Fish eDNA metabarcoding from aquatic biofilm samples: Methodological aspects.

Fish eDNA metabarcoding is usually performed from filtered water samples. The volume of filtered water depends on the study scope and can rapidly become time consuming according to the number of samples that have to be processed. To avoid time allocated to filtration, passive DNA samplers have been used to recover fish eDNA from marine environments faster. In freshwater ecosystems, aquatic biofilms were used to catch eDNA from macroinvertebrates. Here, we test the capacity of aquatic biofilms to entrap fish eDNA in a large lake and, therefore, the possibility to perform fish eDNA metabarcoding from this matrix compared to the traditional fish eDNA approach from filtered water samples. Methodological aspects of the use of aquatic biofilms for fish eDNA metabarcoding (e.g. PCR replicates, biological replicates, bioinformatics pipeline, reference database and taxonomic assignment) were validated against a mock community. When using biofilms from habitats sheltered from wind and waves, biofilm and water approach provided similar inventories. Richness and diversity were comparable between both approaches. Approaches differed only for rare taxa. Our results illustrate the capacity of aquatic biofilms to act as passive eDNA samplers of fish eDNA and, therefore, the possibility to use biofilms to monitor fish communities efficiently from biofilms. Furthermore, our results open up avenues of research to study a diversity of biological groups (among which bioindicators as diatoms, macroinvertebrates and fish) from eDNA isolated from a single environmental matrix reducing sampling efforts, analysis time and costs.

Exploring the capacity of aquatic biofilms to act as environmental DNA samplers: Test on macroinvertebrate communities in rivers.

Aquatic biofilms are heterogeneous assemblages of microorganisms surrounded by a matrix of extracellular polymeric substances (EPS). Recent studies suggest that aquatic biofilms can physically act as sorptive sponges of DNA. We took the opportunity from already available samples of stone biofilms and macroinvertebrates specimens collected in parallel at the same sites to test the capacity of biofilms to act as DNA samplers of macroinvertebrate communities in streams. Macroinvertebrate communities are usually studied with metabarcoding using the DNA extracted from their bodies bulk samples, which remains a time-consuming approach and involves the destruction of all individual specimens from the samples. The ability of biofilms to capture DNA was explored on 19 rivers sites of a tropical island (Mayotte Island, France). First, macroinvertebrate specimens were identified based on their morphological characteristics. Second, DNA was extracted from biofilms, and macroinvertebrate communities were targeted using a standard COI barcode. The resulting morphological and molecular inventories were compared. They provided comparable structures and diversities for macroinvertebrate communities when one is working with the unassigned OTU data. After taxonomic assignment of the OTU data, diversity and richness were no longer correlated. The ecological assessment derived from morphological bulk samples was conserved by the biofilms samples. We also showed that the biofilm method allows to detect a higher diversity for some organisms (Cnidaria), that is hardly accessible with the morphological method. The results of this study exploring the DNA signal captured by natural biofilms are encouraging. However, a more detailed study integrating more replicates and comparing the biodiversity signal based on both morphological and molecular bulk macroinvertebrate samples to the one captured by biofilms will be necessary. Better understanding how the DNA signal captured by natural biofilms represents the biodiversity of a given sampling site is necessary before considering its use for bioassessment applications.

DNA metabarcoding and microscopic analyses of sea turtles biofilms: Complementary to understand turtle behavior.

Sea turtles are distributed in tropical and subtropical seas worldwide. They play several ecological roles and are considered important indicators of the health of marine ecosystems. Studying epibiotic diatoms living on turtle shells suggestively has great potential in the study of turtle behavior because diatoms are always there. However, diatom identification at the species level is time consuming, requires well-trained specialists, and there is a high probability of finding new taxa growing on turtle shells, which makes identification tricky. An alternative approach based on DNA barcoding and high throughput sequencing (HTS), metabarcoding, has been developed in recent years to identify species at the community level by using a DNA reference library. The suitabilities of morphological and molecular approaches were compared. Diatom assemblages were sampled from seven juvenile green turtles (Chelonia mydas) from Mayotte Island, France. The structures of the epibiotic diatom assemblages differed between both approaches. This resulted in different clustering of the turtles based on their diatom communities. Metabarcoding allowed better discrimination between turtles based on their epibiotic diatom assemblages and put into evidence the presence of a cryptic diatom diversity. Microscopy, for its part, provided more ecological information of sea turtles based on historical bibliographical data and the abundances of ecological guilds of the diatom species present in the samples. This study shows the complementary nature of these two methods for studying turtle behavior.