Why is Snake Venom Composition So Variable, bu Wolfgang Wuster at the 8th Bio-Ken International Snakebite Seminar, Watamu - November 2012

1. WHY IS SNAKE VENOM COMPOSITION SO
VARIABLE?
Wolfgang Wüster
Bangor University, School of Biological Sciences, Bangor LL57 2UW, UK

2. What is snake venom?
 Complex cocktails
of numerous
bioactive proteins

3. Variation in venom composition
 Occurs at all
taxonomic levels
 Within individual in
time
 Between individuals
within populations
 Between populations
 Between species &
above
 Can result in
profound clinical
differences and
treatment difficulties

4. Symptoms after Asiatic cobra
bites
% with % with Ratio
neurotoxicity necrosis neurotoxicity
: necrosis
Luzon (Naja
philippinensis ) 97 % 7% 14:1
NW Malaysia –
(Naja kaouthia ) 12 % 44 % 1:4

5. What causes variation in venom
composition?
 Long-standing controversy
 Function of venom: overpowering and killing
prey, ?defence, ?digestion

6. What causes variation in venom
composition?
 Long-standing controversy
 Function of venom: overpowering and killing
prey, ?defence, ?digestion

7. What causes variation in venom
composition?
 Long-standing controversy
 Function of venom: overpowering and killing
prey, ?defence, ?digestion
 Potential causes
 Selection for different prey
 Evolutionary history

8. Electrophoretic profiles of individual
Calloselasma rhodostoma
Daltry, Wüster & Thorpe, 1996

9. Association between venom composition
and causal hypotheses in Calloselasma
rhodostoma
Geographic Phylogeny Diet
distance (gene
flow)
Generalised
composition No No YES
Daltry, Wüster & Thorpe, 1996

10. Variability of venom composition:
Selection pressure or “Overkill”?

11. Variability of venom composition:
Selection pressure or “Overkill”?
 “Overkill-Hypothesis”:
 Snakes have far more venom than required for
killing their prey
 Hence no selective pressure for greater lethality
 BUT: Lethality only known in white mice!!!
 Some prey known to be resistant against snake
venoms

12. Resistance against venom
 Example: Pacific rattlesnake (Crotalus oreganus )
and ground squirrel (Spermophilus beecheyi)
 Squirrels from rattlesnake areas display
increased resistance to venom
Spermophilus beecheyi –
Crotalus oreganus - Pacific rattlesnake California ground squirrel

13. Overkill in snakes?
 Example: Crotalus oreganus – Pacific Rattlesnake
 Venom quantity: ~ 90 mg dry venom
 LD50 in mice: 2.84 mg/kg
 Median lethal potential: 31.7 kg of mice
• LD50 in ground squirrels: ~ 40 mg/kg => 2.25 kg
• => per 250g ground squirrel: ~ 10 mg => 9 ground
squirrels
BUT:
• Venom needs to be lethal in minutes, not 24 hrs
• Reserves?

14. Sea krait
Laticauda
Moray eel
Gymonothorax sp.

15. Sensitivity of prey and non-prey
moray eels to Laticauda sea snake
venoms
Sympatric Allopatric
Gymnothorax Gymnothorax
survived 45-75 died after 0.1
mg/kg mg/kg
Heatwole & Poran, 1995

16. Why fine-tune venom?
 Evidence of the energetic cost of venom:
 Increased metabolic rate of snakes after venom
extraction
 O2 consumption increased by ~ 11% after venom extraction in 3
pitviper species (McCue, 2006)
 Venom metering in snakes
 Loss of venomous function in snakes feeding on
undefended prey

17. Venom metering in Crotalus oreganus
mg venom injected
20
18
16
14
12
Small prey
10
8 Large prey
6
4
2
0
Medium Snakes Large Snakes
Hayes et al., 1995

18. Venom loss in snakes feeding on
undefended prey
 E.g., Aipysurus eydouxii
 Feeds exclusively on fish eggs
 Venom of very low toxicity
 Toxins non-functional
 Venom delivery apparatus
atrophied

19. Saw-scaled Vipers (Echis) as a model system
for studying snake venom variation
 One of the main causes of
snakebite mortality
worldwide
 Documented variability in
venom composition,
incompatible antivenoms
% fatality rates after
Echis ocellatus bites
25
20
15
10
5
0
E.ocellatus E. carinatus
antivenom antivenom

20. ? ?
? ?

23. Echis
ocellatus
Echis
carinatus
Echis
pyramidum
Echis
coloratus
Pook et al. 2009

24. Diet and venom activity in
saw-scaled vipers (Echis)
T. Mazuch

25. Do saw-scaled viper venoms show
evidence of adaptive evolution?
T. Mazuch
Diet recorded from museum
specimens

26. Diet of the four Echis species groups
E. carinatus E. ocellatus
Arthropods
Vertebrates
E. pyramidum E. coloratus

27. Do saw-scaled viper venoms show
evidence of adaptive evolution?
T. Mazuch
Diet recorded from museum
specimens
LD50 of 4 representative
venoms estimated for Scorpio
maurus
Functional significance: time to
incapacitation and death after
realistic venom dose

28. LD50 of 4 Echis venoms on Scorpio maurus
µg venom / g scorpion
n/s P < 0.001 P < 0.001 P < 0.05

29. Arthropod Scorpion
feeding lethality
Bitis arietans – –
Echis carinatus ++ ++
1.00 Echis ocellatus + +
Origin of
arthropod 1.00 Echis pyramidum ++ ++
diet +
arthropod-lethal
venom 0.92
Echis coloratus – –
Loss of arthropod diet
+ arthropod-lethal Repeated co-evolution of diet and specific
venom venom lethality => evidence of selection

30. Functional significance of
specific venom lethality
 More rapid prey death?
 Reduction of metabolic cost of venom?
Test:
 Injection of biologically realistic amounts of venom
(3 mg/g = 25-575 x LD50)
 Record time to loss of coordination and death

31. 80
Death
Loss of coordination
60
Time (minutes)
Time (minutes)
40
20
0
E. p. leakeyi E. c. sochureki E. ocellatus E. coloratus B. arietans

32. Staged encounters
Are these experiments biologically realistic?
 Vipers and scorpions placed together
 Video recording
 Time 1st bite – beginning of feeding

36. Staged encounters
Results:
 3 successful trials with E. c. sochureki
 In all cases 1-2 further bites
 Time bite - feeding: 40-48 Min.

37. Functional significance of
venom variation in Echis
 Greater lethality ≠ more rapid prey death!
 Functional significance:
 Venom economy => reduced metabolic expense?
 Facilitation of scorpion feeding for juveniles?

38. How do they do it?
The genetics of venom adaptation
 Venoms consist of several multigene toxin
families
 How does gene diversity correlate with diet?

39. PIII/PIV
SVMP
sub-classes Induce haemorrhage, inhibit
platelet aggregation
Serine
proteases
Procoagulant, fibrinolytic,
inhibit platelet aggregation
=> Link between diet, toxin gene
Casewell et al., in prep. diversity and toxin function

40. Conclusions: why is venom so
variable?
 Evidence of natural selection on venom
composition – specific lethality to prey
 Resistance in some prey => predator-prey arms
races
 Looking beyond lethality: economics of venom
 Genetic mechanism in saw-scaled vipers:
 Importance of diversification of toxin families
=> Extreme variation in venom composition at
all levels

41. Thank you for your attention !
Cheers!