First report on histology and ultrastructure of an intrahemocytic paramyxean parasite (IPP) from tunicate Halocynthia roretzi in Korea

Abstract

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 ...

Mass mortality of cultured ascidians Halocynthia roretzi associated with softening of the tunic and flagellate-like cells

“…Similar granular inclusions are found in tunic cells and in hemocytes of healthy ascidians (Ohtake et al 1994, Hirose et al 2009). In the present study, the hemocytes that are exactly like 'paramyxean-infected cells' in light microscopy reported by Choi et al (2006) were also found in the internal organs of all ascidians observed, irrespective of the health condition of the animals. Therefore, these cells are most likely a type of normal hemocyte of the ascidian, possibly the vacuolated cell (Dan-Sohkawa et al 1995).…”

“…In some cases, the clusters of hemocytes were formed in the basal area of the tunic. Among the hemocytes, there were strongly eosinophilic cells, which had large intracellular granules of varying sizes, showing marked resemblance to the cells that were reported to be infected with a paramyxian parasite by Choi et al (2006). These eosinophilic hemocytes were, however, found in all ascidians observed, including apparently healthy animals.…”

“…Furthermore, we cannot completely discard the possibility that the flagellate-like cells are a secondary invader and that the true causative agent of the disease is yet to be found. Choi et al (2006) identified certain cytoplasmic granules or inclusions found in eosinophilic hemocytes as paramyxean parasites, and suggested a possibility that they are the causative agent of soft tunic syndrome. However, they did not show any evidence to justify their claim that the observed cytoplasmic granules, in which no intracellular organelles were observed in electron microscopy, are parasitic organisms.…”

Azumiobodo hoyamushi gen. nov. et sp. nov. (Euglenozoa, Kinetoplastea, Neobodonida): a pathogenic kinetoplastid causing the soft tunic syndrome in ascidian aquaculture

“…In the diseased ascidians, the tunics become abnormally thin and soft; the ascidian then dies when the tunic tears. Several attempts have been made to identify the causative agent, and the pathogenic organism of the disease has recently been identified (Jung et al 2001, Choi et al 2006, Azumi et al 2007a,b, Hirose et al 2009, Kitamura et al 2010. Kumagai et al (2010) induced experimental infections by incubating healthy individuals with pieces of softened tunics and found flagellates in the softened tunic.…”

Quantitative assessment of Azumiobodo hoyamushi distribution in the tunic of soft tunic syndrome¿affected ascidian Halocynthia roretzi using real-time polymerase chain reaction

“…Due to low fiber density, the tunics become thin and soft and finally rupture leading to leakage of internal tissues and death [6][7][8][9]. Breakdown of the tunics is mediated by a metalloprotease enzyme secreted by A. hoyamushi [10].…”