C S Wang

PCR amplification and sequence analysis of the major capsid protein gene of megalocytiviruses isolated in Taiwan.

Viruses belonging to the genus Megalocytivirus in the family Iridoviridae are one of the major agents causing mass mortalities in marine and freshwater fish in Asian countries. Outbreaks of iridovirus disease have been reported among various fish species in Taiwan. However, the genotypes of these iridoviruses have not yet been determined. In this study, seven megalocytivirus isolates from four fish species: king grouper, Epinephelus lanceolatus (Bloch), barramundi perch, Lates calcarifer (Bloch), silver sea bream, Rhabdosargus sarba (Forsskal), and common ponyfish, Leiognathus equulus (Forsskal), cultured in three different regions of Taiwan were collected. The full open reading frame encoding the viral major capsid protein gene was amplified using PCR. The PCR products of approximately 1581 bp were cloned and the nucleotide sequences were phylogenetically analysed. Results showed that all seven PCR products contained a unique open reading frame with 1362 nucleotides and encoded a structural protein with 453 amino acids. Even though the nucleotide sequences were not identical, these seven megalocytiviruses were classified into one cluster and showed very high homology with red sea bream iridovirus (RSIV) with more than 97% identity. Thus, the seven iridovirus strains isolated from cultured marine fish in Taiwan were closer to the RSIV genotype than the infectious spleen and kidney necrosis virus genotype.

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.

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.

Macrobrachium rosenbergii nodavirus infection in M. rosenbergii (de Man) with white tail disease cultured in Taiwan.

White tail disease (WTD) is a serious problem in Macrobrachium rosenbergii hatcheries and nursery ponds in Asia. The causative agents have been identified as M. rosenbergii nodavirus (MrNV) and its associated extra small virus. This is the first report demonstrating MrNV virus in M. rosenbergii displaying WTD signs in Taiwan by reverse transcriptase-polymerase chain reaction (RT-PCR). Amplified fragments of 850 and 425 bp for RNA-1 and RNA-2 of MrNV, respectively, were obtained by RT-PCR. RT-PCR products of about 850 and 1121 bp for RNA-1 and RNA-2 of MrNV were also obtained using different primer pairs. The amplicons were individually cloned into pGEM-T vector and sequenced. Using this recombinant plasmid of MrNV RNA-2 as DNA template, the non-radioactive DNA probes were prepared by PCR amplification with DIG-11-dUTP. The probes were used to successfully detect MrNV infection in the striated muscle tissues of WTD-diseased prawns using in situ hybridization. The 1121 bp genomic fragment of RNA-2 of MrNV consisted of a unique open reading frame with 1116 nucleotides, and it encoded a structural protein with 371 amino acids. The nucleotide sequence of the partial genome of MrNV RNA-2 revealed a 97% identity with an Indian isolate. A phylogenetic tree constructed using the nucleotide sequence of the viral capsid gene from insect and fish nodaviruses revealed that the MrNV Taiwan isolate could be interpreted as a new genus within the family Nodaviridae. However, its position showed more affinity with Alphanodavirus than with Betanodavirus. The study confirmed the presence of MrNV infection in freshwater prawns cultured in Taiwan suffering from WTD.

Viral susceptibility, transfection and growth of SPB--a fish neural progenitor cell line from the brain of snubnose pompano, Trachinotus blochii (Lacépède).

This study investigates the susceptibilities of the SPB cell line to fish viruses including giant seaperch iridovirus (GSIV-K1), red sea bream iridovirus (RSIV-Ku), grouper nervous necrosis virus (GNNV-K1), chum salmon reovirus (CSV) and eel herpesvirus (HVA). GSIV-K1, RSIV-Ku and CSV replicated well in SPB cells, with a significant cytopathic effect and virus production. However, the cells were HVA and GNNV refractory. To examine the ability of SPB cells to stably express foreign protein, expression vectors encoding GNNV B1 and B2 fused to enhanced green fluorescent protein (EGFP) and GSIV ORF35L fused to DsRed were constructed and introduced by transfection into SPB cells. Stable transfectants displayed different morphologies compared with SPB and with each other. EGFP-B1 was predominantly localized in the nuclei, EFPF-B2 was distributed throughout the cytoplasm and nucleus, and granular 35L-DsRed was localized with secreted vesicles. The expression of EFPF-B2 in SPB cells produced blebs on the surface, but the cells showing stable expression of EGFP, EGFP-B1 or 35L-DsRed showed normal morphologies. Results show the SPB cells and the transfected cells grow well at temperatures between 20 and 35 °C and with serum-dependent growth. SPB cells are suitable for studies on foreign protein expression and virology.

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.

Immunocytochemical characterisation of neural stem-progenitor cells from green terror cichlid Aequidens rivulatus

In this study, cultures of neural stem-progenitor cells (NSPC) from the brain of green terror cichlid Aequidens rivulatus were established and various NSPCs were demonstrated using immunocytochemistry. All of the NSPCs expressed brain lipid-binding protein, dopamine- and cAMP-regulated neuronal phosphoprotein 32 (DARPP-32), oligodendrocyte transcription factor 2, paired box 6 and sex determining region Y-box 2. The intensity and localisation of these proteins, however, varied among the different NSPCs. Despite being intermediate cells, NSPCs can be divided into radial glial cells, oligodendrocyte progenitor cells (OPC) and neuroblasts by expressing the astrocyte marker glial fibrillary acidic protein (GFAP), OPC marker A2B5 and neuronal markers, including acetyl-tubulin, βIII-tubulin, microtubule-associated protein 2 and neurofilament protein. Nevertheless, astrocytes were polymorphic and were the most dominant cells in the NSPC cultures. By using Matrigel, radial glia exhibiting a long GFAP+ or DARPP-32+ fibre and neurons exhibiting a significant acetyl-tubulin+ process were obtained. The results confirmed that NSPCs obtained from A. rivulatus brains can proliferate and differentiate into neurons in vitro. Clonal culture can be useful for further studying the distinct NSPCs.