Description of Discocotyle ciray n. sp. (Monogenea: Discocotylidae) from Parahucho perryi (Brevoort) from Hokkaido, Japan, with a redescription of D. sagittata (Leuckart, 1842).

Discocotyle sagittata (Leuckart, 1842) (Monogenea: Discocotylidae) is redescribed, based on specimens collected from the type host, Salmo trutta Linnaeus, from the type locality, Freiburg, Germany, supplemented with specimens from S. trutta and rainbow trout, Oncorhynchus mykiss (Walbaum) reared in an Austrian aquarium. The diagnosis of the genus Discocotyle Diesing, 1850 is emended. Discocotyle ciray n. sp. is described, based on immature, preadult and adult specimens from the salmonid, Parahucho perryi (Brevoort) at Eniwa, Hokkaido, Japan. Adult specimens of the new species were about twice as large as those of D. sagittata from S. trutta. When the type specimens of D. ciray n. sp. were examined together with museum specimens from P. perryi at Tsurui, Hokkaido, the body and clamp sizes were positively correlated to the host size. Their measurements from a smaller P. perryi at Tsurui overlapped with those of D. sagittata, showing that these size differences were not suitable differentiating keys. Discocotyle ciray n. sp. can be separated from D. sagittata by the morphologies of the female genital system (relatively anteriorly positioned ovary, short joint vaginal duct and much more strongly winding uterus). The genetic distances of COI mtDNA sequence between D. ciray n. sp. and D. sagittata were 18.0-18.6%. These remarkable genetic divergences also supported the distinct taxonomic status of D. ciray n. sp.

Comparison of skin mucus lectins among longtooth grouper Epinephelus bruneus and giant grouper E. lanceolatus as well as the hybrid, Kue-Tama, and their binding abilities to the skin fluke Benedeniaepinepheli.

Interspecific hybrids of farm-raised fish are becoming popular in aquaculture owing to their advantages over pure species, including improved growth and higher resistance to infectious diseases. Kue-Tama is a recently established hybrid grouper derived from the longtooth grouper Epinephelus bruneus (♀) × giant grouper E. lanceolatus (♂). In our previous study, this hybrid showed significantly higher resistance against the skin fluke Benedenia epinepheli, a problematic parasite in grouper farming, than the longtooth grouper. In the present study, we explored lectins in the skin mucus of hybrids and their parent species. While C-type lectins of approximately 15 kDa were obtained from longtooth groupers, additional C-type lectins with molecular masses of approximately 20 and 30 kDa, as well as 45-kDa F-type lectin, were also detected in Kue-Tama and giant groupers. Semi-quantitative reverse transcript-polymerase chain reaction (RT-PCR) demonstrated that the gene expression levels of both C-type and F-type lectins were significantly higher in the skin of the hybrid and giant groupers than that of the longtooth grouper. In addition, some skin mucus lectins of the hybrid and giant groupers were bound to the fluke, suggesting that these lectins conferred resistance to parasitic infections.

Synopsis of myxosporean species (Cnidaria: Myxozoa) parasitizing fishes from Vietnam.

This paper provides an updated checklist of species-level identified myxosporeans from marine and freshwater fishes in Vietnam. The list includes 51 nominal species (38 marine and 13 freshwater) belonging to 9 genera: Myxobolus Bütschli, 1882 (26 species); Kudoa Meglitsch, 1947 (6 species); Henneguya Thélohan, 1892 (6 species); Thelohanellus Kudo, 1933 (5 species); Unicapsula Davis, 1924 (2 species); Ceratomyxa Thélohan, 1892 (2 species), Zschokkella Auerbach, 1909 (2 species); Auerbachia Meglitsch, 1960 (1 species), and Meglitschia Kovaleva, 1988 (1 species). For each parasite species, information on myxospore morphology, line drawings, fish hosts, infection sites, and collection locality in Vietnam are reported. Where available, we also provide GenBank accession numbers for nucleotide sequence data. In addition, taxonomic status of several species was discussed and Myxobolus eszterbaueri nom. nov. is proposed as a junior homonym for Myxobolus hakyi Baska, Voronin, Eszterbauer, Müller, Marton & Molnár 2009, which is preoccupied.

Sex-inducing effects toward planarians widely present among parasitic flatworms.

Various parasitic flatworms infect vertebrates for sexual reproduction, often causing devastating diseases in their hosts. Consequently, flatworms are of great socioeconomic and biomedical importance. Although the cessation of parasitic flatworm sexual reproduction is a major target of anti-parasitic drug design, little is known regarding bioactive compounds controlling flatworm sexual maturation. Using the planarian Dugesia ryukyuensis, we observed that sex-inducing substances found in planarians are also widespread in parasitic flatworms, such as monogeneans and flukes (but not in tapeworms). Reverse-phase HPLC analysis revealed the sex-inducing substance(s) eluting around the tryptophan retention time in the fluke Calicophoron calicophorum, consistent with previous studies on the planarian Bipalium nobile, suggesting that the substance(s) is likely conserved among flatworms. Moreover, six of the 18 ovary-inducing substances identified via transcriptome and metabolome analyses are involved in purine metabolism. Our findings provide a basis for understanding and modifying the life cycles of various parasitic flatworms.

Molecular and morphological description of a novel microsporidian Inodosporus fujiokai n. sp. infecting both salmonid fish and freshwater prawns.

A new microsporidian disease of cultured rainbow trout Oncorhynchus mykiss has recently been confirmed in Japan, and the causative species was tentatively designated as Microsporidium sp. RBT-2021. Involvement of common prawn Palaemon paucidens in its transmission was suggested based on the previous feeding trials, although the microsporidian infection in P. paucidens was not confirmed. In this study, P. paucidens in Lake Biwa, Japan was investigated for microsporidian infection and 4 types of spores (types 1-4) were newly found. The nucleotide sequence of the small subunit ribosomal RNA gene was identical between type 1 and Microsporidium sp. RBT-2021, indicating they are conspecific. However, intriguingly, the spore morphology and the mode of development in fish and prawn were strikingly different. Morphological observations revealed type 1 in the prawn possesses characteristics of the genus Inodosporus Overstreet and Weidner, 1974, while Microsporidium sp. RBT-2021 in the trout exhibited the characteristics of the genus Kabatana Lom, Dyková and Tonguthai, 2000. In the phylogeny, type 1 was placed within a clade comprising Kabatana spp. and Inodosporus octosporus. Based on the morphological and molecular analyses, we describe Microsporidium sp. RBT-2021 as Inodosporus fujiokai n. sp. Together with the success of the previous prawn-feeding trials, this study strongly suggests I. fujiokai n. sp. has a multi-host life cycle utilizing fish and crustacean hosts and different modes of development in each host. Such polymorphic life cycle has barely been known among fish microsporidians. This study also suggests that the genus Kabatana is a junior synonym of the genus Inodosporus.

Description of Myxobolus hoabinhensis n. sp. (Myxosporea: Myxobolidae), infecting the trunk muscles of goldfish Carassius auratus (Linnaeus, 1758) (Cypriniformes: Cyprinidae) in northern Vietnam.

During a survey of myxosporean parasites of freshwater fishes in northern Vietnam, myxospores resembling those of the genus Myxobolus (Myxosporea: Myxobolidae) were found in the trunk muscle of 6 out of 35 specimens (17.14%) of wild goldfish Carassius auratus (Cypriniformes: Cyprinidae). The mature spores were 12.0 ± 0.4 (11.4 - 12.6) µm long, 8.5 ± 0.2 (7.9 - 9.0) µm wide and 6.1 ± 0.2 (5.8 - 6.3) µm thick, containing two pyriform-shaped polar capsules unequal in size. The larger polar capsule was 7.6 ± 0.3 (7.1 - 8.4) µm long and 3.5 ± 0.1 (3.3 - 3.8) µm wide, and the smaller polar capsule was 6.2 ± 0.3 (5.5 - 6.7) µm long and 2.9 ± 0.2 (2.6 - 3.4) µm wide. Each polar capsule contained a polar filament with 3-5 coils. A phylogenetic analysis based on the small subunit rDNA (SSU rDNA) sequence revealed that this Myxobolus species forms a distinct branch in the phylogenetic tree sister to Myxobolus artus and Myxobolus cyprini, with DNA sequence similarity at 97.6% to M. artus and 97.5% to M. cyprini. A combination of the morphological characteristics and molecular data suggest that this is an undescribed species, and we propose the name Myxobolus hoabinhensis n. sp.

A world of taxonomic pain: cryptic species, inexplicable host-specificity, and host-induced morphological variation among species of Bivesicula Yamaguti, 1934 (Trematoda: Bivesiculidae) from Indo-Pacific Holocentridae, Muraenidae and Serranidae.

The taxonomy of species of Bivesicula Yamaguti, 1934 is analysed for samples from holocentrid, muraenid and serranid fishes from Japan, Ningaloo Reef (Western Australia), the Great Barrier Reef (Queensland), New Caledonia and French Polynesia. Analysis of three genetic markers (cox1 mtDNA, ITS2 and 28S rDNA) identifies three strongly supported clades of species and suggests that Bivesicula as presently recognized is not monophyletic. On the basis of combined morphological, molecular and biological data, 10 species are distinguished of which five are proposed as new. Bivesicula Clade 1 comprises seven species of which three are effectively morphologically cryptic relative to each other; all seven infect serranids and four also infect holocentrids. Bivesicula Clade 2 comprises three species of which two are effectively morphologically cryptic relative to each other; all three infect serranids and one also infects a muraenid. Bivesicula Clade 3 comprises two known species from apogonids and a pomacentrid, and forms a clade with species of Paucivitellosus Coil, Reid & Kuntz, 1965 to the exclusion of other Bivesicula species. Taxonomy in this genus is made challenging by the combination of low resolving power of ribosomal markers, the existence of regional cox1 mtDNA populations, exceptional and unpredictable host-specificity and geographical distribution, and significant host-induced morphological variation.

Three new species of Helicometroides Yamaguti, 1934 from Japan and Australia, with new molecular evidence of a widespread species.

We report specimens of monorchiids infecting Haemulidae from the waters off Japan and Australia; these specimens represent five species of Helicometroides Yamaguti, 1934, three of which are unambiguously new. Helicometroides murakamii n. sp. infects Diagramma pictum pictum from off Minabe, Japan; Helicometroides gabrieli n. sp. infects Plectorhinchus chrysotaenia from off Lizard Island, Australia; and Helicometroides wardae n. sp. infects Plectorhinchus flavomaculatus and Plectorhinchus multivittatus from off Heron Island, Australia. Helicometroides murakamii n. sp. and H. gabrieli n. sp. conform to the most recent diagnosis of Helicometroides in lacking a terminal organ, but H. wardae n. sp. possesses a terminal organ with distinct, robust spines; despite this morphological distinction, the three form a strongly-supported clade in phylogenetic analyses. We also report specimens morphologically consistent with Helicometroides longicollis Yamaguti, 1934, from D. pictum pictum from off Minabe, Japan, and Diagramma pictum labiosum on the Great Barrier Reef, Australia. Genetic analyses of ITS2 rDNA, 28S rDNA and cox1 mtDNA sequence data for the Japanese specimens reveal the presence of two distinct genotypes. Specimens of the two genotypes were discovered in mixed infections and are morphologically indistinguishable; neither genotype can be associated definitively with H. longicollis as originally described. We thus identify them as H. longicollis lineage 1 and 2, pending study of further fresh material. Genetic analyses of specimens from the Great Barrier Reef are consistent with the presence of only H. longicollis lineage 1. This species thus has a range that incorporates at least Australia and Japan, localities separated by over 7000 km.

Praziquantel use in aquaculture - Current status and emerging issues.

Parasitic diseases are major constraints in fish mariculture. The anthelmintic praziquantel (PZQ) can effectively treat a range of flatworm parasites in a variety of fish species and has potential for broader application than its current use in the global aquaculture industry. In this review we report on PZQ's current use in the aquaculture industry and discuss its efficacy against various flatworm parasites of fish. Routes of PZQ administration are evaluated, along with issues related to palatability, pharmacokinetics and toxicity in fish, while PZQ's effects on non-target species, environmental impacts, and the development of drug-resistance are discussed.

Morphological and molecular characterization of Ceratomyxa binhthuanensis n. sp. (Myxosporea: Ceratomyxidae) from the gall bladder of blacktip grouper Epinephelus fasciatus (Perciformes: Serranidae) in the East Sea of Vietnam.

A new myxozoan species, Ceratomyxa binhthuanensis n. sp. (Myxosporea: Ceratomyxidae), was found in the gall bladder of blacktip grouper Epinephelus fasciatus (Perciformes: Serranidae) in the East Sea of Vietnam. Myxospores were observed floating free in the gall bladder of 3 out of 20 fish examined (15%). Mature myxospores were elongate and slightly crescent-shaped and measured 12.2 ± 1.3 (10.8-16.0) µm in thickness and 5.8 ± 0.6 (4.8-6.9) µm in length, with two smooth equal shell valves. The two polar capsules were spherical and equal in size, measuring 2.6 ± 0.3 (2.3-2.9) µm in diameter. The posterior angle was slightly concave, 153.7° ± 5.6° (148.9°-166.0°). Molecular analysis of SSU rDNA sequence showed that Ceratomyxa binhthuanensis n. sp. differs from other Ceratomyxa spp. available in GenBank. Phylogenetic analysis indicated that C. binhthuanensis n. sp. was closely related to three species, Ceratomyxa nolani, Ceratomyxa yokoyamai, and Ceratomyxa cutmorei, which also infect fish hosts of the genus Epinephelus.

Phototactic responses in four monogenean oncomiracidia.

Phototaxis is the common behavioral response exhibited by the oncomiracidia of various monogeneans. However, the changes in the oncomiracidial swimming behavior in response to light cues are not well understood. Here, we investigated the light responses of four monogeneans that are important pathogens in mariculture, namely Benedenia epinepheli, Benedenia seriolae, Neobenedenia girellae, and Heteraxine heterocerca. The swimming trajectory and speed of oncomiracidia of each species were assessed in a glass Petri dish with an LED light placed adjacent to it, based on three different light responses: LED light in the off position (normal swimming), LED light in the on position (phototactic behavior), and immediately (< 5 s) after switching the LED light off (photophobic behavior). The oncomiracidia of all four species exhibited positive phototactic and photophobic responses; however, the change in swimming speed between each response differed among the species. The oncomiracidia of three species (B. epinepheli, N. girellae, and H. heterocerca) exhibited high swimming speed, as a phototactic response; in contrast, the oncomiracidia of B. seriolae exhibited reduced swimming speed when moving toward the light source. Benedenia epinepheli and H. heterocerca exhibited the highest swimming speed during the phototaxis phase, whereas B. seriolae and N. girellae exhibited the highest swimming speed during the photophobic phase. These light responses are considered adaptive traits to increase the chance of encountering and infecting suitable hosts in nature, and such responses could potentially be applied to the control of parasite infections in aquaculture.

A new myxosporean species, Henneguya lata n. sp. (Myxozoa: Myxobolidae), from the gills of yellowfin seabream Acanthopagrus latus (Perciformes: Sparidae) in the Gulf of Tonkin, Vietnam.

During a survey of myxosporean parasites of marine fish in the coastal region of Vietnam, a species of the genus Henneguya (Myxosporea: Bivalvulida) was found in the gill of yellowfin seabream Acanthopagrus latus (Perciformes: Sparidae). White and oval cysts, measuring 145-220 µm in diameter, were detected in the gill lamellae of 4 of 15 fish examined (26.7%). Mature myxospores were elongate, with smooth valves, two similar polar capsules, and having the following dimensions: spore body length 9.9 ± 0.5 (8.9-12.5) µm, body width 6.7 ± 0.3 (6.1-7.6) µm, thickness 5.1 ± 0.2 (4.8-5.4) µm, caudal appendage length 10.0 ± 1 (8.3-11.6) µm, and total myxospore length 19.3 ± 1.4 (16.5-21.5) µm. The polar capsules were ovoid, measuring 3.2 ± 0.2 (2.8-3.9) µm long and 1.9 ± 0.2 (1.5-2.3) µm wide. Each polar capsule has a polar filament with 4-5 coils. Histological analysis revealed plasmodia in the connective tissues of the gill lamellae, but inflammation and deformation of the gills were not observed. In the phylogenetic tree reconstructed from the small subunit ribosomal DNA (SSU rDNA), sequences of the Henneguya specimens found in this study form a distinct branch. Morphological characteristics and molecular data identified a new species, namely Henneguya lata n. sp.

Metazoan Parasite Life Cycles: Significance for Fish Mariculture.

Marine fish are a nutritious and high-value food commodity, but many wild-capture fisheries are in decline. Thus, marine fish culture is expected to expand greatly in coming years. Management of disease is a major problem in this industry, and metazoan parasites are among the most significant disease agents in terms of economic loss and animal welfare. Current methods for controlling metazoan parasites are mostly reactionary and rely on chemical treatment. Such methods are ultimately unsustainable. Here, we summarise the life cycles of marine metazoan parasites and how this knowledge can be used for nonchemical management and control. To aid the sustainable growth of marine fish culture, we advocate for a renewed research focus on the basic life history characteristics of parasites.

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.

Production of a novel monoclonal antibody applicable for an immunochromatographic assay for Kudoa septempunctata spores contaminating the raw olive flounder (Paralichthys olivaceus).

Kudoa septempunctata, a myxosporean parasite of the olive flounder (Paralichthys olivaceus), causes foodborne gastroenteritis after ingestion of contaminated raw flounder. Available methods to detect K. septempunctata require expensive equipment, well-trained personnel, and lengthy procedures. Here we generated a novel monoclonal antibody (MAb 15G11) against K. septempunctata and used it to produce a prototype immunochromatographic assay (prototype Kudoa-ICA). Within 15min, the prototype Kudoa-ICA detected ≥1.0×105spores/mL in a spore suspension and ≥2.0×104spores/g of P. olivaceus muscle. The prototype Kudoa-ICA weakly cross-reacted with spores of K. lateolabracis and K. iwatai. cDNA sequence, expression, and western blot analyses revealed that MAb 15G11 detected an approximately 24-kDa protein encoded by a 573bp mRNA. The cDNA nucleotide and predicted amino acid sequences were not significantly similar to any sequence in the GeneBank database. Immunoelectron microscopy revealed that MAb 15G11 reacted with the sporoplasmic cells and mainly with the capsulogenic cells of the K. septempunctata spore. Although the Kudoa-ICA was weakly cross-reactive with two other Kudoa species, it detected >1.0×106spores/g of K. septempunctata in P. olivaceus muscle, which is the criterion used to indicate a violation of the Food Hygiene Law of Japan. We conclude that MAb 15G11 may be suitable for use in an immunochromatographic assay for screening P. olivaceus muscle contaminated with K. septempunctata at food distribution sites such as food wholesalers, grocery stores, and restaurants.

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.

Developmental stages of fish blood flukes, Cardicola forsteri and Cardicola opisthorchis (Trematoda: Aporocotylidae), in their polychaete intermediate hosts collected at Pacific bluefin tuna culture sites in Japan.

Farming of Pacific bluefin tuna (PBT), Thunnus orientalis, is a rapidly growing industry in Japan. Aporocotylid blood flukes of the genus Cardicola comprising C. orientalis, C. opisthorchis and C. forsteri are parasites of economic importance for PBT farming. Recently, terebellid polychaetes have been identified as the intermediate hosts for all these parasites. We collected infected polychaetes, Terebella sp., the intermediate host of C. opisthorchis, from ropes and floats attached to tuna cages in Tsushima, Nagasaki Prefecture, Japan. Also, Neoamphitrite vigintipes (formerly as Amphitrite sp. sensu Shirakashi et al., 2016), the intermediate host of C. forsteri, were collected from culture cages in Kushimoto, Wakayama Prefecture, Japan. The terebellid intermediate hosts harbored the sporocysts and cercariae in their body cavity. Developmental stages of these blood flukes were molecularly identified using species specific PCR primers. In this paper, we describe the cercaria and sporocyst stages of C. opisthorchis and C. forsteri and compare their morphological characteristics among three Cardicola blood flukes infecting PBT. We also discuss phylogenetic relations of the six genera of the terebellid intermediate hosts (Artacama, Lanassa, Longicarpus, Terebella, Nicolea and Neoamphitrite) of blood flukes infecting marine fishes, based on their morphological characters.

Immune reactivity in early life stages of sea-cage cultured Pacific bluefin tuna naturally infected with blood flukes from genus Cardicola (Trematoda: Aporocotylidae).

Pacific bluefin tuna (PBT), Thunnus orientalis, due to its high average price on the market is an economically valuable fish species. Infections by blood flukes from the genus Cardicola (Trematoda: Aporocotylidae) represent a growing concern for the cage culture of bluefin tuna in Japan, Australia and Southern Europe. The accumulation of numerous Cardicola eggs in the fish gills causes severe pathology that has been linked to mortality in PBT juveniles up to one year old. The only effective treatment used to mitigate the infection is the oral administration of the antihelminthic drug praziquantel (PZQ) to the affected fish. However, with the need to minimise therapeutic drug use in aquaculture it is hoped that immunoprophylaxis can provide a future alternative to protect the PBT juveniles against Cardicola infection. Currently, little is known of the host immune response to these parasites and of their infection dynamics. In this study, using real-time qPCR we aimed to quantitatively detect C. orientalis and C. opisthorchis DNA within the gills and heart of cultured PBT juveniles and to investigate the host immune response at the transcriptional level in the gills. The research focused mainly during early stages of infection soon after young PBT were transferred to culture cages (from 14 to 77 days post-transfer). An increase (up to 11-fold) of immune-related genes, namely IgM, MHC-I, TCR-ß and IL-1ß was observed in the PBT gills infected with Cardicola spp. (28-77 days post-transfer). Furthermore, IgM (19-fold increase) and MHC-I (11.5-fold increase) transcription was strongly up-regulated in gill samples of PBT infected with C. orientalis relative to uninfected fish but not in fish infected with C. opisthorchis. Cardicola-specific DNA was first detected in the host 14 days post-transfer (DPT) to sea-cages which was 55 days earlier than the first detection of parasite eggs and adults by microscopy. Oral administration of PZQ did not have an immediate effect on parasite DNA presence in the host and the DNA presence started to reduce after 24 days only in the host heart. The results provide evidence of an immune response in early age sea-cage cultured juveniles of PBT naturally infected with C. orientalis and C. opisthorchis. This response, whilst not protective against primary infection, provides evidence that immunisation at an early age may have potential as a health strategy.

Phylogenetic study of the genus Kudoa (Myxozoa: Multivalvulida) with a description of Kudoa rayformis sp. nov. from the trunk muscle of Pacific sierra Scomberomorus sierra.

Kudoa rayformis n. sp. (Myxozoa; Multivalvulida) was observed in the trunk muscle of Pacific sierra Scomberomorus sierra caught off the coast of Tonosi, Panama. The species formed pseudocysts in myofibers and infection was subclinical. The myxospores possessed four polar capsules and spore valves, one of which had a distinct filamentous extension. This unique morphological characteristic of the myxospore validated this as a new species of Kudoa. Genetically, K. rayformis n. sp. is closest to K. inornata, with 98% and 91% similarity in 18S and 28S rDNA, respectively, but its spore shape was clearly distinct. The 18S rDNA and concatenated sequences from K. rayformis were used in molecular phylogenetic analyses of kudoids to examine the congruence of phylogeny with infection site tropism, spore morphology and cyst/pseudocyst formation. The results demonstrated that the phenotypic traits were correlated with the phylogeny of Kudoidae, and that the biological features of K. rayformis originated from the ancient Kudoidae as exhibited by the non-specific infection site tropism and the ability to infect muscle and form pseudocysts.

Discovery of intermediate hosts for two species of blood flukes Cardicola orientalis and Cardicola forsteri (Trematoda: Aporocotylidae) infecting Pacific bluefin tuna in Japan.

Fish blood flukes (Aporocotylidae) are important pathogens of farmed finfish around the world. Among them, Cardicola spp. infecting farmed tuna are considered to be serious threats to tuna farming and have received tremendous attention. We conducted periodical samplings at a tuna farming site in Japan between January and May, 2015 to determine the life cycle of Cardicola spp. We collected over 4700 terebellid polychaetes from ropes, floats and frames of tuna culture cages and found nearly 400 infected worms. Sporocysts and cercariae found in Nicolea gracilibranchis were genetically identified as Cardicola orientalis by 28S and ITS2 ribosomal DNA sequences. This was the first discovery of the intermediate host for this parasite species. Infection prevalence and the abundance of N. gracilibranchis significantly varied between sampling points and the highest number of infected terebellids were collected from ropes. We also demonstrated morphologically and molecularly that asexual stages found in a single Amphitrite sp. (Terebellidae) and adult worms isolated from farmed juvenile tuna were Cardicola forsteri. This is the first report of C. forsteri in Pacific bluefin tuna (PBT) Thunnus orientalis in Japan. Our results demonstrated that all three species of Cardicola orientalis, C. forsteri and Cardicola opisthorchis exist in Japanese farmed PBTs and that they all use terebellid polychaetes as the intermediate hosts.