Identifying the roles of different hosts and vectors is a major challenge in studies of the ecology of diseases caused by multi-host pathogens. Intensive field studies in northern Colorado suggested that northern grasshopper mice (Onychomys leucogaster) help spread the bacterium that causes plague (Yersinia pestis) in prairie dog colonies by sharing fleas with black-tailed prairie dogs (Cynomys ludovicianus) and transporting infected fleas across colonies; yet conclusive evidence that prairie dog fleas (Oropsylla hirsuta) feed on grasshopper mice is lacking. We used stable isotope analysis and molecular genetic methods (PCR) to identify the blood meals of fleas combed from rodents and swabbed from prairie dog burrows.
Stable nitrogen isotope values of the blood of prairie dogs, which are herbivores, were significantly depleted in 15N compared to those of carnivorous grasshopper mice. The intermediate nitrogen isotope values of pooled O. hirsuta combed from grasshopper mice suggested that these fleas fed on both grasshopper mouse and prairie dog blood. Nitrogen isotope values of O. hirsuta from burrows indicated that they contained diminishing blood meals from prairie dogs. Amplification of the cytochrome b gene of grasshopper mice and prairie dogs, using species-specific primers in a standard PCR assay, revealed that 57% of individual O. hirsuta combed from mice contained mouse DNA, indicating definitively that O. hirsuta can feed on grasshopper mice. However, no host DNA could be identified in 43% of O. hirsuta combed from mice, or from 230 O. hirsuta swabbed from burrows. We speculate that O. hirsuta digests blood of prairie dogs quickly, resulting in the degradation of host DNA that precludes its identification using conventional PCR assays unless fleas are collected soon after feeding. Our results suggest that, by providing information on older or time-integrated feeding events, stable isotope analysis can complement other molecular or immunological approaches in studies of host-parasite relationships.