Abstract
The annual killifish Austrolebias charrua is an endangered species, endemic to the southern region of South America, which inhabits temporary ponds that emerges in the rainy season. The main anthropogenic threat driving the extinction of A. charrua stems from extensive agriculture, primarily due to the widrespread use of glyphosate-based herbicides near their habitats. Annual killifishes have been used as models for ecotoxicological studies but, up to now, there are no studies about reference genes in any Austrolebias species. This represents an obstacle to the use of qPCR-based technologies, the standard method for gene expression quantification. The present study aimed to select and validate potential reference genes for qPCR normalization in the annual killifish Austrolebias charrua considering different tissues, gender and environmental conditions. The candidate reference genes 18 s, actb, gapdh, ef1a, shox, eif3g, and the control gene atp1a1 were evaluated in male and female individuals in three different tissues (brain, liver, and gills) under two experimental conditions (control and acute exposition to Roundup Transorb®). The collected tissues were submitted to RNA extraction, followed by cDNA synthesis, cloning, sequencing, and qPCR. Overall, 18 s was the most stable reference gene, and 18 s and ef1a were the most stable combination. Otherwise, considering all variables, gapdh and shox were the least stable candidate genes. Foremost, suitable reference genes were validated in A. charrua, facilitating accurate mRNA quantification in this species, which might be useful for developing molecular tools of ecotoxicological assessment based on gene expression analysis for environmental monitoring of annual killifish.
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Authors contributions
Matheus Volcan, Tony Silveira, Mariana Remião, Antônio Pagano, and Vinicius Campos conceived and designed the experiments. Tony Silveira and Mateus Kütter collected the animals. Antônio Pagano and Natiéli Gonçalves maintained the animals. Antônio Pagano, Lucas de Souza, William Domingues, Patrícia Costa and Adalto Bianchini carried out the laboratorial analyses. Antônio Pagano, Eduardo Blödorn, and Vinicius Campos contributed to the interpretation of the results. Antônio Pagano took the lead in writing the manuscript and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.
Funding
This study was supported by Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul (FAPERGS-FAPESP #19/2551-0001323-0 and FAPERGS/SICT 06/2022 INOVA AGRO # 22/2551-0001645-6), Conselho Nacional de Desenvolvimento Científico e Tecnológico (Chamada CNPq/MCTI/CT-BIOTEC N° 31/2022, Processo # 440636/2022-1) and was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nivel Superior – Brasil (CAPES) Finance Code 001 and AUXPE #2537/2018.
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The collection of animals was carried out through permit IBAMA/SISBIO 71072. The animal use and all handling practices were approved by the Ethics Committee on Animal Experimentation of the Federal University of Rio Grande (process no. 23116.003876/2019-24).
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Pagano, A.D., Blödorn, E.B., Domingues, W.B. et al. Validation of qPCR reference genes in the endangered annual killifish Austrolebias charrua considering different tissues, gender and environmental conditions. Ecotoxicology (2024). https://doi.org/10.1007/s10646-024-02752-0
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DOI: https://doi.org/10.1007/s10646-024-02752-0