First environmental DNA (eDNA) record of central Amazon in a floodplain lake: extraction method selection and validation / Primeiro registro de DNA ambiental (eDNA) da Amazônia central em um lago de várzea: seleção e validação de método de extração

Danniel Rocha Bevilaqua, Sabrina Araújo de Melo, Carlos Edwar de Carvalho Freitas, Ana Caroline Viana da Silva, Jacqueline da Silva Batista


In recent years, environmental DNA (eDNA) has emerged as an effective method for detecting aquatic organisms. The current study tested a method of DNA extraction from water samples from Central Amazonian floodplain lake. Water collections were performed in a floodplain lake on Paciência Island, on the Solimões River (Amazon Basin, Brazil) during the high water period. Seven commercial DNA extraction methods/kits were tested with samples collected from Lake Cacau. After quantification, eDNA samples were amplified with 12S and 16S mitochondrial gene universal primers. The DNeasy Blood & Tissue kit (Qiagen), with modifications developed during the current study (adapt 03), gave the best overall result showing expected gene amplification, and intense agarose gel banding. eDNA concentrations ranged from 0.00 to 22.90 ng/µL for the other methods, with either lower intensity of the amplicon size to target gene correspondence when compared to that obtained with the DNeasy Blood & Tissue kit adapt 03 method, or there was an absence of amplification.The concentration of eDNA extracted from the lake sample was 2.68ng/µL. Prochilodus nigricans and Potamorhina altamazonica genomic DNA (gDNA) samples were extracted for use as a positive controls. The 12S and 16S genes from eDNA were amplified and sequenced with next generation Sequencing (NGS). Using a metabarcode approach, we used the 12S (12S-V5) and 16S (L2513/H2714) minibarcode universal primers to detect fish species DNA traces present in water samples from the Cacau lake. The NGS produced 18 and 492,454 12S and 16S minibarcode readings, respectively, for three taxonomic groups (Actinopterygii, Mammalia and Squamata). Adding the two genes, a total of 492,472.00 readings, with 15 readings with 12S and 4,431.082 readings with 16S for fish. Fish (Actinopterygii) represented 83.33% and 0.60% (three Orders, five families and five genus identified according to BLASTN), mammals had 11.11% and 99.40% and reptiles with 5.56% and 0.00% for the total readings of the 12S and 16S respectively, with more than 97% similarity to NCBI-available reference sequences. Taxonomic classification at the level of species was not very resolute when comparing OTU's with sequences published in NCBI.This study sets a milestone in the methodology of DNA extraction from floodplain lake, and shows the method can be used to monitor Amazon basin ichthyofauna, as it was possible to detect the presence of fish species DNA in collected water samples.


Monitoring, Environmental DNA, DNA extraction

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Aras S, Duran A, Yenilmez G (2003). Isolation of DNA for RAPD analysis from dry leaf material of some Hesperis L. specimens. 21, 461-462.

Barthem R, Goulding M (1997). The catfish connection: ecology, migration, and conservation of Amazon predators. Columbia University Press.

Batista V, Petrere M (2003). Characterization of the commercial fish production landed at Manaus, Amazonas State, Brazil. 33, 53-66.

Bayley P (1988). Factors affecting growth rates of young tropical floodplain fishes: seasonality and density-dependence. 21, 127-142.

Campos C, Freitas CEC, Amadio S (2015). Growth of the Cichla temensis Humboldt, 1821 (Perciformes: Cichlidae) from the middle rio Negro, Amazonas, Brazil. 13, 413-420.

Castello L, Mcgrath DG, Hess LL, Coe MT, Lefebvre PA, Petry P, Macedo MN, Renó VF, Arantes CC (2013). The vulnerability of Amazon freshwater ecosystems. 6, 217-229.

Cerdeira RGP, Ruffino ML, Isaac VJ (1997). Consumo de pescado e outros alimentos pela população ribeirinha do Lago Grande de Monte Alegre, PA-Brasil. 27, 213-228.

Dejean T, Valentini A, Duparc A, Pellier-Cuit SP, Pompanon F, Taberlet P, Miaud C (2011). Persistence of environmental DNA in freshwater ecosystems. PloS One, 6, e23398.

Dejean T, Valentini A, Miquel C, Taberlet P, Bellemain E, Miaud C (2012). Improved detection of an alien invasive species through environmental DNA barcoding: the example of the American bullfrog Lithobates catesbeianus. Journal of Applied Ecology, 49, 953-959.

Duponchelle F, Pouilly M, Pécheyran C, Hauser M, Renno JF, Panfili J, Darnaude AM, GARCÍA‐Vasquez A, Carvajal‐Vallejos F, García‐Dávila CJ (2016). Trans‐Amazonian natal homing in giant catfish. 53, 1511-1520.

Fabré N, Saint‐Paul UJ (1998). Annulus formation on scales and seasonal growth of the Central Amazonian anostomid Schizodon fasciatus. 53, 1-11

Fernandes CCJ (1997). Lateral migration of fishes in Amazon floodplains. 6, 36-44.

Freitas CEC, Siqueira-Souza FK, Guimarães AR, Santos FA, Santos IL (2010). Interconnectedness during high water maintains similarity in fish assemblages of island floodplain lakes in the Amazonian Basin. Zoologia, 27.

Gotelli NJ, Colwell RK (2001). Quantifying biodiversity: procedures and pitfalls in the measurement and comparison of species richness. 4, 379-391.

Hopkins G, Freckleton RP (2002). Declines in the numbers of amateur and professional taxonomists: implications for conservation. 5, 245-249.

Kitano T, Umetsu K, Tian W, Osawa M (2007). Two universal primer sets for species identification among vertebrates. 121, 423-427.

Knoppel H (1970). Food of Central Amazonian fishes; contribution to the nutrient-ecology of Amazonian rain-forest-streams.

Kramer D, Lindsey C, Moodie G, Stevens E (1978). The fishes and the aquatic environment of the central Amazon basin, with particular reference to respiratory patterns. 56, 717-729.

Loubens G, Panfili J (1997). Biologie de Colossoma macropomum (Teleostei: Serrasalmidae) dans le bassin du Mamoré (Amazonie bolivienne). 8, 1-22.

Mackenzie DI, Nichols JD, Sutton N, Kawanishi K, Bailey LL (2005). Improving inferences in population studies of rare species that are detected imperfectly. Ecology, 86, 1101-1113.

Nakatani S, Burger M, Assef M, Brockelt S, Cogo L, Messias-Reason I, Diseases I (2004). Efficient method for mycobacterial DNA extraction in blood cultures aids rapid PCR identification of Mycobacterium tuberculosis and Mycobacterium avium. 23, 851-854.

Reis RE, Albert JS, Didario F, Mincarone MM, Petry P, Rocha LA 2016. Fish biodiversity and conservation in South America. Journal of Fish Biology, in press

Riaz T, Shehzad W, Viari A, Pompanon FO, Taberlet P, Coissac E (2011). ecoPrimers: inference of new DNA barcode markers from whole genome sequence analysis. Nucleic Acids Research, 39, e145-e145.

Ruffino M., Isaac V.J. (1994) The fisheries of the lower Amazon: questions of management and development. 15, 37-46.

Sambrook J, Fritsch EF, Maniatis T. (1989). Molecular cloning: a laboratory manual. 2.ed. Cold Spring Harbor.

Sousa R, Humston R, Freitas C (2016). Movement patterns of adult peacock bass Cichla temensis between tributaries of the middle Negro River basin (Amazonas–Brazil): an otolith geochemical analysis. 23, 76-87.

Thomsen P., Kielgast J, Iversen LL, Møller PR, Rasmussen M, Willerslev E (2012a). Detection of a diverse marine fish fauna using environmental DNA fromseawater samples. PLoS ONE 7, e41732.

Venter JC, Remington K, Heidelberg JF, Halpern AL, Rusch D, Eisen JA, Wu D, Paulsen I, Nelson KE, Nelson WJS (2004). Environmental genome shotgun sequencing of the Sargasso Sea. 304, 66-74.

Vié JC, Hilton-Taylor C, Stuart SN (2009). Wildlife in a changing world: an analysis of the 2008 IUCN Red List of threatened species. IUCN.

Wheeler QD, Raven PH, Wilson EO (2004). Taxonomy: impediment or expedient? American Association for the Advancement of Science.

Yamamoto S, Masuda R, Sato Y, Sado T, Araki H, Kondoh M, Minamoto T, Miya M (2017). Environmental DNA metabarcoding reveals local fish communities in a species-rich coastal sea. Scientific Reports, 7, 40368.



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