Estimation of chronological age of Risso’s dolphin (Grampus griseus) based on DNA methylation frequency

Abstract

Age of cetaceans is an important data set for their conservation and management. Although the chronological age of a toothed whale has traditionally estimated from the number of annual tooth growth layer groups (GLGs), this method has potential difficulties such as errors due to the subjectivity of the age-readers and time-consuming sample processing, in addition to lethal tooth sampling in many cases. To overcome these difficulties through a non-lethal approach, considerable amount of efforts has been made in recent years to construct chronological age estimation models based on the correlation between chronological and biological age expressed as the frequency of DNA methylation at CpG (cytosine-phosphate-guanine) sites. The present study, by using next-generation sequencing on DNA extracted from the skin tissues of 30 individuals (15 males and 15 females), performed methylation analysis on multiple CpG sites in the three genes previously reported as age-linked in cetaceans (GRIA2, TET2, and CDKN2A) to construct an age estimation model for Risso’s dolphins. Key aspects of this study are: (1) the chronological ages of Risso’s dolphins of wild origins used in this study had been determined by the GLGs of teeth prior to this study, and (2) as Risso’s dolphins are said to have a lifespan of 30 years, samples were collected from individuals aged 1–29 years, covering from young to old ages. Among the 11 CpG sites in GRIA2, 6 CpG sites in TET2, and 7 CpG sites in CDKN2A analyzed, the CpG sites in GRIA2 showed a statistically significant correlation with age, whereas those in TET2 and CDKN2A did not show such a strong correlation. Using multiple regression analysis on the CpG sites in the three genes, the best fitted model was expressed as follows: y=-0.7548 + 5.6164x(GRIA2_CpG3)+2.6187x(TET2_CpG6)-1.5907x(CDKN2A_CpG3) (R²=0.7062, residual mean=2.98, standard deviation=2.3). With the mean error of 2.98 years between the estimated and known chronological ages, the accuracy of this model was found to be comparable to or higher than that of previously reported age estimation models for cetaceans. This study demonstrated for the future that the age of wild Risso’s dolphins can be estimated by using their skin samples collected with minimal invasiveness.