Occurrence and distribution of yellow grub trematodes (Clinostomum complanatum) infection in Taiwan.

Digenetic trematodes of the genus Clinostomum are widely distributed in many species of freshwater fish and are known to cause the zoonotic disease Halzoun. Humans may become accidental hosts if they ingest raw freshwater fish containing metacercaria of Clinostomum complanatum, which causes pharyngitis or laryngitis. The yellow grub parasitizing cultivated ayu (Plecoglossus altivelis) and loach (Misgurnus anguillicaudatus) was one of the most serious problems in fish farms from 1977 to 1979 in Taiwan. The present study revealed, for the first time, frequent occurrence of C. complanatum metacercaria in various fish species in a natural environment, the Dahan River, in Taiwan. We examined 1503 fish specimens belonging to four families and 162 snails representing six species. C. complanatum metacercaria was mainly from the fish family Cyprinidae, and only cercaria from the snail Radix swinhoei was recovered. The prevalence and mean intensity of metacercaria were 9.8% and 9.35 parasites/infected fish, respectively. The prevalence of cercaria was low, 0.62%, in snails. Acrossocheilus paradoxus, Zacco barbata, Zacco pachycephalus, Zacco platypus, Onychostoma barbatula, and Hemibarbus labeo are new host records. Metacercariae were primarily found in the operculum, mandible, muscle, and oral cavity of fish. Morphological description and molecular analysis with 18S rDNA sequencing allowed for rapidly identifying C. complanatum. Encysted and excysted metacercariae cultivated at 22 °C in physiological saline died within 60 h. The mean intensity of infection increased with an increasing length of Z. pachycephalus. We found no association between monthly parasite prevalence and mean intensity at each sampling location. No C. complanatum metacercaria survived after 8 h of salting. The Dahan River has suitable conditions and hosts (snails, fish, and fish-eating birds) for maintaining the life cycle of C. complanatum.

Scavenging hagfish as a transport host of Anisakid nematodes.

Hagfish are the most primitive craniates and scavengers, feeding on dead organisms as well as fish and invertebrates. Hagfish play an important ecological role in recycling nutrients, helping to recycle biomass from the upper water column. We investigated 265 specimens of four hagfish species, including Eptatretus burgeri, Eptatretus yangi, Eptatretus sheni and Eptatretus taiwanae from northeastern Taiwanese waters of the northwestern Pacific from November 2013 to June 2014. Eight species of Anisakid nematodes were identified: Anisakis pegreffii, Anisakis simplex s.s., a recombinant genotype of A. pegreffii and A. simplex s.s., Anisakis typica, Anisakis sp., Anisakis brevispiculata, Anisakis physeteris and Hysterothylacium amoyense. Anisakis sp. and H. amoyense represented new locality records. The prevalence, mean intensity and mean abundance of anisakid nematodes for all specimens were 21.51%, 5.39 larvae per fish and 1.16 larvae per fish, respectively. A. pegreffii was the most frequent species in E. burgeri, E. yangi and E. taiwanae, whereas in E. sheni, the dominant species was Anisakis sp. The number of nematodes was significantly related to the host length for E. burgeri and E. sheni, but was not related to the sex of the four hagfish species. This report of scavenging hagfish infected with Anisakid larvae suggests hagfish as a transport/paratenic host between cetaceans and piscivores. Anisakiasis may be caused by the consumption of infectious third-stage larvae in raw or undercooked hagfish.

Effects of the acanthocephalan Polymorphus minutus and the microsporidian Dictyocoela duebenum on energy reserves and stress response of cadmium exposed Gammarus fossarum.

Amphipods are commonly parasitized by acanthocephalans and microsporidians and co-infections are found frequently. Both groups of parasites are known to have severe effects on their host. For example, microsporidians can modify host sex ratio and acanthocephalans can manipulate the behavior of the amphipod to promote transmission to the final host. These effects influence host metabolism in general and will also affect the ability of amphipods to cope with additional stressors such as environmental pollution, e.g., by toxic metals. Here we tested the effects of sub-lethal concentrations of cadmium on glycogen and lipid levels, as well as on the 70kDa heat shock protein (hsp70) response of field collected Gammarus fossarum, which were naturally infected with microsporidians and the acanthocephalan Polymorphus minutus. Infected and uninfected G. fossarum were exposed to a nominal Cd concentration of 4 µg/L, which resembled measured aqueous Cd concentration of 2.9 µg/L in reconstituted water for 7 d at 15 °C in parallel to an unexposed control. After exposure gammarids were snap frozen, weighed, sexed and tested for microsporidian infection by PCR. Only individuals containing the microsporidian Dictyocoela duebenum were used for the further biochemical and metal analyses. P. minutus infected amphipods were significantly smaller than their uninfected conspecifics. Mortality was insignificantly increased due to cadmium exposure, but not due to parasite infection. Microsporidian infection in combination with cadmium exposure led to increased glycogen levels in female gammarids. An increase of glycogen was also found due to interaction of acanthocephalan and microsporidian infection. Elevated lipid levels were observed in all groups infected with microsporidians, while acanthocephalans had the opposite effect. A positive correlation of lipid and glycogen levels was observed. The general stress response measured in form of hsp70 was significantly increased in microsporidian infected gammarids exposed to cadmium. P. minutus did not affect the stress response of its host. Lipid levels were correlated negatively with hsp70 response, and indicated a possible increased stress susceptibility of individuals with depleted energy reserves. The results of our study clearly demonstrate the importance of parasitic infections, especially of microsporidians, for ecotoxicological research.

Expression patterns and structural modelling of Hsp70 and Hsp90 in a fish-borne zoonotic nematode Anisakis pegreffii.

Heat shock proteins (HSPs) are essential molecular chaperones that are highly conserved across organisms. They have a pivotal function in responding to thermal stress and are responsible for many cellular functions. Here, we aimed to elucidate the possible roles of Hsp70 and Hsp90 in the life cycle of the parasitic nematode Anisakis, particularly third- and fourth-stage larvae, from cold-blooded fish to warm-blooded marine mammals or accidentally to human hosts. We examined the expression profiles of Hsp70 and Hsp90 in different developmental stages of Anisakis pegreffii. The open reading frame of Hsp70 of A. pegreffii was 1950 bp, and deduced amino acid sequence showed high homology with those of other nematodes. Heatmap analysis revealed sequence identity of Hsp70 and Hsp90 in 13 important parasitic species, human and yeast. On heatmap and phylogenetic analysis, ApHsp70 and ApHsp90 shared the highest amino acid sequence identity with other nematodes and formed a monophyletic clade. The three-dimensional (3D) structure prediction of the newly characterized ApHsp70 and known ApHsp90 gene showed highly conserved motifs between A. pegreffii and other species. Quantitative real-time PCR and western blot analysis revealed higher mRNA and protein expression for ApHsp70 and ApHsp90 in fourth- than third-stage larvae, with higher mRNA and protein expression for ApHsp70 than ApHsp90. ApHsp70 and ApHsp90 may play important roles in Anisakis in response to thermal stress and might be important molecules in the development of A. pegreffii, which has implications for its control.

Occurrence and prevalence of fish-borne Anisakis larvae in the spotted mackerel Scomber australasicus from Taiwanese waters.

Anisakid nematodes have been found in a variety of marine fishes throughout the world and they are known to cause anisakiasis in human hosts. The present study investigated the prevalence of potentially zoonotic anisakid larvae in spotted mackerel caught from Taiwanese waters where fish represents an important food sources. Anisakis third-stage larvae (L3, n=502) were isolated from 250 spotted mackerel Scomber australasicus. Anisakis L3 larvae were divided morphologically into two types, Anisakis type I larvae had a longer ventriculus and mucron while type II larvae had a shorter ventriculus and no mucron. Anisakis species were identified by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of the internal transcribed spacer (ITS) regions of ribosomal DNA and direct sequencing. A simple molecular taxonomic key, utilizing RFLP by two restriction enzymes HinfI and HhaI, enabled the differentiation of the genus Anisakis. The prevalence, mean intensity and mean abundance of Anisakis nematodes recorded for the total specimens were 72.8%, 2.8 (1-15) and 2.0 (0-15), respectively. Anisakis pegreffii was determined to be the dominant species (prevalence=57.2%) and important agent of human anisakiasis. A recombinant genotype (Anisakis simplex sensu stricto × A. pegreffii) was identified as the subdominant species (25.3%) followed by Anisakis typica (10%), Anisakis physeteris (4.0%), Anisakis paggiae (3.0%) and Anisakis brevispiculata (0.5%). The topology of the maximum likelihood and neighbor-joining trees show two well supported clades: one includes the species of A. pegreffii and the other includes A. paggiae, A. physeteris and A. brevispiculata, while A. typica has basal position to all other Anisakis spp. analyzed. This study advances our knowledge of the prevalence of different Anisakis spp. in the spotted mackerel from Taiwanese waters, which is helpful for monitoring the fish populations throughout a diverse array of aquatic ecosystems. More importantly, we provide the concise characterization of multiple Anisakis spp. by PCR-RFLP, which could also be applicable for the rapid diagnosis of human anisakiasis.

Effect of different temperatures on the expression of the newly characterized heat shock protein 90 (Hsp90) in L3 of Anisakis spp. isolated from Scomber australasicus.

Anisakid nematodes are distributed worldwide in a wide variety of marine fishes and they are known to cause the zoonotic disease, anisakiasis. The temperature control is commonly applied for prevention and control of anisakiasis. To analyze the cellular response to temperature stress in Anisakis, the heat shock protein 90 (Hsp90) was chosen in the present study, as it plays a key role in many cellular processes and responds to stress conditions such as heat or cold shock. Anisakids were sampled from spotted mackerel Scomber australasicus caught from the coastal waters of Yilan, in northeastern Taiwan (25 °N, 121 °E). Anisakid nematodes were pre-identified morphologically and later molecularly by PCR-RFLP. In total, we obtained six species of the genus Anisakis, A. typica, A. pegreffii, A. paggiae, A. brevispiculata, A. physeteris, and a recombinant genotype between A. pegreffii and A. simplex sensu stricto. Thereby we provide new host and locality records for A. paggiae, A. brevispiculata and A. physeteris. The Hsp90 genes of five species (except the recombinant genotype) were cloned by rapid amplification of cDNA ends (RACE) and their deduced amino acid sequences were further characterized. Quantitative real-time PCR and Western blot analysis were used to examine the expression levels of the Hsp90 in A. pegreffii under different temperature conditions. Quantitative RT-PCR showed that Hsp90 transcript levels increased slightly under heat shock (50 °C) treatment, and increased gradually during the first 3h, and thereafter, returned to its baseline value at 37 °C. Under cold shock (4 °C) treatment, the mRNA expression of Hsp90 did not change significantly. In addition, we found a clear time-dependent Hsp90 protein expression pattern of A. pegreffii exposed to high temperature. Our results suggest that the mRNA and protein expression patterns of Hsp90 are related to the temperature, and are especially significantly increased under heat stress.

A new species of chondracanthid copepod parasitic in the pharynx of hagfishes (Myxiniformes: Myxinidae) from off Taiwan.

A new species of Acanthochondria Oakley, 1927 (Copepoda, Poecilostomatoida, Chondracanthidae), parasitic in the pharynx of hagfishes Eptatretus burgeri (Girard), E. sheni (Kuo, Huang & Mok) and E. yangi (Teng), from off Taiwan is described and illustrated. The new species can be distinguished from its congeners by having the neck region longer than wide (about 2-3 times as long as wide), the moderately long protopod of leg 2 (Type E), the endopod of leg 2 as large as exopod, the cephalosome wider than long, the armature formula of antennule (1, 1, 2, 2, 9), the relatively long trunk (6.7 mm) and egg-sacs (14.4 mm). This is the first record of a parasitic chondracanthid from hagfishes. The findings may provide insights into the phylogenetic relationships and interactions among chondracanthids, hagfishes and demersal fishes, but call for further molecular studies.

Parasitism between Anisakis simplex (Nematoda: Anisakidae) third-stage larvae and the spotted mackerel Scomber australasicus with regard to the application of stock identification.

The nematode fauna of 369 spotted mackerel of the species Scomber australasicus, collected off the northeastern Taiwanese coast of the northwestern Pacific, was investigated monthly from April 2004 to March 2005. The following nematode species were recorded: Anisakis simplex complex, Hysterothylacium aduncum, Porrocaecum decipiens and Raphidascaris trichiuri. The seasonal variation in the infection with A. simplex third stage larva (L3) was studied throughout the 12 months. The prevalence of A. simplex L3 recorded for total fish samples was 93.6%, varying between 86.7 and 100%. There was an increase in the abundance of this nematode in spring, with the peak occurring in April. To reveal whether intrinsic factors of the spotted mackerel host contributed to infection with this nematode, fish were grouped according to their body weight, age and gonad development (reported as gonadoosomatic index, GSI), respectively, and infection parameters (i.e., prevalence, abundance and intensity) were analyzed. Results showed that abundance was significantly higher in both larger (>450 g) and older (>3 years old) fish. The gonad development of the host fish was not correlated with the intensity of the larval infection in both female and male fish. Two distinct Anisakis species were identified by PCR-RFLP, namely A. pegreffii and a recombinant genotype of A. pegreffii and A. simplex sensu stricto. These species occurred with frequencies of 97% and 3%, respectively. The usefulness of using parasites as biomarkers for spotted mackerel stock identification around Taiwanese waters was confirmed herein. A second group of 58 spotted mackerel were obtained from the coastal waters off southwestern Taiwan. In addition to the two species, A. pegreffii and the recombinant one, which were found with frequencies of 63% and 9%, respectively, an additional Anisakis species A. typica was identified with a frequency of 28% from these fish. Two spotted mackerel stocks could thus be identified based on their infrastructure of Anisakis species community and their frequency. To the best of our knowledge, this is the first report of stock identification of spotted mackerel using endoparasite biomarkers.

Effects of various treatments on egg hatching of Dendromonocotyle pipinna (Monogenea: Monocotylidae) infecting the blotched fantail ray, Taeniurops meyeni, in Taiwan.

The monocotylid monogenean Dendromonocotyle pipinna infects the dorsal skin of the blotched fantail ray, Taeniurops meyeni, and is problematic for Hualien Farglory Ocean Park, an aquarium in Taiwan. Over the last 2 years, eight rays have died due to heavy infections with this parasite. In this study, we found that the epidermis of T. meyeni with attached D. pipinna was not markedly thinner but contained decreased numbers of mucous cells and numerous vacuoles. We examined the effects of temperature (from 16 to 30 degrees C in 2 degrees C increments), salinity (from 10 to 50 per thousand in 5 per thousand increments), desiccation (from 1 to 10 min) and sodium hypochlorite (from 5 to 20 ppm in 5 ppm increments) treatment on the embryonation period and hatching success of D. pipinna eggs, with the goal of disinfecting equipment used in aquaria. Temperature strongly influenced embryonation period: eggs first hatched 4 days after being laid at 30 degrees C and 16 days after being laid at 16 degrees C. However, hatching rate was not significantly influenced by incubation temperature, since the final hatching rates under the incubation temperatures tested herein were not significantly different from one another. Hyposalinity had a lethal effect on D. pipinna eggs, completely preventing the hatching of eggs cultured at 10 and 15 per thousand salinity. Hypersalinity was only partially effective, with a hatching rate close to 7% at 50 per thousand salinity. Desiccation was effective at preventing hatching, and the effectiveness increased with increasing treatment duration. The hatching rate of D. pipinna eggs was significantly decreased when incubated under desiccating conditions for even 1 min. Furthermore, a complete inhibition of hatching was achieved by desiccating eggs for 10 min. Sodium hypochlorite treatment completely prevented hatching at concentrations higher than 10 ppm after 18 h of exposure, but a concentration of 5 ppm was ineffective at preventing hatching even after 24 h of treatment. We therefore propose effective combinations of sodium hypochlorite and exposure time as a means to sterilize tanks and equipment containing D. pipinna eggs.

Anisakis simplex (Nematoda: Anisakidae) third-stage larval infections of marine cage cultured cobia, Rachycentron canadum L., in Taiwan.

The first confirmed case of Anisakis simplex infection of the marine cage cobia, Rachycentron canadum (L.), was recorded in Taiwan. The case investigation revealed the presence of third-stage larvae (L3) in either the stomach lumen or abdominal cavity of the cobia but never within the musculatures. Larvae were mainly encapsulated in the peritoneal mesentery on the outer surface of the stomach wall and occasionally on the liver surface. Part of the diet fed to the cobia includes chopped raw fish, and of these, seven species were found to harbor these larvae (as paratenic hosts), indicating that these particular fish might be the larval sources for this infection. To illustrate the course of infection and distribution of this parasite inside cobia, both juvenile and adult cobia were experimentally infected with live L3 by oral transmission. The prevalence of infection reached 100% at the end of all trials. The course of the infection was assessed after necropsy by histological and ultrastructural observations. A. simplex L3 recovered from various locations within juvenile cobia at different post-infection (p.i.) times were at the L3 stage and did not grow significantly. The L3 either adhered to or penetrated into the gastric mucosa of cobia by 2 h p.i. By 25 d p.i., many were trapped within the submucosa and encapsulated by fibroconnective tissue. This phenomenon was more apparent in adult cobia, such that 37.5-86.0% of the injected L3 were primarily found encapsulated within the gastric submucosa. Based upon a PCR-RFLP assay, the larvae encountered in this study were identified as having a recombinant genotype of A. simplex sensu stricto and A. pegreffii. Based upon the results of this study, strategies to ensure the safety of seafood manufactured from cobia and to prevent the potential risks of anisakiasis or allergies risk to consumers were suggested.

Parasitic helminth fauna of the cutlass fish, Trichiurus lepturus L., and the differentiation of four anisakid nematode third-stage larvae by nuclear ribosomal DNA sequences.

The helminth fauna of the gastrointestinal tract and abdominal cavity of cutlass fish, Trichiurus lepturus L., off the Taiwanese coast of the north-western Pacific was investigated. The following helminths were found: (1) nematodes--Anisakis simplex, Hysterothylacium aduncum, Porrocaecum decipiens, Raphidascaris trichiuri; (2) digeneans--adult Lecithochirium trichiuri; and (3) cestodes-plerocercoids of Proteocephalus spp. The third-stage larvae of these four anisakid nematodes were characterized genetically using a molecular approach. The nuclear ribosomal DNA region spanning the first internal transcribed spacer (ITS-1), the 5.8S gene and the second internal transcribed spacer (ITS-2) was amplified and sequenced. Based on the sequence differences, a PCR-based restriction fragment length polymorphism method was established for the unequivocal delineation of the four species. Phylogenetic analysis showed that H. aduncum clustered with P. decipiens, whereas A. simplex was not closely related to these according to the nucleotide sequences of all rDNA.

Geographic variations among infectious hypodermal and hematopoietic necrosis virus (IHHNV) isolates and characteristics of their infection.

Nucleotide sequence variations of a 2.9 kb fragment of infectious hypodermal and hematopoietic necrosis virus (IHHNV) isolated from samples of Penaeus monodon were determined and compared with an isolate from Hawaii. The infection characteristics of these isolates were examined by histology, in situ hybridization, and laboratory challenge studies with P. vannamei. Isolates of IHHNV were obtained from samples collected from the SE Asia region (the Philippines, Thailand, and Taiwan). Isolates of putative IHHNV were obtained from African samples (Tanzania, Madagascar, and Mauritius). The Philippine isolate had a very high nucleotide sequence identity (99.8%) to Hawaii IHHNV. The Thailand isolate showed a slightly lower identity (96.2%). The putative IHHNV sequences collected from Tanzania and Madagascar showed greater divergence from Hawaii IHHNV, 8.2% difference for Tanzania and 14.1% difference for Madagascar. A phylogenetic analysis showed that the Philippine IHHNV clustered with IHHNV found in the western hemisphere. This supports the theory that the Philippines was the origin of IHHNV that was first detected in Hawaii. In the laboratory infection study, both the Philippine and Thailand IHHNV were passed into P. vannamei, and the infected shrimp did not suffer any mortalities. In another laboratory infection, P. vannamei injected with a tissue homogenate of P. monodon from Madagascar, which tested positive for IHHNV by PCR, did not demonstrate IHHNV infection, suggesting that this putative IHHNV is not infectious to P. vannamei.