Mie University develops a method to estimate dolphin age based on fecal DNA using methylation rates

Estimating the age of dolphins and other long-lived wild animals is extremely difficult because the methods commonly used for such estimations involve observational studies over a time period longer than the animal's life span and also requires capture of the animals. A research group comprising PhD Student Genfu Yagi, Professor Tadamichi Morisaka, and Professor Motoi Yoshioka (currently Executive Director of Mie University) of the Graduate School of Bioresources at Mie University, Professor Miho Inoue-Murayama of the Wildlife Research Center and PhD Students Huiyuan Qi and Kana Arai of the Graduate School of Science at Kyoto University, Associate Professor Yuki F. Kita of the Department of Marine Biology and Sciences of the School of Biological Sciences at Tokai University, and Mr. Kazunobu Kogi of Mikurashima Tourism Association have developed a method for estimating the age of dolphins from the DNA information contained in their feces.

This is the first successful case of epigenetic clock analysis on DNA from non-invasive samples, such as feces, from wild aquatic animals. In addition to helping to clarify and conserve the ecology of the Indo-Pacific bottlenose dolphin, this could be a first step toward application of the technique to other wildlife. The study results have been published online in Molecular Ecology Resources.

Studies in humans have shown that there are aging-related changes that occur in the epigenetic mechanisms that regulate gene transcription and translation without alteration of the DNA sequence. Among other methods, DNA methylation can be used to estimate human age based on the fact that its abundance changes with age. This method has recently been applied to wildlife. However, there were few examples of studies using samples that could be collected without touching the individuals.

The research group applied this method to DNA extracted from the feces of wild dolphins. Unlike blood or skin, non-invasive samples such as feces have lower concentrations of host DNA, are more easily fragmented, and contain substances that inhibit analysis. Additionally, it was not even known if this method could be used on wild dolphins, since DNA degradation is more likely to occur in an underwater environment.

To estimate age, feces should be collected from individuals of known actual age, and the methylation rate of DNA extracted from the feces should be measured to determine its correlation with age.

Over the past 29 years, approximately 150 wild Indo-Pacific bottlenose dolphins living around Mikurashima, Tokyo, have been individually identified through body scars and other clues. This information is maintained by the Mikurashima Tourist Association. Using this identification information, the actual age of dolphins born during this period can be calculated since the year of birth is generally known. Additionally, dolphin swims are held on Mikurashima, and people can see scenes of dolphins defecating in the water.

For the study, the researchers went into the water with a small action camera and a polyethylene tube and photographed the dolphins defecating as well as collected their feces. In the laboratory, DNA was then extracted from the fecal sample, and the methylation rates of two gene regions (GRIA2 and CDKN2A) were measured. Of the DNA extracted from 61 fecal samples, methylation rates were available for 36 samples from 30 individuals.

The research group observed a correlation between the methylation rate and the actual age of the animals. Age estimation was achieved for Indo-Pacific bottlenose dolphins, with an average error of approximately 5 years for a life span of close to 50 years. The fact that age estimation could be accomplished using feces collected in an environment unsuitable for DNA preservation suggests that the method is feasible not only for dolphins and whales but also for land mammals.

Such age estimation methods using non-invasive samples are expected to provide age information from rare species as well as large, difficult-to-capture ones. The ability to obtain age information will enhance our understanding of the life history and other ecology of the species. By combining the life history of animals with their population pyramids, the future extinction risk of the species can be statistically predicted and conservation measures considered. It is hoped that age estimation from feces will promote understanding of the ecology of wild animals and, thereby, better coexistence with humans.

Journal Information
Publication: Molecular Ecology Resources
Title: Non-invasive age estimation based on faecal DNA using methylation-sensitive high-resolution melting for Indo-Pacific bottlenose dolphins
DOI: 10.1111/1755-0998.13906