· AndreaMassaro3· PaoloSartor4· AlessandroLigas4·
CarloPretti1· GianfrancaMonni1· FrancescaCecchi1· MonicaCaara2
Received: 24 February 2021 / Accepted: 27 May 2021
© The Author(s) 2021
Abstract
This study investigated the distribution of nematode larvae of Anisakidae and Raphidascarididae (genera Anisakis and Hys-
terothylacium) in Trachurus trachurus (Linnaeus, 1758) in the Ligurian and central-northern Tyrrhenian Seas. The relation-
ship 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.
Keywords Anisakis spp.· Hysterothylacium spp.· Trachurus trachurus· Infection drivers· Fish condition· Mediterranean
Sea
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
and Biocca, 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 para-
sites in different marine and freshwater fish species (Bezerra
etal. 2020). H. aduncum Rudolphi, 1802 and H. fabri Rudol-
phi, 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 conse-
quences of Hysterothylacium infection on fish health is lim-
ited: parasites of this genus are considered only mildly patho-
genic for adult fish (Ishikura etal. 1993; Yagi etal. 1996;
Valero etal. 2003; Cavallero etal. 2012); however, mortal-
ity episodes in larval and juvenile fish have been reported
Francesca Cecchi and Monica Caffara equally contributed to this
work.
Section Editor: Federica Marcer
* Fabio Macchioni
fabio.macchioni@unipi.it
1 Department ofVeterinary Sciences, University ofPisa, Pisa,
Italy
2 Department ofVeterinary Medical Sciences, Alma Mater
Studiorum, University ofBologna, Bologna, Italy
3 APLYSIA, Livorno, Italy
4 CIBM, Inter-University Center ofMarine Biology
andApplied Ecology “G. Bacci”, Livorno, Italy
/ Published online: 14 August 2021
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1 3
(Bristow 1990; Balbuena etal. 2000). Although generally
not listed among fish-borne zoonotic agents, preliminary evi-
dence 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 Hysterothy-
lacium in wild fish for human consumption is therefore nec-
essary, particularly regarding selected species (Debenedetti
etal. 2019) considered at higher risk of infection. Further-
more, the high parasite load reported in susceptible fish spe-
cies (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 Anisak-
ids and Raphidascaridids, the Atlantic horse mackerel Tra-
churus 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
(Abaunza etal. 2008), and supports large fisheries (Abaunza
etal. 2003), both as target and by-catch species. This spe-
cies 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.
Material andmethods
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 implemen-
tation of the EU-funded Mediterranean international trawl
survey (MEDITS project, Spedicato etal. 2019). After cap-
ture, samples were frozen immediately on board and trans-
ported 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 = aTLb, 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.
Fig. 1 Map of the study area in the Ligurian and northern and central Tyrrhenian Seas (FAO-GFCM GSA9)
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1 3
For the parasitological examination, the abdominal cav-
ity was examined by visual inspection, while the internal
organs were observed under a stereomicroscope (magnifi-
cation 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 micros-
copy. 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 manufactur-
er’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 Scien-
tific, 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 restric-
tion 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 con-
trols 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 per-
formed to assess possible significant differences in the
prevalence of Anisakid and Raphidascaridid parasites
between male and female fish and also to test the relation-
ship 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 para-
sites), 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 pos-
sible effect of parasites on the condition factor was evaluated
by Student’s t-test.
The prevalence of single or multiple infections of nema-
todes 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 Chi-
square test, and was considered significant at P < 0.05.
Results
A total of 190 specimens of T. trachurus were collected dur-
ing 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.
Table 1 Number of fish (NF), number of parasitized fish (NPF), prevalence (%), CI 95% confidence interval, range of intensity (I), min–max
(average) (RI), abundance (A), number of parasites (NP)
NF NFP % CI RI A NP
Males 83 30 36.14 29.20–43.09 1–6 (2.2) 1.12 93
Females 107 40 37.38 30.39–44.37 1–8 (2.36) 0.63 68
Total 190 70 37.89 29.87–43.81 1–8 (2.29) 0.85 161
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1 3
Statistical analysis showed that the fish were more signifi-
cantly infected with Anisakis larvae than with Hysterothy-
lacium 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 signif-
icant differences in the condition factor emerged between
males and females (t = 0.190; p > 0.05) and between
Table 2 Number of fish parasitized by Anisakis (FPA), number of fish
parasitized by Hysterothylacium (FPH), prevalence (%), CI 95% con-
fidence intervals, range of intensity (RI), min–max (average), abun-
dance (A), number of Anisakis (NA), number of Hysterothylacium
(NH). P = 0.032
FPA % CI RI/min-max A NA FPH % CI RI/min–max A NH
21 25.30 19.02–31.58 1–6 (2.47) 0.57 47 9 10.84 2.22–19.46 1–4 (2.33) 0.253 21
34 31.77 25.05–38.50 1–8 (2.24) 0.77 83 6 5.61 1.64–9.58 1–3 (1.67) 0.0935 10
55 28.95 22.39–35.50 1–8 (2.35) 0.68 130 15 7.89 3.24–12.55 1–4 (2.07) 0.1632 31
Fig. 2 a Length–weight rela-
tionship in males of Trachurus
trachurus. b Length–weight
relationship in females of Tra-
churus trachurus
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1 3
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 relation-
ship 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).
The prevalence of nematode parasites was significantly
higher (P < 0.0001) in horse mackerels caught at depths
below 250m (47.6%) compared to those captured above
250m (23.5%). All (100%) the parasitized fish from deeper
waters (> 250m) were infected with Anisakis spp., while
only one fish (1.9%) showed coinfection with Hysterothy-
lacium spp. In contrast, the parasitized fish from shallower
waters (< 250m) were more frequently infected with Hys-
terothylacium spp. (70%) and less by Anisakis spp. (25%).
With regard to molecular analyses, all the specimens sub-
jected 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 spe-
cies 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 (west-
ern Mediterranean), correlating the infection data with the
biological and biometric features of the hosts.
Our results highlighted that the genus Anisakis (28.95%)
was more prevalent than Hysterothylacium (7.89%). This
coinfection pattern is in accordance with other parasitologi-
cal investigations on T. trachurus from the Mediterranean
and Extra-Mediterranean regions. Fioravanti etal. (2003)
reported a higher prevalence of Anisakis (33.7%) compared
to Hysterothylacium (12.2%) in T. trachurus from the cen-
tral Adriatic Sea. With respect to the Ligurian Sea, in horse
mackerels Serracca etal. (2013) reported a prevalence of
15.6% for Anisakis and 9.3% for Hysterothylacium larvae.
Manfredi etal. (2000) reported higher prevalence values
(80–100%) only for Anisakis spp. In a survey carried out on
Trachurus spp. caught off the coast of Sardinia, Angelucci
etal. (2011) reported prevalences of 52.5% for Anisakis spp.
Table 3 Length–weight relationship parameters for males and
females of Trachurus trachurus. a and b are the parameters of the
power function; SE(b), the standard error of b; r2, the coefficient of
determination; and t-value, the value of the t-test
a b SE(b) r2t-value
Males 0.0102 2.937 0.052 0.977 56.35
Females 0.0053 3.145 0.038 0.985 83.28
Fig. 3 Restriction fragment length polymorphism patterns (molecular
weight marker 100 base pairs) obtained with a restriction enzymes
HinfI (lanes 1, 3, 5, 7, 9, and 11) and HaeIII (lanes 2, 4, 6, 8, 10, and
12), lanes 1–6 Anisakis pegreffii, lanes 7–8 Anisakis pegreffii/Anisakis
simplex hybrid, lanes 9–10 positive control (K +) = A. pegreffii, lanes
11–12 K + = Anisakis simplex; b restriction enzymes HinfI (lanes
1, 3, 5, 7, 9, and 11) and HaeIII (lanes 2, 4, 6, 8, 10, and 12), lanes
9–10 K + = Hysterothylacium aduncum, lanes 11–12 K + H. fabri;
and c restriction enzymes HinfI (lanes 1, 3, 5, 7, and 9) and AluI
(lanes 2, 4, 6, 8, and 10), lanes 1–2 H. aduncum, lanes 3–6 H. fabri,
lanes 7–8K + = H. aduncum, lanes 9–10K + = H. fabri
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1 3
and 77.9% for Hysterothylacium spp. In T. trachurus fished
off the coasts of Sicily, Costa etal. (2016) found a 6.7%
prevalence for H. aduncum. Goffredo etal. (2019) reported
prevalence values of 50.8% for Anisakis and 0.54% for Hys-
terothylacium in T. trachurus from the Ionian Sea. MacKen-
zie etal. (2008) analyzed the parasite fauna of T. trachurus
in different sampling stations across the northeastern Atlan-
tic and Mediterranean Seas, reporting that Anisakis spp. and
H. aduncum were the most common parasites detected in
horse mackerel. Their results also highlighted the usefulness
of Anisakis spp. and Hysterothylacium spp. as biological
tags for distinguishing different horse mackerel stocks and
identifying migration patterns.
The high prevalence of Anisakis is probably also related
to the common practice of local fishermen, who discard the
fish viscera directly at sea. These viscera then become a food
source for a variety of fish, cetaceans, and seabirds, which
can thus ingest any larvae of Anisakis that may be present
(Oro and Ruiz 1997; Morton and Yuen 2000; Arcos etal.
2001; Bozzano and Sardà 2002). In the Ligurian Sea, it is
also possible that the high prevalence of Anisakis is linked
to the presence of the “Pelagos Sanctuary,” a marine pro-
tected area with a high density of marine mammals which
are definitive hosts of this genus (Mattiucci etal. 2004; Mat-
tiucci and Nascetti 2006, 2008).
Concerning the molecular analysis, the PCR–RFLP iden-
tified 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
(Meloni etal. 2011).
The results are in agreement with the evidence that A.
pegreffii is the dominant Anisakis species in the Mediter-
ranean 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; 2014).
The reason for the spread of hybrid genotypes in the Medi-
terranean is still unclear (Meloni etal. 2011). In the north-
eastern Atlantic and in the western Mediterranean, A. simplex
(s.s.) and A. pegreffii are known to occur in sympatry (Abollo
etal. 2001; 2003) and may undergo interspecific hybridiza-
tion. 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 inves-
tigations 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, isomet-
ric 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 (Lembo etal. 2012;
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 Raphi-
dascaridid 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 rela-
tion 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 matu-
ration 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 differ-
ent 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 tempera-
ture 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
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1 3
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 nema-
tode 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 cur-
rents, 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 infec-
tion 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 sup-
port 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 infec-
tion levels with Anisakid and Raphidascaridid nematodes.
Author contribution Conceived the study: FM; designed the experi-
ment: FM, CP, PS, AM, MC; performed the field activities and sam-
pling: VC, AM; performed the laboratory work: FM, VC, PT, AM,
GM; analyzed and interpreted the data: FM, FC, VC, PT, MC, AM,
AL; wrote the original draft of the manuscript: FM, PT, AM, MC, AL;
reviewed and edited the final version of the manuscript: FM, PT, VC,
AM, PS, AL, CP, MG, FC, MC; supervision: FM.
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.
Consent to participate Not applicable.
Consent for publication Not applicable.
Conflict of interest The authors declare no competing interests.
Open Access This article is licensed under a Creative Commons Attri-
bution 4.0 International License, which permits use, sharing, adapta-
tion, distribution and reproduction in any medium or format, as long
as you give appropriate credit to the original author(s) and the source,
provide a link to the Creative Commons licence, and indicate if changes
were made. The images or other third party material in this article are
included in the article’s Creative Commons licence, unless indicated
otherwise in a credit line to the material. If material is not included in
the article’s Creative Commons licence and your intended use is not
permitted by statutory regulation or exceeds the permitted use, you will
need to obtain permission directly from the copyright holder. To view a
copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.
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