Improved eDNA Primer Enhances Rockfish Species Identification in Alaska

A groundbreaking new environmental DNA (eDNA) tool is set to transform how scientists monitor rockfish populations. Rockfish are important to both commercial and recreational fisheries in Alaska. By analyzing DNA collected from water samples, this tool provides unprecedented accuracy in identifying a wide range of rockfish species. This- includes those that are closely related and hard to tell apart using previously available genetic methods.

Finding and identifying different species of rockfish in the ocean can be surprisingly tricky. Many of them have patchy distributions and live in rocky habitats making them difficult to survey using nets. While eDNA has become a popular method to sample fish biodiversity, previous methods were unable to tell apart many of the most abundant rockfish species in Alaska waters. But a new genetic tool, the SebastesD-loop primer set, is changing that.

DNA primers match a target sequence in the eDNA and allow researchers to amplify species of interest. Primers are designed to identify multiple species of fishes, marine mammals, invertebrates, sharks, or any other organisms that might be in a water sample. In a recent study, NOAA Fisheries scientists demonstrate that the primer set can successfully identify nearly all rockfish species in Alaska waters. It offers a more accurate and efficient way to monitor these commercially important fish. A few closely related species still require extra effort to distinguish. But, this new approach marks a big step forward in monitoring rockfish populations and protecting their habitats.

Using eDNA to Identify Rockfish

Rockfish are a highly diverse group with more than 110 species worldwide. They are a vital component of nearshore and offshore ecosystems in the Eastern North Pacific. In Alaska, species like Pacific Ocean perch, northern rockfish, and dusky rockfish support significant fisheries. However, monitoring these populations has been challenging, especially in rocky, "untrawlable" habitats where conventional survey methods like trawl nets cannot be used.

"A key goal of eDNA research is to use genetic data to inform management and conservation of wild populations," explains Kim Ledger, research biologist with NOAA's Auke Bay Laboratories and lead author on the study. "While eDNA metabarcoding holds immense promise, a frequent limitation has been its inability to differentiate closely related species of interest."

Earlier eDNA tools often had trouble telling certain rockfish species apart. For instance, they could not reliably distinguish between black and yellowtail rockfish, or between northern, dusky, and dark rockfish-all key species in Alaska's rich rockfish diversity. The new "SebastesD-loop" primer set changes that by focusing on a highly variable region of mitochondrial DNA.

Mitochondrial DNA is much more abundant than nuclear DNA because there are many mitochondria in each cell, but only one copy of nuclear DNA. It provides an abundant genetic marker that increases the chances of detection from environmental samples. Some sequences of mitochondrial DNA change relatively quickly over time, creating distinct genetic markers that act like a fingerprint for each species. This makes it especially useful in eDNA studies, such as those used to identify and track closely related rockfish species.

Verifying Effectiveness of the New Method

The research team validated the effectiveness of the new primer set through a series of tests. Using tissue samples from adult rockfish, they were able to accurately identify nearly all of the 42 rockfish species tested-showing the tool's precision for species identification. Using eDNA extracted from water samples, the primer set also successfully amplified rockfish DNA from controlled (aquarium) and uncontrolled (trawl survey) environments.

"Our results show that the SebastesD-loop primer set can accurately detect different rockfish species," says Ledger. "This makes it a powerful tool for improving species identification and relatedness in eDNA studies, and reliably monitoring and assessing rockfish populations."

The study also addressed a crucial aspect of eDNA analysis: species-specific amplification bias. Species-specific amplification bias happens when the DNA primers used in testing bind and amplify the DNA of some species more efficiently than others. As a result, the final DNA sample may not accurately reflect the true mix of species in the environment. This makes it harder for scientists to estimate the relative proportion of each species present. By using "mock community" samples with known proportions of different rockfish DNA, the researchers quantified species-specific biases. This step is essential for future quantitative applications, allowing scientists to correct for these biases and obtain more accurate estimates of species abundance.

The SebastesD-loop primer set will immediately offer opportunities for eDNA to aid fisheries management. The study authors acknowledge that some species pairs, such as dusky and dark rockfish, remain indistinguishable at this specific genetic marker because they are closely related. However, the authors suggest that incorporating additional genetic data could further refine identification for these challenging groups.

This new eDNA primer set represents a powerful addition to the fisheries management toolkit. It offers a less-invasive and more comprehensive way to assess rockfish populations, particularly in hard-to-reach habitats. The ability to accurately identify these vital species at a higher resolution will provide critical data for ensuring the long-term sustainability of Alaska's rockfish fisheries.