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β-Glucan and triterpenes are two important derivative compounds from traditional medicinal mushroom, such as Ganoderma lucidum and Antrodia cinnamomea. β-glucan and triterpenes are considered to have immunoregulatory properties in disease treatment for long years. The immunoregulatory effects are usually activated through some transcription of pro-inflammatory genes and possess immunomodulatory activity. Difficulty in healing wound now is a common condition that occurred in diabetic patients, and the physiological hyperglycemic status of diabetic patients resulting in the wounds continue to produce an inflammatory response. Thus, we hope to use β-glucan and triterpenes for difficult wound healing that possess immunomodulatory activity on the wound micro-environment and stimulate the positive effects on healing. In this chapter, these two important derivative compounds from traditional medicinal mushroom were examined by diabetic mammal’s wound healing models. In these models, the skin wounds’ microenvironment is expected close to diabetic foot, suffering in hyperglycemic and inflammatory status. The results are clearly presented, with the immunomodulatory effects from mushroom β-glucan and triterpenes that involved in modulating the cell-mediated immune system to cause cellular proliferation and further to introduce healing performance of the chronic inflammation wounds.

In order to simplify the survey of pesticide (including carbamate, organochlorine, organophosphate and synthetic pyrethroid pesticides) residues in fishery products (including bivalve, crustacean, fish and cuttlefish), multiresidue determination methods were developed through a solid phase extraction (SPE) technique. In the present procedures, samples were extracted with acetonitrile except for bivalve and cuttlefish samples. Mixed solvent of water, acetone and acetonitrile was added to bivalve prior to homogenization, whereas water was needed in cuttlefish samples for blending. For both kinds of sample, an additional procedure of salting-out was needed during extraction. Tandem SPE cartridges of C18 and aminopropyl, using acetonitrile as the only solvent, were used to clean up extracts from either method. A total of 91 pesticides in four major pesticide groups were tested in this study Gas chromatography (GC, equipped with electron capture detector and flame photometric detector) and high-performance liquid-chromatography (HPLC) equipped with fluorescence detector were used for analysis. The validation of the method was evaluated for each fishery product using samples spiked with all pesticide standards at three concentration levels. The results indicated percentage of recovery ranged from 60%-120% and coefficients of variation ＜20% for all but 10 of the pesticides analyzed (including 1-naphthol, 3-hydroxy carbofuran, aldicarb sulfoxide, heptachlor, trifuralin, acephate, dichlorvs, methamidophos, monocrotophos and omethoate). Residue extraction techniques described in this report are rapid and suitable for screening of pesticide residues in monitoring programs.

In this study, diverse haemocytes from Pacific white shrimp Litopenaeus vannamei were spread by flow cytometer sorting system. Using the two commonly flow cytometric parameters FSC and SSC, the haemocytes could be divided into three populations. Microscopy observation of L. vannamei haemocytes in anticoagulant buffer revealed three morphologically distinct cell types designated as granular cell, hyaline cell and semigranular cell. Immune genes, which includes prophenoloxidase (proPO), lipopolysaccharide-β-glucan binding protein (LGBP), peroxinectin, crustin, lysozyme, penaeid-3a and transglutaminase (TGase), expressed from different haemocyte were analysed by quantitative real time PCR (qPCR). Results from the mRNA expression was estimated by relative level of each gene to β-actin gene. Finally, the seven genes could be grouped by their dominant expression sites. ProPO, LGBP and peroxinectin were highly expressed in granular cells, while LGBP, crustin, lysozyme and P-3a were highly expressed in semigranular cells and TGase was highly expressed in hyaline cells. In this study, L. vannamei haemocytes were firstly grouped into three different types and the immune related genes expression in grouped haemocytes were estimated.

The present study aims to investigate the effects of mushroom beta glucan (MBG) on wound recovery in partial hepatectomy (PH) in Nile tilapia (Oreochromis niloticus) and in rat skin wound healing examination. Following PH, we focussed on the effects on liver repair ability using in vitro and in vivo tests. In vitro, we examined whether the MBG has an impact on liver cell proliferation, mainly through 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assays and bromodeoxyuridine (BrdU) cell proliferation assay detection method. Results showed that MBG treatment was remarkable in enhancing cell proliferation of hepatocytes and in maintaining the cellular viability. Immunohistochemical staining to analyse Wnt/β-catenin signalling also showed that MBG has the effect of promoting cell proliferation of liver tissues after PH surgery.

The present study showed that oral mushroom beta-glucan treatment significantly increased IFN-γmRNA expression but significantly reduced COX-2 mRNA expression within the lung. For LLC tumor model, oralGanoderma lucidumorAntrodia camphoratapolysaccharides treatments significantly reduced TGF-βproduction in serum. In addition, IL-12 and IFN-γmRNA expression were significantly increased, but IL-6, IL-10, COX-2, and TGF-βmRNA expression were substantially following oral mushroom polysaccharides treatments. The study highlights the efficacious effect of mushroom polysaccharides for ameliorating the immune suppression in the tumor microenvironment. Increased M1 phenotype of tumor-associated macrophages and attenuated M2 phenotype of tumor-associated macrophages could be achieved by ingesting mushroom polysaccharides.

Background Binding of beta 1,3/1,6 glucan of Ganoderma lucidum (G. lucidum) with the receptor results in a series of signal transfers (signalling cascades), which activates the transcription factors for regulating inflammation. Excess cholesterol intake leads to an increase in the distance between fat cells and capillaries, which may cause hypoxia in the fat tissue of obese mice. This hypoxia induces the death of fat cells, resulting in the inflammation of adipose tissue or an increase in the inflammatory gene expression associated with obesity. Methods The current study examined the immunomodulation effect of G. lucidum beta 1,3/1,6 glucan according to immunoglobulin, poly-Ig receptor expression, Nature Killer cell (NK cell) activity, lymphocytes proliferation and cytokines expression. Results Our present study shows that feeding G. lucidum beta 1,3/1,6 glucan to mice induces IgA or IgG expression in the serum and small intestine washing fluid and enhances poly-Ig receptor expression in the small intestine moreover, the observation of the IL-2 and Nature killer cell activity were exchanged. Conclusions The effect of a high-cholesterol diet in the inflammatory response was observed in heart, liver, kidney, spleen, and colon tissues through histopathological evaluations. The presented evidence demonstrates that the inflammation response in the high-cholesterol diet group was much higher than in the other groups and the beta 1,3/1,6 glucan reduces inflammation in obese mice fed a high-cholesterol diet.

A continuous cell line (KF-101) derived from the caudal fin of the koi carp Cyprinus carpio was established and characterized. The KF-101 cell line multiplied abundantly in Leibovitz's L-15 medium containing 10% foetal bovine serum at 25° C, and was subcultured for >90 passages over a period of 3 years. Immunocytochemistry revealed that the KF-101 cells contain keratin, junction proteins connexin-43 and occludin, and ectodermal stem-cell marker Pax-6, but not vimentin. Furthermore, the KF-101 cells reacted with anti-human DARPP-32 and anti-human GATA-4 antibodies, and the labelling was regulated according to the cell cycle. The labels of the DARPP-32 and GATA-4 antibodies in the KF-101 cells were the suggested phosphatase-1 inhibitor-1 and GATA-3, respectively. In addition, the KF-101 cells were susceptible to koi herpesvirus but were resistant to eel herpesvirus, iridovirus, grouper nodavirus and chum salmon (Oncorhynchus keta) virus. The results indicate that the KF-101 cells are suitable materials for investigating biological and virological development.

This research evaluated the effects of triterpenes on the regulation of STZ-induced hyperglycaemic diabetes through an anti-inflammatory response. Diabetic mice were orally administered various concentrations of triterpenes on a daily basis. Weight gain, volume of drinking water, and liver and spleen weight were recorded and evaluated. These evaluations presented a positive regulation to the abnormal metabolism appearance compared to the diabetic mice. In the diabetic mice, the detection of adiponectin production or elevated levels of inflammatory factors such as CCL1 and TPO expression were found to reduce hyperglycaemia and thereby induce an inflammatory response. Moreover, to the best of our knowledge, hyperglycaemia impairs the tissue healing associated with an increased and prolonged inflammatory response. An investigation of the anti-inflammatory response in wound healing as affected by the triterpenes verified the promotion of wound recovery.

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.

Prevention and therapy of thrombotic diseases have attracted much attention in developed countries during recent years. Investigators have been looking for cheaper and safer thrombolytic agents for therapy of thrombotic diseases. Recently, we have discovered a fibrinolytic protease from Schizophyllum commune. In this study, the protease was proven to degrade blood clot effectively. Seven divalent metal ions were used to test the selectiveness on enhancing protease activity. The treated rat blood was traced by thromboelastography to assess the viscoelastic properties of whole blood. As the result, fibrinolytic activity of the protease was enhanced remarkably by Mg2+ in reducing the strength of blood clot and showed the innovative anti-thrombotic effects. This is the first study of anti-thrombotic effects from fungal-derived fibrinolytic protease using thromboelastography and delineates the efficacy of magnesium supplementation in enhancement of thrombolytic activity from S. commune fibrinolytic protease.

The fisheries products in this study comprise fish, bivalve, crustacean and cephalopod collected from different types of markets in Taiwan between the years 2001 and 2003. A total of 91 pesticide residues belonging to four major pesticide groups were tested and analyzed. The test results show that 65.40% of fish, 93.55% of shellfish, 84.92% of crustacean and 98.33% of cephalopod samples contain no detectable residues. There are only two kinds (organochlorine and organophosphate) of totally six pesticides (DDTs, dieldrin, chlorpyrifos, fenitrotion, fenthion and prothion) that have been detected from the fisheries products in this study. For there were pesticides present in the fish products, consumption of fisheries product there was no zero risk. But the exposure of consumer did not exceed the acceptable daily intakes (ADI). For male, there was the highest risk in exposure to dieldrin, which the percentage of ADI was 93.56%. This study also suggests that a yearly monitoring program for organophosphate pesticide residues in fish is necessity.

We consider that inflammatory substances play an important role in the carcinogenesis process. In the process, cytokines and growth factor secreted by cells can actively recruit immune cells in the carcinogenesis microenvironment, further, promote carcinogenesis progression. The carcinogenesis microenvironment even subverted the immune system, moreover, enhanced the carcinogenesis through immune suppressive mechanisms within the carcinogenesis microenvironment.

Wound healing is a complex process involving soluble mediators, blood cells, extracellular matrix, and parenchymal cells in a response that occurs after surgical procedure or traumatic injury. The present study aims to investigate the ROS producing from the injury that involved in the wound healing using the ZFL (zebrafish liver cell) and tilapia partial hepatectomy model. In the ZFL, we observed that while over-inhibition of NADPH activity leading to reduce the wound healing moreover, experiment of the oxidative stress by the extracellular hydrogen peroxide exactly presented to increase the PCNA, BrdU and Ki-67 histopathological repair response of tilapia liver follow partial hepatectomy. We conclude that over inhibition of the NADPH oxidase by DPI may reduce the cell even the tissue in the progress of healing after the injury.

The total haemocyte count (THC), differential haemocyte count (DHC), respiratory bursts, and phenoloxidase (PO) activity were determined in the Pacific white shrimp Litopenaeus vannamei which administrated by feeding with diets containing mushroom beta glucan (MBG) at 0.05% and 0.1%. Results showed that shrimp fed a diet containing 0.05% MGB had significantly increased THC and semi-granular cells ratio at 28 days. Intracellular superoxide anion (O2-) production were significantly increased at 14 days which shrimp fed a diet containing 0.05% MBG and PO activity were significantly increased at 14 days which shrimp fed a diet containing 0.1% MGB but equal with control at 28 days. In previously study, semi-granular cells were found as the primary immune activator with their high levels of granules, enzymes and proteins. In this study, the effectively enhance immune responses and compare to high induced semi-granular cells in Pacific white shrimps concluded that MBG is an effective and powerful immunostimulants for shrimp. For application in aquaculture, MBG administration through dietary administration is also a convenient and useful practical technique in the future.

Recently studies performed on mushroom isolated polysaccharides demonstrated thatβ-(1,3)-glucan may affect the balance of Th1/Th2 cell response. Using ovalbumin (OVA) as a hypersensitivity inducer, we evaluated the ability of mushroom beta-glucan (MBG) in modulating Th1/Th2 cell responses in B6 mice. As compared to the control group, administration of MBG resulted in an increase of phagocytic activities, Th1 cytokine productions, immunoglobulins including IgG2A and IgA, and a significant expression of the splenic surface markers including CD3, CD4, CD8, and F4/80. In contrast, administration of MBG has significantly suppressed IgE and IgG1 levels and Th2 cytokines including IL-4, IL-5, and IL-6. Histopathological observation of MBG-treated followed by OVA-treated mice showed less filtration of eosinophil in pulmonary tissue sections. Our data suggested that administration of MBG treatments alters the natural course of the IgE-mediated hypersensitivities. In this investigation, we realize the mushroom beta glucan alter the Th2 response toward the Th1 in the allergic, resulting in a reduction in IgE productions which played a substantive role in reducing the severity of IgE-mediated hypersensitivity.

This study evaluates the effect of mushroom beta-glucans (MBGS) derived from solid culture ofGanoderma lucidumon tumor inhibition by examining size of the primary tumor and rate of metastasis in Lewis lung carcinoma (LLC) bearing mice (C57BL/6), given oral administration of MBGS with radiation therapy. A previous result showed that MBGS enhances NK cell-mediated cytotoxicity in mice without LLC bearing in advance. Furthermore, applications of MBGS in conjunction with radiation therapy were effective in controlling tumor growth, and rate of metastasis, life threatening, and can potentially serve as a protective factor for wounds and hair loss that resulted from the overgrowth of primary tumor in LLC bearing mice.

We consider that from the wound to the healing process, the physiology point key to linkage of the process is still unclear. The process from inflammation to the wound healing is divided into three phases: (1) inflammation process, (2) tissue formation, and (3) tissue remodeling. The inflammation program includes cell produced related factors and immune cells infiltration. We thought the inflammation factors that may be also involved in the followed healing process. But the question is “what kind of factor is the major key involved in the end of the inflammation then to initiate the healing.” We suspect that the apoptosis of immune cell may be the major key to end of inflammation and to initiate the healing.

Red-parrot fish (male midas cichlid Cichlasoma citrinellum × female redhead cichlid C. synspilum) is one of the most important tropical fish in Taiwan. In high-density culture, poor water quality often induces emaciation, gill filament ulceration, gill opercula malformation, and high mortality. In an effort to solve these problems in a high-density culture environment, two bacteria, Bacillus subtilis and B. megaterium, were added to the recirculating-water systems of the grow-out facilities. The addition of Bacillus held total ammonia nitrogen concentrations to 5.4 mg/L or less and chemical oxygen demands to 40.8 mg/L or less; it also maintained the transparency of the recirculated aquarium water at 30–50 cm. Survival rate and quality of fish were dramatically improved with this microbial water quality management process. This method is easy to perform and can be adapted effectively in the commercial culture of red-parrot fish.

Reactive oxygen species (ROS) may act as signaling molecules in the physiology responses and the present study aims to investigate the effect of extracellular hydrogen peroxide on macrophages cellular response. The results obtained in the present study showed that the extracellular hydrogen peroxide affectively alter the membrane potential of the cell membrane and ion exchange channels in the cell membrane through intracellular NAD turnover that may lead to an intracellular calcium ion concentration alteration and subsequently induce the downstream signal activation.

This study describes apoptosis induced in vivo and in vitro in tilapia, Oreochromis aureus Steindachner, by Staphylococcus epidermidis. In an in vivo experiment, tilapia were challenged using viable S. epidermidis and its cultured supernatant, respectively. Apoptosis was predominantly detected in lymphocytes and macrophages in spleen and kidney. Apoptotic figures were observed occasionally in the brain, liver, gonad, mesentery, stomach, intestine and skeletal muscle of infected fish. In an in vitro experiment, TO-2 cells treated with brain heart infusion broth supernatant of the bacteria after 48 h revealed that the supernatant was able to induce the cell apoptosis. Fragmented DNA was also detected 48 h after treatment using 1.5% agarose gel electrophoresis. The smallest multimeric DNA fragment was approximately 180-bp in length. Results of both the in vitro and in vivo experiments showed that products of the bacteria could induce tilapia cell apoptosis.

Six species of copepods of the genus Caligus (Caligidae, Siphonostomatoida) parasitic on marine fishes of Taiwan are reported. They are: C. absens n. sp. from Priacanthus blochii and P. macracanthus; C. epinepheli Yamaguti, 1936 from Scolopsis vosmeri; C. kanagurta Pillai, 1961 from Decrapterts kurroides; C. laticaudus Shiino, 1960 from Lutjanus vitta and Parapristiopoma trilineatum; C. nengai Rangnekar, Rangnekar & Murti, 1953 from Triacanthus biaculeatus; and C. rotundigenitalis Yu, 1933 from Drepane punctata, Liza macrolepis and Terapon jarbua. C. distortus Pillai & Natarajan, 1977 is relegated to a synonym of C. nengai and C. multispinosus Shen, 1957 reported by Lin et al. (1994) from the cultured sea bream Acanthopagrus schlegeli is a misidentification for C. rotundigenitalis Yu, 1933.

Monolayer cultures were established from ovary, heart, lymphoid tissue and peripheral hemocytes of penaeid shrimps including Penaeus monodon, P. japonicus and P. penicillatus. The most favorable conditions for the culture of penaeid shrimp cells in vitro was in CMRL and L-15 tissue culture media when used within an osmolarity range of 620--760 mmol/kg. The optimal maintenance temperature was 25 °C for tissues of P. japonicus and 28 °C for tissues of P. monodon and P. penicillatus. Among the four tissues tested, lymphoid tissue, or 'Oka organ', was superior to the other tissues for the formation of confluent cell monolayers. Cell cultures from lymphoid tissue and ovary have been subcultured up to three times. When peripheral hemocytes and heart were cultured, a maximum survival of 4 days was obtained. In contrast, cell cultures derived from ovary and lymphoid tissue were maintained alive for at least 20 days in appropriate culture systems. Neither confluent cell sheet nor adherence of cells was obtained in cultivation of hepatopancreas using the present culture systems. The results obtained from the present study also revealed that ovary extract, muscle extract and lobster hemolymph enhanced the survival of the cultured cells of penaeid shrimp in vitro. When the 'Oka organ' cell monolayer was incubated with either white spot disease virus (WSDV) or yellow head virus (YHV), no cytopathic effect (CPE) was obtained. However, at 5--7 days after establishment, significant CPE (a few foci) was observed in cell monolayers derived from WSDV- and YHV-infected Oka tissue. By electron microscopy, virions of WSDV and YHV were observed in the nuclei and cytoplasm of cultured cells. The CPE foci developed further with increased incubation time.

The present study attempts to establish cell culture systems for the oyster, Crassostrea gigas Roding and the hard clam, Meretrix lusoria Thunberg. Treatment with collagenase was better than trypsin at dissociating mollusc tissue fragments for in vitro culture. Heart tissue of oyster and hard clam proved to be the most promising target tissue for the establishment of cell lines in vitro. Primary cultures of clam heart were established and successfully maintained for more than 5 months. Collagenase at a concentration of 100 microg/ml may enhance the growth of oyster and hard clam heart cell cultures in vitro.

Since 1987, farmers in southern Taiwan have reported significant disease-related mortalities in freshwater prawns. Most mortalities have occurred during the winter, and usually almost all the adult prawns die within 4-5 days after clinical signs appear. Histopathological studies show that cells in hepatopancreatic ducts and tubules are vacuolized and degenerated. Large numbers of membrane-bound yeast aggregates are observed in the affected tissues. Two hundred and seventeen yeast isolates were obtained from diseased prawns, pond water and sediments from six areas. DNA restriction fragment length polymorphism (RFLP) was used to analyse the yeast genomes and to categorize them into five groups. Conventional biochemical and metabolic methods were then used to identify the yeasts of each group. The results showed that two strains of Candida sake (I and II), Pichia anomala, Endomyces fibuliger and Candida famata were present. In addition, the RAPD (random amplified polymorphic DNA) method was used to determine their genomic similarities. Two strains (I and II) of C. sake were the most similar (72%). C. sake I appears to be the primary causative agent of disease, based on frequencies of occurrence of the yeasts found in the diseased prawns.

The development of Caligus multispinosus was studied using instars reared from eggs in the laboratory and specimens obtained from the gills of pond-cultured black sea bream. Nine stages were identified, namely, two nauplii, one copepodid, four chalimus stages, one preadult and the adult. The distinctive feature of the spinulose outer spines on leg 4 appeared late in development (preadult stage) and the frontal filament produced in chalimus I is kept throughout the chalimus stages.

Since 1992, mass mortalities among cultured giant tiger shrimp, Penaeus monodon (Fabricius), and kuruma shrimp, Penaeus japonicus (Bate), have been observed in Taiwan. The condition is known as ‘white spot disease’ (WSD), based on the characteristic white spots on the cuticle of diseased shrimp. With the scanning electron microscope, two sizes of white spots were observed. Each spot represented a protrusion on the inside surface of the carapace. The composition of white spots was similar to that of the cuticule, most calcium, as determined with an energy dispersive spectrometer. Histological studies of moribund, infected specimens revealed degenerated cells, characterized by hypertrophied nuclei, in various meso- and ectodermal tissues. Infected tissues included cuticular epidermis, connective tissue, lymphoid organ, antennal gland, and haematopoietic, gill and nervous tissue. Nuclei were Feulgen-positive and no occlusion body was found in the necrotic tissue. Transmission electron microscopy revealed the presence of rod-shaped and enveloped virions in the hypertrophied nuclei. The virions measured 298 ± 21 × 107 ± 8 nm in the giant tiger shrimp and 248 ± 12 × 104 ± 8 nm in the kuruma shrimp. In an experimental infection trial, cumulative mortality was 40% within 14 days under stress conditions. No mortality was observed in controls or in non-stressed infected shrimp. Experimental infections show that environmental stressors such as ammonia may enhance the severity of WSD virus infections in cultured shrimp.

The purification and characterization of an extracellular protease produced by pathogenic luminous Vibrio harveyi strain 820514, originally isolated from diseased tiger prawn (Penaeus monodon), was presented in this paper. The purification steps included ammonium sulfate precipitation, with columns of hydrophobic interaction chromatography and anion exchange on fast protein liquid chromatography. The protease is an alkaline cysteine protease, heat labile, inhibited by iodoacetamide, iodoacetic acid, N-ethylmaleimide, p-chloromercuribenzoate, and p-chloromercuribenzene-sulfonic acid, and showed maximal activities at pH 8 and 50 degrees C, having a molecular mass of 38 kDa as estimated by SDS-PAGE and gel filtration column. In addition, the protease was also completely inhibited by CuCl2 and HgCl2, but not or only partially inhibited by other inhibitors tested. Furthermore, 2-mercaptoethanol was the most effective reducing agent in the activation of the enzyme. The present protease is the first cysteine protease found in Vibrio species.

Mass mortalities of hatchery-reared juvenile groupers have occurred in southern Taiwan. The diseased fish swam in a darting, corkscrew fashion. Light microscopy revealed vacuolation in the brain tissue. Electron microscopy showed numerous non-enveloped, cytoplasmic viral particles (20–25 nm in diameter) in the brain cells, and many virions were enclosed in the membrane-bound organelles of the cells. Two structural proteins of the purified grouper virus, with molecular weights of 44 and 43 kDa, were revealed by SDS-PAGE. Moreover, the results of RT-PCR and nested PCR diagnosis using primers specific to the T2 and T4 target segments of striped jack nervous necrosis virus (SJNNV) RNA2 genes suggest that this virus is a fish nodavirus, and is designated as GNNV 9410 strain (grouper nervous necrosis virus strain 9410). This is the first case report of viral nervous necrosis among marine fish in Taiwan.

The present study attempted to investigate the white spot disease virus (WSDV) infection in the wild-captured greasy back shrimp, Metapenaeus ensis. The shrimp showed no symptom of white spots in the carapace that usually occur in the other cultured shrimps. In histopathological studies, the hypertrophied nuclei of the necrotic cells were found in the various tissues originated from ectoderm and mesoderm which were similar to those observed in the giant tiger shrimp (Penaeus monodon) and kuruma shrimp (P. japonicus). Neither occlusion bodies nor cytoplasmic inclusions were found in the infected greasy back shrimp. Transmission electron micrographs revealed rod-shaped and enveloped viruses in the hypertrophied nuclei. The virions measured 221±6nm by 107±7nm in size, and were comparatively smaller than those found in WSDV infected P. monodon and P. japonicus When in situ hybridization using DIG-labelled WSDV probe was used, positive reaction was found in the hypertrophied nuclei. DNA amplification by polymerase chain reaction (PCR) with two oligonucleotide primers developed in our laboratory demonstrated that the virus found in M. ensis is geneticallysimilar to WSDV in the other penaeid shrimp.

The morphogenesis of a highly organized cellular membrane structure, a membranous labyrinth (ML), in the cells of penaeid shrimp infected with Penaeus monodon baculovirus (MBV) is described. The ML possibly originates from dilated Golgi apparatus, endoplasmic reticulum (ER) or the outer nuclear membrane. The ML apparently grows by proliferation of the cellular membranes of these systems. In addition, its degree of development was correlated with stages of MBV reproduction.

The larval development of Caligus epidemicus comprises ten stages: two nauplii, one copepodid, six chalimus, and one preadult (young adult) stages. The morphology of each of these ten instars is described based on material reared from eggs in the laboratory. Addition of fifth and sixth chalimus stages in the ontogeny of C. epidemicus is discussed in the light of the developmental changes in the leg armature. Some peculiarities about the structure of the frontal filament in various chalimus stages are also discussed.

Two species of caligid copepods, Lepeophtheirus atypicus n. sp. and Caligus oviceps Shiino, 1952, are described based on the specimens obtained from moribund rabbit fish (Siganus fuscescens) cultured in a cage-net at the Penghus Hatchery Station on Pescadores Island, Taiwan. The new species bears close resemblance to L. goniistii Yamaguti, 1936, but can be easily distinguished from it by the armature on leg 3. The antennule, sternal furca, and leg 4 of these two species also exhibit difference. Lepeophtheirus species from rabbit fish in Japan reported as L. goniistii by Shiino (1952) is reidentified as L. atypicus n. sp. Caligus truncatogenitalis Roubal, 1981 is proposed to be relegated to the synonym of C. oviceps.

The present study demonstrates the first discovery of a yellow head disease-like virus (YHDLV) in Penaeus japonicus that were diagnosed as white spot disease and describes the histological changes and the characters of the virus under electron microscopical observation. In histological studies, cells with the basophilic inclusion bodies and hypertrophied nuclei were found in various tissues, including the lymphoid organ, interstitial hepatopancreatic tissue, cardiac tissue, gill tissue, connective tissue, cuticular epidermis and hematopoietic tissue. Transmission electron micrographs revealed the presence of rod-shaped, enveloped virions in the cytoplasm of the necrotic cells. The virus was 150-200 nm in length and 40-50 nm in width, but extraordinarily long viruses were also detected in the cells. The features of the YHDLV were very similar to yellow head disease virus of Penaeus monodon in Thailand.

This study attempts to describe the effects of innate immunity responses and field application of mushroom beta-glucan mixture (MBG) in cultured orange-sported grouper, Epinephelus coioides. Chemical analysis for MBG showed that the mixture contains 34.06% of macro-molecular polymers with bio-active linkage such as 3-; 3,4- and 4,6-glucopyranosyl and 6-linked galactopyranosyl residues. Study performed on the innate immunity showed that oral ingestion of MBG at 1.0 g and 2.0 g per kilogram of feed levels may significantly enhance the lysozyme activity, alternative complement activity, phagocytic activity and respiration burst of the experimental groupers. Observation on the experimental challenge of pathogen showed that uses of MBG at 0.1% and 0.2% levels in feed might significantly enhance the protection of grouper against Vibrio alginolyticus. Field trials performed on short and long-term culture showed that feeding of diet containing 0.1% or 0.2% of MBG may significantly enhance the survival of cultured groupers up to 16% when compared with those obtained from controls.

A digoxigenin-labelled DNA probe was used for in situ detection of the Penaeus monodon-type baculovirus (MBV) derived from cloned MBV polyhedrin genome in cultured Penaeus monodon Fabricius. First, the specificity of the probe against MBV DNA with dot blot hybridization analysis was verified. This probe indicated that cloned MBV polyhedrin fragment can be used as an MBV-specific probe. This was then used to microscopically examine sections of MBV-infected tissues for a blue-purple precipitate indicative of a positive reaction for MBV. MBV-positive cells were located only in the epithelium of the hepatopancreatic tubules and of the midgut. Furthermore, comparison of the susceptibility to MBV infection among several life-stages of the shrimp showed that the MBV genome was found in the zoea, mysis, post-larva, and adult stages, whereas MBV DNA was not detected in either eggs or nauplii. The results were quantified from in situ hybridization with an image analyser to compare the degree of cell infection among groups of cultured P. monodon collected from various farms in Taiwan.

Clam virus (CV HB-1) isolated from hard clam Meretrix lusoria , typed as infectious pancreatic necrosis virus (IPNV) Ab serotype, was employed for the present study. The structural polypeptides of purified virus generated by serial undiluted and diluted passaging were analyzed using sodiumdodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The results indicated that in VP2, the virus particles of serial undiluted passaging had viral polypeptides smaller than those of serial diluted passaging. Furthermore, a polypeptide with a molecular mass of 105 kDa was found in the serial undiluted virus preparation. Further analysis by monoclonal antibodies showed that VP105 might be a polyprotein produced by genomic RNA segment A. To investigate the sequential changes of viral polypeptides of the virus preparation at high multiplicity infection, the viral polypeptides at each undiluted passage were analyzed by SDS-PAGE followed by immunoblotting. The results indicated that VP105 was present by undiluted passage 7, while the smaller VP2 was present by undiluted passage 10. The amount of smaller VP2 and VP105 increased as the number of passages increased.

Mushrooms, a special group of macrofungi, are not plants and thus do not use photosynthesis for nutrient acquisition. Mushrooms are fleshy, have the spore-bearing fruiting body of fungi, and typically grow above ground on soil, or other food sources. Most mushrooms are Basidiomycota or Agaricomycetes. The fruiting body of mushrooms is an important food and is used in many cuisines worldwide. Additionally, several species have been consumed extensively as a crude drug or folk tonics; in East Asia, these mushrooms are considered as medicinal mushrooms. Some medicinal mushrooms can be cultured and are an abundant source of natural proteins and polysaccharides. These mushrooms have garnered considerable attention for clinical research and for modern scientific and medicinal researches investigating their biological functions (Kino et al., 1989).

The effect of dietary cholesterol on the growth and survival of juvenile tiger shrimp ( Penaeus monodon ) was tested using semi-purified diets. Twenty-four shrimp per treatment, with an initial mean weight of 268.2 mg, were individually housed in black nylon mesh pots in 40-liter aquaria containing 30 ppt seawater. Diets containing 0, 0.2, 0.4, 0.5, 0.8, and 1.0% supplemental cholesterol were evaluated. The duration of the experiment was 63 days. Shrimp fed diets containing 0% and 1% cholesterol had significantly lower weight gain and poorer survival than those fed the other diets. There was no significant difference in weight gain of P. monodon fed diets containing 0.2–0.8% cholesterol. A diet containing 1% cholesterol had an adverse or toxic effect on P. monodon growth.

Vitellogenesis can be induced in the ovaries of the penaeid shrimp, Penaeus monodon, by eyestalk ablation; 80% of shrimp spawned within 7 days after ablation. Using immunofluorescence, it was observed that vitellin commences to accumulate in the yolk globular stage oocytes. The vitellin of atretic oocytes is reabsorbed and transferred to the newly matured oocytes. Four vitellin-like peptides are synthesized in vitro by the ovaries with mol. wt 220, 168, 130 and 74 kDa, respectively. Amongst them, the 168 and 74 kDa peptides are secreted into the culture medium. The results of immunostaining showed that the anti-Ep antisera is able to react with the ovary extracts of Penaeus japonicus. Each antiserum may react to the compatible peptide of the P. monodon vitellin peptide.

Two species of parasitic copepods are described based on the materials obtained from the pondcultured black sea bream (Acanthopagrus schlegeli) in Tainan County, Taiwan. They are : Caligus acanthopagri n. sp. found on the host's body surface and Caligus multispinosus on the host's gills and in the gill cavities. The new species is very close to Caligus latigenitalis but can be distinguished from it by the fine structures on the exopod of leg 4. This study shows that Caligus dieuzeidei reported by Shiino in 1954 from Sparus macrocephalus (=Acanthopagrus schlegeli) was a misidentification for C. latigenitalis.

Eighty-seven, 72 and 30 kDa heat-shock proteins from color carp testis cell line, CCT, were purified, and rabbit antibodies were raised against them. After heat shock at 37°C, hsp87 appeared mainly in the cytoplasm and hsp72 and hsp30 appeared in the nucleus. When cells were restressed at 37°C for 8 hr after three recovery periods, the intensity and localization of the three anti-CCT hsps staining changed from those following an initial stress for 8 hr. The anti-CCT hsp87 and hsp72 antibodies crossreacted with proteins of similar molecular weights in all tested fish, lizard, mouse and human cell lines, but they showed various degrees in antigenic relevance.

1. 1. The results obtained from the present study show that vitellin (yolk protein), a glycolipoprotein derived from the giant tiger prawn, Penaeus monodon , consists of four polypeptides, Ep1, Ep2, Ep3 and Ep4, with molecular weights of 168, 104, 83 and 74 kDa, respectively. This protein was purified using SDS-PAGE and gel-elution. 2. 2. Antisera against Ep2, Ep3 and Ep4 fractions showed very strong specific binding reactivity when reacted with shrimp ovaries at developing stages. 3. 3. No crossreaction was observed among Ep2, Ep3 and Ep4 fractions of vitellin. However, anti-Ep2 and anti-Ep3 were demonstrated to be able to react with Ep1 fraction. The homogenates obtained from ripe ovaries and hemolymph of vitllogenesis females showed an immuno-identical pattern to those obtained from egg extracts. 4. 4. The proteolysis mapping for each fraction showed that Ep1, the high molecular weight fraction, contained Ep2 and Ep3 fragments. These results suggest that Ep1 may be a precursor for Ep2 and Ep3.