Kudoa tanakai n. sp. (Myxozoa: Myxosporea: Multivalvulida), a new kudoid species with spheroid myxospores from the scalpel sawtail (Actinopterygii: Prionurus scalparum) from western Japan.

We describe a new kudoid species, Kudoa tanakai n. sp., in the scalpel sawfish, Prionurus scalprum (Actinopterygii: Acanthuriformes: Acanthuridae), from the natural water around western Japan. The plasmodia were filamentous, localized in pseudocysts in the myofibers of the trunk muscles. The occurrence of plasmodia in the trunk muscle showed no site preference. Its myxospores were spheroid, measuring 6.6-7.6 (7.0) µm by 5.8-6.9 (6.3) µm in apical view (width) and 5.7-6.6 (6.2) in length (n = 30), with four shell valves and a corresponding number of spheroid polar capsules. Shell valves lacked apical protrusions, but scanning electron microscopy revealed that one of the four shell valves had two semi-lunar flaps at its apical terminus. Nucleotide sequencing of the small and large subunit ribosomal RNA genes of the present isolate showed phylogenetic affinities to kudoid species characterized by spheroid myxospores, such as K. musculoliquefaciens, K. hemiscylli, and K. carcharhini, but was molecularly and morphometrically distinct from these and other kudoid species. For direct comparison, Kudoa hemiscylli was collected from the Pacific spadenose shark, Scoliodon macrorhynchos (Elasmobranchii: Carcharhiniformes: Carcharhinidae), in the South China Sea off Guangdong Province, China, and the myxospore surface of the species was observed using scanning electron microscopy. Our study describes the new host and distribution record of this kudoid species originally described from a variety of elasmobranchs in the Australian Coral Sea.

Four carangid fish species as new host records for Kudoa trachuri Matsukane, Sato, Tanaka, Kamata et Sugita-Konishi, 2011 (Myxozoa: Multivalvulida), and description of a new species, Kudoa longichorda sp. n., forming pseudocysts in the muscle of Decapterus tabl Berry.

Multivalvulid myxosporeans of the genera Kudoa Meglitsch, 1947 and Unicapsula Davis, 1924 (Cnidaria: Myxozoa) are often the cause of unsightly cyst formation or postmortem myoliquefaction in the trunk muscle of commercial marine fish, which reduces the market value of infected individuals. Twenty species (18 Kudoa spp. and two Unicapsula spp.) have been recorded from carangid fish, although the majority of them, excluding polyxenous species, such as K. amamiensis Egusa et Nakajima, 1980, K. iwatai Egusa et Shiomitsu, 1983, K. nova Naidenova, 1975, K. quadratum (Thélohan, 1895) and K. yasunagai (Hsieh et Chen, 1984), are limited to a single or a few fish species. We report the occurrence of macroscopic cysts of Kudoa trachuri Matsukane, Sato, Tanaka, Kamata et Sugita-Konishi, 2011 in the trunk muscle of four new host fish species, i.e., Pseudocaranx dentex (Bloch et Schneider), Decapterus akaadsi Abe, D. muroadsi (Temminck et Schlegel) and Decapterus tabl Berry, fished from the Philippine Sea (Northwest Pacific Ocean), off southwestern of Japan. Myxospore morphology and genetic characteristics of the ribosomal RNA gene (rDNA) of these isolates were consistent with previous records of K. trachuri from Trachurus japonicus (Temminck et Schlegel) from around Japan. In addition, a new species of Kudoa that forms long filamentous pseudocysts in trunk myofibres was found in four of the six D. tabl collected in this study. We describe Kudoa longichorda sp. n. for this new isolate, based on its morphology of subquadrate myxospores with four shell valves and polar capsules and with small dimensions (length 4.3-5.5 µm, width 6.0-6.8 µm, thickness 4.8-6.3 µm, polar capsule length 2.3-3.1 µm, polar capsule width 1.1-1.7 µm), as well as 18S and 28S rDNA sequences distinct from those of known species.

Occurrence of Kudoa prunusi and K. lateolabracis (Myxozoa: Myxosporea: Multivalvulida) in Philippine-Sea Japanese parrotfish (Calotomus japonicus).

Multiple Kudoa spp. (Myxozoa: Myxosporea: Multivalvulida) have been recorded in Japanese parrotfish (Calotomus japonicus) from the Philippine Sea (Northwest Pacific Ocean), off southwestern Japan; Kudoa yasunagai in the brain, and K. igami, K. lateolabracis, and K. thalassomi in the muscles. This study examined eight Philippine Sea Japanese parrotfish samples collected in January and February 2019 and found K. prunusi in the brain (3-57 plasmodia/fish; average 17.9) and K. lateolabracis plasmodia in the trunk muscle of all fish individuals examined. The K. prunusi in this study was characterized by myxospores predominatetly with six shell valves (SVs) and a corresponding number of polar capsules (PCs), contrasting with the original description of the species from farmed Pacific bluefin tuna (Thunnus orientalis) brain that characterized the species as having predominately five SVs/PCs. Molecular-genetic characterization of 18S and 28S ribosomal RNA genes and mitochondrial DNA genes (cytochrome c oxidase subunit 1 and small and large ribosomal RNA subunits) clearly differentiated the K. prunusi isolate from K. yasunagai, commonly characterized by six or seven, but rarely five, SVs/PCs myxospores. The Japanese parrotfish is a new host record for K. prunusi and speculated to be an important reservoir host in its natural waters. Kudoa lateolabracis myxospores isolated from pseudocysts in the myofiber were morphologically and phylogenetically close to a clade of the Kudoa spp. that exhibit cruciform myxospores similar to K. thyrsites. This study is the first to sequence a mitochondrial DNA of small and large subunit ribosomal RNA of K. lateolabracis.

Novel hemagglutinin-binding sulfated oligofucosides and their effect on influenza virus infection.

Among a series of chemically synthesized fucoidan derivatives (1-9), 5 was found for the first time to bind to influenza virus hemagglutinins (HAs) and inhibit hemagglutination activity. In addition, a designed C3-symmetric tripodal ligand 10, synthesized with three sulfated oligofucoside moieties of 5, exhibited much greater hemagglutination inhibition activity than 5. A plaque assay using MDCK cells demonstrated that 10 effectively inhibited influenza virus infection.

Four Myxobolus spp. (Myxosporea: Bivalvulida) from the gill lamellae of common carp (Cyprinus carpio) and Japanese silver crucian carp (Carassius langsdorfii) in the western part of Japan, with the description of three new species (M. tanakai n. sp., M. paratoyamai n. sp., and M. ginbuna n. sp.).

Approximately three dozen Myxobolus spp. (Myxozoa: Myxosporea: Bivalvulida) have been described to parasitize the gills of carp of the genera Cyprinus and Carassius. Hitherto, these fish were often introduced to temperate waters worldwide as food and ornamental fish from Asia, their place of origin. The present study examined the myxosporean infection of seven common carp (Cyprinus carpio) and seven Japanese silver crucian carp (Carassius langsdorfii) collected from the Fushinogawa River around the university in Yamaguchi City, Japan, during the period April 2015 to October 2016. In total, four Myxobolus spp. were detected in the gill lamellae of Cy. carpio and Ca. langsdorfii, i.e., two species in each species of fish. The four species were characterized morphologically and genetically based on the 18S ribosomal RNA gene (rDNA). A new species, Myxobolus tanakai n. sp., from four individuals of Cy. carpio had an elongated pyriform spore (15.4-18.6 µm by 6.3-8.4 µm), resembling the spore shape of Myxobolus koi from Cy. carpio or Carassius auratus in Japan, China, and the USA, but bigger than it (13.2-15.6 µm by 6.6-7.8 µm). The new species formed a clade with M. koi but was distinct from any of the isolates of this species (nucleotide identities less than 98.6%). The second new species, Myxobolus paratoyamai n. sp., from a single Cy. carpio with its one prominent and one rudimentary polar capsule closely resembled the spore morphology of Myxobolus toyamai from Cy. carpio or Carassius gibelio in Japan, China, and the USA. However, the isolate formed a clade with Myxobolus longisporus from Cy. rubrofuscus in China rather than with M. toyamai isolates (nucleotide identities less than 97.9% with known species). Another new species, Myxobolus ginbuna n. sp., from two individuals of Ca. langsdorfii had similar-shaped spores to Myxobolus wulii, but the dimensions were smaller (11.7-13.9 µm by 8.5-9.8 µm vs. 17.6-18.5 µm by 8.9-10.0 µm). This new species formed a clade with M. wulii but was distinct from any of the M. wulii isolates from Ca. gibelio in China (nucleotide identities less than 99.1%). An additional species, Myxobolus pyramidis, from six individuals of Ca. langsdorfii was morphologically and genetically similar to the previous record from Ca. gibelio in China (99.6% nucleotide identity of the 18S rDNA). Two of these six individuals were mix-infected with M. ginbuna n. sp. This is a new host and geographical distribution record for M. pyramidis.

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.

Morphological and genetic characterization of Kudoa whippsi (Myxosporea: Multivalvulida) from Cheilodactylus zonatus in the western Pacific Ocean off Japan, and two new Kudoa spp. (K. akihitoi n. sp. and K. empressmichikoae n. sp.) from Acanthogobius hasta in the Sea of Ariake, Japan.

Molecular genetic characterization using the ribosomal RNA (rDNA) gene accrues a wealth of knowledge regarding the true nature of species diversity of Kudoa Meglitsch, 1947 (Myxozoa: Myxosporea: Multivalvulida) and the biogeographical relationships of isolates from different host fish and sea areas. In the present study, we characterized morphologically and genetically three Kudoa spp. with four shell valves and polar capsules (SV/PC), forming pseudocysts in the myofiber of trunk muscles of Cheilodactylus zonatus or Acanthogobius hasta in the natural seawater around Japan. Myxospores from C. zonatus fished in the western Pacific Ocean off Kochi, Japan, were unequal quadrangular pyramids with one large and three smaller SV/PC, morphologically closest to Kudoa whippsi recorded in various pomacentrid and apogonid fish from the Australian Coral Sea. The 18S and 28S rDNA nucleotide sequences of the Japanese isolate were highly similar to some Australian K. whippsi isolates, but also displayed less similarity to other K. whippsi isolates from the same sea mainly due to instability of nucleotides at certain base positions and/or segments of different isolates. All the K. whippsi isolates including the present Japanese isolate, however, were distinct from Kudoa gunterae, K. whippsi's closest kudoid species in morphology, molecular phylogeny, and biogeography. Our detection of K. whippsi from C. zonatus in the natural seawater around Japan is a new host and geographical record. Kudoid myxospores from A. hasta from the Sea of Ariake, a deep bay of the western part of Japan, exhibited two morphotypes, one resembling K. whippsi and the other Kudoa quadricornis with distinct posteriolateral SV projections. However, rDNA nucleotide sequencing revealed that these two Kudoa spp. were distinct from any known congeners; thus, Kudoa akihitoi n. sp. and Kudoa empressmichikoae n. sp. were erected. The morphological differentiation of K. akihitoi n. sp. from multiple Kudoa spp. with scalene stellate myxospores containing one large and three smaller SV/PC was difficult, whereas K. empressmichikoae n. sp. with spherical spore bodies extending small posteriolateral SV projections was distinct from known congeners with similar but elongated spore bodies and PC, i.e., K. quadricornis and Kudoa paraquadricornis, found in the trunk muscle of carangid fish from the Australian Coral Sea.

New host records of monacanthid fish for three Kudoa spp. (K. septempunctata, K. thyrsites, and K. shiomitsui) prevalent in the olive flounder (Paralichthys olivaceus), with the description of K. parathyrsites n. sp. from a black scraper (Thamnaconus modestus).

Kudoa septempunctata (Myxosporean: Multivalvulida) is known as a cause of foodborne disease associated with consumption of raw flesh of the olive flounder (Paralichthys olivaceus). Knowledge of its life cycle, particularly alternate annelid hosts and reservoirs or susceptible fish hosts in natural waters, may facilitate disease control in aquaculture farms. Our recent survey of myxosporean infection in monacanthid fish in natural waters around Japan revealed infection with three kudoid species prevalent in the olive flounder, i.e., K. septempunctata, Kudoa thyrsites, and Kudoa shiomitsui. Of the 51 black scrapers (Thamnaconus modestus) examined, five fish were infected: two fish with K. septempunctata and three with K. thyrsites. One of the fish infected with K. septempunctata was also infected with a K. thyrsites-like species. One of the 17 threadsail filefish (Stephanolepis cirrhifer) and two of four unicorn leatherjackets (Aluterus monoceros) were parasitized with K. shiomitsui. Three modest filefish (Thamnaconus modestoides) had no kudoid infection. K. septempunctata from a black scraper fished in the Inland Sea of Japan off Yamaguchi had 6-8 (predominantly 7) shell valves/polar capsules, whereas K. septempunctata found in another black scraper from the Sea of Japan off Tottori had 5 or 6 (predominantly 6). However, the two isolates displayed identical 18S and 28S ribosomal RNA gene (rDNA) nucleotide sequences, which were also identical to the isolates from the olive flounder. K. thyrsites from the Inland Sea of Japan off Yamaguchi and Sea of Japan off Tottori and K. shiomitsui from the Sea of Japan off Shimane and western Pacific Ocean off Kochi were also morphologically and genetically characterized. They were found to be coincident with the previous reports from olive flounders. Furthermore, the K. thyrsites-like species found in a black scraper from the Inland Sea of Japan off Yamaguchi was morphologically and genetically characterized; a new species, Kudoa parathyrsites n. sp., is erected for this species. The relationships of the new species with K. thyrsites and related species as well as those of K. shiomitsui with Kudoa pericardialis and related species parasitizing the pericardium are briefly discussed.

Morphological and molecular genetic characterization of two Kudoa spp., K. musculoliquefaciens, and K. pleurogrammi n. sp. (Myxosporea: Multivalvulida), causing myoliquefaction of commercial marine fish.

Genetic characterization of myxosporean species, including members of the genus Kudoa, has advanced dramatically throughout the last decade. This is in stark contrast to those species described further back in time. Kudoa musculoliquefaciens described from the jellied muscle of swordfish, Xiphias gladius, in the western Pacific Ocean off the Sanriku Coast, northern Japan, is one such species. In the present study, multiple pseudocysts (0.66-1.35 mm average length and 0.06-0.10 mm average width) containing K. musculoliquefaciens spores were collected from three host groups: muscle blocks of swordfish caught in the western Pacific Ocean off the Sanriku Coast, or the northern Indian Ocean, and Indo-Pacific sailfish, Istiophorus platypterus, in the western Pacific Ocean off Kochi, western Japan. Subspherical K. musculoliquefaciens spores, 8.0-10.3 µm in width, 7.3-10.0 µm in thickness, 6.4-7.9 µm in sutural thickness, and 5.5-8.1 µm in length, had four subspherical polar capsules, 2.8-4.0 µm in length by 2.2-3.2 µm in width. The kudoid spores found in the different host groups showed morphometric variations to some extent but had essentially identical nucleotide sequences of the ribosomal RNA gene (rDNA), closest to those of Kudoa hemiscylli or Kudoa carcharhini recorded from elasmobranchs in the Indo-Pacific Ocean. Another kudoid species, Kudoa pleurogrammi n. sp., was recorded from the jellied and normal muscles of Atka mackerel, Pleurogrammus monopterygius and Pleurogrammus azonus, fished in the northern Pacific Ocean or northern Sea of Japan. Subquadrate spores found in round-ended pseudocysts (1.15-3.85 mm in length and 0.11-0.26 mm in width) in myofibers were 8.2-9.1 µm in width, 7.1-8.2 µm in thickness, 5.4-7.7 µm in sutural thickness, and 5.6-6.8 µm in length, with four ovoid polar capsules, 2.7-2.9 µm in length by 1.4-2.0 µm in width. Kudoid spores from both jellied and normal muscles or different host fish species had identical 18S or 28S rDNA nucleotide sequences. Thus, molecular genetic characterization of kudoid species with the potential to induce post-mortem myoliquefaction will facilitate the reliable and specific identification of myxosporeans found in either jellied or normal muscles of important commercial fish.

Systematic synthesis of low-molecular weight fucoidan derivatives and their effect on cancer cells.

Low-molecular weight type I and II fucoidan derivatives with different sulfation patterns were designed and systematically synthesized from the corresponding common key intermediate and their anti-proliferative activities and apoptosis-inducing activities against human breast cancer (MCF-7) and human cervical epithelioid carcinoma (HeLa) cells were evaluated. Our results demonstrated that one of the type II fucoidan derivatives, 9, effectively reduced the number of viable MCF-7 and HeLa cells in a dose-dependent manner without causing cytotoxicity toward normal WI-38 cells, and that the anti-proliferative activity of 9 was comparable to that of fucoidan 2 isolated from Fucus vesiculosus. Moreover, it was found that both 2 and 9 exhibited similar apoptosis-inducing activities through activation of caspase-8 and -9 on MCF-7 and HeLa cells, respectively.