Wednesday, January 13, 2016

New Year, New Research: The Ecology of Venom Resistance

My field site, the Blue Oak Ranch Reserve, in winter

I will be graduating with my Ph.D. this spring, but that doesn’t mean I will be done working on the squirrel-rattlesnake system. One aspect that deserves further attention is plasticity in ground squirrel venom resistance—in other words, does resistance to rattlesnake venom change within a squirrel based on its environment?  First, let me give you a quick recap.

California ground squirrels, along with many other small mammals that are preyed upon by or that eat venomous snakes (woodrats, mongoose, opossum, honey badger), have evolved innate resistance to venom toxins. Resistance consists of proteins that circulate in the prey’s blood. These proteins latch onto different types of venom toxins in different ways, thereby inhibiting the negative effects associated with envenomation: hemorrhaging, tissue digestion, and disruption of blood pressure. There are some common misconceptions regarding venom resistance in ground squirrels, which I will dispel here.

  1. Resistance is acquired through repeated non-lethal bites by snakes. In small mammals the only resistance factors that have been isolated to date are innately expressed factors (meaning the animals are born with them without having been previously exposed to venom). In large domestic mammals (pigs, sheep, cows) you can get an induced response that is the basis for antibody production for use in antivenom. There is no evidence for this in small mammals that are preyed upon by snakes.
  2. Adult squirrels are essentially immune to venom and squirrel pups are the ones vulnerable to snake bites.  Because of their large body sizes, adults are better at fighting off the effects of venom than pups, but they can still die or become seriously injured if bit. Envenomation is a major threat to any squirrel, even if it doesn’t kill them. Resistance factors minimize damage from venom toxins, but do not completely eliminate their effects. Thus, envenomation can result in a temporary reduction in bodily function, enhanced risk of secondary infection, and increased predation risk from other animals such as coyotes, foxes, or birds of prey. At my field site, I’ve seen several adult squirrels being consumed by rattlesnakes, so they are definitely not immune to venom.
Now you know more about me!

Now that we have that out of the way, onto new and exciting work! To date, we know little about the presence or pattern of variation in resistance within or among prey populations because most studies focus on the molecular basis of venom neutralization. This means that we don’t know what’s going on in the wild—how does the environment affect venom resistance? All we really know is that squirrels’ ability to withstand envenomation varies among populations depending on rattlesnake density (squirrels from high snake density populations are more resistant than squirrels from low snake density populations), and that squirrels are better able to fight off the venom of their local rattlesnake species than foreign species. But what about within a population? Is resistance effectively the same for all squirrels within a population and does it change over time?



Populations of California ground squirrels that have been studied for venom resistance levels. On the right, we have a cladogram which shows
genetic distances between populations, whether they experience high, medium, or low snake densities (represented by arrows to the right of
city names), and their ability to inhibit rattlesnake venom (numbers to the right of arrows). Populations from high snake density areas (in red)
tend to have higher resistance than populations from low snake density areas (in blue). Taken from Biardi (2008).


These are some questions I am interested in answering. In 2013, I collected some preliminary data as part of a collaborative project I am doing with Matt Holding in the Gibbs Lab at Ohio State University. I drew blood from several of my study subjects, which we already had data on their behavior and stress hormone levels (measured as fecal glucocorticoid metabolites). We first found that individuals significantly differed in resistance levels: some had high levels of resistance whereas others had low levels of resistance (one squirrel had basically no resistance at all). The question emerging from this finding was, why? Why are some squirrels “better” than others?


Individual squirrels vary in venom resistance levels--some greatly inhibit venom activity
(bars lower on the graph) while others do not (bars higher on the graph).


So I dug deeper into my data and found an interesting negative relationship between stress hormone production and resistance, meaning that squirrels with higher stress hormones were less able to withstand envenomation. This seems like a logical finding as we often see animals having compromised immune function when they are stressed, although venom resistance is not part of the immune system.

This was an exciting finding, but we can’t really make any grand conclusions from it because we need to determine whether this pattern persists over time. To find one correlation at one point in time doesn’t mean much because stress hormones are constantly changing; for instance they regulate both daily and annual rhythms, and fluctuate based on perceived or encountered challenges. Furthermore, our finding suggests that resistance could be a costly defense to produce. If it is costly, do squirrels reduce venom resistance levels during times when it is not needed? Rattlesnakes mainly feed in the summer in temperate climates, whereas squirrels can be active throughout the year, so I wonder whether venom resistance is lower in winter when snakes are not a threat. Stress hormone levels change seasonally in squirrels so could they be responsible for altering venom resistance levels too?

Negative relationship between stress hormone production (y-axis) and ability to inhibit the
effects of rattlesnake venom metalloproteases in a population of California ground squirrel.
All data taken from wild adults squirrels in summer 2013.

Thus, I went back to my field site after Christmas to collect data on stress hormones (measured as fecal corticosterone metabolites from squirrel feces) and venom resistance (measured from blood samples). I assembled a team of some of my good friends to help me out: Lauren who was one of my field assistants in 2013, her girlfriend Stephanie, and my friend from college Francesca. We were an awesome girl-power field team that rocked at trapping and bleeding squirrels. Because it was so cold, I was initially worried that the squirrel activity would be low and we would have a hard time trapping, but we ended up doing quite well! We trapped a total of 13 unique squirrels over 5 days (we lost one of these days to rain), and we were able to successfully get both fecal and blood samples from 12 of these individuals. One squirrel I had such a hard time drawing blood from, even after repeated attempts, that my collaborator determined it must have a heart two sizes too small (and I agree it was a Grinch). 


Setting up traps to capture squirrels. We baited them with peanut butter and sunflower
seeds, but any type of nut will really work.

Waiting for the squirrels to come. And here's our coordinates in case your interested lol. 

Our girl-power team ready to process squirrels! Once a squirrel is trapped, we usher it into a handling bag (aka pillow case), then knock it
out with anesthetic. Once it is asleep, I draw blood from its tiny little heart. It sounds scary, but it's actually an easy process. We then take
body measurements and give it an ear tag so we can ID it later.

Now all that is left to do is process the samples to get numbers. I work with Dr. Jennifer Smith at Mills College in Oakland, whose lab will extract and assay the fecal corticosterone metabolites, and Matt Holding at Ohio State University, who will measure the inhibition of snake venom proteins from the squirrel blood serum. We hope to see another negative relationship between stress hormone levels and resistance, and to see lower levels of resistance at this time of year. Cross your fingers for good results!



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References:


Biardi, J. E. 2008. The Ecological and Evolutionary Context of Mammalian Resistance to Rattlesnake Venoms. Pages 557–568 in W. K. Hayes, K. R. Beaman, M. D. Cardwell, and S. P. Bush, editors. The Biology of Rattlesnakes. Loma Linda University Press, Loma Linda.

Biardi, J. E., D. C. Chien, and R. G. Coss. 2005. California ground squirrel (Spermophilus beecheyi) defenses against rattlesnake venom digestive and hemostatic toxins. Journal of Chemical Ecology 31:2501–2518.

Biardi, J. E., C. Y. L. Ho, J. Marcinczyk, and K. P. Nambiar. 2011. Isolation and identification of a snake venom metalloproteinase inhibitor from California ground squirrel (Spermophilus beecheyi) blood sera. Toxicon 58:486–493.

Domont, G. B., J. Perales, and H. Moussatche. 1991. Natural anti-snake venom proteins. Toxicon 29:1183–1194.

Moussatche, H., and J. Perales. 1989. Factors underlying the natural resistance of animals against snake venoms 84:391–394.

Perez, J. C., W. C. Haws, V. E. Garcia, and B. M. Jennings. 1978. Resistance of warm-blooded animals to snake venoms. Toxicon 16:375–383.

Poran, N. S., and R. G. Coss. 1990. Development of antisnake defenses in California ground squirrels (Spermophilus beecheyi): I. Behavioral and immunological relationships. Behaviour 112:222–244.  

Poran, N. S., R. G. Coss, and E. Benjamini. 1987. Resistance of California ground squirrels (Spermophilus beecheyi) to the venom of the northern Pacific rattlesnake (Crotalus viridis oreganus): a study of adaptive variation. Toxicon 25:767–777.


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