Species composition and infection levels of Anisakis (Nematoda: Anisakidae) in the skipjack tuna Katsuwonus pelamis (Linnaeus) in the Northwest Pacific.

Parasites can be used as biological tags to assess stock structures in various marine fish species. In the present study, the species composition and infection levels of parasitic nematodes of the genus Anisakis in the skipjack tuna Katsuwonus pelamis were examined in the Northwest Pacific and adjacent seas. A total of 867 third-stage larvae of Anisakis were collected from 112 skipjack tunas captured around Japan and in other subtropical localities. All larvae were identified as A. berlandi, A. pegreffii, A. simplex (s.s.), A. typica, and A. physeteris (s.l.) by the direct sequencing of the mitochondrial cox2 gene and real-time PCR assays targeting the nuclear ITS region. Anisakis species composition differed among northeastern Japan, the Sea of Japan, and other areas (central Japan, the Nansei Islands, and subtropical region), which is largely concordant with previous stock discrimination of skipjack tuna. Molecular phylogenetic analysis resulted in two intraspecific genetic groups in A. simplex (s.s.), one of which occurred almost exclusively in northeastern Japan. This could be a useful indicator for stock discrimination. Skipjack tunas from northeastern Japan were also characterized by a remarkable variety in the intensity of A. simplex (s.s.), suggesting the commingling of individuals with different migration patterns. This idea might be further justified by the geographic distribution of two genetically distinct groups of A. physeteris (s.l.).

First report of Kudoa thunni and Kudoa musculoliquefaciens affecting the quality of commercially harvested yellowfin tuna and broadbill swordfish in Eastern Australia.

Recent anecdotal reports from seafood processors in eastern Australia have described an increased occurrence of post-mortem myoliquefaction ('jellymeat') in broadbill swordfish Xiphias gladius, and macroscopic cysts throughout the musculature of yellowfin tuna Thunnus albacares. A genus of parasitic cnidarians, Kudoa (Myxosporea, Multivalvulida), species of which are known to occur in economically important wild-caught fish species globally, can cause similar quality-deterioration issues. However, Kudoa sp. epizootiology within commercially harvested, high-value fish caught within Australia is poorly understood, despite the parasite's economic importance. To determine the causative agent responsible for the observed quality deterioration in swordfish and yellowfin tuna, muscle-tissue samples from seafood processors in Mooloolaba, Australia, collected from October 2019-February 2020, were examined for parasitic infection. Kudoid myxospores were identified from both hosts and were subquadrate in shape, with four equal-sized polar capsules. The SSU rDNA sequences from both fish shared > 99% identity to Kudoa species. Kudoa musculoliquefaciens was isolated from 87.1% of swordfish sampled, suggesting that it is a widespread parasite in swordfish from the southwest Pacific Ocean. This study provides the first molecular and morphological characterisation of Kudoa thunni in yellowfin tuna and K. musculoliquefaciens in swordfish harvested from the waters of eastern Australia, expanding the geographical distribution of K. thunni and K. musculoliquefaciens to include the Coral and Tasman Seas. We demonstrate that not all infected swordfish progress to jellymeat, show the usefulness of molecular tools for reliably identifying infection by Kudoa spp., and add to the overall knowledge of kudoid epizootiology in wild-caught fish.

In vivo and in vitro assessment of host-parasite interaction between the parasitic copepod Brachiella thynni (Copepoda; Lernaeopodidae) and the farmed Atlantic bluefin tuna (Thunnus thynnus).

The Atlantic bluefin tuna (ABFT; Thunnus thynnus) today represents one of the economically most important species for Croatian fisheries industry. Although the most diverse and abundant parasitofauna is usually found in the largest specimens of wild ABFT, the opposite was observed in captivity where parasite populations significantly decline by the end of the farming cycle. Copepod Brachiella thynni, is a skin parasite frequently parasitizing tuna, whose population also decreases in number throughout the rearing process. In order to better understand the immunity mechanisms underlying ABFT reaction to B. thynni infection, we studied expression profiles of immunity related genes; interleukin 1ß (il1ß), tumour necrosis factors (tnfα1, tnfα2), complement component 4 (c4) and caspase 3 (casp3), in peripheral blood leukocytes (PBLs) during in vitro stimulation by B. thynni protein extracts (i.e. antigens) and in infected tissues at B. thynni parasitation site. Finally, a histopathological analysis of semi-thin and ultra-thin sections of tissues surrounding B. thynii attachment site was performed to evaluate the severity of parasite-induced lesions and identify involved cell lineages. In vitro stimulation of ABFT PBLs with B. thynii antigens caused a dose-depended upregulation of selected genes, among which tnfα1 showed the highest induction by both concentrations of B. thynni protein extract. However, targeted genes were not significantly upregulated in the infected tissue. Also, no significant alterations in ultrastructure of epithelial layers surrounding B. thynii attachment site were noticed, except local tissue erosion, necrosis of squamous epithelium and proliferation of rodlet and goblet cells. Our results suggest that B. thynii has evolved strategies to successfully bypass both innate immune response and the connective-tissue proliferation processes. Therefore, the observed disappearance of this copepod by the end of the rearing process is more likely related to its limited lifespan on the host and its inability to complete the life cycle in the rearing cages, rather than host's reaction.

Relationship between Southern Bluefin Tuna, Thunnus maccoyii, melanomacrophage centres and Cardicola spp. (Trematoda: Aporocotylidae) infection.

Southern Bluefin Tuna (SBT), Thunnus maccoyii, is ranched off Port Lincoln, South Australia and is Australia's second largest economic finfish aquaculture industry. The biggest threats to SBT health identified by the industry are the blood flukes Cardicola forsteri and C. orientalis (Trematoda: Aporocotylidae). Melanomacrophage centres (MMCs) are aggregations of pigmented macrophage like cells present in spleen, kidney and liver of teleost fish. The aim of this study was to quantify MMCs in SBT anterior kidney, liver and spleen to investigate changes in relation to Cardicola spp. Infection. Samples were collected at the end of ranching from pontoons where SBT were treated with PZQ and pontoons with untreated SBT. SBT MMC percentage of surface area cover was highest in SBT spleen and lowest in the liver. Significant positive correlations were identified between SBT MMC area and SBT size in all three organs (p < 0.05). MMC area and parasite infection showed significant positive correlations in the kidney and spleen for Cardicola spp. gill egg counts, and in the kidney for C. forsteri DNA from SBT hearts and gills (p < 0.05). MMCs area increased with increased intensity of Cardicola spp. Infection and MMCs have the potential to be used as an indicator to assess health effects that Cardicola spp. have on SBT.

Remarkable morphological variation in the proboscis of Neorhadinorhynchus nudus (Harada, 1938) (Acanthocephala: Echinorhynchida).

The acanthocephalans are characterized by a retractible proboscis, armed with rows of recurved hooks, which serves as the primary organ for attachment of the adult worm to the intestinal wall of the vertebrate definitive host. Whilst there is a considerable variation in the size, shape and armature of the proboscis across the phylum, intraspecific variation is generally regarded to be minimal. Consequently, subtle differences in proboscis morphology are often used to delimit congeneric species. In this study, striking variability in proboscis morphology was observed among individuals of Neorhadinorhynchus nudus (Harada, 1938) collected from the frigate tuna Auxis thazard Lacépède (Perciformes: Scombridae) in the South China Sea. Based on the length of the proboscis, and number of hooks per longitudinal row, these specimens of N. nudus were readily grouped into three distinct morphotypes, which might be considered separate taxa under the morphospecies concept. However, analysis of nuclear and mitochondrial DNA sequences revealed a level of nucleotide divergence typical of an intraspecific comparison. Moreover, the three morphotypes do not represent three separate genetic lineages. The surprising, and previously undocumented level of intraspecific variation in proboscis morphology found in the present study, underscores the need to use molecular markers for delimiting acanthocephalan species.

Incidence of three Kudoa spp., K. neothunni, K. hexapunctata, and K. thunni (Myxosporea: Multivalvulida), in Thunnus tunas distributed in the western Pacific Ocean.

A variety of tunas of the genus Thunnus are consumed daily in Japan as sliced raw fish (sashimi and sushi). The consumption of fresh sliced raw fish, i.e., unfrozen or uncooked, can sometimes cause food poisoning that is manifested by transient diarrhea and vomiting for a single day. One of the causes of this type of food poisoning has been identified as live Kudoa septempunctata (Myxosporea: Multivalvulida) in the olive flounder (Paralichthys olivaceus). Furthermore, raw slices of fresh tunas are highly suspected to be a possible causative fish of similar food poisoning in Japan. In the present study, we conducted a survey of kudoid infections in tunas (the yellowfin tuna Thunnus albacares, the Pacific bluefin tuna Thunnus orientalis, and the longtail tuna Thunnus tonggol) fished in the western Pacific Ocean off Japan and several East Asian countries and characterized morphologically and genetically the kudoid myxospores in pseudocysts or cysts dispersed in the trunk muscles. Pseudocysts of solely Kudoa hexapunctata were identified in the Pacific bluefin tuna (four isolates), whereas in the yellowfin tuna (21 isolates) pseudocysts of Kudoa neothunni and K. hexapunctata were detected at a ratio of 15:6, respectively, in addition to cyst-forming Kudoa thunni in five yellowfin tunas. In the trunk muscles of six longtail tunas examined, pseudocysts of K. neothunni (all six fish) and K. hexapunctata (two fish) were densely dispersed. The myxospores of K. neothunni found in these longtail tunas had seven shell valves and polar capsules (SV/PC) instead of the more common six SV/PC arranged symmetrically. Nucleotide sequences of the 18S and 28S ribosomal RNA gene (rDNA), some with the internal transcribed spacer regions as well, of K. hexapunctata and K. neothunni from the three Thunnus spp., including the seven-SV/PC morphotype, were very similar to previously characterized nucleotide sequences of each species, whereas the 18S and 28S rDNA of four isolates of K. thunni from yellowfin tunas showed a range of nucleotide variations of 99.0-99.9% identity over 1752-1763-bp long partial 18S rDNA and 97.4-99.9% identity over 797-802-bp long partial 28S rDNA. Therefore, this rather high variation of the rDNA nucleotide sequences of K. thunni proved to be contrary to the few variations of K. neothunni and K. hexapunctata rDNA nucleotide sequences. The present study provides a new host record of the longtail tuna for K. neothunni and K. hexapunctata and reveals a high prevalence of the seven-SV/PC myxospore morphotype of K. neothunni in this tuna host.

Cross-fertilization as a reproductive strategy in a tissue flukes Didymosulcus katsuwonicola (Platyhelmintes: Didymozoidae) inferred by genetic analysis.

Mitochondrial DNA locus cytochrome oxidase I was used to asses intraspecific genetic diversity of a didymozoid species Didymosulcus katsuwonicola. Adult forms of this species live encapsulated in pairs in the gills of the reared Atlantic bluefin tuna (Thunnus thynnus). The life cycle of this food-borne parasites and its migration in the host tissues after releasing from the digestive tract to the definitive site in the gills are unknown. Our goal was to assess whether two encysted didymozoids share the same haplotype, indicative of a common maternal origin, as well as the extent of cross- in respect to self-fertilization strategy. Intraspecific comparison showed high haplotype diversity, while the presence of two matching haplotypes within a single cyst encompassed only 17% of sampled individuals. This infers that cross-fertilization between paired individuals within the cyst is more common mechanism than self-fertilization. Such hermaphroditic parasite's trait suggests the existence of intricate infection and reproduction mechanisms, presumably as an adaptation for successful fulfillment of their indirect life cycle through dissemination of genetically more diverse and consequently more fit offspring.

Antigenicity of Anisakis simplex s.s. L3 in parasitized fish after heating conditions used in the canning processing.

BACKGROUND: Some technological and food processing treatments applied to parasitized fish kill the Anisakis larvae and prevent infection and sensitization of consumers. However, residual allergenic activity of parasite allergens has been shown. The aim here was to study the effect of different heat treatments used in the fish canning processing industry on the antigen recognition of Anisakis L3. Bigeye tuna (Thunnus obesus) and yellowfin tuna (Thunnus albacares) were experimentally infected with live L3 Anisakis. After 48 h at 5 ± 1 °C, brine was added to the muscle, which was then canned raw (live larvae) or heated (90 °C, 30 min) (dead larvae) and treated at 113 °C for 60 min or at 115 °C for 90 min. Anisakis antigens and Ani s 4 were detected with anti-crude extract and anti-Ani s 4 antisera respectively. RESULTS: Ani s 4 decreased in all lots, but the muscle retained part of the allergenicity irrespective of the canning method, as observed by immunohistochemistry. Dot blot analysis showed a high loss of Ani s 4 recognition after canning, but residual antigenicity was present. CONCLUSION: The results indicate that heat treatment for sterilization under the conditions studied produces a decrease in Ani s 4 and suggest a potential exposure risk for Anisakis-sensitized patients.

Morphological characterisation and identification of four species of Cardicola Short, 1953 (Trematoda: Aporocotylidae) infecting the Atlantic bluefin tuna Thunnus thynnus (L.) in the Mediterranean Sea.

Blood flukes of the genus Cardicola Short, 1953 are considered the most potentially pathogenic parasites in bluefin tuna cultures. Morphological study and genetic analyses of the ribosomal internal transcribed spacer ITS-2 and the mitochondrial cytochrome c oxidase 1 (cox1) gene fragments revealed the occurrence of four aporocotylid species (C. forsteri Cribb, Daintith & Munday, 2000, C. orientalis Ogawa, Tanaka, Sugihara & Takami, 2010, C. opisthorchis Ogawa, Ishimaru, Shirakashi, Takami & Grabner, 2011 and Cardicola sp.) in 421 Thunnus thynnus (L.) from the Western Mediterranean (274 fished from the wild and 147 from sea-cages). Cardicola opisthorchis was the most abundant species, with higher prevalence in the cage-reared fish than in those fished in the wild (21 vs 6%, p < 0.05). Adults of three species were recovered: C. forsteri from both gills and heart, C. opisthorchis from heart and C. orientalis from gills. The secondary gill lamellae were profusely infected by eggs of C. orientalis. A fourth species was found in four tunas, based on the molecular analyses of eggs apparently indistinguishable in size and shape from the eggs of C. orientalis. The findings provided evidence that infections with Cardicola spp. differed in relation to locality, host origin (wild vs cage-reared) and site of infection. It is necessary to estimate the possible different pathogenic effects of each species of Cardicola in order to take appropriate control measures.

Anisakid larvae in the skipjack tuna Katsuwonus pelamis captured in Japanese waters: Two-year monitoring of infection levels after the outbreak of human anisakiasis in 2018.

In 2018, human anisakiasis caused by the ingestion of the skipjack tuna Katsuwonus pelamis occurred frequently in Japan. This may be attributable to a heavy infection of A. simplex (s.s.) in the host's muscle tissue. In this study, we investigate infection levels of anisakid L3 larvae in skipjack tuna captured in Japanese waters afterward (2019-2020) to contribute to predict and prevent the outbreak of human anisakiasis. A total of 476 larvae were detected from 78 out of 85 skipjack tuna captured at 14 stations of the Pacific and East China Sea. The present parasitological survey suggests that infection levels in 2019-2020 were low, comparing that in 2018; in total only seven larvae were found from the host's muscle tissue. The collected larvae were identified by molecular methods to Anisakis berlandi, A. pegreffii, A. simplex (s.s.), A. typica and Skrjabinisakis physeteris (s.l.). Not only larvae of A. simplex (s.s.) but also those of A. berlandi were found from the muscle tissue and thus the latter species may also be a causative agent of human anisakiasis. In addition, this study confirmed the geographic distribution pattern that A. simplex (s.s.) is abundant in the Pacific, while A. pegreffii is dominant in the East China Sea. Our results contribute to understanding the risk of food poisoning and stock delimitation of host animals.

Characterization of the ribosomal RNA gene of Kudoa neothunni (Myxosporea: Multivalvulida) in tunas (Thunnus spp.) and Kudoa scomberi n. sp. in a chub mackerel (Scomber japonicus).

Kudoa neothunni is the first described Kudoa species having six shell valves and polar capsules, previously assigned to the genus Hexacapsula Arai and Matsumoto, 1953. Since its genetic analyses remain to be conducted, the present study characterizes the ribosomal RNA gene (rDNA) using two isolates from a yellowfin tuna (Thunnus albacares) with post-harvest myoliquefaction and a northern bluefin tuna (Thunnus thynnus) without tissue degradation. Spores of the two isolates localized in the myofiber of trunk muscles, forming pseudocysts, and showed typical morphology of K. neothunni with six equal-sized shell valves radially arranged in apical view: spores (n = 15) measuring 9.5-11.4 µm in width, 7.3-8.6 µm in suture width, 8.9-10.9 µm in thickness, and 7.3-7.7 µm in length; and polar capsules measuring 3.6-4.1 µm by 1.8-2.3 µm. In lateral view, the spores were pyramidal in shape without apical protrusions. Their 18S and 5.8S rDNA sequences were essentially identical, but variations in the ITS1 (62.4 % similarity across 757-bp length), ITS2 (66.9 % similarity across 599-bp length), and 28S (99.0 % similarity across 2,245-bp length) rDNA regions existed between the two isolates. On phylogenetic trees based on the 18S or 28S rDNA sequence, K. neothunni formed a clade with Kudoa spp. with more than four shell valves and polar capsules, particularly K. grammatorcyni and K. scomberomori. Semiquadrate spores of a kudoid species with four shell valves and polar capsules were detected from minute cysts (0.30-0.75 mm by 0.20-0.40 mm) embedded in the trunk muscle of a chub mackerel (Scomber japonicus) fished in the Sea of Japan. Morphologically, it resembled K. caudata described from a chub mackerel fished in the southeastern Pacific Ocean off Peru; however, it lacked filamentous projections on the shell valves of spores. Additionally, it morphologically resembled K. thunni described from a yellowfin tuna also fished in the Pacific Ocean; spores (n = 30) measuring 8.2-10.5 µm in width, 7.0-8.8 µm in thickness, and 6.1-6.8 µm in length; and polar capsule measuring 2.5-3.4 µm by 1.3-2.0 µm. The similarities of the 18S and 28S rDNA sequences between these two species were 98.5 % and 96.3 %, respectively. Simultaneously, the dimensions of cysts in the trunk muscle formed by K. thunni are clearly larger than those of the present species from a chub mackerel: 1.3-2.0 mm by 1.1-1.4 mm (n = 14) vs. 0.30-0.75 mm by 0.20-0.40 mm (n = 7), respectively. Thus, Kudoa scomberi n. sp. is proposed for this multivalvulid species found in the chub mackerel.

Effects of company and season on blood fluke (Cardicola spp.) infection in ranched Southern Bluefin Tuna: preliminary evidence infection has a negative effect on fish growth.

Aporocotylid blood flukes Cardicola forsteri and C. orientalis are an ongoing health concern for Southern Bluefin Tuna (SBT), Thunnus maccoyii, ranched in Australia. Therapeutic application of praziquantel (PZQ) has reduced SBT mortalities, however PZQ is not a residual treatment therefore reinfection can occur after the single treatment application. This study documents the epidemiology of Cardicola spp. infection in ranched SBT post treatment over three ranching seasons (2018, 2019 and 2021). Infection prevalence (percentage of SBT affected) and intensity (parasite load) was determined by adult fluke counts from heart, egg counts from gill filaments and the use of specific quantitative polymerase chain reaction (qPCR) for detection of C. forsteri and C. orientalis ITS-2 DNA in SBT hearts and gills. SBT Condition Index decreased as intensity of Cardicola spp. DNA in SBT gills increased, suggesting blood fluke infection had a negative effect on SBT growth (Spearman's r = -0.2426, d.f. = 138, p = 0.0041). Prevalence and intensity of infection indicated PZQ remained highly effective at controlling Cardicola spp. infection in ranched SBT, 10 years after PZQ administration began in this industry. Company A had the highest prevalence and intensity of Cardicola spp. infection in 2018, and Company G had the highest in 2019. No consistent pattern was seen in 2021. Overall, intensity of infection did not increase as ranching duration increased post treatment. Results from this study improve our knowledge of the biology of blood flukes and helps the SBT industry to modify or design new blood fluke management strategies to reduce health risks and improve performance of SBT.

Metazoan parasites on the gills of the skipjack tuna Katsuwonus pelamis (Osteichthyes: Scombridae) from the Alboran Sea (western Mediterranean Sea).

The gills of 31 skipjack tuna Katsuwonus pelamis (L.) caught in the Alboran Sea (western Mediterranean Sea) were examined for metazoan parasites, and the gills of 4 specimens from the Balearic Sea (also western Mediterranean Sea) were analysed for comparative purposes. Nine -species of parasites were found, including 8 didymozoid trematodes (Atalostrophion cf. bio-varium, Didymocylindrus filiformis, Didymocylindrus simplex, Didymocystis reniformis, Didymoproblema fusiforme, Didymozoon longicolle, Koellikeria sp. and Lobatozoum multisacculatum) and 1 caligid copepod (Caligus bonito). Koellikeria sp. and L. multisacculatum were not recorded in the Balearic Sea. Most of the parasites (79.2% of all specimens) were didymozoids. Didymozoon longicolle was the dominant species; A. cf. biovarium, D. simplex, D. fusiforme and L. multisacculatum are reported from the Mediterranean Sea for the first time. No correlation was found between the intensity of infection of any parasite species and host size or sex. Most of the parasites, particularly didymozoids, showed a high site-specificity. Significant differences were found between the parasite assemblages of K. pelamis from the Alboran Sea and from the Atlantic Ocean. D. fusiforme, D. longicolle and L. multisacculatum are suggested as potential tags to follow skipjack tuna migrations between the Atlantic Ocean and Mediterranean Sea.

A new genus and species of the Didymozoidae (Digenea) from the skipjack tuna Katsuwonus pelamis (L.) (Scombridae).

A new genus and species of didymozoid digenean is described from the skipjack tuna Katsuwonus pelamis (Linnaeus) in the Southwest Atlantic Ocean off Brazil. Pozdnyakovia gibsoni n. g., n. sp. is placed in the Gonapodasmiinae Ishii, 1935. The new genus differs from other genera in the morphology of the posterior regions of the fused pair; this consists of an unlobed, rounded mass fused only dorsally and with a large, elliptical ventral aperture opening into a longitudinal deep cavity from which emerge the two elongate anterior regions. It also differs in the form of the testes, which form two sets of three to four branches in each partner.

The Complete Mitochondrial Genome of Pennella sp. Parasitizing Thunnus albacares.

In the study, the parasite from the yellowfin tuna (Thunnus albacares) was separated, and morphological observation and molecular identification were carried out. Our results showed that the parasite was similar to Pennella sp. Its cephalothorax was covered by spherical to spherical non-branched nipples of almost the same size, which were very similar in shape and arrangement. A pair of slightly larger, the unbranched antenna was present on the outer margin of the small papillae-covered area. The gene sequence of COX1 with a length of 1,558 bp in the mitochondria of the parasite was 100% similar to Pennella sp. (MZ934363). The mitochondrial genome had a total length of 14,620 bp. It consisted of 36 genes (12 protein-coding, 22 transfer RNAs and 2 ribosomal RNAs) and a dummy control region, but the mitochondrial genome had no ATP8 gene. Morphological observation showed that Pennella sp. was dark red, with a convex cephalothorax, with a total length of 8.42 cm, parasitic on the dorsal side of yellowfin tuna. Pennella sp. included the cephalothorax, neck, trunk, abdomen and egg belt. This study was the first report on the mitochondrial genome of Pennella sp. The results provide basic data for further identifying the parasites of Pennella genus.

Profiling the glycome of Cardicola forsteri, a blood fluke parasitic to bluefin tuna.

Infections by blood flukes (Cardicola spp.) are considered the most significant health issue for ranched bluefin tuna, a major aquaculture industry in Japan and Australia. The host-parasite interfaces of trematodes, namely their teguments, are particularly rich in carbohydrates, which function both in evasion and modulation of the host immune system, while some are primary antigenic targets. In this study, histochemistry and mass spectrometry techniques were used to profile the glycans of Cardicola forsteri. Fluorescent lectin staining of adult flukes indicates the presence of oligomannose (Concanavalin A-reactive) and fucosylated (Pisum sativum agglutinin-reactive) N-glycans. Additionally, reactivity of succinylated wheat germ agglutinin (s-WGA) was localised to several internal organs of the digestive and monoecious reproductive systems. Glycan structures were further investigated with tandem mass spectrometry, which revealed structures indicated by lectin reactivity. While O-glycans from these adult specimens were not detectable by mass spectrometry, several oligomannose, paucimannosidic, and complex-type N-glycans were identified, including some carrying hexuronic acid and many carrying core xylose. This is, to our knowledge, the first glycomic characterisation of a marine platyhelminth, with broader implications for research into other trematodes.

Detection and characterization of Kudoa thunni from uncooked yellowfin tuna (Thunnus albacares) in Southeast Asia.

Myxosporean parasites Kudoa spp. have been reported in several marine fish species worldwide. However, little is known about the contamination of these parasites in raw fish in Southeast Asia, where the consumption demand of uncooked fish is increasing. In 2019, the occurrence of several cases of raw yellowfin tuna (Thunnus albacares) obtained from retail shops with the presence of unknown white, nodular cysts within the musculature have raised public health concerns for the consumption of raw marine fish in Vietnam. Microscopic examination revealed numerous myxospores with the quadratic shape of the Kudoidae. Morphologically, stained spores detected in this study are suspected to Kudoa thunni. To confirm the suspected Kudoa species, further examination of the 18S small-subunit (SSU) was conducted and the results of nucleotide sequence analysis obtained from nodular cysts revealed 99.18-100% identity to that of Kudoa thunni sequences available in GenBank. Detection of K. thunni infection in tuna in Southeast Asia highlights the need for appropriate surveillance and control measures to ensure high quality standards and safety on raw fish production and consumption.

Effects of immunostimulants on ranched southern bluefin tuna Thunnus maccoyii: immune response, health and performance.

Ranched southern bluefin tuna Thunnus maccoyii were fed baitfishes supplemented with vitamins (predominantly E and C) or vitamins and immunostimulants, nucleotides and ß-glucans, over 12 weeks after transfer and monitored for enhancement in immune response, health and performance through their 19 week grow-out period. Fish from two different tows were sampled separately at three different sampling points: at transfer to grow-out pontoons, at 8 weeks post-transfer and at harvest, 19 weeks post-transfer. Lysozyme activity was enhanced during vitamin supplementation compared to control fish. Performance (i.e. survival, condition index and crude fat), health (i.e. blood plasma variables including pH, osmolality, cortisol, lactate and glucose) and alternative complement activity were not commonly improved through diet supplementation. There were some tow-specific improvements in performance through vitamin supplementation including survival, selected parasite prevalence and intensity, and alternative complement activity. Immunostimulant supplementation also showed a tow-specific improvement in plasma cortisol level. Tow-specific responses may suggest that life history, previous health condition and husbandry can affect the success of vitamin and immunostimulant enhancement of immune response, health and performance of ranched T. maccoyii.

Probable association between Anisakis infection in the muscle of skipjack tuna (Katsuwonus pelamis) and human anisakiasis in Tokyo, Japan.

Anisakiasis is a gastrointestinal disease caused by parasitic anisakid nematodes, mainly Anisakis simplex sensu stricto (A. simplex). Anisakiasis is prevalent in Japan and approximately 40% of anisakiasis cases in Tokyo occur through the consumption of raw or marinated mackerel. However, in 2018, there was a sudden increase in the number of the food poisoning cases in Tokyo caused by consumption of skipjack tuna (Katsuwonus pelamis). Therefore, we investigated anisakiasis cases resulting from ingestion of skipjack tuna in Tokyo, and surveyed the presence of Anisakis larvae in skipjack tuna in 2018 and 2019. Nineteen samples from 15 patients (13 in 2018 and 2 in 2019) with anisakiasis surely caused by ingestion of skipjack tuna were all identified as A. simplex. The higher mean abundance of Anisakis simplex larvae in skipjack tuna muscle in May 2018 (1.30; 13 larvae/10 fishes) compared to that in the other periods was regarded as a contributing factor in the increase in anisakiasis cases by ingesting skipjack tuna in 2018. To verify whether Anisakis larvae migrate from the visceral organs to the muscle during the period from fishing on the boat until processing for sale, the number of Anisakis larvae in skipjack tuna caught from August to November 2018 was investigated by removing the visceral organs at three different timings, i.e., immediately after catching, after landing, and after transport to the laboratory. Anisakis larvae were detected in the muscle irrespective of the timings at which visceral organs were removed. All larvae from the muscle were detected only from the ventral part and were identified as A. simplex. We thus consider that avoiding raw consumption of the ventral muscle should be an effective measure to prevent anisakiasis.

Expression of cytokines IL-1beta and TNF-alpha in tissues and cysts surrounding Didymocystis wedli (Digenea, Didymozoidae) in the Pacific bluefin tuna (Thunnus orientalis).

Tuna long distance migrations and exposure to wide range of ambient water temperatures facilitate infections with several parasitic groups. This is reflected in the remarkable diversity of tuna parasite communities, especially members of Didymozoidae superfamily (Poche, 1907) (Trematoda, Digenea). Didymocystis wedli is the most frequent species encountered in bluefin tuna parasitizing gill filaments, therefore suggested as a biological marker to differentiate between discrete tuna Atlantic stocks. Because of its high occurrence in gill tissue and inflammatory reaction as the consequence, the aim of our study was to asses if inflammatory madiation through expression of IL-1beta and TNF-alpha is present locally at the site of D. wedli encystment, as well as if the systematic expression of cytokines can be detected in different tissues of infected versus uninfected fish. Quantification of localized cytokine expression was done on paraffine embedded gill sections by in situ hybridization, while quantitative PCR was used to mesured cytokine transcripts in skin mucus, kidney, spleen, gills and liver. Our results suggest that tuna constitutive expression of IL-1beta and TNF-alpha in gills and skin implies a well-adapted innate immunity present at the barrier between the organism and environment. Upregulation of both cytokines in Didymocystis-infected gills not followed by a systematic response evidences the ongoing of an inflammatory process specific for the parasitation site. However, the lack of intensive cytokines response to D. wedli observed by molecular and histological data that fails to eliminate the parasite, could be related to the "old" age of the parasitic process.