Ontogenetic Variation in Biological Activities of Venoms from Hybrids between Bothrops erythromelas and Bothrops neuwiedi Snakes

Abstract

Lance-headed snakes are found in Central and South America, and they account for most snakebites in Brazil. The phylogeny of South American pitvipers has been reviewed, and the presence of natural and non-natural hybrids between different species of Bothrops snakes demonstrates that reproductive isolation of several species is still incomplete. The present study aimed to analyze the biological features, particularly the thrombin-like activity, of venoms from hybrids born in captivity, from the mating of a female Bothrops erythromelas and a male Bothrops neuwiedi, two species whose venoms are known to display ontogenetic variation. Proteolytic activity on azocoll and amidolytic activity on N-benzoyl-DL-arginine-p-nitroanilide hydrochloride (BAPNA) were lowest when hybrids were 3 months old, and increased over body growth, reaching values similar to those of the father when hybrids were 12 months old. The clotting activity on plasma diminished as hybrids grew; venoms from 3- and 6-months old hybrids showed low clotting activity on fibrinogen (i.e., thrombin-like activity), like the mother venom, and such activity was detected only when hybrids were older than 1 year of age. Altogether, these results point out that venom features in hybrid snakes are genetically controlled during the ontogenetic development. Despite the presence of the thrombin-like enzyme gene(s) in hybrid snakes, they are silenced during the first six months of life.

Fig 1. Illustrative geographical distribution of Bothrops erythromelas (yellow area) and Bothrops neuwiedi (red area) in Brazil.

Areas of sympatria are shown in cyan. Distribution data were based on [39]. Brazil map was obtained from OpenStreetMap contributors (http://www.openstreetmap.org/).

Fig 2. Photographs of hybrid snakes.

At the top of the figure, the B. erythromelas (Be, ♀) and the B. neuwiedi (Bn, ♂) that mated and originated F1 hybrids (Hy) (photographed at 36 months old). At the bottom of the figure is shown two 2-month-old individuals originated from the mating between Hy# 11 and Hy# 03.

Fig 3. Growth of F1 hybrids determined by weight gain over time.

The weights for the father and mother are also shown in the figure for comparison.

Fig 4. Enzymatic activities of venom samples from the mother (B. erythromelas), the father (B. neuwiedi) and hybrid progeny over time.

Venom samples were tested for coagulant activity on plasma (blue bars) and fibrinogen (red bars), amidolytic activity (orange bars) and collagenolytic activity (green bars). Data from the mother and father are expressed as mean ± standard error of mean of individual results obtained from venom samples extracted over the period of hybrid growth.

Fig 5. Comparative electrophoretic profile of non-reduced venom samples (20 μg/lane) run in 12% SDS-PAGE gels from individual hybrid snakes over their development.

For comparison, the venom pools from the mother, father and hybrids at 3 and 6 months old (mo), and at >2 years old (yo) are also shown. Molecular mass markers are shown on the right. Gels were silver stained.

Fig 6. Comparative distribution of protein spots in two-dimensional gel electrophoresis of individual male (Hy# 03, Hy# 08 and Hy# 15) and female (Hy# 06, Hy# 10 and Hy# 11) hybrids at 3 and 24 months old (mo).

The images of the biological mother (B. erythromelas) and father (B. neuwiedi) pooled venoms are depicted in Fig 7. Gels were run under identical conditions and silver stained. For comparison, boxes were drawn over images of Hy# 03 to facilitate comparison: green dashed boxes: acidic proteins (molecular mass: 50–125 kDa, pI 4.0–6.0); blue dashed box: neutral proteins (molecular mass: 23–25 kDa, pI 6.5–7.0).

Fig 7. Two-dimensional gel electrophoresis profiles of pooled venoms from the mother (B. erythromelas), adult hybrids (> 2 years old) and the father (B. neuwiedi).

Gels were run under identical conditions and silver stained. Selected spots were excised from the gels and analyzed by MS.

Fig 8. Analyses of venom samples (15 μg, non-reduced conditions) by SDS-PAGE (a) and Western blotting (b).

Venom samples: mother pool (B. erythromelas, lane 2); pooled venoms from hybrids at 3 and 6 months old (lane 3), and adult hybrids (>2 years old) (lane 4); father pool (B. neuwiedi, lane 5); pooled venom from B. atrox (lane 6, positive control); and individual venom samples from hybrid # 15 at 6 (lane 7), 12 (lane 8), 24 (lane 9), and 36 (lane 10) months old. SDS-PAGE gels were silver stained [45]. Numbers on the left correspond to the position of molecular mass markers. For Western blotting, nitrocellulose membranes were incubated with anti-recombinant batroxobin (1/2500), and developed as described in Materials and Methods. The arrow indicates the band with molecular mass equivalent to native batroxobin.