Characterisation of cathepsin B-like cysteine protease of Lepeophtheirus salmonis Eleanor Cunningham a, ⁎, Elaine McCarthy a , Lorraine Copley b , Dave Jackson b , David Johnson c , John P. Dalton d , Grace Mulcahy a a Veterinary Science Centre, School of Agriculture, Food Science and Veterinary Medicine, University College Dublin, Ireland b Marine Institute, Rinville, Oranmore, Co. Galway, Ireland c Biomedical Parasitology Division, Department of Zoology, Natural History Museum, Cromwell Road, London, UK d Mc Gill University, Montreal, Canada abstract article info Article history: Received 27 September 2010 Accepted 11 October 2010 Keywords: Atlantic salmon Lepeophtheirus salmonis Cathepsin B Lepeophtheirus salmonis, otherwise known as the salmon louse, is an ectoparasite that feeds on Salmo salar (Atlantic salmon), causing large losses to the aquaculture industry. The parasite feeds on the skin and mucus tissues of its host causing extensive damage and bleeding. A cDNA clone encoding a cathepsin B cysteine protease was isolated from adult female L. salmonis (LsCtB). The nucleotide sequence encoded a preproenzyme and a mature-enzyme. The mature portion shares 69% identity to human cathepsin B and contains all of the conserved regions of cathepsin B including the occluding loop, the double histidine sequence and active site residues. Additionally, cathepsin B activity in L. salmonis was identified and characterised using fluorogenic peptide substrates. The pH optimum for hydrolytic activity was 3.5, and temperature optimum 45 °C. © 2010 Elsevier B.V. All rights reserved. 1. Introduction Lepeophtheirus salmonis (Krøyer) is one of the most widespread pathogenic marine parasites (Costello, 2006). The parasite is otherwise known as the salmon louse and is an ectoparasite that survives on the skin and mucus of the host, the Atlantic salmon (Salmo salar). It has become more common in recent times with the advent of fish farming, as the confined spaces in which the fish are housed favour the transmission of the parasite. It has a detrimental effect on the farming of S. salar (Atlantic salmon) and also has been implicated in the decreased survival of wild salmon; according to some sources may actually cause the extinction of the wild Atlantic salmon in the near future (Krkosek et al., 2007). The parasite has a direct life cycle and includes planktonic stages (nauplius I and II), the infectious copepodid stage that attaches to the fish, attached non-mobile stages (chalimus I to IV) and attached mobile preadult and adult stages. Once it attaches to its host the parasite's feeding activity causes skin lesions due to mechanical damage and also due to the release of digestive enzymes. This skin damage causes physiological stress to the salmon and an inability to maintain osmotic balance (Pike and Wadsworth, 1999) which can further lead to secondary infections, decreased growth and even death (Costello, 2006). Current treat- ments include the use of chemotherapeutic agents such as cyperme- thrin and emamectin benzoate, but environmental contamination with these compounds can affect biodiversity and in-feed treatment can lead to unequal distribution of the chemical in the fish (Skilbrei et al., 2008). Cathepsin B (E.C. 3.4.22.1) is a cysteine protease expressed in all cells and is a member of the papain family of proteases, peptidase C1 superfamily (Dalton et al., 1996; Sajid and McKerrow, 2002). They are important in various parasitic species including the root worm Diabrotica virgifera (Bown et al., 2004), the horse parasites of the Cyathostominae subfamily (Kinsella et al., 2002), the porcine tape worms Taenia solium and T. saginata (Zimic et al., 2007), the human hookworm Nector americanus (Duffy et al., 2006) and the ruminant parasite Haemonchus contortus (Jasmer et al., 2001). It has been extensively studied in the helminth parasites Fasciola hepatica (Beckham et al., 2006) and Schistosoma mansoni (Correnti et al., 2005). Cathepsin B has also been localised in the gut of schistosomes (Correnti et al., 2005), F. hepatica (Beckham et al., 2006) and N. americanus (Ranjit et al., 2008) indicating a function in digestion. The protease has been shown to have multiple functions including an ability to degrade tissues and digest haemoglobin in Ancylostoma caninum and S. mansoni and to digest the host's blood in H. contortus (Williamson et al., 2003). It is thought to be involved in degrading host protein and iron acquisition in Trypanosoma brucei (O'Brien et al., 2008) and an important factor in the ability of F. hepatica to migrate within the host (McGonigle et al., 2008). Cathepsin B is also important for both parasite development and successful survival of the host; if inhabited, development is stunted as shown in both S. mansoni (Correnti et al., 2005) and Toxoplasma gondii (Que et al., 2002). Therefore, we surmised that cysteine proteases may play an important Aquaculture 310 (2010) 38–42 ⁎ Corresponding author. E-mail address: elliecunningham@gmail.com (E. Cunningham). 0044-8486/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2010.10.013 Contents lists available at ScienceDirect Aquaculture journal homepage: www.elsevier.com/locate/aqua-online