Unraveling Scent Signals to Protect African Wild Dogs
Although chromatographys versatility leads to its application to a host of diverse problems, helping to protect endangered African wild dogs from conflicts with people is perhaps not one that you would expect. With a grant from the Paul G. Allen Family Foundation, the Botswana Predator Conservation Trust (BPCT) has established a GC/MS laboratory to identify the chemical signals that African wild dogs use to mark their territory boundaries. The ultimate aim is to use artificial scent marks as "BioBoundaries" to limit movements by wild dogs into areas where they come into conflict with people and their livestock.
The BPCT BioBoundary project is led by Dr. John "Tico" McNutt, who has been studying wild dogs since 1989, on the fringe of the Moremi Game Reserve and the Okavango Delta in northern Botswana. The GC/MS laboratory is located in the village of Maun, just 65 km from the BPCT study area, so that it can keep in close contact with field operations.
Figure 1 African wild dogs: highly social superpredators.
African wild dogs (Lycaon pictus) are intensely social predators. They live in packs of up to 27 adults and yearlings, in which usually only one pair breeds but everyone cares diligently for the pups. Numbering less than 6,000, they are one of Africas most endangered carnivores, and their habitats are increasingly threatened by the expansion of human activities. Because wild dog packs have huge territories, only the very largest of protected wildlife areas can sustain viable populations. In Africa, wildlife areas with free-ranging carnivores are often separated from people and their livestock by only a line on a map or fences that are easily penetrated. Predators in livestock areas threaten peoples livelihoods and the dogs usual fate is to be shot, snared, or poisoned. The aim of the BPCT BioBoundaries project is to deploy artificial territorial scent marks, formulated with chemicals identified in natural wild dog marks, along protected area boundaries to create "virtual" neighboring packs that will deter dogs from crossing into areas where they are at risk. The stakes are high—population models predict that wild dogs will be extinct in the wild in 50 years, unless new ways are found to protect them.
Wild dogs, like nearly all mammals, live in a world dominated by odors. Airborne chemical signals, known as semiochemicals, play critical roles in their sexual and social behavior. The packs dominant pair assiduously overmark each others feces and urine, and these double marks stake out the packs territory.
Chemically, mammal scents are bafflingly complex, with the active messenger compounds at trace levels among hundreds of other components. Quantities of active compounds range down to picograms and concentrations of 10-18 molar. Nonetheless, mammal chemical signals are within range of gas chromatography and mass spectrometry, as long as the technology is used to its full potential. Maximum resolution and reproducibility along with minimum contamination, discrimination, and limits of detection are required so that biological differences are not obscured by analytical artifacts and variability.
Sample preparation is both the most critical step and the Achilles heel. To preserve the integrity of the signal I have to sample what the dogs do: the volatiles in the air around a scent mark. Solid phase microextraction (SPME) and adsorption/thermal desorption looked promising, but yielded too many peaks from contaminants and too few from wild dogs. A simpler system was required to reduce contamination, variability, and analytical artifacts. Direct thermal desorption from urine-marked soil and cryotrapping with sample flow paths of glass and fused silica has provided the cleanest chromatograms so far. In nature, the scent marks are still active on hot, dry sand; therefore, samples can be dried prior to desorption to prevent icing of the cryotrap and then desorbed at 60°C.
The complexity of most mammal odors puts them well inside the Giddings zone, where at least 20% of chromatographic peaks overlap; not surprisingly, a dog mark chromatogram is so complex it has no clean baseline. Overlapping peaks cannot be properly quantified or identified and most failures to find an MS library match are due to coelutions that produce a mixed mass spectrum—only a minority of those without matches are new and, therefore, exciting compounds. To get cleanly resolved peaks I will be using two-dimensional GC to transfer incompletely separated peaks from one column to another column with complementary selectivity.
Identifying everything in scent mark odor is unnecessary and impractical; the spotlight needs to fall on the few compounds that send the message. The critical challenge then is to differentiate the biologically relevant signal from the chemical noise, and this is where close links between the laboratory and the field operations play an absolutely critical role. Only dominant dogs produce territorial marks, so the signaling compounds will be present in their marks, but absent from subordinates marks. The marks withstand 65K temperature differences in the soil substrate between midwinter midnights and summer afternoons. The marks last for at least six weeks and their emissions of territorial semiochemicals should be stable for at least as long. Without a detailed behavioral and social context for each sample it would be impossible to recognize the semiochemicals among the forest of extraneous peaks.
The wild dog boundary semiochemicals have to stand out against a background of the millions of natural chemicals that permeate the environment, and so I expect them not to be common constituents of mammal scent marks, feces or urine, or volatiles from plants or soil. Library searches of integer resolution mass spectra will eliminate compounds that are known to come from these sources.
Now that the sampling and separation conditions are worked out, in the months to come I will be running scent mark samples from several dogs in different packs searching for a peak, or a pattern of peaks that is present only in the marks of dominant animals, that stays the same with time and temperature, and that is not part of the environmental background. When I find it (or them) the next challenge will be to identify the compound(s). That will be a story for another time.
Peter Apps runs the BPCT Paul G. Allen Family Foundation Wildlife Chemistry Laboratory in Maun, northern Botswana. He is a zoologist with a long career in chromatography, a rare combination that led him back to his zoological roots to set up the laboratory in July 2008.