Molecular Immunology 44 (2007) 2303–2314 Response of the rainbow trout monocyte/macrophage cell line, RTS11 to the water molds Achlya and Saprolegnia Stephen C. Kales, Stephanie J. DeWitte-Orr, Niels C. Bols, Brian Dixon ∗ Department of Biology, University of Waterloo, 200 University Avenue West, Waterloo, Ontario, Canada N2L 3G1 Received 14 July 2006; received in revised form 20 October 2006; accepted 6 November 2006 Available online 3 January 2007 Abstract The Saprolegniales are responsible for various fish mycoses worldwide and considered the most important fungi afflicting fresh water fish. Saprolegniosis leads to massive epidermal destruction and macrophage recruitment, yet little is known regarding the cytological response of their piscine hosts. The objective of this study was to explore the response of fish macrophage to members of the Saprolegniales using the rainbow trout monocyte/macrophage cell line, RTS11. After 48h in co-culture, RTS11 demonstrated chemotaxis, adherence and homotypic aggregation to both live and heat-killed fungal spores and mycelia. This aggregation was enhanced when using conditioned media from co-cultured RTS11 and Achlya, suggesting the presence of synergistic effectors of aggregation. Although fungal toxins were not evident, as cells remained viable throughout fungal overgrowth, phagocytosis was inhibited due to large fungal spore size, allowing these molds to evade macrophage defenses. Although class I MH and other viral response genes showed no significant change in expression, calreticulin and interleukin-8 were moderately up-regulated implicating calcium modulation and chemotactic response, respectively. Cyclooxygenase (COX-2) and the cytokines IL-1 and TNF were strongly up-regulated in the presence of Achlya, while gene expression of the class II major histocompatibility (MH II) receptor and associated molecules appeared down-regulated, suggesting fungal interference of immune function. Previous studies have shown an increased dependence of macrophage in immune function at low temperatures; based upon data presented here, this reduction of macrophage MH II receptor expression and inability to phagocytose spores may limit host response thereby providing increased susceptibility to these opportunistic pathogens. © 2006 Elsevier Ltd. All rights reserved. Keywords: Achlya; Saprolegnia; RTS11; Trout; Macrophage; Arachidonic acid; COX-2; MHC; Homotypic aggregation 1. Introduction The Saprolegniales are an aquatic order of the Oomycota (Egg–fungi) which includes notorious terrestrial pathogens such as the Irish potato blight fungus, Phytophthora infestans, the grapevine downy mildew, Plasmopara viticola and mam- malian “swamp cancer”, Pythium insidiosum. As their name implies, the Saprolegniales are considered mainly saprophytic, serving simply as organic decomposers of aquatic ecosystems, however like their terrestrial counterparts several are considered highly opportunistic pathogens. Although their membership in the Kingdom Fungi is still of much debate, this group is considered to be the most important fungal group affecting both wild and farmed fishes (Bruno and Wood, 1994). Species ∗ Corresponding author. Tel.: +1 519 888 4567x32665; fax: +1 519 746 0614. E-mail address: bdixon@uwaterloo.ca (B. Dixon). of Aphanomyces, Saprolegnia and Achlya, all members of the Saprolegniales, are major pathogens of many fish species (Jeney and Jeney, 1995). Achlya debaryana was demonstrated as the causative agent of epizootic mycoses of channel catfish in India (Khulbe et al., 1994). In that same year, Saprolegnia was isolated as the cause of “winter kill” in Mississippi catfish farms (Bly et al., 1992) and more recently, caused an epizootic mycosis in Japanese salmon farms, further demonstrating this organism’s ubiquity (Hatai & Hoshiai, 1992). Fish serve as an excellent source of protein worldwide. To satisfy global demand, a seven-fold increase in fish production is anticipated within the next 15 years, yet because natural sources are being quickly exploited, aquaculture is becoming the chief source of this valuable resource (Fletcher et al., 1999). Unfortunately, current farming conditions often lead to increased frequency of disease. Saprolegniosis outbreaks have led to considerable economic damage and many attempts have been made to deal with this pathogen. 0161-5890/$ – see front matter © 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.molimm.2006.11.007