Contents lists available at ScienceDirect Comparative Biochemistry and Physiology, Part C journal homepage: www.elsevier.com/locate/cbpc Biochemical characterization of the venom of Central American scorpion Didymocentrus krausi Francke, 1978 (Diplocentridae) and its toxic efects in vivo and in vitro Daniela Rojas-Azofeifa a,b,1 , Mahmood Sasa b,c , Bruno Lomonte b , Elia Diego-García d,e,2 , Natalia Ortiz a,b,1 , Fabián Bonilla b , Renato Murillo f , Jan Tytgat e , Cecilia Díaz a,b, ⁎ a Departamento de Bioquímica, Escuela de Medicina, Universidad de Costa Rica, San José, Costa Rica b Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San José, Costa Rica c Escuela de Biología, Universidad de Costa Rica, San José, Costa Rica d Cátedras CONACYT-El Colegio de la Frontera Sur (ECOSUR), Tapachula, Chiapas, Mexico e Department of Pharmaceutical and Pharmacological Sciences, University of Leuven, Belgium f Centro de Investigaciones en Productos Naturales, Universidad de Costa Rica, San José, Costa Rica ARTICLEINFO Keywords: Cytotoxicity Myotoxicity Paralysis Peptides Toxins Scorpion venom ABSTRACT Venoms of medically important scorpions from Buthidae family have been intensively studied, in contrast to non-buthid venoms, for which knowledge is scarce. In this work, we characterized the venom of a Diplocentridae species, Didymocentrus krausi, a small fossorial scorpion that inhabits the Tropical Dry Forest of Central America. D. krausi venom soluble fraction contains proteases with enzymatic activity on gelatin and casein. Mass spec- trometry and venomic analysis confrmed the presence of elastase-like, cathepsin-O-like proteases and a ne- prilysin-like metalloproteinase. We did not detect phospholipase A 2 , C or D, nor hyaluronidase activity in the venom. By homology-based venom gland transcriptomic analysis, NDBPs, a β-KTx-like peptide, and other pu- tative toxin transcripts were found, which, together with a p-benzoquinone compound present in the venom, could potentially explain its direct hemolytic and cytotoxic efects in several mammalian cell lines. Cytotoxicity of D. krausi venom was higher than the efect of venoms from two buthid scorpion species distributed in Costa Rica, Centruroides edwardsii and Tityus pachyurus. Even though D. krausi venom was not lethal to mice or crickets, when injected in mouse gastrocnemius muscle at high doses it induced pathological efects at 24 h, which in- clude myonecrosis, weak hemorrhage, and infammatory infltration. We observed an apparent thrombotic efect in the skin blood vessels, but no in vitro fbrinogenolytic activity was detected. In crickets, D. krausi venom induced toxicity and paralysis in short periods of time. 1. Introduction Living scorpions comprise > 2200 species whose origin probably goes back to the Upper Carboniferous (Jeram, 1998). These chelicerates share a series of characteristics, such as a unique body plan, pedipalps that end in large claws, viviparity, and adaptations to nocturnal ac- tivity; but their most distinctive feature is the presence of specialized venom glands and a stinger in the last segment of their abdomen (Hjelle, 1990). Traditionally, living scorpions are divided into buthids and non- buthids. The former belong to a single family that includes almost all species of medical importance. Scorpions from this group have narrow pedipalps and chelae and a relatively large and efcient stinger. The non-buthids cover all the rest of scorpion families, which have strong pedipalps (Sunagar et al., 2013), but with stingers commonly small, to the point that in some species they seem almost inefective. Phylogenetic reconstructions based on morphological characters (Fet and Soleglad, 2005; Prendini and Wheeler, 2005; Sharma et al., 2015) support this division, with buthids often separated from the common trunk of other scorpion groups. Recently, however, Sharma and collaborators (2018) propose a phylogenetic reconstruction based on a robust set of molecular characters. Their new arrangement raises https://doi.org/10.1016/j.cbpc.2018.11.021 Received 16 October 2018; Received in revised form 27 November 2018; Accepted 28 November 2018 ⁎ Corresponding author at: Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San José 11501, Costa Rica. E-mail address: cecilia.diaz@ucr.ac.cr (C. Díaz). 1 Present address: Departamento de Bioquímica, Escuela de Medicina, Universidad de Costa Rica, San José, Costa Rica. 2 Present address: Cátedras CONACYT-El Colegio de la Frontera Sur (ECOSUR), Tapachula, Chiapas, Mexico. Comparative Biochemistry and Physiology, Part C 217 (2019) 54–67 Available online 02 December 2018 1532-0456/ © 2018 Elsevier Inc. All rights reserved. T