In 2004, epizootiological studies were conducted on mass mortalities of tunicates Halocynthia roretzi in Goje, Korea. The clinical characteristics of infected H. roretzi were weakness of the tunic, loss of elasticity, and finally death involving a rupture of the tunic. Histological studies revealed severe hemocyte infiltration in the connective tissue surrounding the intestine and mantle of infected H. roretzi. Hypertrophied eosinophilic hemocytes containing several cytoplasmic vacuoles were observed in the connective tissue surrounding the intestine, gill and mantle. Ultrastructural examination revealed the presence of a parasite in the cytoplasm of hemocytes. Secondary cells were observed in the primary cell of the parasite. Spore formation within primary cells suggests that the parasite may be an intrahemocytic paramyxean parasite (IPP) and may cause mass mortality of H. roretzi.
KEY WORDS: Intrahemocytic paramyxean parasite · Tunicate · Halocynthia roretzi · Histology · UltrastructureResale or republication not permitted without written consent of the publisher Dis Aquat Org 72: [65][66][67][68][69] 2006 Halocynthia roretzi is an important aquaculture species in Korea and Japan. It is an edible tunicate as well as a source of collagen. Mass mortalities of H. roretzi have occurred over the last 2 decades along the southern and eastern coasts of Korea causing serious economic damage.Mortalities of more than 50 000 t of cultured Halocynthia roretzi were reported from Korea in 2003, and this occurence was the incentive for the present epidemiological study. Diseases causing mortalities in urochordates have rarely been reported (Monniot 1990). In the present study, we describe histological and ultrastructural characteristics of an intrahemocytic paramyxean parasite (IPP) that is associated with the mass mortality of H. roretzi. This paper provides the first description of an IPP infecting the hemocytes of edible tunicates H. roretzi cultured on the southern coast of Korea.
MATERIALS AND METHODSTunicates (n = 30) Halocynthia roretzi 1 to 2 yr old were collected from private aquaculture farms located in Goje, Korea, in June 2004.Histological examination. Halocynthia roretzi tissues were fixed in Davidson's solution (Howard & Smith 1983), processed for paraffinembedding and cut into 4 µm thick sections. Sections were stained with Harris's hematoxylin and eosin (H&E) for light microscopy examination.Ultrastructural examination. Halocynthia roretzi tissues were fixed in 2.5% glutaraldehyde in 0.2 M cacodylate buffer at pH 7.2 for 1 h at 4°C. After 2 washes in cacodylate buffer, the tissues were post-fixed in osmium tetroxide in the same buffer at 4°C. Samples were dehydrated through graded alcohols, rinsed twice for 15 min in propylene oxide, and embedded in Epon resin compound (Ouken). Semi-thin sections (200 µm) were stained with toluidine blue, and ultra-thin sections (60 µm) were stained with uranyl acetate and lead citrate. Ultra-thin sections were carefully examined with a Hitachi 7100 transmission ...
A long double-stranded RNA (dsRNA)-producing vector driven by fugu double U6 promotors, in which the two promoters were arranged in a head-to-head fashion, was newly constructed. To determine whether the DNA-vector-based long dsRNAs can induce sequence-specific RNA interference (RNAi), Epithelioma papulosum cyprini (EPC) cells and chinook salmon embryonic (CHSE-214) cells were transfected with the long dsRNA vector targeting the G gene of VHSV, and its effect on expression of the G gene and viral proliferation was investigated. The sequence-specific inhibitory effect was further confirmed by analysis of interferon (IFN)-triggered Mx1 gene expression and cross-protection against infectious hematopoietic necrosis virus (IHNV). The fugu double U6 promoter-driven vector successfully produced long dsRNAs in EPC cells, a system that allows continuous production of long dsRNAs in transfected cells. The plasmid-based long dsRNAs targeting the VHSV G gene effectively suppressed G gene expression, but control dsRNAs targeting the EGFP gene did not. Furthermore, there was no significant difference in Mx gene expression between cells transfected with the long dsRNA-producing vector and those transfected with the control empty vector. These results suggest that G gene expression was suppressed not by type-I-IFN-mediated nonspecific inhibition but in a sequence-specific manner. Both EPC and CHSE-214 cells transfected with plasmids producing long dsRNAs targeting the VHSV G gene were protected against VHSV infection but were not protected against IHNV infection, suggesting sequence-specific RNAi-mediated inhibition of viral proliferation. In conclusion, we show, for the first time, long-dsRNA-mediated RNAi in fish cells. The DNA-vector-based long dsRNAs may provide an efficient tool for analysis of gene function in fish cells without preliminary burdensome work for selection of effective siRNA clones, and it may be applied as an antiviral measure in cultured fish.
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