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.

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.

Title: 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. Abstract: Les poissons sont souvent parsemés de « points noirs ¼ 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 ¼ parmi les poisons d'importance commerciale. Par ailleurs, des observations de « points noirs ¼ 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 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.

Optimization of tools for the detection and identification of Cryptocotyle metacercariae in fish: Digestion method and viability studies.

Some trematode metacercariae, including marine digeneans belonging to the genus Cryptocotyle, induce black spots in target tissues due to the attraction of fish host melanophores. To promote precise quantification of infection, the counting of black spots has to be confirmed by reliable quantification of metacercariae after tissue digestion. This process ensures the isolation of undamaged parasites for morphological and molecular identification. The aim of this work was to optimize the pepsin digestion protocol and to assess the duration of viability of Cryptocotyle metacercariae in fish post-mortem (pm). Four digestion protocols were compared by measuring the viability rate of metacercariae. The present study shows that the orbital digestion method was the least destructive for metacercariae and allowed better quantification of Cryptocotyle infection. Moreover, morphological identification seemed reliable up to 8 days pm for Cryptocotyle infection.

Metazoan parasite communities in Alosa alosa (Linnaeus, 1758) and Alosa fallax (Lacépède, 1803) (Clupeidae) from North-East Atlantic coastal waters and connected rivers.

Metazoan parasites were studied in 96 Alosa alosa and 78 Alosa fallax from North-East Atlantic coastal waters and connected rivers (among them three sympatric sites) in order to increase knowledge on these anadromous endangered fish and measure the parasitic impact on host condition. All shads were infected by one to six metazoan parasite taxa among the 12 identified in the whole sampling, with a mean abundance of parasites higher for A. alosa (167 ± 10) than for A. fallax (112 ± 11). Helminths, mostly trophically transmitted, were the best represented (eight taxa, prevalence up to 99%) in contrast with crustaceans and Petromyzontidae that rarely occurred (four taxa, prevalence <6%). Despite some quantitative differences, metazoan parasite communities of A. alosa and A. fallax remained stable in composition whatever the host developmental stage, sex, sample site, and salinity. Among the nine parasite taxa harbored by each Alosa species, six were shared with some differences in distribution patterns including in sympatric conditions, suggesting increasing dissimilarities between A. alosa and A. fallax with the age. Information on feeding ecology provided by trophically transmitted helminths confirmed euryphagous opportunistic diet of immatures and adults of both shad species, and assessed feeding of adults during spawning migrations. Our study also revealed the significant negative impact of Hemiurus appendiculatus on A. alosa and Pronoprymna ventricosa on A. fallax. Because helminth parasites are omnipresent in the shads and decrease their fitness, parasitological data must be included in further investigations and management programs on A. alosa and A. fallax.