Post-mortem 'soft flesh' in three commercial fish species from off Atlantic Morocco associated with the myxosporean parasites Kudoa thyrsites and K. encrasicoli (Myxozoa).

Small pelagic fishes represent one of the most important food resources off the Northwest coast of Africa. Despite their economic significance, little is known about the infections with flesh invading myxosporean parasites of genus Kudoa (Cnidaria, Myxozoa). Heavy infections in the flesh may be associated with post-mortem myoliquefaction, commonly known as 'soft flesh'. This condition may reduce the quality and marketability of the fish fillet, resulting in both economic losses to the fishing industry and loss of consumer confidence. In this study, we investigated Kudoa-induced 'soft flesh' occurrence in European anchovy Engraulis encrasicolus, European pilchard Sardina pilchardus, and Atlantic chub mackerel Scomber colias caught in 2019 off the Moroccan Atlantic coast. Five hundred specimens of each fish species were examined for 'soft flesh' by texture testing and visual inspection 48 h post-catch. 'Soft flesh' occurred in 0.2 % of the European anchovies, 1.4 % of the European pilchard, and in 4.4 % of the Atlantic chub mackerel. Microscopic examination of muscle samples revealed that 'soft flesh'-affected fish were infected with myxospores of K. thyrsites-like morphotype. Analysis of the kudoid SSU rDNA sequence obtained from European pilchard and the Atlantic chub mackerel identified these as K. thyrsites (100 % identity), whereas analysis of the sequence from European anchovy identified the presence of K. encrasicoli (100 % identity). Even if there are no known human health consequences associated with the ingestion of these Kudoa species, the unsightly appearance of some infected fillets is a food quality issue, that can eventually lead to reduced marketability and value.

Resurrection of genus Phocanema Myers, 1959, as a genus independent from Pseudoterranova Mozgovoi, 1953, for nematode species (Anisakidae) parasitic in pinnipeds and cetaceans, respectively.

Species of the genus Pseudoterranova, infect kogiid cetaceans and pinnipeds. However, there is mounting molecular evidence that those from cetaceans and pinnipeds are not congeneric. Here, we provide further evidence of the non-monophyly of members of Pseudoterranova from phylogenetic analyses of the conserved nuclear LSU rDNA gene, entire ITS rDNA region and mtDNA cox2 gene, and identify morphological characters that may be used to distinguish the members of the two clades. We propose the resurrection of the genus Phocanema, with Ph. decipiens (sensu stricto) as the type species, to encompass Ph. decipiens, Ph. azarasi, Ph. bulbosa, Ph. cattani and Ph. krabbei, all parasites of pinnipeds. We propose to restrict the conception of genus Pseudoterranova, which now harbours two species infecting kogiid whales; Ps. kogiae (type species) and Ps. ceticola. Members of the genera Phocanema and Pseudoterranova differ by the shape and orientation of the lips, relative tail lengths, adult size, type of final host (pinniped vs. cetacean) and phylogenetic placement based on nuclear rDNA and mtDNA cox2 sequences.

Anisakid parasites (Nematoda: Anisakidae) in 3 commercially important gadid fish species from the southern Barents Sea, with emphasis on key infection drivers and spatial distribution within the hosts.

Northeast Arctic cod, saithe and haddock are among the most important fisheries resources in Europe, largely shipped to various continental markets. The present study aimed to map the presence and distribution of larvae of parasitic nematodes in the Anisakidae family which are of socioeconomic and public health concern. Fishes were sourced from commercial catches during winter or spring in the southern Barents Sea. Samples of fish were inspected for nematodes using the UV-press method while anisakid species identification relied on sequencing of the mtDNA cox2 gene. Anisakis simplex (s.s.) was the most prevalent and abundant anisakid recorded, occurring at high infection levels in the viscera and flesh of cod and saithe, while being less abundant in haddock. Contracaecum osculatum (s.l.) larvae, not found in the fish flesh, showed moderate-to-high prevalence in saithe, haddock and cod, respectively. Most Pseudoterranova spp. larvae occurred at low-to-moderate prevalence, and low abundance, in the viscera (Pseudoterranova bulbosa) and flesh (Pseudoterranova decipiens (s.s.) and Pseudoterranova krabbei) of cod, only 2 P. decipiens (s.s.) appeared in the flesh of saithe. Body length was the single most important host-related factor to predict overall abundance of anisakid larvae in the fish species. The spatial distribution of Anisakis larvae in the fish flesh showed much higher abundances in the belly flaps than in the dorsal fillet parts. Trimming of the flesh by removing the belly flaps would reduce larval presence in the fillets of these gadid fish species by 86­91%.

Characterization of Pseudoterranova ceticola (Nematoda: Anisakidae) larvae from meso/bathypelagic fishes off Macaronesia (NW Africa waters).

The genus Pseudoterranova includes parasite species of cetaceans and pinnipeds. The third stage larva (L3) of seal-infecting species occur in second intermediate or paratenic fish hosts mainly in neritic waters. This study firstly describes a Pseudoterranova L3 from meso/bathypelagic fishes off Macaronesia. L3s were morphologically and genetically studied by light microscopy and sequencing of the mtDNA cox2 and entire ITS rDNA genes. Bayesian inferences were performed with sequences from the larvae and selected sequences from GenBank. The nematode L3s were molecularly identified as Pseudoterranova ceticola, a parasite of kogiid whales. Such larvae were collected from Bolinichthys indicus, Chauliodus danae, Eupharynx pelecanoides, Diaphus rafinesquii, D. mollis, Diretmus argenteus and Maulisia argipalla. They mainly occurred in the viscera of these fishes. Pseudoterranova ceticola L3 were small (< 12 mm) and whitish, and a prominent characteristic is a circumoral ridge extending from the ventral boring tooth which differentiate them from Pseudoterranova spp. L3 maturing in pinnipeds and Terranova sensu lato larvae that mature in poikilotherms. The shape of the tail: conical, long, pointed, ventrally curved and lacking mucron also distinguish these larvae from those of the pinniped-infecting Pseudoterranova spp. Phylogenetic analyses based on mtDNA cox2 and ITS rDNA sequences suggest that P. ceticola is closely related to Skrjabinisakis spp., and not with Pseudoterranova spp. parasitizing pinnipeds. The related species Skrjabinisakis paggiae, S. brevispiculata and S. physeteris (until recently belonging to genus Anisakis), are as P. ceticola also parasites of physeteroid cetaceans. The morphology and morphological variation of the larvae of the cetacean parasite P. ceticola is thoroughly described for the first time. These L3 can readily be morphologically distinguished from those of the pinniped-infecting Pseudoterranova spp. The parasite likely completes its life cycle in the mesopelagic and bathypelagic realm, with meso/bathypelagic fish as 2nd intermediate or paratenic hosts and kogiids as final host. Thus, Pseudoterranova from cetaceans appear to be morphologically, genetically, and ecologically differentiated to those from pinnipeds, suggesting that they are not congeneric.

Anisakid nematodes in Trichiurus lepturus and Saurida undosquamis (Teleostea) from the South-West Indian Ocean: Genetic evidence for the existence of sister species within Anisakis typica (s.l.), and food-safety considerations.

Nematode parasites of the genus Anisakis (Nematoda, Anisakidae) are considered among the most important biological hazards in seafood products worldwide. In temperate and tropical waters, the most common species appears to be Anisakis typica, generally found around the viscera and sporadically in the flesh of various fish host species. This study investigated the infection sites and genetic diversity of A. typica infecting commercial fishes from the South-West Indian Ocean. Largehead hairtail (N = 20) and brushtooth lizardfish (N = 72) fished off Tanzania were inspected for anisakid nematodes by UV-press. A subsample of 168 nematodes were identified by sequence analyses of the cox2 mtDNA gene and ITS region of rDNA. The species A. typica (s.l.) (N = 166), Pseudoterranova ceticola (N = 1) and Anisakis paggiae (N = 1) were molecularly identified. Phylogenetic analysis of A. typica (s.l.) sequences based on both genes, indicated the existence of two distinct phylogenetic lineages forming two well-supported clades. The first clade comprised 12 A. typica specimens including individuals from its type locality (central Atlantic Ocean). The second clade comprising 154 specimens, clustered with reference sequences retrieved from GenBank including one apparently undescribed taxon, i.e., Anisakis sp. 1, and A. typica var. indonesiensis. The two reciprocally monophyletic clades are closely related and correspond to two distinct sister species within A. typica (s.l.), presently indicated as A. typica sp. A and A. typica sp. B. Two and four fixed alternative nucleotide substitutions (SNPs), i.e., diagnostic positions, between the two taxa, respectively, were found at the mtDNA cox2 and the ITS region of rDNA. The genetic data, as well as their occurrence in sympatry, strengthens the hypothesis that the actual specimens represent two distinct gene pools. The occurrence of both A. typica sp. A and A. typica sp. B in the musculature of freshly examined T. lepturus and S. undosquamis, suggests that both species can migrate intra-vitam into the flesh. Although the zoonotic potential of A. typica s.l. is still unclear, the presence of these parasites in the musculature, edible part of the fish, raises health concerns for consumers.

Ascaridoid nematodes infecting commercially important marine fish and squid species from Bangladesh waters in the Bay of Bengal.

Parasitic ascaridoid nematodes occur in a wide range of marine organisms across the globe. Some species of the anisakid family (Ascaridoidea: Anisakidae) can cause gastrointestinal disease in humans (i. e. anisakidosis). Despite their importance as potentially hazardous parasites, the occurrence and infection characteristics of ascaridoids are still poorly known from many host species and geographical areas. This study investigated the diversity and infection levels of ascaridoid parasites in various commercial fish and squid host species off Bangladesh. Fish and squid specimens were visually inspected for nematodes using the UV-press method. Nematodes were assigned to genus level based on morphology and identified by sequence analyses of the entire ITS region and partial 28S rDNA and mtDNA cox2 genes. Third-stage larvae (L3) of Anisakis typica occurred at low prevalence (P = 10% and 8%, respectively) in the viscera of Selar crumenophthalmus and Trichiurus lepturus, while Hysterothylacium amoyense occurred in the viscera of Sardinella fimbriata (P = 1%) and the viscera and muscle of Harpadon nehereus (P = 32%) and T. lepturus (P = 76%). Lappetascaris sp. Type A L3 occurred in the mantle of the squid Uroteuthis duvaucelii (P = 11%). Anisakis and Lappetascaris species, and H. amoyense were firstly identified in the Bay of Bengal. The potentially zoonotic A. typica was only found in fish viscera. Hysterothylacium amoyense and Lappetascaris sp., both generally regarded as non-zoonotic, occurred at low prevalence in the muscle or mantle of fish or squid, respectively. Since consumption of raw or lightly processed seafood seems to be rare in Bangladesh, the risk of acquiring anisakidosis from consuming fishery products from off Bangladesh appears to be low. Due to its reddish appearance, the visual presence of H. amoyense larvae in fish flesh may represent a food quality issue.

Anisakis simplex (s.s.) larvae (Nematoda: Anisakidae) hidden in the mantle of European flying squid Todarodes sagittatus (Cephalopoda: Ommastrephidae) in NE Atlantic Ocean: Food safety implications.

Few reports exist upon the occurrence and localization of zoonotic anisakid nematodes in T. sagittatus, especially in the mantle of the squid. The occurrence and site of infection of larval anisakids in 98 T. sagittatus caught West off St. Kilda, NE Atlantic Ocean, were investigated. Squids were examined for anisakids using the UV-Press method. In total, 689 nematodes were detected in the viscera and mantle. According to morphology, all the larvae (L3) were assigned to genus Anisakis. Diagnostic allozymes and mtDNA cox2 sequence analysis permitted to genetically identify all larvae as Anisakis simplex (s.s.) (N = 100). Overall prevalence (P = 81%) and mean intensity (mI = 8.6) of infection with A. simplex are provided. Most of the larvae present in the mantle cavity were embedded in the stomach wall or attached in the outer layer of the stomach and caecum (49%). Over a third of squids (37%) hosted A. simplex (s.s.) larvae in the mantle. A novel schematized representation of larvae distribution in the mantle is provided, showing where they were mostly located. According to the results obtained, the risk of anisakiasis associated with consumption of raw or undercooked T. sagittatus should be considered.

Occurrence of larval ascaridoid nematodes in the Argentinean short-finned squid Illex argentinus from the Southwest Atlantic Ocean (off Falkland Islands).

The Argentinean short-finned squid (Illex argentinus) is an oceanic, neritic species widely distributed off the east coast of South America, representing the most abundant commercially exploited squid species in these waters. Despite the great commercial importance of Argentinean short-finned squid as a food resource, and as frozen product exported to Europe, the presence of zoonotic anisakid nematodes, especially in the mantle of the squid, is poorly known. The occurrence and site of infection of larval ascaridoid nematodes in 70 I. argentinus caught off the Falkland Islands were investigated. Squids were examined using the UV-Press method. In total, 30 nematodes were detected in the viscera and mantle. According to morphology, 27 were third-stage larvae (L3) belonging to genus Anisakis, while three were L3 assigned to Hysterothylacium. Anisakis pegreffii (n = 27) were identified by sequence analysis of the mtDNA cox2 and the partial EF1 α-1 region of nDNA genes; Hysterothylacium aduncum (N = 3) were identified by sequence analysis of the ITS rDNA region. These findings represent the first molecular identification of A. pegreffii and H. aduncum in I. argentinus. Both prevalence (P = 15.7%) and abundance (A = 0.39) of infection with A. pegreffii were low, and even lower values of infection were recorded for H. aduncum (P = 2.1%, A = 0.04). Only 3 out of 70 (4.3%) squids hosted A. pegreffii larvae in the mantle. Larvae infecting viscera were coiled and mainly attached to outer surface of visceral organs. Mantle-infecting larvae were situated in the posterior half. Thus, these results suggest that - although low - the risk of acquiring anisakiasis from consumption of raw, marinated and/or undercooked short-finned squid products still exists.

Morphological and molecular identification of a new Kudoa thyrsites isolate in Mediterranean silver scabbardfish Lepidopus caudatus.

Myxozoans of the genus Kudoa (Myxosporea, Multivalvulida) infect marine and estuarine fish species worldwide. Some Kudoa species are of concern to the seafood industry since they may generate macroscopic cysts in the fish host's musculature, or cause post mortem myoliquefaction, commonly known as 'soft flesh'. One of the economically most important species is K. thyrsites, a myoliquefactive myxosporean parasite that occurs in many wild and cultured marine fish species worldwide. Here we identified a K. thyrsites isolate as the causative agent of myoliquefaction in silver scabbardfish Lepidopus caudatus from the Alboran Sea (western Mediterranean Sea). For comparative and validation purposes, the morphological and molecular characteristics of the isolate were compared with fresh spores of a K. thyrsites isolate infecting Atlantic mackerel Scomber scombrus from the Norwegian Sea. Myxospores of both isolates shared a stellate appearance and contained 4 unequal pyriform polar capsules (1 large, 1 small and 2 intermediate). These morphological traits were consistent with all other previously described K. thyrsites isolates. Moreover, the small subunit rDNA sequences of the Mediterranean and Norwegian Sea isolates revealed 100% similarity, and matched 100% with K. thyrsites isolates previously recorded in myoliquefactive Atlantic mackerel from the North Sea and off southern England. The findings suggest that K. thyrsites is the primary cause of myoliquefaction in silver scabbardfish from the Alboran Sea. This report represents the first morphological and molecular characterization of K. thyrsites in the Mediterranean Sea. A set of new allometric characters is proposed as additional descriptors for more accurate and specific description of kudoid myxospores.

Assessing the risk of an emerging zoonosis of worldwide concern: anisakiasis.

Anisakiasis is an emerging zoonosis caused by the fish parasitic nematode Anisakis. Spain appears to have the highest reported incidence in Europe and marinated anchovies are recognised as the main food vehicle. Using data on fishery landings, fish infection rates and consumption habits of the Spanish population from questionnaires, we developed a quantitative risk assessment (QRA) model for the anchovy value chain. Spaniards were estimated to consume on average 0.66 Anisakis per untreated (non-frozen) raw or marinated anchovy meal. A dose-response relationship was generated and the probability of anisakiasis was calculated to be 9.56 × 10-5 per meal, and the number of annual anisakiasis cases requiring medical attention was predicted between 7,700 and 8,320. Monte Carlo simulations estimated post-mortem migration of Anisakis from viscera to flesh increases the disease burden by >1000% whilst an education campaign to freeze anchovy before consumption may reduce cases by 80%. However, most of the questionnaire respondents who ate untreated meals knew how to prevent Anisakis infection. The QRA suggests that previously reported figures of 500 anisakiasis per year in Europe is a considerable underestimate. The QRA tool can be used by policy makers and informs industry, health professionals and consumers about this underdiagnosed zoonosis.