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.

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.

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.

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.

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.

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.).

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.

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.

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.

The discovery of Caligus quadratus Shiino, 1954 (Copepoda: Caligidae) in the Mediterranean Sea, with a new host record and supplementary morphological information obtained using confocal laser scanning microscope (CLSM) and 3D image processing.

In this study, supplementary information on the morphology of the siphonostomatoid copepod Caligus quadratus Shiino, 1954 (Copepoda: Caligidae) is given based on the new material collected from the gills of the Atlantic bluefin tuna, Thunnus thynnus (Linnaeus, 1758) caught in the Gulf of Antalya, Turkey. The morphology of C. quadratus is re-examined for the first time by adopting a recently developed visualisation technique by Kamanli et al. [1]. Appendages of Congo red stained specimens of C. quadratus were dissected and scanned using confocal laser scanning microscope (CLSM) and the CLSM images were processed using Dristhi software programme. 3D reconstructions of some confusing appendages were visualised using Drishti. Line drawing was used to depict the habitus of both female and male C. quadratus. Key diagnostic characters of C. quadratus are presented together with the newly observed additional taxonomic characters. In addition, previously misinterpreted and simply overlooked features in the previous descriptions of C. quadratus are also re-described. This is the first report of C. quadratus from the Mediterranean Sea and the Atlantic bluefin tuna constitutes a new host record for this caligid copepod.

Bloody flukes: a review of aporocotylids as parasites of cultured marine fishes.

Fish blood flukes (Digenea: Aporocotylidae) are important pathogens of fishes in aquaculture. Severe infections have been associated with mass mortality events in cultured marine species of teleosts in Australia, Asia and Europe, leading to significant socio-economic losses. Here we review recent advances towards understanding the biology and ecology of fish blood flukes, and the integral role molecular techniques have played in this development. Techniques include molecular matching of aporocotylid life stages using ITS-2 rDNA, and targeting ITS-2 rDNA to distinguish aporocotylid species using quantitative PCR (qPCR). These approaches have facilitated the elucidation of multiple life cycles for species of Cardicola infecting bluefin tunas Thunnus spp. cultured in Australia and Japan. Continued work to identify intermediate hosts of fish blood flukes is critical to improve understanding of their life cycles and help inform aquatic animal health management e.g. through site selection and/or separation of intermediate and definitive hosts. As praziquantel is the only known treatment option for infected fish, its continued efficacy will need to be monitored and other possible solutions may need to be identified as aquaculture continues to grow and diversify.

In vivo cultivation of tuna blood fluke Cardicola orientalis in terebellid intermediate hosts.

Some fish blood flukes of the genus Cardicola (Aporocotylidae) are considered important pathogens of farmed/ranched tuna, Thunnus spp. Infections with Cardicola spp. might obstruct the blood flow in the gills via massive accumulations of eggs and often lead to mass mortalities in captive tuna. At present, oral administration of an anthelminthic drug, praziquantel is the most effective treatment, but the tuna farming industries are seeking non-drug control measures. Development of prophylactic and holistic measures have been difficult, owing to a lack of basic knowledge about these parasites. Unlike other trematodes which use molluscs, blood flukes of marine actinopterygian fish use terebellid polychaetes as intermediate hosts. However, information about the development of Cardicola spp. within intermediate hosts is very limited. Recent success in Cardicola opisthorchis sporocyst transplantation into the host polychaete has opened possibilities for the cultivation of Cardicola in the laboratory. Here, we conducted several transplantation trials with another tuna blood fluke, Cardicol orientalis, into its natural and surrogate polychaete hosts. Cardicola orientalis sporocysts were injected into a total of 195 Nicolea gracilibranchis, the natural host, and clear sporocyst development and reproduction was observed in 32 recipients (overall success rate 16.4%). The production of daughter sporocysts in the transplanted polychaete occurred within 14 days post injection, and one sporocystogenous cycle took approximately 4 weeks. Serial passage culture via transplantation of in vivo-cultured sporocysts was also achieved, but with limited sporocyst reproduction. In addition, sporocysts were successfully retrieved from six and one individuals of the surrogate hosts, Thelepus setosus (n = 10) and Thelepus japonicus (n = 5), respectively. These results indicate that the in vivo cultivation of C. orientalis sporocysts is possible, not only in its natural host but also in other terebellids, although the problems of high mortality and inconsistency in successful transplantation need to be resolved.

Detection of Kudoa hexapunctata and Kudoa neothunni from retail raw tuna in Japan using a novel duplex polymerase chain reaction.

Kudoa hexapunctata was taxonomically separated from Kudoa neothunni, but their main host is tuna. K. hexapunctata has been identified as causative agent of foodborne diseases associated with the ingestion of raw Pacific bluefin tuna (PBT) in Japan, but K. neothunni has not. Therefore, it is clinically and epidemiologically important to detect and distinguish these two species. In the present study, we developed a novel duplex polymerase chain reaction (dPCR) targeting the 28S rRNA gene sequences of K. hexapunctata and K. neothunni. The dPCR amplified the desired genetic regions of each species, and the detection limit was 10 copies/reaction. A total of 36 retail tuna samples from different fishing ports were purchased and tested by dPCR. Thirty-one tested positive for K. hexapunctata and four tested positive for K. neothunni. Several retail PBT samples were examined in some of the fishing ports, and among these samples, the detection rates of K. hexapunctata was higher than 85%, and the rates were similar between wild and farmed PBT. The detection rates of K. hexapunctata in wild and farmed retail PBT were 75% and 71%, respectively, in May. However, the rates in June and July were 100% for both. K. hexapunctata and K. neothunni myxospores were not observed in the dPCR-positive samples, except in juvenile PBT, suggesting that the number of parasites was insufficient to cause foodborne disease. Thus, dPCR is a useful method for detecting and distinguishing K. hexapunctata and K. neothunni, and can be used in epidemiological studies of these parasites.

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.

Morphological and molecular study of Neorhadinorhynchus nudus (Harada, 1938) (Acanthocephala: Cavisomidae) from Auxis thazard Lacepede (Perciformes: Scombridae) in the South China Sea.

In the present study, Neorhadinorhynchus nudus (Harada, 1938) is reported from the frigate tuna Auxis thazard (Lacepéde) (Perciformes: Scombridae), in the South China Sea for the first time. The detailed morphology of N. nudus was studied using light and scanning electron microscopy based on the newly collected material. The results showed some morphometric variability between our specimens and previous studies, including the number of hooks per longitudinal row and the size of copulatory bursa and eggs. Our SEM observations also revealed all proboscis hooks emerged from elevated round rims on proboscis surface. In addition, N. nudus was firstly characterised using molecular methods by sequencing and analysing the ribosomal ITS and mitochondrial cox1 regions. There is no nucleotide divergence found in the ITS sequences, but a low level of nucleotide variability detected in the cox1 regions (the level of intraspecific nucleotide variability being 0.75% to 2.54%). The DNA sequence data obtained herein will indeed be a useful reference for rapid and accurate species identification of Neorhadinorhynchus.

Descriptions of Neorhadinorhynchus nudum (Cavisomidae) and Heterosentis paraholospinus N. SP. (Arhythmacanthidae) (Acanthocephala) From Fish Along the Pacific Coast of Vietnam, With Notes On Biogeography.

Two species of acanthocephalans are described from fishes caught along the Pacific coast off eastern Vietnam in 2016: (1) Neorhadinorhynchus nudum ( Harada, 1938 ) Yamaguti, 1939 (Cavisomidae) from the frigate tuna Auxis thazard (Lacépède) (Scombridae) in Nha Trang, Pacific south Vietnam, and (2) Heterosentis paraholospinus n. sp. (Arhythmacanthidae) from 3 species of fish: the common ponyfish Leiognathus equulus (Forsskål) (Leiognathidae) and the torpedo scad Megalaspis cordyla (Linn.) (Carangidae) off Nha Trang and Binh Thuân, respectively, and the yellowspotted ponyfish Nuchequula flavaxilla Kimura, Kimura, and Ikejima (Leiognathidae) at Quang Ninh in Pacific waters of Vietnam. Females of N. nudum are fully described for the first time in the Pacific Ocean. Heterosentis paraholospinus n. sp. is similar to Heterosentis holospinus Amin, Heckmann, and Ha, 2011, in having an unspined anterior trunk cone, nucleated pouch at the posterior end of the receptacle, and spines covering the entire trunk, but it differs in having a long cylindrical trunk, lemnisci much longer than the receptacle, more and different-sized proboscis hooks, and subterminal female gonopore. Biogeographical notes are discussed for both genera.

Diagnostic accuracy of the light microscope method to detect the eggs of Cardicola spp. in the gill filaments of the bluefin tuna.

Trematode blood flukes of the genus Cardicola are potentially lethal in bluefin tuna cultures. The present study proposed a new method to detect aporocotylid eggs in tuna gills. Aporocotylid eggs were detected by analysing a pair of gill filaments of five transversal areas of the eight holobranches of one hundred Atlantic bluefin tuna and observed with glycerol and a stereomicroscope with an oblique brightfield. Data were gathered according to holobranches, transversal areas and their combination. Eggs were uniformly distributed among the holobranches, but they had the highest prevalence in the second and fifth transversal areas, which is controversial with respect to previous studies of egg distribution. An abbreviated method called the T-two test, which had the highest sensitivity (96.8%), is proposed for the detection of Cardicola spp. infections instead of the analysis all the holobranches. The T-two test limits the time and cost of the egg parasite screening analysis. The analyses of ten samples could be sufficient to detect the presence of parasites in farmed bluefin tuna; fish from the wild are expected to be less infected and more samples (45) would therefore be necessary.

Spatial and Temporal Changes in the Distribution of Blood Fluke Infection in Nicolea gracilibranchis (Polychaeta: Terebellidae), the Intermediate Host for Cardicola orientalis (Digenea: Aporocotylidae), at a Tuna Farming Site in Japan.

Fish blood flukes of the genus Cardicola (Digenea: Aporocotylidae) are important pathogens in tuna aquaculture. Recent advances in marine blood fluke research have led to the elucidation of the lifecycles of 3 Cardicola spp. infecting tuna; all 3 flukes utilize terebellid polychaetes as the intermediate host. In our survey, we obtained large numbers of Nicolea gracilibranchis infected by larval Cardicola orientalis at our tuna farming site. To determine the spatial and temporal changes in the distribution of N. gracilibranchis surrounding tuna culture cages and their infection by C. orientalis, we conducted monthly sampling for a period of 1 yr. Terebellids were most abundant on the floats and ropes of culture cages, but a significantly higher proportion of infected N. gracilibranchis was detected on ropes, particularly up to 4 m in depth. Cardicola orientalis infection in N. gracilibranchis was clearly seasonal, with a higher infection rate between April and July. Our findings indicate that the infected terebellids inhabit specific microhabitats, and both abiotic and biotic factors likely influence blood fluke infection in the intermediate terebellid host. This information is important to better understand the general biology of marine aporocotylids and may be useful to develop a control strategy for blood fluke infection in tuna aquaculture.

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.

Transplantation of Cardicola opisthorchis (Trematoda: Aporocotylidae) sporocysts into the intermediate host, Terebella sp. (Polychaeta: Terebellidae).

Cardicola opisthorchis is a blood fluke pathogen significantly affecting cultured Pacific bluefin tuna Thunnus orientalis in Japan. It is known that the intermediate host of C. opisthorchis is a terebellid polychaete Terebella sp. In order to study the intrapolychaete larval development of C. opisthorchis, we transplanted sporocysts, which contained a large number of cercariae, of C. opisthorchis obtained from Terebella sp. into sporocyst-free Terebella sp., which had been maintained at 20°C. The transplanted sporocysts switched from cercarial to sporocystal production by 17days after transplantation (d.a.t.) and daughter sporocysts were released into the polychaete body cavity at 25d.a.t. Subsequently, the released daughter sporocysts produced daughter sporocysts again. Thereafter, daughter sporocysts that contained cercariae appeared at 38d.a.t. and gradually increased. At 51d.a.t., 136 sporocysts that had multiplied from the original two transplanted sporocysts were observed in the body of one polychaete, and cercariae were released from daughter sporocysts inside the polychaete body cavity. Subsequently the cercariae were found to be released outside the polychaete at 57d.a.t. This is the first successful case of in situ observation of the development of a blood fluke within the intermediate host.