The environmental DNA (eDNA) era for reef fish surveys?
Every time I get to SCUBA dive in Vava’u for my PhD fieldwork, I have to pinch myself to make sure it is for real. Vava’u is the most northern island group in the beautiful Kingdom of the Tonga (Image 1). This archipelago sets halfway between the hotspot for fish diversity called the Coral Triangle, and the south Pacific islands of French Polynesia. I chose this location as the focal point for my research because of the gap in knowledge about their reef fish fauna, and more specifically about cryptobenthic reef fishes (CRFs). Cryptic, because they are good at hiding (Image 2), and benthic because they live on or near a substrate. But the most striking characteristic of these fishes is their very small size. Adults are 1-2 cm long, which is a great adaptation if you want to make use of all the crevices that a coral reef ecosystem provides.
CRFs are extremely abundant, diverse, and form a link in the food-web by consuming the micro-invertebrates and algae in the reef ecosystem, thus transferring this energy to larger reef fishes. While CRF are an essential food resource in coral reef ecosystems, they have not received much attention because they are hard for researchers to see while conducting traditional underwater visual censuses. Currently the only way to determine the CRF community composition is to use ichthyocide or anesthetic stations and collect all the fishes in the habitats. Environmental DNA (eDNA) sampling would be a much easier and non-lethal way to determine what species of CRFs are present. But will this method work?
Environmental DNA sampling is an exciting and relatively new technique which is gaining extreme popularity for a multitude of studies that require detection of organisms from DNA fragments that are shed into the environment. This involves collecting samples of soil, water, even air, and using complex laboratory and bioinformatic analyses to detect what species of organisms are present in the sample. Studies of this kind can be very specific, like detecting presence of invasive species, to very large in scope, like inventorying all the organisms present in a specific environment. For my research question, I collected water samples directly from the reef substrate and concentrated on detecting the presence of a specific taxa (CRFs). This is the first study of its kind, as in situ eDNA samples have never been collected from coral reef habitats in conjunction with fish samples.
My PhD research revolves around phylogenetic and ecological aspects of CRFs in general, with a focus on the genus Eviota, family Gobiidae. During the last visit to Vava’u in December 2018 through January 2019, in part funded by the AAUS Doctoral Scholarship, I wanted to compare how many species of the CRFs I could detect from eDNA samples collected underwater with the actual CRFs I collected from ichthyocide stations.
The most exciting and fun part of this research is that I not only go to a beautiful tropical place, but get to SCUBA dive to collect my samples. Since obtaining my diving certification in the Canary Islands, Spain after my undergraduate degree in Marine Sciences, I have participated in many research projects involving diving, from cold waters of Alaska and Puget Sound, to tropical ones like the Caribbean and Micronesia. I feel very comfortable and safe working underwater. It is a very peaceful feeling, even when you are doing work that sometimes it may be very physically demanding. I feel that all the training I have received, first for my first recreational diving certification from the European organization CMAS, and more recently, AAUS scientific diver status through the training program at the University of Washington, has prepared me very thoroughly for my research endeavors.
I also enjoy the intensity of sampling in the field. One of the things I wanted to test about this new eDNA sampling was the feasibility of conducting this type of operation in remote locations with no scientific research stations. We had to setup our own lab facilities and protocols that would work for this type of research. A typical day in the field would start by getting in the water with my diving partner (and hubby, Dr. Ray Buckley) and all the gear (a lot of gear!) and heading to one of our main
sampling sites. Once we got to an area with suitable coral bommies or coral rubble microhabitats, we would set up our gear on the bottom, and then our very choreographed underwater sampling ballet would start. The first thing was to select a desired microhabitat (either coral bommie or coral rubble), then we would get the modified syringes and extract a water sample right from the interstices of the coral head or the rubble (Image 3). Once we had collected the eDNA sample, we would set up an enclosure surrounding the microhabitat, quinaldine anesthetic was delivered inside the enclosure, and after a couple of minutes the collection would start. An airlift device designed for this project was used to suction the specimens (Image 4), some of them as small as 4 mm! inside labeled jars for each of the microhabitats (Image 4). This way we could make sure we knew which eDNA samples corresponded with each microhabitat and the fishes in it. Once we were back to the surface, the eDNA and fish samples were placed in a cooler with ice and processed as quickly as possible (Image 5).
The results from this testing of eDNA sampling, after processing the samples back at the labs at the University of Washington and the sequencing lab where the final analysis was done, indicated that this eDNA sampling was a poor method for detecting CRFs. Of the overall number of CRF species collected with the ichthyocide stations, about 40, the eDNA samples detected only four. Although it was disappointing to have such low level of detectability with the in situ eDNA sampling technique, these are very important results that define the potential limits of this emerging sampling technique. Negative results in science can sometimes be equally as important as positive findings. Nevertheless, it was encouraging to see that we were able to detect some of the species of CRFs that we collected with the traditional method. We also detected several of larger coral reef fish species, as well as a multitude of invertebrate’s species and other metazoans. My analysis of this eDNA sampling is still ongoing, and the process of going over every step of the procedures followed, from collection to the bioinformatic analysis of the sequenced data, is being scrutinized to determine how future projects using this eDNA technique could improve the detectability of this very important group of fish fauna, the cryptobenthic reef fishes.
I am extremely thankful for the support I received from AAUS for this research. I am also very lucky to have found very collaborative partners from the strong grassroots NGO, Vava’u Environmental and Protection Association, and the Government of Tonga. After I complete my PhD, I intend to continue our long-term collaboration with the local groups in Tonga doing underwater research, and to contribute to the knowledge of the functioning of our changing coral reef ecosystems from the perspective of the smallest fish inhabitants!
University of Washington
2018 AAUS Doctoral Scholarship Recipient