And Sex-Dependent Variations in the Venom Proteins of Montivipera Albizona (Nilson, Andrén & Flärdh, 1990) (Ophidia: Viperidae) in Anatolia

Home , Montivipera, Montivipera raddei, Montivipera xanthina

BIHAREAN BIOLOGIST 14 (1): 30-32 ©Biharean Biologist, Oradea, Romania, 2020 Article No.: e191307 http://biozoojournals.ro/bihbiol/index.html

Preliminary study on the age- and sex-dependent variations in the venom proteins of Montivipera albizona (Nilson, Andrén & Flärdh, 1990) (Ophidia: Viperidae) in Anatolia

Ahmet, MERMER1, Hüseyin, ARIKAN1, Kerim, ÇİÇEK1 and Cemal Varol, TOK2

1. Section of Zoology, Department of Biology, Faculty of Science, Ege University, 35100, Izmir, Turkey. 2. Department of Biology, Faculty of Arts and Sciences, Çanakkale Onsekiz Mart University, 17100, Çanakkale, Turkey * Corresponding author, A. Mermer, E-mail: [email protected]

Received: 10. July 2019 / Accepted: 22. July 2019 / Available online: 25. July 2019 / Printed: June 2020

Abstract. In this study, polyacrylamide disc gel electrophoresis and densitometry methods were used to analyze venom extracts of three Montivipera albizona specimens collected from Sivas (Central Anatolia, Turkey). The venom secretion was colorless in juvenile and light yellow in mature individuals. The venom proteins of M. albizona could be separated into 10, 11, 12 fractions or fraction groups. Besides, the electropherograms of the venom protein samples showed both sex-based and age-dependent qualitative and quantitative variations.

Key words: Montivipera albizona, polyacrylamide disc gel electrophoresis, venom proteins, Mountain Viper, Turkey.

Introduction an elevation of approx. 1600 m. Thereafter, the specimens were transported to the laboratory alive and kept in a terrarium in a brief The Mountain vipers are included in the family of Viperidae period. Primarily, their lengths were measured using a ruler. Snout- vent length (SVL): 68.3 cm, tail length (TL): 5.1 cm for female, SVL: and are represented by five valid species [Montivipera xan- 55.8 cm, TL: 4.5 cm for male and SVL: 35.0 cm, TL: 3.3 cm for juvenile thina, M. albizona, M. bulgardaghica, M. wagneri, M. raddei] in specimens. Anatolia (Mulder 1994, Göçmen et al. 2009, 2014). The Ana- Venom samples were extracted from individuals without apply- tolian endemic mountain viper, Montivipera albizona (Nilson, ing any pressure on the venom glands, as described by Tare et al. Andrén and Flärdh 1990), is firstly described from Kulmaç (1986), using a laboratory beaker. Then they were released to the col- Mountain Range, Sivas (Central Anatolia) close to the “Ana- lected habitat. As venom extracts also contain dead cells, they were tolian Diagonal” in Turkey by Nilson et al. (1990) as Vipera centrifuged at 600 g for 5 min, and 4 µl of a venom sample, which albizona. Recent genetical study by Stümpel et al. (2016) sug- was used for each separation. The venom proteins were separated according to methods given by Davis (1964), slightly modified by gested that M. albizona should be treated as a subspecies of Arıkan et al. (2006, 2014). Accordingly, a pH 6.7 stacking gel was M. bulgardaghica. Since more data from a higher number of layered above the pH 9 separation gels of 7.5 % polyacrylamide, to- specimens is needed and their taxonomy is still in question, gether with pH 8.3 tris-glycine buffer system, using a Canalco Model we considered this taxon as a full species in this study. 1200 electrophoresis apparatus. Separation gels were stained with Snake venoms are typically one of the most complex 0.5% Amido black (Naphthol Blue Back 10-B), later destained pas- mixtures and the venom of some species might contain more sively with repeated 7% acetic acid baths and the densitometric than 100 different toxic and non-toxic proteins and peptides curves of the separations were obtained by means of a Gelman ACD- 15 Model 39430 densitometer at 500 nm. (Chippaux 2006, Mackessy 2010). Various studies (Chippaux

2006, Mackessy 2010) have reported that variation of sources in venom composition, which includes phylogeny and taxo- Results nomic relationships, age, geography, diet and other possible sources of variation (seasonal variation and sex-based varia- The venom extracts of M. albizona specimens had a viscosity tion). Age-dependent variations in biological and biochemi- higher than that of water. Although venom secretion of ju- cal features of venom extracts have been frequently reported venile specimen was colorless, they were light yellow in ma- by different researchers (Fiero et al. 1972, Theakston & Reid ture individuals. The gel photographs of the venom protein 1978, Meier & Freyvogel 1980, Mackessy 1988, Furtado et al. samples of M. albizona specimens are presented in Fig. 1. Ac- 1991, Tun-Pe et al. 1995, Arıkan et al. 2006, 2014, Guércio et cording to the electrophoretic and densitometric analyses, al. 2006). Recently, in addition to the studies related to tax- the venom proteins of M. albizona could be separated into 10, onomy on snake venoms (Arıkan et al. 2003, 2005, 2008, 11, 12 fractions or fraction groups (respectively in juvenile, Göçmen et al. 2006), its biological and proteomic characteri- male and female). Our comparative results of electrophoretic zation have been done in Turkey (İğci & Demiralp 2012, Nalbantsoy et al. 2013, Yalçın et al. 2014). The present study contains data on venom proteins of M. albizona collected from Sivas, Central Anatolia. All venom extracts, obtained from 3 different specimens having different lengths and sex, were analyzed with polyacrylamide disc gel electrophoresis, and their venom electrophoretic patterns were compared.

Figure 1. Polyacrylamide gel electrophoresis of venom proteins of M. Material and Methods albizona of different lengths. A. 38.3 cm (juvenile), B. 60.3 cm (male), C. 73.4 cm (female) (S: Start, junction between the stacking and Montivipera albizona specimens were collected from Gürün (Sivas) at separation gels. The venom proteins of Montivipera albizona 31

protein fractions and densities of the three specimens re- vealed significant both qualitative and quantitative differences (Figs 2, 3, 4).

Discussion

Tun-Pe et al. (1995) found that the venom of juveniles of Russell’s viper, Daboia russelli siamensis was colorless. Similar results were reported in studies on the venom of various viperids (Theakston & Reid 1978, Furtado et al. 1991, Arıkan et al. 2006, 2014). Our observations on the color of the venom of M. albizona are in accordance with the literature (Tun-Pe et al. 1995, Arıkan et al. 2006, 2014). The yellowish color of viper venoms mainly originates from flavin groups of L-amino

acid oxidases (Chippaux 2006). Figure 2. Gel photograph showing the electrophoretic separation of Many studies have documented different levels of varia- the venom protein sample obtained from the 38.3 cm length of M. tion in the composition of venoms, among major and minor albizona juvenile specimen, together with its densitometric tracing curve (O.D.: Optical density, S: Start, junction between the stacking taxonomic groups, between different parts of the same and separation gels). population of one species, during different ages of the animal, and several other factors such as seasonal variation and sex-based variation (Mackessy 2010). According to Mackesy (2010), because venoms are trophic adaptations that facilitate handling of prey, their effects on different organisms are quite variable. Age affects several parameters of venom, most obviously overall yield. It is determined that volume and total dry weight of venom produced increase exponentially with age/size in several species (Mackessy 1988, Mirtschin et al. 2002). Venom ontogeny has been noted for several species, with lower protease activity noted in venoms from neo- nate/juvenile snakes, and age-related differences in composition are apparently more pronounced among viperids. Various studies on age-dependent variations in snake venom proteins showed that younger individuals had more po-

tent venom toxicity, and procoagulant and defibrinating ac- Figure 3. Gel photograph showing the electrophoretic separation of tivity (Fiero et al. 1972, Theakson & Reid 1978, Meier & the venom protein sample obtained from the 60.3 cm length of M. Freyvogel 1980, Furtado et al. 1991, Tun-Pe et al. 1995, Guér- albizona male specimen, together with its densitometric tracing cio et al. 2006, Mackessy et al. 2006, Arıkan et al. 2006, 2014). curve (For further explanation, see caption of Figure 2). The total protein content of venom may also increase with age (Bonilla et al. 1973, Mackessy et al. 2006). Several studies on age-related differences in the venom proteins of different viperids and crotalids showed that the venom of the youngest individuals had fewer protein fractions and the number of the fractions increased with age (Tun-Pe et al. 1995, Arıkan et al. 2006, 2014). The present study indicated that the number of fractions is higher in old- er individuals than in juveniles. Our results support those of Bonilla et al. (1973), Meier & Freyvogel (1980), Tun-Pe et al. (1995), and Arıkan et al. (2006, 2014) on venom proteins of different viperids and crotalids. An isoelectric focused study of venom from Malayan pit viper, Calloselasma rhodostoma noted that one band was present in venoms from females but absent from male venoms (Daltry et al. 1996). Similar results were found in venom composition that exist between male and female South

Figure 4. Gel photograph showing the electrophoretic separation of American Pit Viper, Bothrops jararaca (Menezes et al. 2006). the venom protein sample obtained from the 73.4 cm length of M. Sex-based variation in the venom of M. albizona has been albizona female specimen, together with its densitometric tracing shown in the present study. In accordance with the litera- curve (For further explanation, see caption of Figure 2). ture, one band was present in venom from female but absent 32 A. Mermer et al. from male venom. It was specified that the biological signifi- Furtado, M.F.D., Maruyama, M., Kamiguti, A.S., Antonio, L.C. (1991): cance of these sex-based differences in venom composition Comparative study of nine Bothrops snake venoms from adult female snakes and their offspring. Toxicon 29: 219-226. are unclear, but it is apparent that at least some sex-based Göçmen, B., Arikan, H., Ozbel, Y., Mermer, A., Çiçek, K. (2006): Clinical, differences may be expected in venoms from other species of physiological and serological observations of a human following a reptiles. venomous bite by Macrovipera lebetina lebetina (Reptilia: Serpentes). Türkiye Parazitoloji Dergisi 30(2): 158-62. Venomic characterization studies of Montivipera bul- Göçmen, B., Arıkan, H., Yıldız, M.Z., Mermer, A., Alpagut Keskin, N. (2009): gardaghica and M. raddei, taxonomically close species to M. Serological characterization and confirmation of the taxonomic status of albizona, resulted in the identification of metalloprotease, ser- Montivipera albizona (Serpentes, Viperidae) with an additional new locality record and some phylogenetical comments. Animal Biology 59: 87-96. in protease, phospholipase A2, L-amino acid oxidase, cyste- Göçmen, B., Mebert, K., İğci, N., Akman, B., Yıldız, M.Z., Oğuz, M.A., Altın, Ç. ine rich secretory protein, C-type lectin and vascular endo- (2014): New locality records for four rare species of vipers (Reptilia: thelial growth factor as major protein families (Nalbantsoy et Viperidae) in Turkey. Zoology in the Middle East 60(4): 306-313. Guércio, R.A.P., Shevchenko, A., López-Lozano, J.I., Paba, J., Sonsa, M.V., al. 2017). Similar protein content of M. albizona could be ex- Ricart, C.A.O. (2006): Ontogenetic variations in the venom proteome of the pected. Individual variations of these proteins may affect the Amazonian snake Bothrops atrox. Proteome 4: art.11. pathology of their venom. Although one individual is not İğci, N., Demiralp, D. (2012): A preliminary investigation into the venom enough to make a reliable venom comparison, our data proteins of Macrolebetina lebetina obtusa (Dwigubsky, 1832) from SoutheasternAnatolia by MALDI-TOF mass spectrometry and comparison could be considered as a preliminary result and shows the of venom protein profiles with Macrovipera lebetina lebetina (Linnaeus, 1758) potential variation. Further studies with larger sample size from Cyprus by 2D-PAGE. Archives of Toxicology 86: 441-451. will reveal the variation in more detail. Mackessy, S.P. (1988): Venom ontogeny in the Pacific rattlesnakes Crotalus viridis helleri and C.v. oreganus. Copeia 1988: 92-101. Mackessy, S.P. (2010): Thrombin-like enzymes in snake venoms. pp. 519-557. In: Kini. R.M., Clemetson, K.J., Markland, F.S., McLane, M.A., Morita, T. (eds.). Toxins and Hemostasis. Springer, Dordrecht Heidelberg. Acknowledgment. The data contained in this study were reached Meier, J., Freyvogel, T.A. (1980): Comparative studies on venoms of the fer-de- within the scope of a project entitled “The species action plan of lance (Bothrops atrox), carpet viper (Echis carinatus) and spitting cobra (Naja Montivipera albizona in Sivas”, funded by the Directorate of Sivas nigricollis) snakes at different ages. Toxicon 18: 661-662. Menezes, M.C., Furtado, M.F., Travaglia-Cardoso, S.R., Carmago, A.C.M., Branch of the 15th Regional Directorate of the Department of Nature Serrano, S.M.T. (2006): Sex-based individual variation of snake venom Protection and National Parks of the Republic of Turkey, Ministry of proteome among eighteen Bothrops jararaca siblings. Toxicon 47: 304–12. Forestry and Water Affairs. We would like to thank the Mirtschin, P.J., Shine, R., Nias, T.J., Dunstan, N.L., Hough, B.J., Mirtschin, M. establishment concerned and Ak-Tel Mühendislik for the support to (2002): Influences on venom yield in Australian tigersnakes (Notechis the study. scutatus) and brownsnakes (Pseudonaja textilis: Elapidae, Serpentes). Toxicon 40: 1581–92. Mulder, J. (1994): Additional information on Vipera albizona (Reptilia, Serpentes,

Viperidae). Deinsea 1: 77-83. Nalbantsoy, A., Baykan-Erel, Ş., Köksal, Ç., Göçmen, B., Yıldız, M.Z., Karabay References Yavaşoğlu, N.Ü. (2013): Viper venom induced inflamation with Montivipera xanthina (Gray, 1849) and the anti-snake venom activities of Artemisia Arıkan, H., Kumlutaş, Y., Türkozan, O., Baran, İ. (2003): Electrophoretic absinthium L. in rat. Toxicon 65: 34-40. patterns of some viper venoms from Turkey. Turkish Journal of Zoology 27: Nalbantsoy, A., Hempel, B-F., Petras, D., Heiss, P., Göçmen, B., Iğci, N., Yildiz, 239-242. M. Z., Süssmuth, R. D. (2017): Combined venom profiling and cytotoxicity Arıkan, H., Göçmen, B., Mermer, A., Bahar, H. (2005): An electrophoretic screening of the Radde's mountain viper (Montivipera raddei) and Mount comparison of the venoms of a colubrid and various viperid snakes from Bulgar Viper (Montivipera bulgardaghica) with potent cytotoxicity against Turkey and Cyprus, with some taxonomic and phylogenetic implications. human A549 lung carcinoma cells. Toxicon 135: 71-83. Zootaxa 1038: 1-10. Nilson, G., Andrén, C., Flärdh, B. (1990): Vipera albizona, a new mountain viper Arıkan, H., Alpagut Keskin, N., Çevik. İ.E., Ilgaz, Ç. (2006): Age-dependent from central Turkey, with comments on isolating effects of the Anatolian variations in the venom proteins of Vipera xanthina (Gray, 1849) (Ophidia: “Diagonal”, Amphibia-Reptilia 11: 284-294. Viperidae). Turkiye Parazitoloji Dergisi (Acta Parasitologica Turcica) 30(2): Pimenta, D.C., Prezoto, B.C., Konno, K., Melo, R.L., Furtado, M.F., Carmago, 163-165. A.C.M., Serrano, S.M.T. (2007): Mass spectrometric analysis of the individual Arikan, H., Göçmen, B., Kumlutaş, Y., Alpagut-Keskin, N., Ilgaz, Ç., Yildiz, M. variability of Bothrops jararaca venom peptide fraction. Evidence for sex- Z. (2008): Electrophoretic characterisation of the venom samples obtained based variation among the bradykinin-potentiating peptides. Rapid from various Anatolian snakes (Serpentes: Colubridae, Viperidae, Elapidae). Communications in Mass Spectrometry 21: 1034–1042. North-Western Journal of Zoology 4(1): 16-28. Stümpel, N., Rajabizadeh, M., Avcı, A., Wüster, W., Joger, U. (2016). Phylogeny Arıkan, H., Göçmen, B., İğci, N., Akman, B. (2014): Age-Dependent Variations and diversification of mountain vipers (Montivipera, Nilsonet al., 2001) In The Venom Proteins of Vipera kaznakovi Nikolsky, 1909 and Vipera triggered by multiple Plio–Pleistocene refugia andhigh-mountain ammodytes (Linnaeus, 1758) (Ophidia: Viperidae). Turkish Journal of topography in the Near and Middle East. Molecular Phylogenetics and Zoology 38: 216-221. Evolution 101: 336-351. Bonilla, C.A., Faith, M.R., Minton, S.A. (1973): L-amino acid oxidase, Tare, T.G., Sutar, N.K., Renapurkar, D.M. (1986): A study of snake venom yield phosphodiesterase, total protein and other properties of juvenile timber by different methods of venom extraction. Amphiba-Reptilia 7: 187-191. rattlesnake (C.v. horridus) venom at different stages of growth. Toxicon 11: Theakston, R.D., Reid, H.A. (1978): Changes in the biological properties of 301-303. venom from Crotalus atrox with aging. Periodicum Biologorum 80(Suppl. 1): Chippaux, J.P. (2006): Snake venoms and envenomations. Krieger Publishing 123-133. Company, Malabar, FL, USA. Tun-Pe, Nu-Nu-Lwin, Aye-Aye-Myint, Kyi-May-Htwe, Aung-Cho (1995): Daltry, J.C., Wüster, W., Thorpe, R.S. (1996): Diet and snake venom evolution. Biochemical and biological properties of the venom from Russell’s Viper Nature 379: 537–540. (Daboia russelii siamensis) of varying ages. Toxicon 33: 817-821. Davis, B.J. (1964): Disc electrophoresis - II. method and application to human Yalçın, H.T., Özen, M.O., Göçmen, B., Nalbantsoy, A. (2014): Effect of Ottoman serum proteins. Annals of the New York Academy of Sciences 121: 404-427. viper (Montivipera xanthina (Gray, 1849) venom on various cancer cells and Fiero, M.K., Seifert, M.W., Weaver, T.J., Bonilla, C.A. (1972): Comparative study on microorganisms. Cytotechnology 66: 87-94. of juvenile and adult prairie rattlesnake (Crotalus viridis viridis) venoms. Toxicon 10: 81-82.