OnThursday, December 8, 2017, Valentin Vasselon, PhD student in Biodiversity, Ecology, and Environment atthe Alpine Center for Research on Trophic Networks in Limnic Ecosystems (CARRTEL), will defend his thesis entitled "Barcoding and Bioindication: Development of Metabarcoding of Diatoms for the Assessment of Watercourse Quality." The defense will take place at 9 a.m. in the videoconference room of the CARRTEL/INRA joint research unit in Thonon-les-Bains.
thesis abstract
Diatoms are microscopic single-celled algae that are excellent indicators of the ecological status of their environment. Under the Water Framework Directive (WFD), diatom communities are used to assess the quality of watercourses. To do this, quality indices based on the sensitivity of species to pollution are calculated from the composition and relative abundance of diatom taxa. Species identification is usually carried out under a microscope, which, in addition to being complex, can be quite time-consuming and costly when dealing with large numbers of samples. A new method recently developed makes it possible to identify species not on the basis of their morphological variability, but on the basis of their genetic variability using short DNA sequences (or DNA barcodes). Combined with high-throughput sequencing technologies, this molecular approach, known as metabarcoding, makes it possible to identify all the species present in an environmental sample and to process several hundred samples in parallel.
These advantages make metabarcoding an attractive alternative to morphological identification methods in the context of the WFD. Although several studies have demonstrated the ability of this approach to correctly identify diatom species found in environmental samples, the lack of reliability in quantifying relative species abundances limits the calculation of reliable indices and the use of metabarcoding as a tool for bioindication. The objectives of this thesis were therefore (i) to identify and optimize the biases affecting the relative quantification of diatoms in metabarcoding; (ii) to apply the molecular approach on a large scale to environmental samples from river networks in order to compare the quality assessments obtained by morphological and molecular approaches.
First, we evaluated the quantification biases associated with DNA extraction from pure cultures of diatoms and environmental samples. Although the choice of extraction method affects the quality and quantity of the extracted DNA, as well as the relative abundances of certain species obtained by metabarcoding, the community composition and quality scores are not significantly affected. We therefore decided to use the GenElute method for the rest of the work, as it produces the largest quantities of DNA at the lowest cost. Secondly, through qPCR experiments carried out on pure diatom cultures, we showed that the number of copies of the rbcL gene (used as a DNA barcode) is proportional to the biovolume of the cells, which results in an overestimation of species with large biovolumes in metabarcoding. Based on this correlation, a correction factor was proposed and applied to metabarcoding data from artificial communities and environmental samples, making it possible to obtain relative species abundances comparable to those obtained by microscopy and to improve the reliability of quality scores.
Finally, applying the molecular approach to the WFD monitoring networks for watercourses in Mayotte and mainland France has shown that metabarcoding is a faster and more economical alternative to the morphological approach, while still providing a good assessment of watercourse quality. Our work confirms that the molecular approach can be used to assess water quality. However, further studies will need to be carried out before considering routine application and implementation in the WFD, particularly in terms of standardization and normalization of the methods used in the molecular approach.