Aprimoramento do protocolo de DNA metabarcoding para aplicação na identificação de áreas prioritárias à conservação da ictiofauna do rio Mogi-Guaçu
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2021-05-21
Autores
Costa, Gabriela Omura da
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Universidade Estadual Paulista (Unesp)
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Apesar do Brasil possuir a maior riqueza de peixes do mundo, esta diversidade encontra-se ameaçada por diversos impactos antrópicos, sendo o barramento de rios um dos mais contundentes. Barramentos podem reduzir a diversidade local de peixes por meio de alterações físico-químicas da água, facilitação de introdução e proliferação de espécies não nativas, reestruturação de comunidades, afetando principalmente espécies migradoras de longa distância e favorecendo as sedentárias por consistir numa barreira física, muitas vezes intransponível, prejudicando o recrutamento de novos indivíduos. O conhecimento de períodos reprodutivos, áreas de desova e crescimento são extremamente importantes para a conservação de peixes, visto que tais informações podem subsidiar a delimitação de áreas prioritárias à conservação, auxiliando ações de manejo executadas por entidades federais ou estaduais. Contudo, a taxonomia tradicional apresenta limitações na identificação do ictioplâncton, visto que ovos e larvas apresentam poucos caracteres diagnósticos formados, dependendo do estágio de desenvolvimento. Num primeiro momento, para sanar esse problema, o uso de análises moleculares, baseada no sequenciamento de Sanger, utilizando o gene mitocondrial citocromo c oxidase subunidade I (COI), se mostrou uma ferramenta altamente resolutiva para essa identificação. Entretanto, essa técnica apresenta alto custo e demanda muito tempo de laboratório, quando necessária aplicação em estudos extensivos. Num segundo momento, com o avanço das técnicas de sequenciamento, o sequenciamento de nova geração, utilizando a plataforma Illumina Miseq apresentou potencial para estudos do ictioplâncton, reduzindo tempo e custo, além de aumentar a capacidade a ser analisado e mantendo a resolutividade, porém ainda carecendo de aprimoramentos. Os principais objetivos deste estudo foram i) estabelecer um protocolo de DNA metabarcoding aplicado a peixes, efetivo e de baixo custo (capítulo 1); ii) identificação resolutiva de todos os indivíduos coletados (capítulo 2); iii) identificar áreas de desova e crescimento de peixes na bacia do rio Mogi-Guaçu, buscando melhor entendimento da dinâmica reprodutiva (capítulo 2). Diante deste cenário, foram conduzidas coletas em seis pontos de um trecho do rio Mogi-Guaçu, em dois ciclos reprodutivos dos anos de 2017 a 2019 (novembro a fevereiro). O material coletado foi submetido a um sequenciamento massivo, utilizando nova metodologia com 4 dois conjuntos de primers, para obtenção do fragmento completo do gene COI (653pb). Com os resultados obtidos pudemos constatar que i) o protocolo aplicado foi capaz de gerar fragmentos com mais de 600 pb que possibilitaram a identificação a nível de espécie, diminuindo expressivamente os custos (capítulo 1); ii) a técnica de DNA metabarcoding possibilitou a identificação de 28 táxons a nível de espécie, com grande resolutividade, permitindo a identificação de três espécies que não estavam presentes em estudos de revisão anteriores para a bacia (Astyanax biotae, Pinirampus pirinampu e Sorubim lima) (capítulo 2); iii) há uma distribuição do ictioplâncton condizente com rios neotropicais, com prevalência de ovos nos trechos superiores e larvas nos trechos inferiores, com preferência de espécies de grandes migradores pelo ponto 2 (capítulo 2). Concluímos que os resultados se mostraram promissores na ampliação do conhecimento sobre a ictiofauna em geral, podendo fundamentar ações de manejo mais eficazes para conservação e também para modelo em estudos futuros nessa área de pesquisa.
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.
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.
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Palavras-chave
Ictioplâncton, NGS, Manejo pesqueiro, Recursos pesqueiros, Ichthyoplankton, Fishing management, Fishing resources