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.
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.
Table 1.
Identification of peptides released from in gel trypsin digestion of venom protein spots shown in Fig 7 from pooled adult hybrid snakes (H), the mother (M, B. erythromelas), and the father (F, B. neuwiedi), by LC-MS/MS and database search.
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.