Proteomic analysis of ontogenetic and diet-related changes in venom composition of juvenile and adult Dusky Pigmy rattlesnakes (Sistrurus miliarius barbouri). 
Snake venom proteins show high levels of variation at the level of the individual yet the environmental and molecular mechanisms that generate this diversity remain unclear.  Here we report the results of a controlled feeding experiment combined with proteomic analyses of periodically collected venom samples to assess the roles of ontogenetic and diet-related effects on venom composition of captive juvenile and adult Dusky Pigmy rattlesnakes (Sistrurus miliarius barbouri).  Juvenile snakes fed from birth with mice, lizards, or frogs showed little evidence for an ontogenetic shift in venom composition from 5 to 26 months in terms of substantial changes in the relative abundance of major classes of venom toxins.  However, there were fine-scale changes in the relative abundance of D49-PLA₂ 15, PI-SVMPs, and PIII-SVMP 28, and a decline in the abundance of other PIII-SVMPs.  Although juveniles raised on different prey exhibited distinct relative toxin compositional change rates, at 26 months old, their venoms showed similar patterns of protein composition suggesting little effect of diet on the overall make-up of venom in snakes this age or younger.  In contrast, adult females raised on different prey over a 26 month period showed prey-related changes in the relative abundance of major protein families from initial to final samples.  Specifically, mouse-fed females showed substantial increases in the relative abundance of total PLA₂s and serine proteinases of 95% and >100%, respectively, whereas comparable values for lizard- (42% and -22%) and frog-fed females (2% and 11%) were distinctly smaller in magnitude.  Venom from adult snakes fed on different prey also showed distinct changes in the abundance of PLA₂ molecules 15, 19a, and 19, which were, respectively, (>100%, 33%, 63%), (>100%, 0%, 35%), and (71%, 20%, -4%) for the mice-, lizard-, and frog-diet.  Venom from snakes raised on frogs contained a small (1.1%) but consistent amount of a PLA₂ molecule (15a) not present in snakes fed on mice or lizards.  This work provides evidence that venom composition is somewhat plastic in both juvenile and adult S. m. barbouri and that, at least in adults, prey consumed may influence the relative abundance of possibly functionally-distinct classes of venom proteins.