An analysis of venom ontogeny and prey-specific toxicity in the Monocled Cobra (Naja kaouthia) Cassandra M. Modahl a , Ashis K. Mukherjee a, b , Stephen P. Mackessy a, * a School of Biological Sciences, University of Northern Colorado, 50120th St., Greeley, CO 80639-0017, USA b Department of Molecular Biology and Biotechnology, Tezpur University, Tezpur, 784028 Assam, India article info Article history: Received 23 March 2016 Received in revised form 25 April 2016 Accepted 28 April 2016 Available online 7 May 2016 Keywords: Enzymes Mass spectrometry Phospholipases A 2 Three-finger toxins Venom variation abstract Venoms of snakes of the family Elapidae (cobras, kraits, mambas, and relatives) are predominantly composed of numerous phospholipases A 2 (PLA 2 s) and three-finger toxins (3FTxs), some of which are lethal while others are not significantly toxic. Currently, the only identified prey-specific toxins are several nonconventional 3FTxs, and given the large diversity of 3FTxs within Monocled Cobra (Naja kaouthia) venom, it was hypothesized that several 3FTxs, previously found to be non-toxic or weakly toxic 3FTxs in murine models, could potentially be toxic towards non-murine prey. Additionally, it was hypothesized that ontogenetic dietary shifts will be correlated with observable changes in specific 3FTx isoform abundance. Adult and juvenile N. kaouthia venom composition was investigated using ion- exchange FPLC, 1D and 2D SDS-PAGE, mass spectrometry, and various enzymatic and LD 50 assays. Alpha-cobratoxin (a-elapitoxin) was the only significantly toxic (LD 50 < 1 mg/g) 3FTx found in N. kaouthia venom and was equally toxic toward both lizard and mouse models. The abundance and diversity of 3FTxs and most enzyme activities did not vary between adult and juvenile cobra venoms; however, total venom PLA 2 activity and specific PLA 2 isoforms did vary, with juveniles lacking several of the least acidic PLA 2 s, and these differences could have both biological (related to predation) and clinical (antivenom efficacy) implications. Nevertheless, the ubiquitous presence of a-cobratoxin in both adult and juvenile cobra venoms, with high toxicity toward both reptiles and mammals, represents a venom compositional strategy wherein a single potent toxin effectively immobilizes a variety of prey types encountered across life history stages. © 2016 Elsevier Ltd. All rights reserved. 1. Introduction The Monocled Cobra (Naja kaouthia) is the most abundant species of Asian cobra, with a range that includes India, Bangladesh, Nepal, Myanmar, southwestern China, and Thailand (Mukherjee and Maity, 2002; Reali et al., 2003). In Thailand, snakebite enve- nomations by N. kaouthia account for the highest number of human fatalities among all venomous snake species (Kulkeaw et al., 2007). Patients who have systemic envenoming by N. kaouthia usually develop neurotoxic symptoms, including ptosis, dysphagia, and increased salivation, followed by coma and death from respiratory paralysis in severe cases (Sells et al., 1994; Reali et al., 2003). Because N. kaouthia is very common and is responsible for human morbidity and mortality, there have been many studies published characterizing specific venom proteins and describing overall venom composition (Hamako et al., 1998; Sakurai et al., 2001; Meng et al., 2002; Mukherjee and Maity, 2002; Doley and Mukherjee, 2003; Kulkeaw et al., 2007; Mordvintsev et al., 2007, 2009; Debnath et al., 2010), including two recent publications on the complete venomics profile of N. kaouthia (Laustsen et al., 2015; Tan et al., 2015). The venom of N. kaouthia is primarily composed of three-finger toxins (3FTxs; neurotoxic and cardiotoxic/cytotoxic) and phospholipase A 2 (PLA 2 ) isoforms (Namiranian and Hider, 1992; Kulkeaw et al., 2007; Laustsen et al., 2015; Tan et al., 2015). Geographic venom variation was also recently documented for N. kaouthia (Tan et al., 2015), but because pooled venoms were used in these studies, there is still a lack of information on individual intraspecific venom variation or ontogenetic venom compositional changes in N. kaouthia. Venom variability has been documented at the family, genus, * Corresponding author. E-mail address: stephen.mackessy@unco.edu (S.P. Mackessy). Contents lists available at ScienceDirect Toxicon journal homepage: www.elsevier.com/locate/toxicon http://dx.doi.org/10.1016/j.toxicon.2016.04.049 0041-0101/© 2016 Elsevier Ltd. All rights reserved. Toxicon 119 (2016) 8e20