Improvement of the DNA metabarcoding protocol for application in the identification of priority areas for the conservation of the ichthyofauna of the Mogi-Guaçu river

Author(s)

Gabriela Omura da Costa

• Document type: Master's Dissertation
• Press: Botucatu. 2021-09-21.
• Institution: Universidade Estadual Paulista (Unesp). Instituto de Biociências. Botucatu
• Defense date: 2021-05-21
• Advisor: Cláudio de Oliveira

Abstract

Despite the fact that Brazil has the greatest richness of fish in the world, this diversity is threatened by several anthropic impacts, with damming of rivers one of the most notable. Dams can reduce the diversity of local fish through physical-chemical changes in the water, facilitating the introduction and proliferation of non-native species, restructuring communities, affecting mainly long distances migratory species and favoring sedentary species, consisting of a physical barrier, frequently impassable, hindering the recruitment of new individuals. The knowledge of reproductive periods, spawning and growth areas are extremely important for the conservation of fish, since such information can subsidize the delimitation of priority areas for conservation, assisting management actions carried out by federal or state entities. However, the traditional taxonomy has limitations in the identification of ichthyoplankton, since eggs and larvae have few formed diagnostic characters, depending on the stage of development. At first, to solve this problem, the use of molecular analysis, based on Sanger sequencing, using the mitochondrial cytochrome c oxidase subunit I (COI) gene, proved to be a highly resolving tool for this identification. In a second step, with the advancement of sequencing techniques, the new generation sequencing, using the Illumina Miseq platform, presented potential for studies on ichthyoplankton, reducing time and cost, in addition to increasing the capacity to be analyzed and maintaining resolvability, but requiring enhancements. The main objectives of this study were i) to establish an effective and low cost DNA metabarcoding protocol applied to fish (chapter 1); ii) resolutive identification of all individuals sampled (chapter 2); iii) identify spawning and fish growth areas in the Mogi-Guaçu River basin, seeking a better understanding of reproductive dynamics (chapter 2).. In view of this scenario, catches were carried out at six points on a stretch of the Mogi-Guaçu River, in two reproductive cycles from 2017 to 2019 (November to February). The collected material was subjected to massive sequencing, using a new methodology with two sets of primers, to obtain the complete fragment of the COI gene (653bp). The results obtained showed that i) the applied protocol was able to generate fragments with more than 600 bp that enabled identification at the species level, significantly reducing costs (chapter 1); ii) the DNA metabarcoding technique enabled the identification of 28 taxa at the species level, with 6 high resolution, allowing the identification of three species that were not present in previous review studies for the basin (Astyanax biotae, Pinirampus pirinampu and Sorubim lima) (chapter 2); iii) there was a ichthyoplankton distribution consistent with Neotropical rivers, with the prevalence of eggs in the upper stretches and larvae in the lower stretches, with preference for species of great migrators by point 3 (chapter 2). We conclude that the results have shown promise in expanding knowledge about ichthyofauna in general, being able to support more effective management actions for conservation and also for models in future studies in this area of research. (AU)

• FAPESP's process: 19/03597-4 - Use of DNA metabarcoding in the identification of priority areas for the conservation of Neotropical ichthyofauna
• Grantee: Gabriela Omura da Costa
• Support Opportunities: Scholarships in Brazil - Master