Asian elephant (Elephas maximus) populations are under significant threat due to poaching, habitat loss, fragmentation, and consequent human-elephant conflict. As a result, they are listed as endangered species on the IUCN Red List since 1986, as the population has declined by at least 50% over the last three generations [1]. To protect the existing populations, intensive conservation efforts are needed which rely on accurate estimation of density and age composition. For endangered species, this is essential for assessing the impact of poaching, habitat, and population management [2]. These mitigation strategies require a reliable estimate of age. Therefore, the ability to determine age is an important ecological tool for conservation and management. Moreover, knowing an individual’s age is useful for understanding ecological traits because as individuals start to age, many biological characteristics such as size, behaviour, and sexual maturity change [3]. Such changes could impact an individual’s ability to survive [4] and reproduce effectively [5]. This in turn affects the population, thus accurately determining age would help estimate population age structure which can indicate past events, and present and future extinction risks [6, 7].
Historically, most age estimation methods required either long-term visual surveys, mark-recapture strategies or examination of dead remains [8–11]. As suggested, most of these methods investigate morphological characteristics and individual tracking. However, this is often time-consuming, and a limited number of species exhibit observable and significant age-related changes.
Currently, the main method to estimate age in Asian elephants is visually estimated by observing their size such as shoulder height [9, 12]. However, this method is prone to gross overestimation especially in long-lived species like the Asian elephants because as they mature slowly, the visual determination of matured adults becomes difficult and subjective [9]. Cranio-dental-based age criteria for Asian elephants have also been developed [13, 14], however, it is only applicable to dead specimens and cannot be applied to living individuals. Other age estimation techniques developed for Asian elephants include eye lens [15], tusk size, ear pigmentation [9], and dung bolus circumference [16, 17]. However, most of these currently employed methods to estimate age in Asian elephant individuals can either be highly subjective, inaccurate, or difficult to carry out in the field. Hence, not a single technique has been adopted universally that is routinely applied to estimate an individual’s age [18]. As such, there is a need to develop an objective and accurate method to estimate the age of Asian elephants. Recently, various molecular ageing markers have been developed to estimate age such as mutation accumulation in mitochondrial or nuclear DNA, changes in mitochondrial DNA copy number, telomere length and DNA methylation [6, 19, 20]. Of these, it is considered that DNA methylation-derived epigenetic clock is a highly promising molecular biomarker of ageing, especially for live animals [21, 22].
DNA methylation is one of the best-studied epigenetic modifications which involves the transfer of a methyl group onto cytosine to form 5-methylcytosine; affecting the activity of a DNA segment without changing the actual sequence [23]. In mammals, it plays a significant role in various biological processes such as regulating gene expression, genomic imprinting, X-chromosome inactivation and ageing [24, 25]. Changes in ageing-associated DNA methylation levels occur in CpG sites (where a cytosine is next to guanine, in the DNA sequence), where clusters of these sites called CpG islands are often present in the gene’s promoter [25]. This change is a factor that determines the level and integrity of gene expression [19, 26] in response to developmental and environmental signals [27]. Most importantly, many studies have reported correlations between DNA methylation levels at specific CpG islands in certain genes and age, mainly in humans [19, 28–30]. However, recently, increasing numbers of studies on other species have been carried out, such as in dogs (Canis familiaris) [31], cats (Felis catus) [32], chimpanzees (Pan troglodytes) [33], bats (Myotis bechsteinii) [34, 35], seabirds (Ardenna tenuirostris) [36], green turtles (Chelonia mydas) [37], fish [38, 39], and several cetacean species [40–43].
DNA methylation studies on Asian elephants are currently lacking. To our knowledge, there is only a single study which used an array-based technology that permits the quantification of methylation levels at specific CpG sites [44]. Although this molecular technique showed an accurate method to estimate age, it may not be the most time- and cost-effective approach, particularly for mammalian species [45], more so when applications to conservation and management strategies, including for existing wild populations are intended. Hence, it is necessary to further develop a reasonably time- and cost-effective molecular technique, specific to Asian elephants.
Methylation-sensitive high-resolution melting (MS-HRM) is a method that measures methylation profiles in a labour-, time- and cost-effective manner [46, 47]. The bisulphite-treated DNA is amplified through PCR followed by melting analysis, where the unmethylated cytosines are converted into uracil during bisulphite conversion while the methylated cytosines remain the same [48, 49]. This allows for different base compositions and information on methylation status is directly converted to the sequence, enabling a quantitative methylation assessment [46, 50]. Using MS-HRM to determine age has only been successfully attempted on humans [46, 50], cats and snow leopards (Panthera uncia) [32]. An age estimation model using MS-HRM specific for Asian elephants would be valuable for future management strategies.
A practical and objective method for the age estimation of Asian elephants would enable a better understanding of their population dynamics [51]. Estimates of these characteristics would overall allow for risk extinction for these iconic, and endangered species to be assessed more accurately [37]. As there is still a lack of information on DNA methylation in Asian elephants, we developed a DNA methylation-based age estimation technique via MS-HRM as an alternative to current age estimation methods. We investigated the relationship between epigenetic modifications in age-related genes and chronological age and its potential to build an age estimation model for Asian elephants. The methylation rate of two candidate epigenetic ageing markers, RALYL and TET2 was measured to assess the following questions: (1) is there a correlation between age and methylation levels? (2) how accurately can chronological age be estimated in Asian elephants using MS-HRM and (3) how does MS-HRM as a method, compare to other species that have also developed MS-HRM based age estimation and other current methods used to estimate age in Asian elephants? Our findings of genes in which the level of methylation relates to age, opens the possibility of using molecular approaches to estimate age in Asian elephants and the future application towards in–situ and ex–situ conservation efforts.