Toxicon 187 (2020) 111–115

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Toxicon

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Short communication Toxicological comparison of ruber lucasensis venom from different ecoregions of the 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, 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 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 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 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. 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). and 8.1 with 50 mM NaOH. Venoms samples were added and pH Hemorrhagic and fibrino (geno)lytic metalloproteinases HT1, HT2 maintained over a value of 8.0 by adding constant volume of 50 mM and HT3 with molecular masses of 60, 24.8 and 25.7 kDa, respectively, NaOH and recording time between additions. Amount of NaOH (μmol) has been described for C. r. ruber venom (Mori et al., 1987b). Proteins were plotted as function of time (min). One enzymatic unit was defined bands at 25 and around 50 kDa observed in electrophoretic profiles

Table 1 Identification and locality data (red triangles) on C. r. lucasensis specimens used in this study. a: Snout-Vent Length. b: CGC: Central Gulf Coast; MP: Magdalena Plains; CDF: Cape Deciduous Forest.

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Fig. 1. Electrophoretic profilesof C. r. lucasensis venom from different location in Baja California Peninsula. Total of 15 μg were loaded per lane under reduced and non-reduced condition. A) Magdalena Plains; B); Central Gulf Coast; C) Cape Deciduous Forest. MWM = Molecular Weight Markers. Identificationnumbers at the top of each lane correspond to those listed in Table 1. could be homologous proteins to those previously mentioned and should due to very low procoagulant activity (>350 μg; no differences were be responsible of high hemorrhagic activities found in C. r. lucasensis found among eco-regions). venoms (Arnaud-Franco et al., 2018; this work) with mild differences in Phospholipases A2 is one of the three main protein families found in concentrations among populations. viper venoms (Calvete et al., 2009; Holzer and Mackessy, 1996; Mack­ The presence of HT1, HT2 and HT3 related proteins along with essy, 2008; Munekiyo and Mackessy, 1998). Hydrolysis of sn-2 glycer­ Rubelase-like component, a non-hemorrhagic fibrinogenolytic metal­ o-phospholipids is related (CCA, 1997) to wide variety of biological loproteases of 23.3 kDa from C. r. ruber (Komori et al., 2011), could be activities (Kini, 2003), including an increase in edema responses responsible of defibrinogenatingactivity. In a modifiedmethod of whole (Montecucco et al., 2008). It is interesting to note that MP sample blood clotting time (human blood) all three venoms provoked unclot­ showed higher values of both PLA2 and edema-forming activities; table blood with 2.5 μg (data not show), nevertheless only CGC sample nevertheless, specificcorrelation between these two activities (as well as show defibrinogenating activity (20 μg) in the mouse model (Table 2). with hemorrhagic activity) must be studied in detail. Important differences on fibrinogen susceptibility to snake venoms has The differences in LD50 values in this study should be related either been demonstrated (Santoro and Sano-Martins, 1993; Teng et al., 1985). to hemorrhagic, edema-forming and PLA2 related toxins, as well as other In this sense, natural resistance of mouse fibrinogen against C. r. luca­ non-studied activities/components (i.e. myotoxic or vasoactive pep­ sensis venoms could explain the low levels of activities of CDF and MP tides) but could also be attributed to local differences on prey con­ samples. sumption, as observed for other snake species (Clark, 2002; Glaudas The edematous response it is multifactorial in origin and can be et al., 2008; Holycross et al., 2002). It is possible that biological activ­ triggered by protease activity (metallo and serine proteases), vasoactive ities found in this work could be correlated to prey availability and or ´ peptides, A2 phospholipases and other venom components (Gutierrez, resistance. However, lack of precise information on rodents/lagomorphs 2010). Proteins responsible for the aforementioned hemorrhagic and populations densities hamper the ability to test this hypothesis. defibrinogenating activities could also be related to the high edema-forming activity of these venoms. Besides, a 31 kDa Kallikrein Authorship Contribution Statement component capable to increase permeability as well as hydrolyze fibrinogen,without formation of clot, was isolated from C. r. ruber (Mori Ivan´ Fernando Pozas-Ocampo: performed project, Formal analysis, and Sugihara, 1988) and could also be present in C. r. lucasensis (proteins Writing - original draft, Writing - review & editing. Alejandro Carbajal- bands around 30 kDa in all samples). Our results suggest that no Saucedo: performed project, Formal analysis, Writing - original draft, thrombin-like enzymes are present in our C. r. lucasensis venoms samples Supervision, Writing - review & editing. Ana Bertha Gatica-Colima:

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Table 2 generating the map. IFPO received a scholarship (number 614163) from Biological and Phospholipase activities of C. r. lucasensis venoms. CONACyT. Collecting permits for venom were issued by the government a a Eco- LD50 (mg/ MHD MED MDD ID PLA2 of Mexico (SEMARNAT) to GAF (07033/19). This research was carried region kg) (μg) (μg) (μg) (U/mg)c out without receiving any specific grant from funding agencies. MP 6.8 1.2 0.4 >30a 05.01.17 168 (6.6–6.9) (±0.4) (±0.04) (±15) References 05.02.17 163 ´ (±9) Arnaud-Franco, G., Cordero-Tapia, A., Ortíz-Avila, V., Moctezuma-Gonzalez,´ C., 05.03.17 321 Tejocote-P´erez, M., Carbajal-Saucedo, A., 2018. Comparison of biological and (±27) biochemical characteristics of venom from rattlesnakes in the southern Baja CGC 1.4 0.8 0.5 20 06.01.16 232 California Peninsula. Toxicon 148, 197–201. https://doi.org/10.1016/j. (1.3–1.5) (±0.3) (±0.01) (±50) toxicon.2018.04.030. 07.02.17 323 Calvete, J., Sanz, L., Cid, P., de la Torre, P., Marietta, F.-D., Dos Santos, M.-C., Borges, A., Bremo, A., Angulo, Y., Lomonte, B., Guti´errez, J.M., 2010. Snake venomics of the (±24) Central American rattlesnake Crotalus simus and the South American Crotalus 07.04.17 239 durissus complex points to neurotoxicity as an adaptive paedomorphic trend along (±10) Crotalus dispersal in South America. J. Proteome Res. 9, 528–544. > a CDF 4.4 1.0 0.3 30 05.06.17 263 Calvete, J.J., Sanz, L., Angulo, Y., Lomonte, B., Gutierrez,´ J.M., 2009. Venoms, venomics, – ± ± ± (4.3 4.5) ( 0.2) ( 0.01) ( 36) antivenomics. FEBS Lett. 583, 1736–1743. https://doi.org/10.1016/j. 05.07.17 280 febslet.2009.03.029. (±52) Campbell, J., Lamar, W., 2004. The Venomous of the Western Hemisphere, 05.08.17 386 Comstock Books in Herpetology. Comstock Pub. Associates. ˇ (±39) CCA, 1997. Regiones ecol—gicas de AmZrica del Norte: hacia una perspectiva común. 05.09.17 200 Quebec´ . (±22) Clark, R., 2002. Diet of the timber rattlesnake Crotalus horridus. J. Herpetol. 36, 494–499. 05.10.17 413 Daltry, J., Ponnudurai, G., Shin, C., Tan, N., Thorpe, R., Wüster, W., 1996. (±84) Electrophoretic pofilesand biological activities: intraspecificvariation in the venom – 02.01.17 456 of malayan pitviper (Calloselasma rhodostoma). Toxicon 34, 67 79. Daniel, W., 2008. Bioestadística, base para el analisis´ de las ciencias de la salud, fourth (±25) ed. Limusa Wiley, M´exico D.F. LD50: Median lethal Dose was definedas the amount of venom required to kill De Roodt, A., Dolab, J., Dokmetjian, J., Litwin, S., Segre, L., Vidal, J., 2000. – A comparison of different methods to assess the hemorrhagic activity of Bothrops 50% mice population (ICR, 18 20 g). Confidence Intervals (95%) are shown in – μ venoms. Toxicon 38, 865 873. https://doi.org/10.1016/S0041-0101(99)00205-6. parentheses. MHD: Minimum Hemorrhagic Dose, is the amount of venom ( g) Dias, G., Kitano, E., Pagotto, A., Santanna, S., Rocha, M., Zelanis, A., Serrano, S., 2013. necessary to produce a 10 mm hemorrhagic halo after 2 h post-inoculation (ICR; Individual variability in the venom proteome of juvenile Bothrops jararaca 20–25 g). MED: Minimum Edema-forming Dose is the amount of venom (μg) specimens. J. Proteome Res. 12, 4585–4598. necessary to produce a 30% increase in paw volume in relation to control. Glaudas, X., Jezkova, T., Rodriguez-Robles, J., 2008. Feeding ecology of the graet basin – Significant difference were found among ecoregions (F2 = 19.62; p < 0.01) rattlesnake (Crotalus oreganus lutosus, Viperidae). Can. J. Zool. 86, 1 12. μ Glenn, J., Straight, R., 1985. Venom propierties of the rattlesnakes (Crotalus) inhabiting MDD: Minimum DefibrinogenatingDose, is the amount of venom ( g) necessary the Baja California region of Mexico. Toxicon 23, 769–775. to produce unclottable blood in all samples (n = 4) after 60 min. Gonzalez-Abraham,´ C.E., Garcillan,´ P.P., Ezcurra, E., 2010. Ecorregiones de la península PLA2 specificactivity was determined on 10% egg-yolk solutions as described in de baja California: una síntesis. Bol. la Soc. Bot. Mex. 87, 69–82. https://doi.org/ Material and Methods. U = μmol NaOH/min. Activities for pooled venoms was 10.17129/botsci.302. also determined and resulted in 368 (±8), 208 (±19) and 179 (±7) for MP, CGC Grismer, L., 2002. Amphibians and Reptiles of Baja California, its PacificIslands, and the Islands in the Sea of Cort´es. University of California Press, London. https://doi.org/ and CDF, respectively. However, these results were not considered in the sta­ 10.1525/j.ctt1pnzvb. tistical analysis. Guti´errez, J., Rojas, G., Lomonte, B., Gen´e, J., Cerdas, L., 1986. Comparative study of the *: Mouse injected with more than 30 μg develops high hemorrhagic lesions in the edema-forming activity of costa rican snake venoms and its neutralization by a tail. MPD: Minimum Procoagulant dose over Human Plasma, is the amount of polyvalent antivenom. Comp. Biochem. Physiol. Part C, Comp. 85, 171–175. https:// μ doi.org/10.1016/0742-8413(86)90069-1. venom ( g) needed to produce a visible clot in 60 s over citrated human plasma. ´ a Gutierrez, J.M., 2010. Snakebite envenomation in Central America. In: Mackessy, S.P. : Values represent the average of three independent determinations. Stan­ (Ed.), Handbook of Venoms and Toxins of Reptiles. CRC Press, Boa Raton, dard deviation is shown in parentheses. pp. 492–507. c Values represent the average of three independent determinations. Standard Holycross, A., Painter, C., Prival, D., Swann, D., Schroff, M., Edwards, T., Schwalbe, C., deviation is shown in parentheses. 2002. Diet of Crotalus lepidus klauberi (banded rock rattlesnake). J. Herpetol. 36. Holzer, M., Mackessy, S.P., 1996. An aqueous endpoint assay of snake venom phospholipase A2. Toxicon 34, 1149–1155. https://doi.org/10.1016/0041-0101(96) gave their valuable and professional suggestions, Writing - original 00057-8. ´ ´ draft, Writing - review & editing. Amaury Cordero-Tapia: gave their Instituto-Clodomiro-Picado, 2008. Determinacion de actividades toxicas de venenos de serpientes y su neutralizacion´ por antivenenos, Manual de. Lara Segura & Asoc. valuable and professional suggestions, Writing - original draft, Writing - Kang, T.S., Georgieva, D., Genov, N., Murakami, M.T., Sinha, M., Kumar, R.P., Kaur, P., review & editing. Gustavo Arnaud-Franco: performed project, Formal Kumar, S., Dey, S., Sharma, S., Vrielink, A., Betzel, C., Takeda, S., Arni, R.K., analysis, Writing - original draft, Methodology, Writing - review & Singh, T.P., Kini, R.M., 2011. Enzymatic toxins from snake venom: structural characterization and mechanism of catalysis. FEBS J. 278, 4544–4576. https://doi. editing. org/10.1111/j.1742-4658.2011.08115.x. Kini, R.M., 2003. Excitement ahead: structure, function and mechanism of snake venom – Declaration of competing interest phospholipase A2 enzymes. Toxicon 42, 827 840. https://doi.org/10.1016/j. toxicon.2003.11.002. Komori, Y., Sakai, K., Masuda, K., Nikai, T., 2011. Isolation and biochemical The authors declare that they have no known competing financial characterization of rubelase, a non-hemorrhagic elastase from Crotalus ruber ruber – interests or personal relationships that could have appeared to influence (Red rattlesnake) venom. Toxins 3, 900 910. https://doi.org/10.3390/ toxins3070900. the work reported in this paper. Kondo, H., Kondo, S., Irezawa, H., Murata, R., 1960. Studies on the quantitative method for determination of hemorrhagic activity of Habu snake venom. Jpn. J. Med. Sci. Acknowledgments Biol. 13, 43–51. https://doi.org/10.7883/yoken1952.13.43. Laemmli, U., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 224, 680–685. We thank Martín Ramírez, CIBNOR Marine Immunogenomics Lab­ Mackessy, S., 2008. Venom composition in rattlesnakes: trends and biological oratory, for his support in freeze-drying the venoms; the Blood Bank of significance. In: Hayes, W., Beaman, K.R., Cardwell, M.D., Bush, S.P. (Eds.), The Biology of Rattlesnakes, pp. 495–510. Baja California Sur, for the donation of human blood plasma; Víctor Mackessy, S.P., 1985. Fractionation of red diamond rattlesnake (Crotalus ruber ruber) Ortíz, Israel Guerrero, Abelino Cota, Franco Cota (†), Patricia Vazquez,´ venom: protease, phosphodiesterase, l-amino acid oxidase activities and effects of Heleodoro Corrales, Cecilia Picos and Fernando Aguirre, for their sup­ metal ions and inhibitors on protease activity. Toxicon 23, 337–340. https://doi. org/10.1016/0041-0101(85)90157-6. port in the field and laboratory work. We thank Tania Perez´ for

114 I.F. Pozas-Ocampo et al. Toxicon 187 (2020) 111–115

Mackessy, S.P., Leroy, J., Mocino-Deloya,˜ E., Setser, K., Bryson, R.W., Saviola, A.J., 2018. Neri-Castro, E., Lomonte, B., Guti´errez, M., Alagon,´ A., Guti´errez, J.M., 2013. Venom ontogeny in the mexican lance-headed rattlesnake (Crotalus polystictus). Intraspecies variation in the venom of the rattlesnake Crotalus simus from Mexico: Toxins 10. https://doi.org/10.3390/toxins10070271. different expression of crotoxin results in highly variable toxicity in the venoms of Minton, S., Weisntein, S., 1986. Geographic and ontogenic variation in venom of the three subspecies. J. Proteomics 87, 103–121. https://doi.org/10.1016/j. western diamondbck rattlesnake (Crotalus atrox). Toxicon 24, 71–80. jprot.2013.05.024. Modahl, C., Mukherjee, A., Mackessy, S., 2016. An analysis of venom ontogeny and prey- Santoro, M.L., Sano-Martins, I.S., 1993. Different clotting mechanisms of Bothrops specific toxicity in the Monocled Cobra (Naja kaouthia). Toxicon 119, 8–20. jararaca snake venom on human and rabbit plasmas. Toxicon 31, 733–742. https:// Montecucco, C., Gutierrez,´ J.M., Lomonte, B., 2008. Cellular pathology induced by snake doi.org/10.1016/0041-0101(93)90379-W. venom phospholipase A2 myotoxins and neurotoxins: common aspects of their Shiloah, J., Klibansky, C., de Vries, A., Berger, A., 1973. Phospholipase B activity of a mechanisms of action. Cell. Mol. Life Sci. 65, 2897–2912. https://doi.org/10.1007/ purified phospholipase A from Vipera palestinae venom. J. Lipid Res. 14, 267–278. s00018-008-8113-3. Sunagar, K., Undheim, E., Scheib, H., Gren, E., Cochran, C., Person, C., Koludarov, I., Mori, N., Nikai, T., Sugihara, H., 1987a. Phosphodiesterase from the venom of Crotalus Kelin, W., W, H., King, G., 2014. Intraspecific venom variation in the medically ruber ruber. Int. J. Biochem. 19, 115–119. https://doi.org/10.1016/0020-711X(87) significant Southern Pacific Rattlesnake (Crotalus oreganus helleri): biodiscovery, 90321-1. clinical and evolutionary implications. J. Proteomics 99, 68–83. Mori, N., Nikai, T., Sugihara, H., Tu, A.T., 1987b. Biochemical characterization of Tan, K., Tan, C., Fung, S., N, T., 2015. Venomics, lethality and neutralization of Naja hemorrhagic toxins with fibrinogenase activity isolated from Crotalus ruber ruber kaouthia (Monocled cobra) venoms from three different geographical regions of venom. Arch. Biochem. Biophys. 253, 108–121. https://doi.org/10.1016/0003-9861 Southeast Asia. J. Proteomics 120, 105–125. (87)90643-6. Teng, C., Ouyang, C., Lin, S., 1985. Species differences in the fibrinogenolyticeffects of a Mori, N., Sugihara, H., 1989. Characterization of kallikrein-like enzyme from Crotalus and b fibrinogenases from Trimeresurus mucrosquamatus snake venom. Toxicon 23, ruber ruber (Red rattlesnake) venom. Int. J. Biochem. 21, 83–90. https://doi.org/ 777–782. 10.1016/0020-711X(89)90030-X. Theakston, R., Reid, H., 1983. Development of simple standard assay procedures for the Mori, N., Sugihara, H., 1988. Kallikrein-like enzymes from Crotals ruber ruber (red characterization of snake venoms. Bull. ofthe WorldHealth Organ. 61, 949–956. rattlesnake) venom. Int. J. Biochem. 20, 1425–1433. World Health Organization, 2010. Guidelines for the Production. control and regulation Munekiyo, S.M., Mackessy, S.P., 1998. Effects of temperature and storage conditions on of antivenom immunoglobulins, Geneve. the electrophoretic, toxic and enzymatic stability of venom components. Comp. Yamakawa, M., Nozaki, M., Hokama, Z., 1976. Fractionation of sakishimahabu Biochem. Physiol. B Biochem. Mol. Biol. 119, 119–127. https://doi.org/10.1016/ (Trimeresurus elegans) venom and lethal, hemorrhagic and edema-forming activities S0305-0491(97)00294-0. of the fractions. Anim. plant Microb. toxins 1, 97–109. Zar, J., 1999. Biostatistical Analysis. Pearson Education, India.

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