Development and reproduction of Saprolegnia species in biofilms Shimaa E. Ali a , Even Thoen a,b , Trude Vra ˚ lstad a , Ralf Kristensen a , Øystein Evensen b , Ida Skaar a, * a Norwegian Veterinary Institute, P.O. Box 750 Sentrum, 0106 Oslo, Norway b Norwegian School of Veterinary Science, P.O. Box 8146 Dep., 0033 Oslo, Norway 1. Introduction Saprolegnia species belong to the class Oomycetes (Baldauf et al., 2000), some of which act as pathogens on fresh water fish, amphibians and crustaceans (Dieguez- Uribeondo et al., 2007; Krugner-Higby et al., 2010). Many salmonid fry raised in fish farms die as a result of oomycete infections, and total infection rates of 10% for salmonid eggs were estimated by Bruno et al. (2011). It is now widely accepted that microbes in nature rarely survive as solitary cells, but rather grow as biofilm (Harding et al., 2009). Biofilms are microbial communities that develop in association with a surface. They form a protected environment where microorganisms adopt a specific physiology. The discovery of biofilm formation by bacteria and yeasts has led to a better understanding of the mechanisms of virulence and persistence of pathogenic microorganisms (Harding et al., 2009). The secretion of extracellular enzymes and apical hyphal growth make fungi regarded as especially adapted for growth on surfaces (Jones, 1994). Fungi are therefore excellent candidates for biofilm formation (Harding et al., 2009). Oomycetes are not true fungi (Burki et al., 2007), but similar to fungi, Saprolegnia spores/cysts possess the ability to secrete adhesive materials that have affinity to bind to lectins (Lehnen and Powell, 1989; Burr and Beakes, 1994; Beakes et al., 1994). Sources of Saprolegnia infection cannot always be identified. Thoen (2011) reported that the Saprolegnia spore counts in effluent water of most Norwegian salmon hatcheries were higher than in inlet Veterinary Microbiology 163 (2013) 133–141 A R T I C L E I N F O Article history: Received 28 May 2012 Received in revised form 6 December 2012 Accepted 8 December 2012 Keywords: Saprolegnia Biofilm CLSM Oomycetes Water mould Virulence factor A B S T R A C T Saprolegnia spp. can cause mortality and economic losses in freshwater fish and eggs. Biofilm formation is generally regarded as a virulence factor, and biofilms can be an important cause of infection recurrence. Evidence of persistent sources of Saprolegnia infections on fish and eggs in fish farms support the assumption that Saprolegnia spp. might be able to form biofilms. In this study, we aimed to test the ability of Saprolegnia to form biofilms where it can survive, reproduce and resist different chemicals used for its control. Naturally formed biofilms were obtained from laboratory aquaria. Saprolegnia growth within these biofilms was demonstrated with light microscopy and confirmed by isolation. Isolates were identified morphologically and molecularly on the basis of ITS- sequences. Two isolates were identified as Saprolegnia parasitica, a species known to be highly pathogenic for fish, while the other belonged to S. australis. Selected Saprolegnia strains obtained from natural biofilms were then used to establish simple methods for in vitro induction of Saprolegnia biofilm. The ability of Saprolegnia isolates to form biofilms with subsequent production of infective motile zoospores within the biofilm was documented by light and confocal laser scanning microscopy. We demonstrate for the first time that isolates of S. parasitica and S. australis can form biofilm communities together with multiple microorganisms, wherein they grow and reproduce. It is therefore likely that natural biofilms constitute incessant Saprolegnia reservoirs in nature and aquaculture. ß 2012 Elsevier B.V. All rights reserved. * Corresponding author. Tel.: +47 23216244; fax: +47 23216202. E-mail address: ida.skaar@vetinst.no (I. Skaar). Contents lists available at SciVerse ScienceDirect Veterinary Microbiology jo u rn al ho m epag e: ww w.els evier.c o m/lo cat e/vetmic 0378-1135/$ – see front matter ß 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.vetmic.2012.12.012