Scientists have discovered new methods for estimating the ages of native fish without harming endangered populations.

Previously, estimating the age of a fish involved removing the fish’s otoliths, the bones in a fish’s ear. Otoliths grow with the fish and form visible bands each year, and so, similar to a tree ring, the bands can be counted to determine the age of a fish. However, in order to age a fish, scientists had to kill it to get the otoliths. This is not ideal when aging threatened or high value fish species and can limit the accuracy of population level studies. That has all changed now with a new non-lethal and simple genetic ageing method.

Otoliths rings can be used to identify the age of fish, however, the process is lethal for endangered fish species. Source: Bill Brazier, Off the Scale.

Understanding the age structure of a threatened fish population is fundamental to conservation projects. The age structure of a population defines important traits such as life span, age at maturity, mortality and recruitment patterns. Knowing these can help scientists develop conservation tools such as population modelling and species management plans, as well as determining what is a sustainable level of fishing. New research has developed a non-lethal, rapid and cost-effective method to accurately age threatened fish species using DNA samples from fin clips. This breakthrough research has important implications for threatened fish species and is hoped to significantly improve how we understand their population age structures and what is needed to improve conservation and management efforts.

The research was conducted on three key threatened species in Australia, the Australian lungfish, the Murray cod and the Mary River cod:

The Australian lungfish (Neoceratodus forsteri) is a threatened species, endemic to southeast Queensland. Often referred to as a “living fossil” due to its extraordinary evolutionary history in the fossil record and shared common ancestor that evolved long before the emergence of land animals. Ageing lungfish has been difficult as they do not have hard otoliths. A successful method to age lungfish using bomb radiocarbon dating of the scales has been published, however, this method is expensive and variable at older ages and in some cases cannot be used at all.

The Australian lungfish is the older known living vertebrate species and is now threatened. Source: OpenCage.info.

Murray cod (Maccullochella peelii) and Mary River cod (M. mariensis) are part of an iconic group of Australian freshwater cod species which also includes the endangered eastern freshwater cod (M. ikei) and trout cod (M. macquariensis). These species are known for their high economic, scientific and cultural values. Historically otoliths have been used to determine age for these fish, however, population level assessments from lethal age sampling are not common due to the risk of negative impacts on wild populations to acquire the required data.

Mary River cod. Photo credit: Stewart Rowland.
Murray Cod. Photo credit: Rudie H. Kuiter / Aquatic Photographics.

So how can DNA be used to predict age?

DNA in animals undergoes a process called DNA methylation, also known as epigenetic modification. DNA methylation is when there is a change to the DNA that does not change the DNA’s sequence but can result in changes in proteins and genes in a cell. It has recently been shown to be an accurate predictor of age, for a wide range of animal groups, as the methylation often occurs at a steady rate overtime. Measuring the methylation in an animal can thus show its age. These new age models are called epigenetic ‘clocks’. DNA can be sourced from any tissue from the animal, importantly for fish, it can be sourced harmlessly from a small fin clip.

Can age models be used for fish?

We first tested if DNA methylation can predict age in zebrafish (Danio rerio), a model organism commonly used in biomedical research due its short lifespan and ease of captive breeding. We then used DNA methylation levels of zebrafish with known ages to identify the DNA markers that would predict age. The DNA methylation markers identified were then transferred across to the DNA of Australian lungfish, Murray cod and Mary River cod to develop the models for predicting age in those species, and it worked… really well. Using known aged and otolith/radiocarbon aged samples of these species, we were able to calibrate and validate the models.

One of the key challenges with the epigenetic clocks is getting accurate results for older fish. The oldest measured lungfish was 77 years old, whilst the oldest measured Maccullochella was 12 years old. These fish species are known to live well past these ages, with Murray cod living up to 50 years, so more research is required for older ageing accuracy.

An equally rare large ‘grandad’ lungfish can exhibit wide variation in length for age. Being able to age these large fish aids in longevity and mortality estimates, past recruitment dynamics and growth rates. Photo credit: David Roberts, Seqwater.

Epigenetic Clocks offer a conservation management boom

We were able to successfully develop a rapid, cost-effective and non-lethal method for accurately determining the age of three iconic Australian fish species: the Australian lungfish, Murray cod and Mary River cod. Importantly, this DNA-based method has the potential to be widely applied across a broad range of other fish species leading to major improvements in the management and conservation of wild fish populations.

We found not only does our method work across species separated by more than 100 million years of evolution, but that there is real potential for the application of epigenetic clocks across closely-related species using a common ‘Genus clock’. The ‘Maccullochella clock’ developed here, accurately predicted the age for both Murray and Mary River cod equally well, and after some testing we are hopeful it will also work for the other species of Maccullochella such as eastern freshwater and trout cod.

Broader application of DNA ageing in wildlife management

Our work on fish ageing is part of ongoing research to identify biomarkers for wildlife management. We have been able to determine the lifespan of species using DNA, in the same way human population demographers use census data to understand and model human populations. We are continuing to develop and test the application of epigenetic ‘clocks’ across other species and within species groups. We will also be exploring other fundamental population characteristics such as age, sex, stress and life expectancy that are difficult to obtain or unknown for most species. In future, these methods may be used more readily by other researchers to better understand and manage wild animal populations.

The research was a collaboration between CSIRO; Department of Regional Development, Manufacturing and Water (Qld); Seqwater; Department of Primary Industries (NSW); Department of Agriculture and Fisheries (Qld); University of Queensland; and University of Western Australia.

The research “Non-lethal age estimation of three threatened fish species using DNA methylation: Australian lungfish, Murray cod, and Mary River cod” is published in Molecular Ecology Resources. 

Featured image: A young of the year Mary River Cod fingerling that is typically stocked into waterways as part of the MRC Recovery Strategy. Being able to age and release for recapture stocked fish is an important advantage of non-lethal ageing protected species. Photo credit: David Roberts, Seqwater.