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Pseudocyst formation in the marine parasitic amoeba Neoparamoeba perurans: a short-term survival strategy to abrupt salinity variation.

Amoebic gill disease (AGD), caused by the freeliving marine amoeba Neoparamoeba perurans, has long been recognized as the major health concern affecting the Atlantic salmon aquaculture industry in Australia (Munday et al. 1993) and is now emerging as a significant threat worldwide (Mitchell & Rodger 2011). Bathing affected fish in freshwater is the current treatment strategy employed by the Australian industry (Parsons et al. 2001). However, despite the effectiveness of the treatment, the number of gill-associated amoebae has been shown to return to prebath levels within 10 days (Clark, Powell & Nowak 2003), in part attributed to a small percentage of amoebae survival following bathing (Adams & Nowak 2004). Although exposure of individual amoebae to freshwater may vary with localized gill structure and mucus cover, an adaptation mechanism to survive sudden freshwater immersion for an extended period of time should also be expected for N. perurans. The occurrence of round cyst-like cells has been reported for the Acanthamoeba T4 genotype in response to organic solvents exposure (Kliescikova, Kulda & Nohynkova 2011). The spherical structures were classified by the authors as pseudocysts as, unlike double-walled true cysts, these cells were coated by a single-layered envelope and survived for shorter periods of time when exposed to the stress factor. Interestingly, pseudocysts were eventually able to revert to viable trophozoites once returned back to optimum culture conditions. The development of non-motile spherical cells has been also described for N. perurans when submitted to in vitro freshwater incubation (Powell & Clark 2003; Nowak, Crosbie & Adams 2010). In both studies, rounded amoebas were counted as dead cells based on either trypan blue or neutral red viability assays which were performed at the end of each in vitro challenge. Therefore, given the frequent reports of amoeba survival following freshwater bathing, the possibility of N. perurans recovering from the apparently dead spherical stage should be accounted for. To investigate this hypothesis, in vitro cultured trophozoites were submitted to freshwater incubation and the dynamics of rounded cells closely monitored by microscopy, once returned to optimum culture conditions. Briefly, N. perurans trophozoites were freshly isolated from gill swabs taken from AGD-affected fish and maintained on seawater agar plates (Crosbie et al. 2012) until reaching 50% confluence. Prior to the freshwater challenge, the cells were evenly spread into 24-well culture plates to a final concentration of 1000 amoebae/well and allowed to adhere at 16 °C overnight (Lima et al. 2014). At this stage, an aliquot of cells was used to Correspondence P C Lima, Integrated Sustainable Aquaculture Production, CSIRO Agriculture and Food, BIRC, 144 North Street, Woorim, QLD 4507, Australia (e-mail: paula.lima@csiro.au)