Parasite 30, 28 (2023)
Ó M. Duflot et al., published by EDP Sciences, 2023
https://doi.org/10.1051/parasite/2023028
RESEARCH ARTICLE
Available online at:
www.parasite-journal.org
OPEN
ACCESS
Black spot diseases in seven commercial fish species from
the English Channel and the North Sea: infestation levels,
identification and population genetics of Cryptocotyle spp.
Maureen Duflot1,2,* , Pierre Cresson3 , Maéva Julien1, Léa Chartier1, Odile Bourgau1, Marialetizia Palomba4
Simonetta Mattiucci5 , Graziella Midelet1 , and Mélanie Gay1,*
1
2
3
4
5
,
ANSES, Laboratory for Food Safety, 62200 Boulogne-sur-Mer, France
University of Littoral Côte d’Opale, Boulogne-sur-Mer, France
Ifremer, RBE/HMMN, Laboratoire Ressources Halieutiques Manche Mer du Nord, 62200 Boulogne-sur-Mer, France
Department of Ecological and Biological Sciences, Tuscia University, Viale dell’Università s/n, 01100 Viterbo, Italy
Department of Public Health And Infectious Diseases, Section of Parasitology, Sapienza University of Rome, P.le Aldo Moro, 5,
00185 Rome, Italy
Received 10 November 2022, Accepted 13 June 2023, Published online 6 July 2023
Abstract – Fish are often speckled with “black spots” caused by metacercarial trematode infection, inducing a host
response. Cryptocotyle spp. (Opisthorchiidae) are among the parasites responsible for this phenomenon. So far, the
impact on human health is still unknown. In addition, few publications dealing with black spot recovery, identification,
distribution and diversity among commercially important fish are available. Moreover, “black spots” have been
observed by fishermen on marine fish, revealing an appreciable but unquantified presence in consumed fish. An
epidemiological survey of 1,586 fish from seven commercial species (herring, sprat, whiting, pout, dab, flounder,
and plaice) was conducted in the Eastern English Channel and the North Sea in January 2019 and 2020. Encysted
metacercariae were found in 325 out of 1,586 fish, with a total prevalence of 20.5%. Intensity of infection varied from
1 to 1,104 parasites. The recorded encysted metacercariae were identified either by microscopic examination or with
molecular tools. Partial sequences of the mtDNA cox1 gene and of the rDNA ITS region were obtained. Two species of
Cryptocotyle, Cryptocotyle lingua (Creplin, 1825) and Cryptocotyle concava (Creplin, 1825) were found. Metacercariae belonging to other trematode families were also identified. Molecular phylogenetic analysis and haplotype
network construction were performed to confirm the identification and to study the potential presence of different
populations of Cryptocotyle spp. This survey enabled us to describe the distribution of two species of Cryptocotyle
in the English Channel and North Sea ecosystems. The observed differences in infestation levels between fish species
and geographical areas will contribute to better understanding of the ecology of these parasites.
Key words: Cryptocotyle, Trematode, Commercial fish species, Epidemiological study, Parasitological descriptors.
Résumé – Maladies des points noirs chez sept espèces commerciales de poissons de la Manche et de la mer du
Nord : niveaux d’infestation, identification et génétique des populations de Cryptocotyle spp. Les poissons sont
souvent parsemés de « points noirs » causés par une infection par des métacercaires de trématodes induisant une
réponse de l’hôte. Les Cryptocotyle spp. (Opisthorchiidae) font partie des parasites responsables de ce phénomène.
Jusqu’à présent, leur impact sur la santé humaine est inconnu. De plus, il existe peu de publications traitant de la
récupération, l’identification, la distribution et la diversité des « points noirs » parmi les poisons d’importance
commerciale. Par ailleurs, des observations de « points noirs » sur les poissons marins ont été constatées par les
pêcheurs révélant une présence assez importante mais non quantifiée dans les poissons consommés. Une enquête
épidémiologique portant sur 1 586 poissons de sept espèces commerciales (hareng, sprat, merlan, tacaud, limande,
flet et plie) a été menée en Manche orientale et en Mer du Nord, en janvier 2019 et 2020. Des métacercaires
enkystées ont été trouvées chez 325 poissons parmi 1 586, avec une prévalence totale de 20,5 %. L’intensité de
l’infection variait de 1 à 1 104 parasites. Les métacercaires enkystées répertoriées ont été identifiées soit par
examen microscopique, soit avec des outils moléculaires. Des séquences partielles du gène cox1 de l’ADNmt et de
la région ITS de l’ADNr ont été obtenues. Deux espèces de Cryptocotyle, Cryptocotyle lingua (Creplin, 1825) et
Cryptocotyle concava (Creplin, 1825) ont été trouvées. Des métacercaires appartenant à d’autres familles de
Edited by Jean-Lou Justine.
*Corresponding authors: melanie.gay@anses.fr; maureen.duflot@outlook.fr
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
M. Duflot et al.: Parasite 2023, 30, 28
2
trématodes ont également été identifiées. Une analyse phylogénétique moléculaire et la construction d’un réseau
d’haplotypes ont été effectuées pour confirmer l’identification et étudier la présence potentielle de différentes
populations de Cryptocotyle spp. Cette étude a permis de décrire la distribution de deux espèces de Cryptocotyle
dans les écosystèmes de la Manche et de la Mer du Nord. Les différences observées dans les niveaux d’infestation
entre les espèces de poissons et les zones géographiques contribueront à une meilleure compréhension de l’écologie
de ces parasites.
Introduction
Fish harbor many pathogens – including parasites – that
may affect cultured or wild fish. Their presence may impact fish
production and, for some of them, they may be a threat to
human health. More than 50 to 60 million people are annually
reported to be infected by foodborne trematode infections
around the world [15]. Humans can be infected by ingestion
of raw, undercooked or pickled fish containing metacercariae.
Infection by members of the superfamily Opisthorchioidea
causes a wide range of impacts on human health, from very
severe human disease, such as in infection by Clonorchis sinensis or Opisthorchis viverrini, to unknown zoonotic potential for
other species [14, 15, 22]. These parasites have a complex life
cycle. Commonly they have a mollusc as first intermediate host,
in which several larval stages sequentially develop (miracidium,
sporocyst, redia, and cercaria). A fish acts as second intermediate host, in which the cercariae evolve into metacercaria.
Vertebrate animals, including mammals and birds, complete
the life cycle [28, 72]. Many trematode species can encyst at
the metacercariae stage in marine and freshwater fish, and some
of them cause black spot disease [1, 5, 24, 44]. Additionally,
some digeneans, such as Apophallus Lühe, 1906 [73], Cryptocotyle Lühe, 1899 [17, 78], Haplorchis Looss, 1899 [61] and
Stellantchasmus Onji & Nishio, 1916 [16] infect marine fish,
their intermediate host. Their swimming cercariae encyst,
develop into the metacercarial stage, and cause immune cutaneous black spot. Black spot disease is an immune response
towards encysted metacercariae, due to the concentration of
melanomacrophages at the infection site [21, 78]. These black
spots may induce esthetic problems that lower the commercial
value of the fish [44]. Some Opisthorchioidea parasites are
known to have zoonotic potential [15, 56, 74]. As Cryptocotyle spp. belong to the Opisthorchioidea superfamily, they
have a potential zoonotic nature, but their impact on human
health is poorly known [13, 15]. In addition, few data are available regarding their distribution among commercially important
fish species. Therefore, one of the first steps in risk assessment
is the acquisition of knowledge and fundamental data on the
distribution of this parasite in different fish species and different
geographical areas, before any zoonotic potential assessment.
Reports on parasite fauna in fish from the English Channel
and North Sea are numerous but, to date, few have dealt with
encysted metacercariae diseases [32, 60]. To study the circulation of Cryptocotyle spp. and other black spot-causing parasites
in fish, the infestation levels of black spot in seven fish species
in five geographical areas, covering the eastern English Channel
and the south of the North Sea (i.e., south of < 55 °N), were
determined by fish dissection to isolate and identify metacercariae. The seven fish species were selected based on their
former description as hosts for Cryptocotyle spp., and their
biology, behavior, and position in these ecosystems. The first
two species (Clupea harengus and Sprattus sprattus) are medium-small sized species that play an important role in the food
web. These zooplankton feeding species are considered pivotal
between primary production and top predators, and thus play a
major role in ecosystem functioning [54]. Herring (C. harengus)
has also been a major commercial fish species in the area for
centuries, while sprat (S. sprattus) abundance and catches have
increased in recent years, potentially because of its larger ecological niche [19, 36]. Two species of gadoids (Merlangius
merlangus and Trisopterus luscus) and three species of flatfish
(Limanda limanda, Platichthys flesus, and Pleuronectes
platessa) were chosen as they are relevant in fisheries. As an
example, annual landings in the North Sea and English Channel
have been between 50 kt and 80 kt for plaice (P. platessa),
between 3 kt and 7 kt for dab (L. limanda), and around 15 kt
for whiting (M. merlangius) since 2010 [35, 37, 38]. In
addition, they occupy different positions in the water column:
herring and sprat are strictly pelagic, gadoids are benthodemersal (i.e., able to move vertically in the column), and flatfish species are considered to be purely benthic.
The selected species have already been described as
Cryptocotyle spp. hosts [7, 32, 59, 62, 65, 67, 91]. The present
study aimed at assessing the prevalence, intensity, and
abundance of encysted metacercariae in the seven selected fish
species collected in the Eastern English Channel and North Sea.
Moreover, preferential anatomical location of black spot disease
was characterized. Metacercariae were identified molecularly or
morphologically. Molecular examinations were completed with
phylogenetic analyses incorporating species of the superfamily
Opisthorchioidea Loss, 1899 and an analysis of genetic
diversity within the species C. lingua. This study provides the
first evidence of Cryptocotyle spp. distribution in the Eastern
English Channel and North Sea.
Material and methods
Fish samples
Seven fish species were selected for this study: herring
Clupea harengus Linnaeus, 1758, sprat Sprattus sprattus
(Linnaeus), whiting Merlangius merlangus Linnaeus, pout
Trisopterus luscus (Linnaeus), dab Limanda limanda
(Linnaeus), flounder Platichthys flesus (Linnaeus), and plaice
Pleuronectes platessa Linnaeus. All species were sampled in
2019 while whiting and pout only were collected in 2020. A
total of 1,163 and 423 specimens were collected by bottom
trawling during the International Bottom Trawl Survey in the
English Channel and North Sea in January 2019 and 2020,
respectively [39, 40]. The initial protocol included the collection of 40 individuals in each geographical area. The sampling
M. Duflot et al.: Parasite 2023, 30, 28
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Figure 1. Sampling locations in the English Channel and North Sea with indications of number of sampled fish at each station. Size of the
circle is proportional to the total number of fish (n fish) of the five species sampled at each station. Stations names with a code beginning with
an X and with full circle were sampled in 2019 and by a Y and empty circle in 2020.
area was divided into five sub-areas defined based on
environmental characteristics (temperature, salinity) (Fig. 1).
Individuals of all species were sampled in all areas, except
for pout in the east North Sea in both years (Table 1). For
2019 sampling, all fish were frozen at 20 °C rapidly on board.
For 2020 sampling, individuals were eviscerated and kept fresh
at 1 °C for a maximum of 11 days until parasite inspection at
the laboratory.
the present study, following definitions from Bush et al. [12]. In
particular, prevalence is the number of fish infected divided by
the total number of host fish examined, abundance is the
number of black spots divided by the number of fish examined,
and intensity is the number of black spots on an infected fish.
The data were analyzed with R software 4.0.2 (R Core Team,
2020) and the ggplot2 package [86].
Isolation of metacercariae
Evaluation of infection and parasitological
descriptors
Parasitological infection was first assessed by macroscopic
examination of fish. The presence of cutaneous black spots was
recorded for each defined body area on both sides of each fish
(Fig. 2), according to the method used by Duflot et al. [21].
Fish that exhibited one or more of the typical black spots
formed around encysted metacercariae were recorded as being
“infected”, and individuals with no visible spots were recorded
as “uninfected”. Three parasitological descriptors were used in
For frozen fish (2019 sampling), the samples were thawed
slowly at 1 °C overnight. For each infected fish, a maximum
of 15 metacercariae were isolated through dissection and/or
partial digestion of skin and subcutaneous muscle (thickness ~ 5 mm). Partial digestion was achieved in a Petri dish
by adding a pepsin/HCl/saline solution in excess until tissues
were fully immersed [7]. The Petri dish was placed on a hot
plate at 37 °C (± 1) for 5–10 min, depending on the fish species.
Metacercariae were visually isolated from the tissues under a
stereomicroscope (SZX16, Olympus Corporation, Tokyo,
M. Duflot et al.: Parasite 2023, 30, 28
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Table 1. Geographical origin and biometric data of fish species, including weight and length ± SD (min–max).
Number of fisha
Species
Fishing
year
Herring
Sprat
Whiting
2019
2019
2019
2020
Pout
2019
2020
39
26
40
68
0
0
13
24
23
39
Dab
Flounder
Plaice
2019
2019
2019
40
26
35
40
38
34
40
36
25
40
15
34
40
2
23
Weight (g)
EEC SBNS ENS CNS WNS
21
40
43
40
39
41.72 ± 36.37 (2–183)
40
42
38
44
43
6.89 ± 3.00 (1–19)
40
40
30
40
40
218.70 ± 136.43 (16–674)
80
39
41
66
40
143.00 ± 109.29 (4–882)
Total length (mm)
Parasite identification
methods
170.93 ± 53.98 (35–306)
99.58 ± 13.57 (54–135)
258.26 ± 64.83 (126–405)
249.68 ± 61.99 (98–450)
Molecular
Molecular
Molecular
Morphological &
Molecular
146.18 ± 109.83 (16–773) 219.64 ± 45.04 (126–367) Molecular
102.68 ± 61.57 (28–421) 204.05 ± 35.79 (140–317) Morphological &
Molecular
91.64 ± 51.82 (8–249)
2020.23 ± 35.78 (100–296) Molecular
207.94 ± 108.65 (18–652) 263.48 ± 47.03 (125–420) Molecular
163.75 ± 188.80 (3–1314) 229.86 ± 98.13 (77–524) Molecular
a
Number of fish per area; EEC: eastern English Channel, SBNS: Southern Bight of the North Sea, ENS: eastern North Sea, CNS: central
North Sea, WNS: western North Sea.
Figure 2. Definition of the different areas of a fish: (1) caudal fin, (2) 2nd dorsal fin, (3) 1st dorsal fin, (4) pectoral fin, (5) pelvic fin, (6) anal
fin, (7) dorsofrontal area, (8) ventrofrontal area, (9) dorsoposterior area, (10) ventroposterior area, (11) opercula, and (12) eye; according to
Buchmann [10].
Japan), with micro-pliers and a scalpel. Each metacercaria was
placed in a separate well on a 96-well plate.
For each fresh individual (2020 sampling), the most
infected area of the skin and of the underskin muscle of each
infected fish was selected for the isolation of metacercariae.
The optimized artificial digestion method described by Duflot
et al. [21] (D4 method) was used to isolate the metacercariae
from the fish tissues. Briefly, skin and subcutaneous muscle
were digested separately in a pepsin solution at 37 °C for
1 h on an orbital agitator. Then encystment of each metacercaria was carried out by trypsin treatment at room temperature
for 1 h.
Identification of metacercariae
All the metacercariae isolated from thawed fish were identified by the molecular method. For the parasites isolated from
fresh fish, one third of the metacercariae were identified based
on morphological criteria (with a maximum of 5 metacercariae
per fish), and the other two thirds were identified using the
molecular method (with a maximum of 15 per fish). Observation of parasites with the same general morphology under an
optic microscope (Ts2 Nikon Eclipse, Nikon, Tokyo, Japan)
enabled us to divide samples between the two types of identification methods, morphological and molecular.
Morphological identification was performed according to
the protocol described by Duflot et al. [20]. Identifications of
metacercariae were based on general microscopic observation
and measurements of classical characteristics of Opisthorchioidea trematodes on each excysted metacercaria mounted
on a slide after staining with hematoxylin. Supplemental measurements were acquired such as the oral and ventrogenital
sucker width, the distance between the oral sucker and the pharynx, and the pharynx length and width. Parasites were characterized (100–200 magnification) under a Leica DLMB
microscope with a Leica DC300 camera (Leica, Wetzlar,
Germany). Identifications were based on the descriptions of
Borges et al. [7], Casalins et al.[13], Gibson [27], Goncharov
et al. [30], Linton [53], Ransom [66], Stunkard [80], and
Tatonova and Besprozvannykh [83].
Molecular identification was carried out according to a protocol of DNA extraction, PCR amplification, and Sanger
sequencing on a partial region of the mtDNA cox1 gene
(350 bp) and the rDNA ITS1-5.8S-ITS2 region (1,200 bp)
described by Duflot et al. [20]. Target regions were amplified
using the primer pairs JB3/JB4.5 [9] and BD1/28S1R [77],
respectively. PCR products of the expected size were sequenced
twice and from both sides (forward and reverse), using Sanger
sequencing (Genoscreen, Lille, France) with the same primers.
Each obtained consensus sequence was subjected to a BLAST
search [3], after visualization in BioEdit 7.0.9.0 software [33],
clarification of ambiguous bases, and Clustal W alignment
using MEGA 10.1.8 [45]. All sequences the diversity of were
submitted to GenBank and assigned accession numbers.
M. Duflot et al.: Parasite 2023, 30, 28
5
Table 2. Molecular sequences used as references.
Species
Clonorchis sinensis
Cryptocotyle concava
Cryptocotyle lata
Cryptocotyle lingua
Cryptocotyle micromorpha
Haplorchis taichui
Opisthorchis sudarikovi
Opisthorchis viverrini
Outgroup
Fasciola hepatica
Fasciola gigantica
Reference
Lee and Huh [50]
Qiu et al. [64]
Gonchar [29]
Tatonova and Besprozvannykh [83]
Borges et al. [7]
Blakeslee et al. [6]
Duflot et al. [20]
Presswell and Bennett [63]
Lee et al. [49]
Le et al. [47]
Suleman et al. [81]
Thaenkham et al. [84]
Reaghi et al. [68]
Le et al. [48]
Le et al. [48]
Phylogenetic analysis and genetic diversity of
Cryptocotyle lingua populations
The alignments were trimmed to the length of the shortest
sequence. Trees were built with reference sequences of digeneans belonging to the superfamily Opisthorchioidea and with
Fasciola sp. as outgroups (Table 2). Phylogenetic analyses
were conducted in MEGA 10.1.8 using the Maximum Likelihood (ML), Neighbor-Joining (NJ) and Minimum Evolution
(ME) methods, with 1,000 bootstrap replications. The most
suitable fit model for each targeted marker was determined
using the corrected Akaike Information Criterion (AICc) and
the Bayesian Information Criterion (BIC) on the 24 models
tested in MEGA 10.1.8. Cox1 and ITS sequences were fitted
to the JC model. Phylogenetic relationships under Bayesian
inference (BI) were also generated in MrBayes v3.2.7 [34].
Two independent runs were performed for 10,000,000 generations and sampled every 500th generation. The burn-in was set
for the first 25% of the sampled trees. Bayesian analyses were
executed online on NGPhylogeny.fr [52].
Two analyses of molecular variance were carried out on
Cryptocotyle lingua samples to assess the presence of different
populations. The first analysis was based on the Cryptocotyle
spp. populations from the different geographical areas, the second on the different fish species. Pairwise genetic differentiation
of C. lingua was estimated with the fixation index (Fst), using
ARLEQUIN 3.5 software [25]. This parameter ranges between
0 and 1, in which Fst = 0 indicates no differentiation between
the populations, and Fst = 1 means complete differentiation
among the sequences of the different populations. Pairwise
comparisons of Fst (assuming that p < 0.05 indicates a significant difference) were based on 1,000 permutations of the data
matrix. Then, Tajima’s D neutrality test [82] and Fu’s Fs [26]
were calculated to verify the null hypothesis of selective neutrality, using DnaSP 6.12.03 software [71].
Estimation of the population genetic diversity of C. lingua
among sampling areas and among fish species was inferred
from mtDNA cox1 gene and ITS region rDNA sequence data
GenBank accession numbers
cox1
AF181889
–
MT422290; MT422303
–
KJ711861–KJ711862
EU876357–EU876411
MW542531–MW542549
OL504983
KF214770
–
–
HQ328544
ITS
–
MK450527
–
MH025622-MH025623
KJ641518–KJ641519
–
MW544135–MW544136
–
–
KX815126
MK227161
–
MT951585–MT951587
–
–
–
MN970007
MN970008
sets with the following parameters: number of haplotypes
(Nh), nucleotide diversity (p), haplotype diversity (Hd), average
number of differences (K), number of polymorphic sites (S). All
parameters were estimated using DnaSP 6.12.03 software [71].
Haplotype network constructions were carried out using
PopART 1.7 software [51] based on cox1 sequences gene
(269 bp). Network calculation was realized with the TCS model
[18].
Results
Black spot infection data
Prevalence of infection
Infection by encysted metacercariae was observed in all
sampled areas and for all fish species considered. Of the
1,586 sampled fish, 325 fish were parasitized, an overall
prevalence of 20.5%. The prevalence of encysted metacercariae
in the different fish species as well as in the different geographical areas was highly variable (Fig. 3). Prevalence values were
the highest for P. flesus (52.1% in 2019), M. merlangius (26.8%
and 27.8% in 2019 and 2020, respectively) and T. luscus
(20.0% and 26.8% in 2019 and 2020, respectively).
Similar prevalence tendencies were observed between the
geographical areas in the two sampling years. For whiting
samplings, the eastern English Channel was the most infected
area, followed by the Southern Bight of the North Sea, the
eastern North Sea, central North Sea, and western North Sea.
For pout, prevalences were similar in the eastern English
Channel and the Southern Bight of the North Sea (about 20%
in 2019, and about 35% in 2020). Again, similar trends
were observed for prevalences in pout in the central and western North Sea, but with slightly more variation. Pelagic species,
Sprattus sprattus and Clupea harengus, were the least commonly infected fish species overall and within all the geographic areas, with always less than 20% of the fish
exhibiting black spots.
6
M. Duflot et al.: Parasite 2023, 30, 28
Figure 3. Prevalence of black spot in each fish species by geographical areas. EEC: eastern English Channel, SBNS: Southern Bight of the
North Sea, ENS: eastern North Sea, CNS: central North Sea, WNS: western North Sea. Error bar = SD. nd: No sampled individual.
Abundance and intensity
The abundances or intensities of encysted metacercariae followed the same trends as prevalence values (Table 3). Regardless of the sampling area, whiting and pout were the most
infected species, with mean abundances of 8.7 and 9.4 black
spots per fish, respectively. Patterns were not consistent
between 2019 and 2020 for whiting and pout. For whiting,
the Southern Bight of the North Sea and the eastern English
Channel were the most infected areas in 2019 (48.7 and 43.2
black spots per fish on average, respectively) while the central
and eastern North Sea were the most infected in 2020 (181.2
and 69.6 black spots per fish, respectively). For pout, the eastern English Channel and central North Sea were the most
infected geographical areas in 2019 (114.4 and 27.8 spots per
fish on an average, respectively), while the central North Sea
and the Southern Bight of the North Sea were the most infected
in 2020 (55.8 and 42.0 spots per fish). All the targeted fish species sampled in the Southern Bight, eastern North Sea, and central North Sea were parasitized. In the eastern English Channel,
herring and plaice were not infected. Likewise, in the western
North Sea, dab was not infested. Looking at maximum black
spot infection, in 2019, the highest infected individuals of whiting and pout were caught in the eastern English Channel in
2019 (43.2 and 114.4 black spots per fish on average, respectively), and in the central North Sea and the Southern Bight
of the North Sea in 2020 (181.2 and 42.0 black spots per fish
on average, respectively).
Site of infection by Cryptocotyle spp.
Some areas of the fish body exhibited more infection by
encysted metacercariae than others (Fig. 2 and Supplementary
data 1), specifically areas 1 (caudal fin), 7 (dorsofrontal
area), 9 (dorsoposterior area), 10 (ventroposterior area) and
11 (opercula) (Supplementary data 1). Areas 2 (second dorsal
fin), 3 (first dorsal fin), 4 (pectoral fin), 5 (pelvic fin), 6 (anal
fin), 8 (ventrofrontal area), and 12 (eye) were spotted with an
average of less than one parasite, while black spots were more
abundant for other areas. Values were markedly higher for
whiting and pout than for the other species. For example, values
higher than 5 were found in the dorsofrontal and dorsoposterior
areas (7 and 9) on both sides for whiting and pout, while 1 parasite was observed for other species. Sides do not appear to play
a major role for pelagic and demersal species, but this aspect
does play a role for flatfishes, which exhibited slightly more
metacercariae on the right side, and particularly for dab L. limanda. The pelvic fin (area 5) is the only area not affected
by metacercariae infection.
Parasite identification
Comparative morphological analysis
Four different morphologies were observed (Fig. 4, Table 5)
(n = 209 parasites). The first one (Fig. 4a) was predominant
(n = 192 metacercariae) and had the morphological traits of
Cryptocotyle spp. Excysted metacercariae were linguiform to
pyriform, according to their state of contraction at fixation in
ethanol, length 0.57 (0.31–0.92) or 0.55 (0.31–0.69) mm for
metacercariae from whiting and pout, respectively (Table 4).
Width at anterior part of the body was 0.23/0.22 mm (metacercariae from whiting/pout) and second width in the posterior part
of the body was 0.14/0.15 mm. The anterior part of metacercariae was covered with scale-like spines, a subterminal oral
sucker of 0.06/0.05 mm in length by 0.02/0.02 mm width.
The prepharynx was short followed by an elliptical pharynx
0.04/0.04 mm in length by 0.02/0.02 mm in width, with a
distance from the oral sucker to the end of pharynx of
0.12/0.11 mm, and distance between the oral sucker and
M
555
3
8
608
23
148
8
1104
306
8.7 ± 15.6
0.1 ± 0.1
0.2 ± 0.3
9.4 ± 23.7
0.4 ± 0.8
4.8 ± 3.9
0.2 ± 0.3
16.7 ± 18.4
8.6 ± 11.4
A
I
M
A
0.6 ± 1.1
0.1 ± 0.4
0.1 ± 0.4
3.8 ± 8.5
0.0
0.5
0.1
0.3 ± 1.1
0.8 ± 6.5
I
1.1 ± 3.3
0.1 ± 0.0
0.5 ± 0.8
8.5 ± 28.6
0.4 ± 1.2
12.2 ± 22.5
0.2 ± 0.2
35.7 ± 2.6
11.6 ± 21.0
2020
M
A
I
7.3 ± 8.5
1.0 ± 0.0
2.6 ± 1.8
27.8 ± 51.5
3.4 ± 3.4
16.6 ± 26.3
1.4 ± 0.5
181.2 ± 5.8
55.8 ± 46.1
68
3
2
–
6
33
3
339
–
M
A
3.2 ± 9.9
0.3 ± 0.8
0.1 ± 0.5
–
0.55 ± 1.4
4.1 ± 4.0
0.4 ± 0.6
20.4 ± 58.1
–
16.0 ± 22.1
1.7 ± 0.8
1.3 ± 0.5
–
2.8 ± 1.4
7.0 ± 4.0
1.4 ± 0.6
69.6 ± 58.1
–
I
M
454
1
3
74
23
64
8
180
306
13.4 ± 42.6
0.0 ± 0.0
0.2 ± 0.3
2.1 ± 8.1
0.6 ± 4.7
5.1 ± 5.1
0.5 ± 0.9
9.1 ± 43.1
14.8 ± 6.2
A
I
48.7 ± 81.2
1.0 ± 0.0
1.5 ± 0.7
10.3 ± 18.2
12.5 ± 21.0
12.0 ± 7.9
2.4 ± 1.9
25.3 ± 72.0
42.0 ± 10.5
555
1
0
608
10
14
0
229
7
M
A
23.8 ± 42.6
0.1 ± 0.0
0.0
20.5 ± 71.0
0.3 ± 1.4
1.6 ± 1.9
0.0
11.2 ± 30.1
1.0 ± 87.0
43.2 ± 57.4
1.0 ± 0.0
0.0
114.4 ± 167.7
3.3 ± 4.5
3.4 ± 2.8
0.0
27.9 ± 47.6
3.0 ± 147.9
Whiting
Sprat
Herring
Pout
Dab
Flounder
Plaice
Whiting
Pout
2019
I
28
1
8
105
10
148
2
1104
257
4.2 ± 2.9
2.0 ± 2.0
1.7 ± 1.3
22.0 ± 20.4
0.0
1.0
3.0
5.5 ± 5.0
7.8 ± 20.4
10
3
3
45
0
1
3
10
25
32.4
1.4
1.9
47.0
4.1
9.3
1.9
60.9
32.0
±
±
±
±
±
±
±
±
±
30.2
0.4
0.8
53.0
2.5
5.3
0.7
35.2
22.1
All areas
WNS
CNS
ENS
SBNS
EEC
Table 3. Mean intensity (I), Abundance (A) and Maximum number of black spots per fish (M) of the different geographic areas, eastern English Channel (EEC), Southern Bight of the North
Sea (SBNS), eastern North Sea (ENS), central North Sea (CNS), and western North Sea (WNS) (mean ± SD). (–) No sampled individual.
M. Duflot et al.: Parasite 2023, 30, 28
7
pharynx of 0.02/0.02 mm. Intestinal bifurcation occurred at
0.16/0.16 mm from the oral sucker. The ventral sucker was
0.02/0.02 mm in length and 0.02/ 0.02 mm in width, located
on the median line from one-half to two-thirds of the total body
length according to contraction. Immature sexual organs were
present, in the posterior part of the body.
The other three morphologies (Figs. 4b–4d) corresponded to
a minority of samples (n = 17). They exhibited the general traits
of family Bucephalidae, such as a translucent, elongated to oval
and small- to medium-sized body. An anterior globular attachment organ was present, like an oral sucker or rhynchus, and a
ventral sucker absent. The excretory pore was terminal. No
further organs could be distinctly observed at this larvall stage.
Molecular identification
Sequence analysis of the mtDNA cox1 gene locus (320 bp)
was successfully obtained on 1,034 metacercariae out of the
1,909 analyzed, allowing the identification of the parasite
species. Likewise, PCR of the ITS region of rDNA from
255 parasites (95 identified parasites) resulted in a product of
approximately 1,500 bp long. Sequences of the cox1 gene
and ITS region were deposited in GenBank under accession
numbers MZ731829–MZ731932 and MZ595783–MZ595830,
respectively.
BLAST search (Supplementary data 2 and Table 5) of the
cox1 fragments (performed in February to May 2021) led to an
average of 99.83% similarity with 4 GenBank sequences corresponding to C. lingua samples from Denmark (928 sequences),
99.41% similarity with 13 GenBank sequences corresponding
to C. lingua from Europe (Ireland, UK, Norway, Denmark,
France, and Sweden), Canada and the USA (72 sequences),
and 98.67% similarity with a GenBank sequence of C. lingua
from Russia (14 sequences). Four sequences were identified as
Cryptocotyle concava with 99.5% similarity with GenBank
accession Nos. MT422312 (C. concava. Russia: White Sea),
and MT422306 (C. concava, Varangerfjord, Norway). Four
sequences showed only a low similarity value (79%) with
sequences of other Heterophyidae (KT883857, Pholeter
gastrophilus and LC422949, Metagonimus sp.). Likewise, three
sequences had a low similarity value (74.94%) with AY504855
and AY504859 (Larval bucephalid sp.).
BLAST analysis (Supplementary data 2 and Table 5) of the
ITS fragments (carried out from April to May 2021) led to an
average of 99.8% similarity with 7 GenBank sequences corresponding to C. lingua from Danish seas and the English Channel, France (76 sequences). 99.81% similarity was found with 1
GenBank sequence corresponding to C. lingua from the White
sea. 92.80% similarity was obtained for 1 sequence with a
GenBank sequence corresponding to Bucephalus margaritae
and an average of 88.46% similarity was found for 14
sequences with a GenBank sequence of Bucephalus polymorphus. Cryptocotyle lingua identifications were confirmed with
both markers for 71 individuals.
The ML, NJ, ME and Bayesian methods produced phylogenetic trees with similar topologies. Only the ML tree is presented
in this manuscript. The cox1 (Fig. 5A) and ITS (Fig. 5B) trees
had the highest log likelihoods of 2466.53 and 5763.65
(Fig. 5), respectively. Phylogenetic trees based on nucleotide
M. Duflot et al.: Parasite 2023, 30, 28
8
Figure 4. Excysted Cryptocotyle lingua metacercariae from whiting of the central North Sea (a) and excysted metacercariae of the family
Bucephalidae from pout of the Southern Bight of the North Sea (b) and whiting (c and d) of the eastern English Channel and central North Sea,
respectively. Scale: 50 lm.
Table 4. Morphometric data of metacercariae with morphological traits of Cryptocotyle spp. (n = 192) from naturally infected whiting (n = 54)
and pout (n = 26) and bibliographic references. Measurements are expressed in mm (Average (min–max)) and round number at 2 digits.
Stage of maturity
Body shape
Total length
Width 1
Width 2
Oral sucker length
Oral sucker width
Ventrogenital complex length
Ventrogenital complex width
Distance from oral sucker
to end of pharynx
Distance between oral
sucker and pharynx
Intestinal branches
Pharynx length
Pharynx wide
Parasites from
whiting
Parasites
from pout
Cryptocotyle
lingua
Cryptocotyle
lingua
Cryptocotyle
lingua
Cryptocotyle
concava
Cryptocotyle
jejuna
Metacercariae
Linguiform to
pyriform
0.57 (0.31–0.92)
0.23 (0.06–0.32)
0.14 (0.05–0.39)
0.06 (0.03–0.24)
0.06 (0.03–0.28)
0.02 (0.01–0.03)
0.02 (0.01–0.03)
0.12 (0.06–0.16)
Metacercariae
Linguiform to
pyriform
0.55 (0.31–0.69)
0.22 (0.13–0.36)
0.15 (0.08–0.21)
0.05 (0.04–0.08)
0.06 (0.04–0.12)
0.02 (0.01–0.05)
0.02 (0.01–0.04)
0.11 (0.06–0.22)
Metacercariae
/
Metacercariae
Linguiform to
pyriform
0.39–0.92
0.11–0.31
0.14–0.25
0.03–0.06
/
0.02–0.05
/
0.07–0.12
Metacercariae
Oval
Metacercariae
Elongated
0.58–0.68
0.18–0.21
/
0.05–0.06
0.05–0.06
0.02–0.03
0.02–0.03
/
Adult
Linguiform to
pyriform
0.55–2.00
0.20–0.90
/
0.07–0.11
/
0.12–0.25
/
0.03–0.05
0.42
0.37
/
0.06
/
/
/
0.05 (0.01–0.04)
/
/
/
0.05
/
/
/
0.03 (±0.01)
0.02 (0.00–0.06)
0.02 (0.00–0.07)
/
/
/
/
/
0.16 (0.05–0.31)
0.04 (0.02–0.06)
0.02 (0.01–0.04)
This study
0.16 (0.11–0.23)
0.04 (0.03–0.08)
0.02 (0.01–0.05)
This study
/
0.03–0.04
0.02–0.03
Borges et al. [7]
0.28–0.32
/
/
Ransom [66]
0.06–0.19
/
/
Duflot et al. [20]
/
/
/
Goncharov
et al. [30]
/
/
/
Goncharov
et al. [30]
M. Duflot et al.: Parasite 2023, 30, 28
9
Table 5. Summary of identifications carried out in this study.
Morphological method
C. lingua
C. concava
Bucephalidae
Total
Number of identified
parasites
192
/
17
Related percentage of
identification
91.9%
/
8%
209
sequences of the cox1 gene (Fig. 5A) and ITS region (Fig. 5B)
showed that all the sequences identified as C. lingua by BLAST
analysis clustered together in a highly supported clade, including
C. lingua reference sequences (Fig. 5). The mtDNA cox1
sequences identified as C. concava by BLAST identification also
clustered with C. concava reference sequences in a monophyletic group (Fig. 5A). Since fewer metacercariae were analyzed for the ITS region and C. concava was much rarer than
C. lingua in our sampling, no ITS region sequence was retrieved
for C. concava in the analyzed individuals.
Geographical distribution of black spot disease
and Cryptocotyle spp.
Black spot diseases were observed throughout the eastern
English Channel and the North Sea (Fig. 6). In the eastern English Channel, 75% of fish with black spots hosted at least one
parasite identified as C. lingua. This was the most infected geographic area in this study. The Southern Bight of the North Sea
was the second geographic area, where 70.45% of parasitized
fish hosted at least one parasite identified as C. lingua. In the
North Sea, rates were lower with 44.32%, 35.23% and
18.18% of fish in the central, eastern and western North Sea,
respectively. However, in the eastern and western North Sea,
fish parasitized by at least one C. concava were found too, with
prevalence of 2.27% and 1.14%, respectively (Fig. 6). Only
three fish (0.15%) were parasitized with at least one Bucephalidae parasite, and four fish (0.20%) were found to host at least
one other Heterophyidae genus than Cryptocotyle spp.
Genetic diversity of C. lingua populations
Genetic diversity of C. lingua populations by geographic area
Estimation of the genetic differentiation of the C. lingua
specimens by the geographic areas of sampled fish in this study
was estimated from the fixation index (Fst) (Table 6). No significant level of differentiation was observed among the overall
five geographic areas (p < 0.05). However, significant genetic
differentiation was found between C. lingua from the western
North Sea and the eastern English Channel (Fst = 0.01199;
p = 0.03306 ± 0.0184*).
Based on cox1 (N = 998, 272 bp) sequence analyses from
the different geographical areas (Table 7), C. lingua parasites
exhibited 87 haplotypes (Nh). Among the sequences, 80 polymorphic sites (S) were recorded. The sequences of cox1 from
C. lingua individuals showed low haplotype diversity (on
average Hd = 0.476). The same tendency was observed for
Molecular method
Number of identified
parasites
101
4
3
Related percentage of
identification
98.1%
0.4%
0.3%
1034
nucleotide diversity (pcox1 = 0.00225). The average number
of nucleotide differences was also low (K = 0.610). The highest
haplotype diversity and number of nucleotide differences were
observed for cox1 C. lingua specimens isolated from the western North Sea (Hd = 0.627; K = 00793). Similar values of
nucleotide diversity were observed in all five defined geographical areas.
Tajima’s D and Fu’s Fs values were negative for all fishing geographic areas with significant average: Tajima’s
Dcox1 = 2.61775 (p < 0.001), Fu’s Fscox1 = 211.987. Thus,
the null hypothesis of a constant population size (i.e., the
population evolves according to the infinite site model and all
mutations are selectively neutral) was rejected.
Genetic diversity of C. lingua populations according
to infected fish species
Estimation of the genetic differentiation of the C. lingua
specimens from different infected fish species was estimated
from the fixation index (Fst) (Table 8). No significant level of
differentiation was observed among the seven infected fish species (p < 0.05).
Based on cox1 (n = 998, 272 bp) (Table 9) sequence analyses from the different fish species infected by C. lingua,
parasites exhibited 87 haplotypes (Nh). Among the sequences,
80 polymorphic sites (S) were recorded. The sequences of
cox1 C. lingua specimens showed low haplotype diversity
(on average Hd = 0.476) with a low nucleotide diversity value
(pcox1 = 0.00255). The average number of nucleotide differences was low (K = 0.610). The highest haplotype diversity
and number of nucleotide differences were observed for cox1
C. lingua specimens isolated from plaice (Hd = 0.786;
K = 1.000). Similar values of nucleotide diversity were
observed in all seven fish species. Few sequences of C. lingua
were identified from sprat, plaice, and flounder on the cox1
gene.
Tajima’s D and Fu’s Fs values were negative for all fishing
geographical areas with significant average, Tajima’s
Dcox1 = 2.61775 (P < 0.001), Fu’s Fs cox1 = 211.987. Thus,
the null hypothesis of a constant population size (i.e., the
population evolves according to the infinite site model and all
mutations are selectively neutral) was rejected.
Relationship among haplotypes of C. lingua
The median joining network constructions of cox1 (Fig. 7)
showed the relationship among the 87 haplotypes observed in
the 998 parasites identified as C. lingua.
10
M. Duflot et al.: Parasite 2023, 30, 28
Figure 5. Maximum likelihood trees using 1,000 bootstraps based on cox1 mtDNA (A) and ITS rDNA (B) sequences of Cryptocotyle lingua
(purple) and C. concava (blue) and reference sequences from GenBank (underlined).
There are five major haplotypes (H1 to H5) for C. lingua
cox1 sequences. The major haplotypes were shared by all
geographic areas (Fig. 7A). There was only one haplotype that
was shared by all host fish species of C. lingua, haplotype H1
(Fig. 7B). Equivalent analyses were conducted on the ITS
region sequences, but no significant results were found on the
few sequences available for this analysis.
Discussion
This is the first study investigating the diversity, prevalence
and intensity of encysted metacercariae causing black spots on
fish in the eastern English Channel (EEC) and the North Sea
(NS). Up to now, many Heterophyidae have been described
as responsible for these lesions [43, 53, 75, 76, 88]. In this
study, seven commercial fish species from the eastern English
Channel and the North Sea were characterized by their parasitic
ecology of encysted metacercariae inducing black spots. All the
studied species were susceptible to encysted metacercarial
infection. Encysted metacercariae were found mainly in the skin
layer of the fish, and they were also observed on the flesh close
to the dorsal fin (areas 7 and 9). These observations are consistent with previous descriptions, which have noted major concentrations of black spots above the lateral line and in the
skin layer [7, 20, 21, 59, 85].
M. Duflot et al.: Parasite 2023, 30, 28
11
Figure 6. Prevalence of black spot infection (left) and spatial distribution of Cryptocotyle lingua and C. concava (identified by morphological
or molecular methods) (right) by sampling stations in the English Channel and North Sea in January 2019 and 2020. The proportion reflects the
sampling size (left) and the percentage of metacercariae identified as C. lingua or C. concava (right).
Table 6. Population pairwise Fst from the mtDNA cox1 gene among Cryptocotyle lingua by geographic localities of the eastern English
Channel (EEC), Southern Bight of the North Sea (SBNS), eastern North Sea (ENS), central North Sea (CNS), western North Sea (WNS);
Below the diagonal: conventional Fst from haplotype frequencies; Above the diagonal: p-values of Fst; significance level 0.05, * = significant
value.
EEC
SBNS
ENS
CNS
WNS
EEC
–
0.00171
0.00200
0.00012
0.01199
SBNS
0.09091 ± 0.0262
–
0.00241
0.00168
0.00413
ENS
0.09917 ± 0.0242
0.14050 ± 0.0352
–
0.00027
0.00803
CNS
0.37190 ± 0.0531
0.85124 ± 0.0432
0.38017 ± 0.0544
–
0.00310
WNS
0.03306 ± 0.0184*
0.16529 ± 0.0304
0.03306 ± 0.0136
0.23967 ± 0.0361
–
Table 7. Genetic diversity values of the mtDNA cox1 gene of Cryptocotyle lingua from seven selected fish hosts, by geographic localities of
the eastern English Channel (EEC), Southern Bight of the North Sea (SBNS), eastern North Sea (ENS), central North Sea (CNS) and western
North Sea (WNS).
EEC
SBNS
ENS
CNS
WNS
Total
N
298
278
155
212
55
998
Nh
32
37
28
27
11
87
p
0.00181
0.00236
0.00268
0.00219
0.00292
0.00225
Hd
0.406
0.511
0.491
0.474
0.627
0.476
K
0.493
0.643
0.729
0.594
0.793
0.610
S
31
34
30
25
9
80
Tajima’s D
2,46711***
2,50921***
2,52705***
2,61775***
1,79176 n.s.
2.61775***
Fu’s Fs
56,857
63,891
41,223
40,894
8,171
211.987
Number of analyzed sequences (N); number of haplotypes (Nh); nucleotide diversity (p); haplotype diversity (Hd); average number of
nucleotide differences (K); number of polymorphic sites (S); two neutrality tests (Tajima’s D and Fu’s Fs). *p < 0.05; **p < 0.001;
***p < 0.0001; n.s.: not significant.
Prevalences of infection were highly variable between
the fish species and the geographical areas. Plaice, whiting
and pout were the most infected species. Prevalence, intensity
and abundance trends were not consistent between both sampling years. Nevertheless, the same trends of prevalence
between the five geographical areas were observed for whiting
and pout. Likewise, Van Den Broek [85] found divergence of
intensity and prevalence on pout and whiting samplings
between 1973 and 1975 at Kingsnorth Power Station, USA.
As previously observed by some authors [21, 55], the black
spot immune lesions can be influenced by certain environmental and inherent fish factors. McQueen et al. [59] demonstrated
that pigmented cells in plaice appeared 10 days after experimental infection, and Chapman and Hunter [17] observed
pigmented cells from 10 to 30 days after infection in the cunner
Tautogolabrus adspersus. Black spot intensity depends on the
M. Duflot et al.: Parasite 2023, 30, 28
12
Table 8. Population pairwise Fst from mtDNA cox1 gene among Cryptocotyle lingua by fish species from the English Channel and North Sea.
Below the diagonal: conventional Fst from haplotype frequencies; Above the diagonal: p-values of Fst; significance level 0.05, * = significant
value.
Herring
Sprat
Whiting
Pout
Dab
Flounder
Plaice
Herring
Sprat
Whiting
Pout
–
0.00011
0.00616
0.00351
0.00081
0.00616
0.02595
0.44531 ± 0.0174
–
0.03167
0.03053
0.04370
0.09117
0.01369
0.17773 ± 0.0080
0.09473 ± 0.0103
–
0.00101
0.00109
0.04339
0.01310
0.25977 ± 0.0119
0.10645 ± 0.0090
0.81934 ± 0.0105
–
0.00023
0.04503
0.01114
Dab
Flounder
0.47949
0.07129
0.52148
0.41797
± 0.0149
± 0.0067
± 0.0162
± 0.0152
–
0.03482
0.00376
0.41797
0.07812
0.08008
0.07812
0.14941
± 0.0197
± 0.0094
± 0.0081
± 0.0103
± 0.0098
–
0.03614
Plaice
0.80078
0.42578
0.29688
0.32129
0.45117
0.19043
±
±
±
±
±
±
–
0.0106
0.0155
0.0131
0.0159
0.0126
0.0097
Table 9. Genetic diversity values of the mtDNA cox1 gene of Cryptocotyle lingua by fish species from the English Channel and North Sea.
Herring
Sprat
Whiting
Pout
Dab
Flounder
Plaice
Total
N
31
12
642
251
44
10
8
998
Nh
13
4
61
28
9
3
5
87
p
0.00419
0.00273
0.00216
0.00217
0.00214
0.00204
0.00368
0.00225
Hd
0.735
0.636
0.4630
0.474
0.405
0.378
0.786
0.476
K
1.140
0.742
0.588
0.588
0.582
0.556
1.000
0.610
S
13
3
57
27
10
2
4
80
Tajima’s D
2,23666**
0,82879 n.s.
2,58160***
2,39500**
2,18614**
0,69098 n.s.
1,53470 n.s.
2.61775***
Fu’s Fs
11,230
1,256
131,969
42,541
7,415
0,594
2,800
211.987
Number of analyzed sequenced (N); number of haplotypes (Nh); nucleotide diversity (p); haplotype diversity (Hd); average number of
nucleotide differences (K); number of polymorphic sites (S); two neutrality tests (Tajima’s D and Fu’s Fs); *p < 0.05; **p < 0.001;
***p < 0.0001; n.s. not significant.
Figure 7. Haplotype networks of Cryptocotyle lingua based on cox1 gene sequences (n = 998 sequences) from seven fish species from the
eastern English Channel (EEC), Southern Bight of the North Sea (SBNS), eastern North Sea (ENS), central North Sea (CNS),
western North Sea (WNS) (A) and from fish hosts (B). Circles indicate haplotypes, their frequencies correspond to the circle sizes. The
haplotypes linked by a black line differ by one (no black point) or more (number of black points) substitutions. Colors are consistent with the
map in Figure 1.
location of the metacercariae on the fish [17]. Wood and
Matthews [89] found that melanization of metacercarial cysts
was most intense in fish maintained in constant darkness after
cercarial infection. In the present study, parasitological descriptors were estimated on visible black spot counts. Hence, false
negatives were probably included in this numbering since black
spots can appear only after several days. Recent infestations
could be omitted. Experimental infection challenge tests are
needed to determine the time and the biotic/abiotic conditions
that could influence black spot development in each fish
species. Due to the size of this epidemiological study, based
on more than 1,500 fish, it was technically difficult to isolate
all the encysted parasites from each individual fish. As this
study is designed to describe parasites causing black spot
diseases in marine fish and as Duflot et al. [21] described the
ratio between black spot and recovered metacercariae as close
M. Duflot et al.: Parasite 2023, 30, 28
to one, black spot counting was used to estimate parasitical
descriptors of this substantial sampling. To strengthen these
results, identifications of metacercariae were performed. For
flatfish, few metacercariae were identified in comparison to
the number of black spots observed. As flatfish possess
chromatophores on the dermal layer for differential responsiveness to backgrounds [11], their melanophores are more mobile,
so those around metacercariae could migrate after infection. On
the other hand, the presence of these chromatophores could be
mistaken for black spots due to encysted parasites. This observation could explain the discrepancy between the high intensity
and the low number of identified parasites observed on
flounder.
A total of 1,586 fish were examined and 325 of them were
parasitized by encysted metacercariae. A subsample of the
metacercariae from each sample were isolated and identified
by morphological traits or molecular tools. A total of
209 excysted metacercariae were successfully identified by
the morphological method. Four morphologies were observed
according to the morphological characteristics described by
several authors [7, 13, 27, 30, 53, 66, 80, 83]. The majority
of metacercariae were identified as C. lingua after comparison
with published general traits and measurements of the main
characteristic organs [7, 66]. In fact, observed metacercariae
differed from C. concava and C. jejuna by their body shape
and their width. Three other morphologies were observed.
Some larvae with morphological characteristics of the
Bucephalidae family were observed [27]. Due to the scarcity
of detailed identification-keys for the genus and species of this
family, parasites were only identified to the family level. Additionally, Bower [8] highlighted the lack of studies on their life
cycle and their taxonomy. From the description of Baturo
(1977) in Kinkelin et al. [42] and Al-Zubaidy [2], some metacercariae (Fig. 4, b) could belong to Bucephalus polymorphus or
Bucephalus margaritae. Other metacercariae (Fig. 4, d) could
belong to the Rhipidocotyle genus with regards to the description of Gibson [27] and Bartoli and Bray [4]. The last morphology (Fig. 4, c) observed could not be related to any former
description. However, these observations were consistent with
those of Nicoll [60], who observed Bucephalidae parasites in
different fish species from the English Channel on the British
coast. The morphological identifications were strengthened
and confirmed by molecular identification carried out on different parasites isolated from the same host. Nevertheless, as
excysted metacercariae are small parasites (less than 500 lm),
it was impossible to undertake morphology and molecular identification on the same individual.
A total of 1,034 and 95 specimens were identified by molecular methods with cox1 and ITS markers, respectively. These
two markers were selected because the available sequences targeted mainly the cox1 and ITS region within the superfamily
Opisthorchioidea [22]. Some molecular identifications of isolated metacercariae were not successful. Due to COVID-19
lockdowns, some samples were kept frozen for a long time
and frost could have damaged the DNA. According to BLAST
analysis, three main identifications were obtained with the cox1
marker: C. lingua, C. concava and larval of bucephalid spp.
Results of the ITS region identification confirmed cox1 results
with identifications of C. lingua, Bucephalus polymorphus and
13
B. margaritae. Cryptocotyle lingua identifications were confirmed by both selected markers for 71 metacercariae. The
newly generated sequences of C. lingua on cox1 and ITS markers clustered with strong bootstrap values with the reference
sequences of C. lingua. In both BLAST analysis and phylogenetic trees, the majority of studied sequences with both molecular markers revealed high similarity with both C. lingua
cercariae and metacercariae sampled in the North of Europe
and in North America [6, 7, 20] and some sequences matched
with C. lingua from Littorina littorea isolated in the White
Sea (Kartesh, Russia) [31]. The position of the Opisthorchiidae
reference sequences (Clonorchis and Opisthorchis sp.) used in
the phylogenies confirms the question of the problematic nature
of interrelationships between Opisthorchioidea. The present
phylogenies were consistent with the results of Tatonova and
Besprozvannykh [83], Kuzmina et al. [46] and Sokolov et al.
[79], which pointed out the controversial phylogenetic positions
of the family and genera of the superfamily Opisthorchioidea.
Moreover, Kacem et al. [41] observed that the Heterophyidae
and Opisthorchiidae form an inseparable single clade. The position of the Cryptocotyle genus is also controversial, sometimes
characterized as Heterophyidae [13, 14, 30] and sometimes as
Opisthorchiidae [79, 83]. These data, in addition to those of
the present study, confirm the need for additional descriptions
using both adult stage morphological identification and molecular confirmation to constitute reliable and comparable data for
further studies.
Eventually, the morphological and molecular identifications
were in accordance. Cryptocotyle lingua was the main species
causing black spot diseases identified in the English Channel
and the North Sea ecosystems. Thereby, the prevalence of
Cryptocotyle lingua can be assessed by the prevalences
obtained by counting black spot in these marine ecosystems.
Some Cryptocotyle concava and Bucephalidae larvae were also
obtained, showing that encysted metacercariae of other species
coexist in these ecosystems. The low infestation levels of these
taxa could be due to the selected anatomical sampling. Indeed,
the most visible infected area was chosen here and was mostly
represented by an area of the flesh (Nos. 7, 8, 9 or 10), but the
preferential site of infection for Cryptocotyle concava and
Bucephalidae parasites was not always the one selected for this
study. Cryptocotyle concava predominantly infects the kidney,
the fins, the gills and the skin of fish hosts [30, 91]. The
Bucephalidae have been described as encysting in deeper
tissues in fish, such as cranial nerves [87].
Cryptocotyle lingua was distributed in all the studied
species of the ecosystem of the eastern English Channel and
the North Sea. Cryptocotyle concava was observed on flounder
and plaice near the British and Netherlands coasts. These observations agreed with the detection of C. concava on gobies
Pomatoschistus microps in Stiffkey (Norfolk, UK) by
El-Mayas and Kearn [23] or on P. microps and Pomatoschistus
minutus by Malek [57]. Cryptocotyle concava is a marine
species, but is also observed in brackish, fresh and terrestrial
waters [90]. Cryptocotyle lingua was observed in Littorina
littorea in Cardigan Bay [69, 70] or in Cawsands, Plymouth
[58]. In addition, C. lingua was observed in the North of
Europe, at the metacercariae stage in Chelon labrosus
Plymouth, United Kingdom) [88], Gadus morhua (Danish
14
M. Duflot et al.: Parasite 2023, 30, 28
waters) [7, 20] or Merlangius merlangus (English Channel,
Bristol Channel and Inner Severn Estuary) [20, 62].
Mixed infections were observed in this study. As previously
discussed, the presence of trematodes different from Cryptocotyle might have been underestimated due to the sampling
strategy. Further analysis on other anatomical areas of fish will
make it possible to specify the Bucephalidae and C. concava
infection levels in the eastern English Channel and North Sea
ecosystems. Coinfections of encysted metacercariae could
amplify the severity and the dynamics of fish diseases, where
each parasite has its own effects [56]. Moreover, parasitic coinfection could lead to competition of parasites against each other
for tissue tropism inside the infected host [56].
A total of 998 and 72 specimens of C. lingua were investigated for their genetic diversity on cox1 and ITS, respectively.
The weak genetic differentiation between the five geographic
areas suggests the existence of a low level of genetic differentiation between the C. lingua metacercariae of the English
Channel and North Sea. Moreover, no specific diversity has
been observed within the different intermediate fish hosts of
Cryptocotyle lingua, which could confirm that these parasites
were not specific in the choice of this intermediate host [88].
To our knowledge, only Blakeslee et al. [6] and Gonchar
[29] have previously studied the genetic diversity of cox1
Cryptocotyle spp. cercariae in North America and in the White
Sea (Russia), respectively. Both have found minimal differentiation between location sites. Blakeslee et al. [6] demonstrated a
reduction in genetic diversity in North America vs. Europe. It
would be interesting to expand such a study on the genetic
diversity of C. lingua to broader geographical areas with
ecosystems significantly different from each other.
To conclude, this study described high prevalence and
intensity values of encysted metacercariae infection from both
regions: the eastern English Channel and the North Sea. The
major taxon observed was Cryptocotyle spp. Two species,
C. lingua and C. concava, were identified in these ecosystems.
All examined fish species and all studied geographic areas were
subject to encysted metacercariae infections.
This broad distribution brings into focus a potential risk
from a public health point of view. The Cryptocotyle genus
has been described as zoonotic [15] and fish-borne zoonotic
trematodes are gaining public attention worldwide, with more
than 18 million people infected annually [56]. Moreover, so
far, the fishing industry is not aware of the presence of this parasite and black spots are not considered a parasitological risk.
Thus, although European legislation bans the sale for human
consumption of fishery products that are obviously contaminated with parasites, seafood professionals do not discard fish
speckled with black spots. Furthermore, new sampling needs
to be carried out to assess the potential risk for consumer and
human health. Similarly, from an ecological point of view,
the abiotic and biotic factors influencing Cryptocotyle spp. distribution should be analyzed to have a better understanding of
the circulation of these parasites in a marine ecosystem.
Acknowledgements. Maureen Duflot benefited of a PhD grant
from the French Agency for Food, Environmental and Occupational
Health & Safety (ANSES) and the Hauts de France region of France.
Figure 2 was created with https://www.Biorender.com under agreement number XS22USVOV8. The authors would like to thank the
personnel onboard for both Ifremer scientific campaigns [41, 42]
who helped with the sampling and data collection.
Conflict of interest
The authors declare that they have no conflict of interest.
Supplementary materials
The supplementary material of this article is available at
https://www.parasite-journal.org/10.1051/parasite/2023028/olm.
Supplementary data 1: Infection with encysted metacercariae reporting data of the different infected samples, according to fish areas: (1) caudal fin, (2) 2nd dorsal fin, (3) 1st
dorsal fin, (4) pectoral fin, (5) pelvic fin, (6) anal fin, (7) dorsofrontal area, (8) ventrofrontal area, (9) dorsoposterior area,
(10) ventroposterior area, (11) opercula, and (12) eye. Bold letters indicate cases in which the average numbers of observed
black spots are the 5th highest of each fish species (R: right
side, L: left side).
Supplementary data 2: Summary of BLAST analysis results
on the cox1 gene and ITS region of rDNA sequences; *: number of sequences from parasites isolated in the present study.
Supplementary data 3: Haplotype network groups of C. lingua for cox1 and ITS and their number of sequences.
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Cite this article as: Duflot M, Cresson P, Julien M, Chartier L, Bourgau O, Palomba M, Mattiucci S, Midelet G & Gay M. 2023. Black
spot diseases in seven commercial fish species from the English Channel and the North Sea: infestation levels, identification and population
genetics of Cryptocotyle spp.. Parasite 30, 28.
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