Toxicon 187 (2020) 111–115 Contents lists available at ScienceDirect Toxicon journal homepage: http://www.elsevier.com/locate/toxicon Short communication Toxicological comparison of Crotalus ruber lucasensis venom from different ecoregions of the Baja California Peninsula Ivan´ Fernando Pozas-Ocampo a, Alejandro Carbajal-Saucedo b, Ana Bertha Gatica-Colima c, Amaury Cordero-Tapia a, Gustavo Arnaud-Franco a,* a Centro de Investigaciones Biologicas´ Del Noroeste SC, Instituto Polit´ecnico Nacional, #195 Col. Playa Palo Santa Rita Sur, La Paz, BCS, CP 23096, Mexico b ISBI Biosciences S.C, Alta Tension´ #4, Huitzilac, Morelos, CP 62510, Mexico c Universidad Autonoma´ de Ciudad Juarez,´ Instituto de Ciencias Biom´edicas, Anillo Envolvente Del PRONAF y Estocolmo, S/n. Ciudad Juarez,´ Chihuahua, CP 32310, Mexico ARTICLE INFO ABSTRACT Keywords: The Baja California Peninsula possesses a mosaic of ecoregions that offers a wide variety of environments for the Red diamond rattlesnake species that here inhabit. Here we report biological variations in. Toxicology Crotalus ruber lucasensis venom from arid, semiarid and tropical eco-regions. Lethal (1.4–6.8 mg/kg), ede­ Venom matogenic (0.3–0.5 μg) and defibrinogenating (from non-detectable to 20 μg) activities were found to have Hemorrhage significant differences among eco-regions. Edema PLA2 Snake venoms of the Viperidae family are composed of a complex of the arid Magdalena Plains eco-region (MP); El Comitan,´ representa­ mixture of proteins (Calvete et al., 2010; Kang et al., 2011; Mackessy, tive of semiarid Central Gulf Coast (CGC), and; Santiago, representative 2008), which may vary at an inter- and intra-specific level, which is a of tropical Cape Deciduous Forest (CDF). Venom samples were taken function of the habitat in which the snake lives, time of year, sex, age, from 12 specimens that were subsequently released at the same capture diet and geographical region (Daltry et al., 1996; Dias et al., 2013; point (Table 1). Mackessy, 2008; Minton and Weisntein, 1986; Modahl et al., 2016; Venom samples obtained individually were immediately stored in Neri-Castro et al., 2013; Sunagar et al., 2014; Tan et al., 2015). This liquid nitrogen for transfer and posterior lyophilization using a Freeze geographical variation can affect the toxicity of the venom, and also Plus system (LabConco, MO) and weighted. In order to avoid individual suggests that there may be population differences that cause these var­ variation and obtain a representative sample of each eco-region, pooled iations (Minton and Weisntein, 1986). The Baja California Peninsula samples were constructed by mixing 3–5 mg of lyophilized individual (BCP) is an arid region divided in 14 eco-regions (Gonzalez-Abraham´ venoms and dissolved in normal saline (0.9% NaCl) to give a final et al., 2010), were Crotalus ruber (Red Diamond Rattlesnake) is the most concentration of 10 mg/ml (dry weight). Venoms solutions were divided ◦ widely distributed rattlesnake species; three subspecies are currently into aliquots and stored at 40 C. Albino mice of the strain CD-1 (ICR) recognized: C. r. ruber, C. r. exsul and, C. r. lucasensis, this last distributed of 18–30 gr, of indistinct sex, were used in experimental procedure. in the southern portion of BCP (Campbell and Lamar, 2004; Grismer, Venom components (from individual and pooled samples) were sepa­ 2002). Unlike C. r. ruber (Glenn and Straight, 1985; Komori et al., 2011; rated in 12.5% acrylamide:bisacrylamide (36.5:1) gels using discontin­ Mackessy, 2008, 1985; Mori et al., 1987a; Mori and Sugihara, 1989, uous system described by Laemmli (1970) under reducing (2-mercato 1988), the venom of C. ruber lucasensis has received lesser attention ethanol) and non-reducing conditions. Samples of 15 μg were dissolved (Arnaud-Franco et al., 2018; Glenn and Straight, 1985), especially from in loading buffer, boiled and applied. Runs were performed at 80 V by an ecological perspective. C. r. lucasensis occupy six eco-regions in BCP 30 min (in stacking gel) followed by 100 V for 3 h. Gels were fixedand (Grismer, 2002). In this context, we objective was to identify the bio­ stained with Coomasie brilliant blue R-250 (0.1%). logical characteristics of its venom from three different eco-regions. Lethal doses were determined from groups of six mice (18–20 g, both Three location were selected: Valle de Santo Domingo, representative sexes), which were given different doses of venom through venous * Corresponding author. La Paz, BCS, CP 23096, Mexico. E-mail address: [email protected] (G. Arnaud-Franco). https://doi.org/10.1016/j.toxicon.2020.08.029 Received 13 June 2020; Received in revised form 7 August 2020; Accepted 31 August 2020 Available online 5 September 2020 0041-0101/© 2020 Elsevier Ltd. All rights reserved. I.F. Pozas-Ocampo et al. Toxicon 187 (2020) 111–115 puncture (i.v.) in a total volume of 0.5 ml. Animals were kept in as the amount of NaOH consumed per time unit (μmol/min). Three appropriate environmental conditions with food and water ad libitum. replicates were done per sample. Deaths were recorded 24 h post injection and the median lethal dose A priori tests of normality (Kolmogorov-Smirnov) (Daniel, 2008) and calculated by Spearman-Karber¨ (World Health Organization, 2010). homoscedasticity (Cochran) were used to validate the parametric sta­ To determine Minimum Hemorrhagic Doses (MHD) six different tistics analysis to findsignificant differences between eco-regions based doses of venoms were administered by the intradermal route in the on pools of venom. As the a priori parameters for each group were met, dorsal skin of mice (20–25 g, both sexes) in a total volume of 50 μl (three parametric test of Analysis of Variance (ANOVA) were used. Finally, mice per dose level). Two hours after inoculation animals were sacri­ when necessary, Fisher’s a posteriori test were used (Daniel, 2008; Zar, ficed by CO2 inhalation and skin removed. Hemorrhagic spots were 1999). measured as the average of two perpendicular diameters. MHD were Since sex (Mackessy et al., 2018) and ontogeny (Minton and determined by linear regression of data using Graph Pad Prism 6.0 and Weisntein, 1986) can influencevenom composition in rattlesnakes only defined as the amount of venom that produces a 10 mm diameter spot adult individuals were used and their venoms mixed to minimize bias. (De Roodt et al., 2000; Kondo et al., 1960). Electrophoretic profiles show high level of similarities among sam­ To determine Minimum Edema-Forming Dose (MED) five different ples. Protein band of 20–25 kDa is common to all samples in both doses of venoms were administered subcutaneously at the right foot paw reduced and non-reduced conditions (Fig. 1). Samples form Magdalena of mice (25–30 g, both sexes). For the control normal saline (50 μl) was Plains (MP) and Central Gulf Coast (CGC) also shares prominent bands administered to the left foot. Animals were sacrificed 30 min post around 50 and 12 kDa, followed in abundance by bands located around inoculation and the diameter of the foot paw was measured with a 60 and 14 kDa. Samples from Cape Deciduous Forest (CDF) shows the digital caliper. MED was definedas the amount of venom that produces a most abundant bands around 12 and 14 kDa and, even when present, 30% increase in volume compared to control (Gutierrez´ et al., 1986; band around 50 kDa is not a major component as in the other two Yamakawa et al., 1976). localities. To determine Minimum Defibrinogenating Dose (MDD), different In order to avoid bias, pooled venom samples from each eco-regions amounts of venoms were administered by intravenous route into mice were tested for various biological activities (Table 2). Venoms differ (20–25 g, both sexes) in a total volume of 200 μl. One hour after inoc­ importantly in lethal activity in which CGC shown to be 3.1 and 4.9 ulation blood sample were obtained in capillary tubes and incubated at times more potent than CDF and MP samples, respectively. Edema- ◦ 37 C for 60 min. MDD was definedas the lowest amount of venom that forming dose showed significant difference among the eco-regions, produces unclottable blood in all samples (Instituto-Clodomiro-Picado, with CDF venom (subtropical environment) showing the highest activ­ 2008). ity (F2 = 19.62; p < 0.01). To determine the Minimum Procoagulant Dose (MPD), a bag of Only one (CGC) of the three venoms tested produces unclottable pathogen-free frozen human blood plasma (2.2% sodium citrate + 0.8% blood in the mouse model; Venoms from MP and CFD produce unclot­ citric acid + 2.45% dextrose), was used. Different amounts of venom table blood in only some mice, but doses of 30 and 35 μg produce pro­ ◦ were added in test tubes with 200 μl of plasma (pre-incubated at 37 C) fuse hemorrhage in the tail zone. No further doses were tested. and time to the formation of visible clot was registered. MPD was Hemorrhagic activity was found to be high for all three samples definedas the amount of venom required to obtain a visible clot in 60 s (0.8–1.2 μg) with non-significant difference among eco-regions (F2 = (Theakston and Reid, 1983). 1.22; p = 0.35). Individual and pooled venoms were unable to clot Phospholipase A2 activity (PLA2) was determined over by titrimetric citrated human plasma; only CGC samples produce a mild unstable ag­ assay over 10% egg yolk solution (0.1 M NaCl, 0.01 M CaCl2, 0.5% gregation even at higher doses (350 μg). Finally, CDF show slightly Triton X100) following the method of Shiloah and co-workers (Shiloah higher phospholipase activity, nevertheless non-significant difference et al., 1973). Egg yolk solution was adjusted to pH value between 8.0 was found among eco-regions (Table 2).