Identification and phylogeny of Arabian snakes: Comparison of venom chromatographic profiles versus 16S rRNA gene sequences

Abstract

Identification of snake species is important for various reasons including the emergency treatment of snake bite victims. We present a simple method for identification of six snake species using the gel filtration chromatographic profiles of their venoms. The venoms of Echis coloratus, Echis pyramidum, Cerastes gasperettii, Bitis arietans, Naja arabica, and Walterinnesia aegyptia were milked, lyophilized, diluted and centrifuged to separate the mucus from the venom. The clear supernatants were filtered and chromatographed on fast protein liquid chromatography (FPLC). We obtained the 16S rRNA gene sequences of the above species and performed phylogenetic analysis using the neighbor-joining method. The chromatograms of venoms from different snake species showed peculiar patterns based on the number and location of peaks. The dendrograms generated from similarity matrix based on the presence/absence of particular chromatographic peaks clearly differentiated Elapids from Viperids. Molecular cladistics using 16S rRNA gene sequences resulted in jumping clades while separating the members of these two families. These findings suggest that chromatographic profiles of snake venoms may provide a simple and reproducible chemical fingerprinting method for quick identification of snake species. However, the validation of this methodology requires further studies on large number of specimens from within and across species.

1. Introduction

It has been estimated that about 2.5 million people are annually affected by snake bites around the world and more than 4% of the victims lose their lives (Calvete et al., 2007). There are approximately 2700 species of snakes, of which only 20% are venomous (Mebs, 2002). Different snake species possess different venom profiles in terms of their composition and toxicity. By virtue of evolutionary adaptation, more closely related species of snakes exhibit more similarities in their venom composition. The venoms from Elapidae and Viperidae snakes are complex mixtures containing different components such as metalloproteinases, proteolytic enzymes, phospholipase, serine proteinase, presynaptic and postsynaptic neurotoxins, potassium channel-binding neurotoxins, cytotoxins, cardiotoxins and platelet aggregation inhibitors (Tu, 1988; Meier and Stocker, 1991; Fry, 2005).

Since venom contains a mixture of peptides and proteins secreted by a specific gland, analysis of venom components can produce a valuable fingerprint that can be used as a valuable reference tool in taxonomic analysis, as a complementary method to morphology and behavioral characterization for species identification and classification (Newton et al., 2007). A comparative proteomic analysis has shown that compositional differences between snake venoms can be employed as a taxonomy signature for unambiguous species identification independently of geographic origin and morphological characteristics (Tashima et al., 2008). Proteomics-guided identification of evolutionary and immunoreactivity trends among homologous and heterologous venoms may aid in the replacement of the traditional geographic- and phylogenetic-driven hypotheses for antivenom production strategies by a more rational approach based on a hypothesis-driven system venomics approach (Calvete, 2013). Both SDS–PAGE and PAGE profiles of venoms from different snake species indicate that some proteins and polypeptide components of these venoms have common electrophoretic characteristics suggesting a genetic relationship (Mendoza et al., 1992).

In continuation to a previous work on chemical fingerprinting of scorpion venoms (Al Asmari et al., 2012), we compared the chromatographic profiles of four species from the family Viperidae (Echis coloratus, Echis pyramidum, Cerastes gasperettii and Bitis arietans) and two species from the family Elapidae (Naja arabica and Walterinnesia aegyptia) with special reference to their application in species identification. We also compared to phylogenetic trees constructed using the similarity matrix generated from chromatographic data as well as 16S rRNA gene sequences.

2. Materials and methods

2.1. Snakes and venom collection

We collected 6 species of snakes (Table 1) including E. coloratus, E. pyramidum, C. gasperettii, B. arietans, N. arabica and W. aegyptia from the different regions of Saudi Arabia. The snakes were kept in plastic boxes and fed on mice and water ad libitum. After milking, the crude venom was diluted with distilled water, properly mixed, and centrifuged at 10,000 rpm at 4 °C for 20 min to separate the mucus. The clear supernatant was filtered through a 0.20 μm filter. The protein concentration was determined at Abs 280/260 nm using a spectrophotometer (Shimadzu UV-160A) and the collected filtrate (mucus-free venom) was stored at −20 °C until used.

Table 1.

Taxonomic classification of six snake species used in this study.

Class	Reptilia	Reptilia	Reptilia	Reptilia	Reptilia	Reptilia
Order	Squamata	Squamata	Squamata	Squamata	Squamata	Squamata
Suborder	Serpentes	Serpentes	Serpentes	Serpentes	Serpentes	Serpentes
Family	Viperidae	Viperidae	Viperidae	Viperidae	Elapidae	Elapidae
Subfamily	Viperinae	Viperinae	Viperinae	Viperinae	-	Elapinae
Genus	Bitis	Cerastes	Echis	Echis	Naja	Walterinnesia
Species	arietans	gasperettii	coloratus	pyramidum	arabica	aegyptia

2.2. FPLC

Fast protein liquid chromatography (FPLC) or gel filtration chromatography was used for venom fractionation on a Superdex 200 PC 3.2/30 column. Venom solution was diluted in 0.05 M sodium phosphate buffer containing 0.15 M NaCl (pH 7.0). An aliquot (25 μL) of venom solution (final concentration 10 mg/mL) was loaded in a previously equilibrated column with the same buffer. The venom components were eluted at a flow rate of 0.4 mL/min while the column operational pressure was 1.5 MPa. The protein elution profile was monitored at 280 nm by a UV spectrophotometer (AKTA Micro System). The void volume (V_o) of the column was determined by using Blue Dextran (2 mg/mL in an equilibration buffer containing 3% sucrose). The total volume of elution up to the fraction having maximum absorbance was considered as the elution volume of the protein (V_e). The elution volumes of different toxins were determined under similar conditions. K_av values were calculated using the equation: K_av = V_e − V_o/V_t − V_o. The chromatographic profiles were tested in triplicate to confirm the reproducibility of the method.

All the K_av values were sequentially arranged and a matrix was created using the presence or absence of the corresponding peaks in the venom of different species (Table 2). The similarity/distance matrix was used to create a tree showing the similarity of venom profiles from different snake species.

Table 2.

Similarity/distance matrix based on the chromatographic profiles of snake species.

Peak No.	Kav	Bitis arietans	Cerastes gasperettii	Echis coloratus	Echis pyramidum	Naja arabica	Walterinnesia aegyptia
1	0.32	1	0	0	1	0	1
2	0.37	0	1	1	1	1	0
3	0.50	0	0	0	0	1	0
4	0.57	1	1	1	0	0	0
5	0.60	0	0	1	1	0	1
6	0.67	1	0	0	1	0	1
7	0.73	0	0	0	0	1	1
8	0.77	0	0	1	0	0	1
9	0.90	1	1	1	1	0	0
10	1.03	1	0	0	0	1	1
11	1.14	1	0	0	0	0	0
12	1.46	0	0	0	0	0	1

1 = Peak present; 0 = Peak absent.

2.3. Phylogenetic analysis

We obtained the sequences of 16S ribosomal RNA gene of all the 6 snake species from the GenBank. The GenBank accession numbers are as follows: GQ359726 (E. coloratus), GQ359724 (E. pyramidum), HQ267809 (C. cerastes), GQ359737 (B. arietans), GQ359749 (N. arabica) and HQ267785 (W. aegyptia). The sequences were aligned by Clustal W and subjected to phylogenetic analysis using the neighbor-joining method.

3. Results

All the snake species showed peculiar chromatographic profiles of their venoms depending on the location and height of the peaks (Fig. 1). A total of 12 chromatographic peaks were observed including the unique and common peaks shared by different venoms. The minimum numbers of peaks were observed with the venom of C. gasperettii (3 peaks) and the maximum number of peaks with the venom of W. aegyptia (7 peaks). Both the members of the genus Echis (E. coloratus and E. pyramidum) showed 5 peaks each (3 common peaks) whereas the venoms of N. arabica and B. arietans resulted in 4 and 6 peaks respectively (Fig. 1).

Figure 1.

Chromatographic profiles of venoms from different snake species.

The similarity matrix based on the presence or absence of chromatographic peaks is given in Table 2. Venom profile-based chemical fingerprinting clearly differentiated the two species of the family Elapidae (N. arabica and W. aegyptia) from the members of the family Viperidae (Fig. 2). The venom profile of E. coloratus was more closely related to the venom profile of C. gasperettii instead of E. pyramidum (Fig. 2).

Figure 2.

Relationship among different snake species using their venom profiles. Light and dark shades confine the members of Elapidae and Viperidae families, respectively.

Molecular cladistics using 16S rRNA gene sequences failed to separately group the members of the two families (Fig. 3). Two species (N. arabica and W. aegyptia) of the family Elapidae formed a jumping clade surrounded by the members of the Viperidae family. Within the family Viperidae, the two species of genus Echis (E. coloratus and E. pyramidum) formed a single clade. However, the remaining two species of the family Viperidae, B. arietans and C. cerastes appeared to be more closely related to the members of the family Elapidae rather than the member of the genus Echis (Fig. 3).

Figure 3.

Phylogenetic relationship among different snake species using their 16S rRNA gene sequences. Light and dark shades confine the members of Elapidae and Viperidae families, respectively.

4. Discussion

The results showed distinctive chromatographic profiles of all the six snake venoms studied (Fig. 1). Intraspecific variation in the venom components has been previously studied in different venomous species such as wasps (Mulfinger et al., 1986), scorpions (Abdel-Rahman, 2008) and snakes (Chippaux et al., 1991). John and Kaiser (1990) compared the venoms from the tiger snakes Notechis scutatus scutatus, Notechis ater serventyi, Notechis ater humphreysi and Notechis ater ater using gel filtration resulting in slightly different elution profiles on a Superose-12 gel filtration column. The protein profile of venoms of Elapidae was identified using the electrofocusing technique; the two species could easily be differentiated whereas the differences between the two sub-species were more difficult to evidence (Pichon-Prum et al., 1990). The elution profiles of the venoms of seven Bothrops species fractionated on a Mono-Q FPLC column resulted in reproducible chromatograms however there was a considerable overlap of active proteins in different species venoms (Leite et al., 1992). In this study, we presented a protocol for creation of a similarity or distance matrix (Table 2) using the presence/absence of respective chromatographic peaks and transformation of the cumulative information for construction of cladograms that clearly separated Elapids from Viperids (Fig. 2).

Several investigators have used venom components and venom delivery to construct a phylogenetic pattern in snakes (Minton, 1986; Kochva, 1987; Minton and Weinstein, 1987). The mode of venom injection into the target differs among the groups of snakes; Viperids possess fangs located on short, rotating maxillae in the front of the mouth whereas Elapids contain fixed fangs often followed by several teeth on elongate maxillae. Heise et al. (1995) have suggested that Viperids diverged prior to the separation of Elapids and Colubrids. To address the present phylogenetic distribution of front-fanged venom delivery systems, it is more likely that such a system evolved early in the evolutionary history of the advanced snakes and later was lost in the colubrid lineage (Underwood and Kochva, 1993). An alternative explanation is that Viperids and Elapids independently evolved front-fanged systems (McDowell, 1986; Cadle, 1988; Knight and Mindell, 1994).

Phylogenetic analysis using mitochondrial 16S rRNA gene sequences did not produce distinctive clades for Elapids and Viperids (Fig. 3). The two species of Elapidae were grouped in a sister clade with the two species of the genus Echis (family Viperidae) whereas the remaining two species of the family Viperidae placed distantly (Fig. 3). A high genetic divergence has been reported earlier among vipers despite having low morphological differentiation (Ursenbacher et al., 2008). An earlier phylogenetic study based on 12S rRNA and 16S rRNA gene sequences revealed that Viperids were monophyletic and formed the sister group to the Elapids and Colubrids; within the Viperids, two monophyletic groups were identified as true vipers and pit vipers plus Azemiops (Heise et al., 1995). Lenk et al. (2001) used cytochrome b and 16S rRNA sequences to consistently identify five major monophyletic groups in true vipers including Bitis, Cerastes, Echis, Atherini and Eurasian viperines. Keogh et al. (1998) have pointed out that intrageneric divergences are almost as large as intergeneric divergences among the elapids while the sequence data of cytochrome b and 16S rRNA cannot fully resolve phylogenetic relationships among the Australian elapids, though some close relationship can be observed among the species. In a mammalian study, both cytochrome b and control region segments appeared to be independent indicators of the phylogenetic relationships (Khan et al., 2008a). Mitochondrial DNA (mtDNA) has a relatively fast mutation rate, which results in a significant variation in mtDNA sequences between species and in principle, a comparatively small variance within species (Khan et al., 2008b). Mitochondrial protein-coding genes are regarded as useful markers for genetic diversity analysis at lower categorical levels, including families and genera (Arif and Khan, 2009; Arif et al., 2011) whereas the control region exhibits a higher level of variability than 16S rRNA and protein-coding sequences that render it more suitable for identification of species and subspecies (Arif and Khan, 2009).

In conclusion, each snake’s venom has a unique chromatographic profile that can be used as a fingerprint to differentiate one species from the other. Our findings suggest that FPLC of snake venom is a simple and reproducible method for identification of snake species. We also proposed a protocol for constructing a similarity matrix using the information of the presence/absence of respective peaks and creation of dendrograms for cladistics interpretation. The chromatographic profiles of venoms not only differentiated Elapids from Viperids but also provided distinctive fingerprints for individual species. However, the verification of this protocol requires additional studies using a large number of inter- and intra-specific venom samples.