Tomoyoshi Yoshinaga

Morphological differences between larvae and in vitro-cultured adults of Anisakis simplex (sensu stricto) and Anisakis pegreffii (Nematoda: Anisakidae)☆

Proper identification of Anisakis species infecting host fishes is very important to both human health and fish disease diagnosis. The foremost problem in the identification of Anisakis larvae in fishes is that L3 larvae cannot be easily differentiated morphologically, especially between A. simplex (sensu stricto) (s.s.) (Rudolphi, 1809) and A. pegreffii Campana-Rouget et Biocca, 1955. Instead, molecular means such as allozyme, mitochondrial DNA (mtDNA) cox2 region and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analyses had been successfully used. In this study, morphological differences of L3 larvae collected from fishes and in vitro-cultured L4 larvae and adult A. simplex (s.s.) and A. pegreffii were evaluated. Anisakis larvae were collected from 7 different host fishes within Japan. Undamaged A. simplex (s.s.) and A. pegreffii collected from Oncorhynchus keta (Walbaum) and Scomber japonicus Houttuyn, respectively, were used for in vitro-culture in order to obtain L4 and adult stages. Species identification was confirmed by PCR-RFLP analysis of the ITS region (ITS1-5.8S-ITS2) of ribosomal DNA and by mtDNA cox2 gene sequencing. Results revealed that L3, L4 and adult stages of A. simplex (s.s.) and A. pegreffii are morphologically distinguishable based on ventriculus length, wherein the former has longer ventriculus (0.90-1.50 mm) than the latter (0.50-0.78 mm). For oesophagus/ventriculus ratio, these two species are distinguishable only during L4 and adult stages. Also, adult male A. simplex (s.s.) and A. pegreffii were found to be distinguishable by differences in the distribution pattern of the caudal papillae, particularly the 3rd pair of distal papillae.

Experimental challenge of Anisakis simplex sensu stricto and Anisakis pegreffii (Nematoda: Anisakidae) in rainbow trout and olive flounder.

The third-stage larvae of Anisakis simplex sensu lato (s.l.) are found in many marine fishes. To ensure food safety, it is important to determine whether these larvae are present in the body muscle of commercial fish species. However, there is little information regarding the tissue specificity of Anisakis and two of its sibling species, A. simplex sensu stricto (s.s.) and Anisakis pegreffii, that are common in marine fish in Japanese waters. We orally challenged rainbow trout (Oncorhynchus mykiss (Walbaum)), and olive flounder (Paralichthys olivaceus (Temminck and Schlegel)) with L3 larvae of these two sibling species and monitored infection for 5weeks. In rainbow trout, A. simplex s.s., but not A. pegreffii larvae, migrated into the body muscle. A small number of freely moving A. pegreffii larvae were recovered within the body cavity. In olive flounder, A. simplex s.s. larvae were found in both the body cavity and body muscle. A. pegreffii larvae were found only in the body cavity and primarily encapsulated in lumps. Our results indicate that there are differences in the sites of infection and host specificity between the two sibling species of A. simplex s.l.

Identification of Larval Anisakis spp. (Nematoda: Anisakidae) in Alaska Pollock (Theragra chalcogramma) in Northern Japan Using Morphological and Molecular Markers

Abstract The Alaska pollock, Theragra chalcogramma (Pallas), is an important raw source for surimi and other food products in Japan. However, Alaska pollock caught in the Atlantic and Mediterranean regions has been reported to harbor Anisakis species that pose considerable food safety problems. Here, we identified the third-stage (L3) Anisakis spp. sampled from Alaska pollock caught in northern Japan using a combination of morphological and molecular analyses which included PCR-RFLP and sequencing of the ITS (ITS1-5.8S rDNA-ITS2) region and mtDNA cox2 gene markers. Four Anisakis spp. were confirmed, namely Anisakis simplex (sensu stricto [s.s.]), A. pegreffii, A. brevispiculata, and an Anisakis sp. belonging to the Anisakis Type II group. The identification of 4 different Anisakis spp. occurring in Alaska Pollock, and the identification of A. brevispiculata and an Anisakis sp. (Anisakis Type II) in the northwest Pacific region, are first reports. Anisakis simplex (s.s.) composed the majority of Anisakis spp. in Alaska pollock at 91.0%, followed by A. pegreffii (5.2%), Anisakis sp. (Anisakis Type II) (2.4%), and A. brevispiculata (1.4%).

Distribution of Anisakis species larvae from fishes of the Japanese waters.

Human anisakiasis is caused by the consumption of raw, marinated or undercooked fish and squid infected with nematodes of the genus Anisakis Dujardin, 1845. In view of food safety, this study was carried out to examine the distribution of Anisakis species in marine fishes within Japanese waters. Seven fish species from six localities were collected and examined for Anisakis infection. Morphological and molecular (ITS region and mtDNA cox2 gene) characterization revealed the presence of two, among the three sibling species of Anisakis simplex, viz. A. simplex sensu stricto (s.s.) and A. pegreffii. Distribution data were collated with the results from the previous researches to better understand Anisakis distribution in Japanese waters. Distributions of Anisakis species were found to be locality-specific rather than host-specific, particularly between the two major species, A. simplex s.s. and A. pegreffii. Anisakis simplex s.s. is mainly found in fishes from northern Japan to Pacific sides, whereas A. pegreffii is in fishes from the Sea of Japan to East China Sea sides.

Experimental evaluation of the pathogenicity of Perkinsus olseni in juvenile Manila clams Ruditapes philippinarum.

We evaluated the pathogenicity of Perkinsus olseni towards the Manila clam, Ruditapes philippinarum, by an experimental challenge. For production of prezoosporangia of P. olseni, we injected uninfected Manila clams with cells of a pure strain of P. olseni and reared them for 7d. Prezoosporangia were isolated from the soft tissue of the injected clams after culturing in Rays fluid thioglycollate medium. Hatchery-reared, uninfected juvenile clams (3-10 mm shell length) were challenged by immersion in one of two concentrations of a prezoosporangial suspension of P. olseni for 6d. The challenged clams had significantly higher mortality at both the concentrations than the unchallenged clams. The mortality due to infection dose-dependently began approximately 4 weeks and 7 weeks after challenge in the higher and lower concentrations, respectively. This is the first experimental evidence that P. olseni causes direct mortality in Manila clams. The lethal level of infection was estimated at approximately 10⁷ pathogen cells/g soft tissue weight.

Experimental challenges of wild Manila clams with Perkinsus species isolated from naturally infected wild Manila clams

Manila clams, Ruditapes philippinarum, are widely harvested in the coastal waters in Japan. However, there have been significant decreases in the populations of Manila clams since the 1980s. It is thought that infection with the protozoan Perkinsus species has contributed to these decreases. A previous study demonstrated that high infection levels of a pure strain of Perkinsus olseni (ATCC PRA-181) were lethal to hatchery-raised small Manila clams, however, the pathogenicity of wild strain Perkinsus species to wild Manila clam is unclear. To address this, we challenged large (30-40 mm in shell length) and small (3-15 mm in shell length) wild Manila clams with Perkinsus species isolated from naturally infected wild Manila clams. We report high mortalities among the small clams, but not among the large ones. This is the first report to confirm the pathogenicity of wild isolate of Perkinsus species to wild Manila clams.

ATTACHMENT-INDUCING CAPACITIES OF FISH TISSUE EXTRACTS ON ONCOMIRACIDIA OF NEOBENEDENIA GIRELLAE(MONOGENEA, CAPSALIDAE)

When oncomiracidia of Neobenedenia girellae (Monogenea, Capsalidae) were incubated in wells with lyophilized extracts of fish skin epithelia on the bottom, some attached to the well bottom with the haptor unfolded and shed the ciliated epidermal cells. Based on these morphological changes in oncomiracidia, we developed a new assay method to examine the attachment-inducing capacities of various fish extracts for oncomiracidia. Attachment-inducing capacities were found only in extracts of fish skin epithelium and not in other fish extracts. No significant difference in capacities was observed among extracts of skin epithelia of 4 fish species. Wheat germ lectin and concanavalin A suppressed capacities in extracts of skin epithelia of Japanese flounder and yellowtail, respectively. Suppressed capacities were recovered by adding sugars that bound specifically to these lectins. These results indicate that some sugar-related chemical substances that exist specifically in fish epithelium induce the attachment of N. girellae oncomiracidia.

Anisakis species (Nematoda: Anisakidae) of Dwarf Sperm Whale Kogia sima (Owen, 1866) stranded off the Pacific coast of southern Philippine archipelago

Anisakid nematodes in the Pacific region of the Philippine archipelago still remain unexplored. This study was carried out to identify anisakid species from one of their final hosts, the Kogiid whale (Dwarf Sperm Whale, Kogia sima) stranded off the southern part (Davao Gulf) of the Philippine archipelago. Anisakid worms were initially identified morphologically using light and scanning electron microscopy, whereas identification to species level was carried out molecularly using PCR-RFLP and sequencing of the ITS (ITS1-5.8s rRNA-ITS2) and mtDNA cox2 regions. Parasitological study revealed new geographical records for the presence of two Anisakis species (A. brevispiculata and A. typica) and two unknown Anisakis species that are genetically close, at mtDNA cox2 region, to A. paggiae and A. ziphidarum. Based on the molecular data on both genes, the current findings suggest possible occurrence of local variations or sibling species of A. paggiae and A. ziphidarum in the region. Given that Anisakis species have not been reported in the Philippine archipelago, their presence in the Dwarf Sperm Whale inhabiting this region indicates high possibility of Anisakis infections in the marine fishes, cephalopods and other intermediate hosts within the Philippine waters.

Origin of the diclidophorid monogenean Neoheterobothrium hirame Ogawa, 1999, the causative agent of anemia in olive flounder Paralichthys olivaceus

In the mid-1990s, Neoheterobothrium hirame suddenly appeared as a new species in olive flounder Paralichthys olivaceus in Japanese coastal waters. Anemia caused by the parasite has prevailed in wild and cultured populations of olive flounder since that time. In this study, to clarify the origin of N. hirame, two Neoheterobothrium species, namely unidentified Neoheterobothrium species (tentatively abbreviated as Neoheterobothrium sp. PL) and N. affine, were collected from Paralichthys lethostigma and Paralichthys dentatus, respectively, off the east coast of North America and compared with N. hirame collected in Japan. No substantial differences were detected in the morphology and DNA sequences of ITS1-5.8S rRNA-ITS2 and mitochondrial cytochrome oxidase subunit I (mt COI) regions between N. hirame and Neoheterobothrium sp. PL. On the other hand, the congeneric N. affine was clearly distinguished from both N. hirame and Neoheterobothrium sp. PL in its longer isthmus and the DNA sequences in ITS1 and mt COI. The absence of differences between N.hirame and Neoheterobothrium sp. PL and the clear difference between both of these and N. affine indicate that N. hirame is conspecific with Neoheterobothrium sp. PL infecting P. lethostigma and that N. hirame was recently introduced from North America to Japan.

Life Cycle of Hysterothylacium haze (Nematoda: Anisakidae: Raphidascaridinae)

Natural infections with Hysterothylacium haze in the Japanese common goby, Acanthogobius flavimanus, were observed in detail. In gobies in which no worm eggs were deposited, second-stage larvae were found in the digestive tract wall, and third-stage larvae occurred in the digestive tract wall, mesentery, and body cavity, whereas fourth-stage larvae and adults were found in the body cavity. This stage-habitat relationship demonstrates the infectivity of second-stage larvae to the goby and the larval migration. In heavily infected gobies, eggs and all worm stages, from hatched second-stage larvae to adults, often were found together in the body cavity of one individual host, suggesting that hatched second-stage larvae can develop in the body cavity. It was shown experimentally that H. haze develops to the second stage in the egg and does not hatch spontaneously. When a goby was fed the viscera of heavily infected gobies containing eggs and various stages of worms or artificially incubated eggs containing second-stage larvae, second- and third-stage larvae were recovered from the digestive tract wall, and fourth-stage larvae and adults were found in the body cavity. When polychaetes or crustaceans were placed in contact with infected goby viscera or incubated eggs, only second-stage larvae were recovered from the body cavity of the invertebrates. The experimental results were consistent with observations on natural infections and indicate that the direct life cycle of H. haze may involve invertebrates as transport hosts.