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Abstract The age of an animal, determined by time (chronological age) as well as genetic and environmental factors (biological age), influences the likelihood of mortality and reproduction and thus the animal’s contribution to population growth. For many long-lived species, such as bats, a lack of external and morphological indicators has made determining age a challenge, leading researchers to examine genetic markers of age for application to demographic studies. One widely studied biomarker of age is telomere length, which has been related both to chronological and biological age across taxa, but only recently has begun to be studied in bats. We assessed telomere length from the DNA of known-age and minimum known-age individuals of two bat species using a quantitative PCR assay. We determined that telomere length was quadratically related to chronological age in big brown bats (Eptesicus fuscus), although it had little predictive power for accurate age determination of unknown-age individuals. The relationship was different in little brown bats (Myotis lucifugus), where telomere length instead was correlated with biological age, apparently due to infection and wing damage associated with white-nose syndrome. Furthermore, we showed that wing biopsies currently are a better tissue source for studying telomere length in bats than guano and buccal swabs; the results from the latter group were more variable and potentially influenced by storage time. Refinement of collection and assessment methods for different non-lethally collected tissues will be important for longitudinal sampling to better understand telomere dynamics in these long-lived species. Although further work is needed to develop a biomarker capable of determining chronological age in bats, our results suggest that biological age, as reflected in telomere length, may be influenced by extrinsic stressors such as disease.