Case study

Inventory and detection of elasmobranchii at La Réunion

The Mascareignes Archipelago is one of the main hotspots of biodiversity in the Indian Ocean. Little is known about Elasmobranchii, which are cartilaginous fishes such as sharks and rays, thus limiting conservation mesures to be set up. In order to complete the taxonomic data obtained by classic inventories from the Observatory of Elasmobranchii of the Mascareignes Archipelago (MAEO), the ARBRE association (Research Agency for Biodiversity at La Réunion) called on ARGALY to obtain the most exhaustive information as possible thanks to the environmental DNA method.

ARGALY's mission:

Optimize a non-invasive, reproducible and standardized protocol for the environmental monitoring of sharks and rays around La Réunion.

First, a pilot study was performed to define the optimal analysis strategy:

Sampling methodology

Which marine area to sample to maximize elasmobranchii detection?

Which water volume to collect?

Molecular analysis methodology

Which primers to use for elasmobranchii identification through metabarcoding?

Specific primer design for species detection through qPCR.

To answer these technical questions, water and sediment samples were collected on three stations around the island Réunion. The sediments were sampled on the ocean floor, and water filtrations were performed with 0.45µm Waterra capsules, on the surface and in the water column. Shark tissue samples served as positive controls.

Aquatic sampling on the water surface (left) and in the water column (right).

After extraction of the DNA contained in the samples, three genetic markers of elasmobranchii were tested for the PCR amplification and metabarcoding inventory : Shark_miniCOI (Fields and al. 2015), Elas01 (Miya and al. 2015) et Elas02 (Taberlet and al. 2018). Simultaneously, six specific primer pairs were conceived or optimized in silico then tested in vitro for the qPCR detection of four shark species and two ray species, selected according to their status on the threatened species red list settled by the International Union for the Conservation of Nature (UICN).

Pilot study results

The sediments turned out to be unappropriate as sampling matrix for the detection of elasmobranchii.

2. The filtration of 50L of surface water combined to 50L of column water seems to allow the detection of complementary taxa compared to the filtration on the surface only.

3. A multi_marker approach is necessary for the detection of sharks and rays. Elas01 detects a high diversity of skeletal fishes and cetaceans, as well as some uneasy-observable shark and ray species, such as the white tip shark and the sagrin shark. Elas02 allows to obtain the highest richness in target taxa. Shark_miniCOI showed a weak performance for the detection of elasmobranchii and was not selected for the global inventory phase.

4. The conceived qPCR primers proved to be functional for the specific detection of target species: tiger shark (Galeocerdo cuvier), nurse shark (Nebrius ferrugineus), scalloped hammerhead shark (Sphyrna lewini), bull shark (Carcharhinus leucas), giant guitar ray (Rhynchobatus djiddensis), spotted stingray (Taeniura meyeni).

These preliminary results paved the way for a global inventory of elasmobranchii on 21 stations around the island. The objective was to evaluate the potential of the eDNA technology to map the whole ichtyofauna compared to classic inventories data, and to compare the metabarcoding and qPCR methods for the identification of six target species. In total, 72 water filtrations were performed on three distinct seasons (March, June, September). The metabarcoding analyses were performed using the Elas01 and Elas02 markers.

Localization of sampling stations for the eDNA taxonomic analyses around La Réunion.

Global inventory results

The sequencing data allowed to draw, through bioinformatic analysis, the list of identified taxa for each sample with the associated number of sequences. The maps below represent the Fish and Elasmobranchii detected taxa by the Elas01 and Elas02 markers, according to the station localization:

Fish families identified through eDNA metabarcoding around La Réunion.

Elasmobranchii taxa detected through eDNA metabarcoding around La Réunion. The circle size is proportional to the number of identified sequences.

Elas01 assessed the ichtyologic diversity with the detection of 38 fish families and 114 species. This marker also identified six ray and four shark species.
Elas02 also detects a large number of skeletal fish taxa (21 families with 64 species) as well as a total of fifteen elasmobranchii species (nine ray and six shark species).
There was no significant difference between the detection performances of the target species through metabarcoding and qPCR, the data obtained by the two methods were consistent.
The inventories from direct observation and from eDNA gave similar results for common species such as eagle rays, black spotted stingrays and whip-rays. However, the two methods are complementary concerning the less common species of the waters of La Réunion.
The elasmobranchii distribution around the island seems to vary according to the East/West orientation and the seasons. For instance, the whip-rays were more detected between January and March, and the presence of the guitar rays was higher during austral winter despite detections all year long.

Study conclusion

The eDNA tool confirms and completes the data obtained from the MAEO program: the majority of visually observed species were detected by eDNA and some species that were invisible during diving or by cameras are identified thanks to eDNA. This project, in collaboration with ARGALY,therefore provided an eDNA expertise for the monitoring of rays and sharks at La Réunion to participate to a better knowledge of elasmobranchii biodiversity in the Mascareignes Archipelago. It is thus paving the way for the implementation of preservation actions .

Hammerhead shark