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Partnerships for enhanced engagement in research (PEER) SCIENCE
Cycle 2 (2012 Deadline)

Biodiversity and adaptations of CYP enzymes in the Amazon Loricariidae fishes

PI: Thiago Parente (, Fundação Oswaldo Cruz (Fiocruz), (formerly at Universidade Federal do Rio de Janeiro)
U.S. Partner: Mark Hahn (Woods Hole Oceanographic Institution)
Project Dates: September 2013 to July 2015

2-435 Zebra Fish
The Hypancistrus zebra, an endangered Loricariidae species (Photo: Dr. Parente)
CYP1 enzymes are responsible for the biotransformation of natural compounds and anthropogenic pollutants. Usually the reactions catalyzed by CYP1 enzymes lead to detoxification, when the compound is eliminated from the body without causing harm. CYP1 enzymes, however, are also known to catalyze bioactivation reactions, in which one of the reaction products is more toxic than its parent compound. The equilibrium between the detoxification (beneficial) and bioactivation (detrimental) roles of CYP1 enzymes has been fine tuned for each and every vertebrate species over the course of evolution. This PEER Science project is closely aligned with the National Science Foundation-supported work of the U.S. partner, Dr. Mark Hahn, as both involve the study of different naturally evolved and selected solutions for the same issue: the balance between detoxification and bioactivation by CYP1 enzymes using fish species as vertebrate models. The adaptation of Killifish (studied by Dr. Hahn) is a well documented event classified as dramatic, rapid, convergent, and triggered by anthropogenic pollutants that balance the dual role of CYP1 enzymes at the gene expression level. However, the adaptation of Loricariidae fish is poorly known and most probably can have the opposite classification: gradual, slow, divergent, and triggered by chemicals naturally present in the fish microhabitat. The goals of this project are to determine whether the adaptations of CYP1 enzymes in Loricariidae fishes are convergent or divergent and how they change the susceptibility of this species to the toxic effects of petrogenic compounds.

Dr. Parente and his research team will sequence the CYP1 genes of 100 Loricariidae species from the Amazon, and these gene sequences will be used to determine the enzyme sequences, which in turn will be aligned and compared for amino acids substitutions and interaction with classical CYP1 substrates. Selected Loricariidae species will be used for biological assays to evaluate the toxic effects of petrogenic derivates and their molecular mechanisms of action. Due to the current and future prospects for crude oil drilling activities in the Amazon region, it is imperative to understand the metabolism of petrogenic hydrocarbons by Amazonian biota. This is especially true in the case of Loricariidae fishes, as it is already known that those species have CYP1 enzymes with distinct affinity for substrates. It needs to be determined whether these changes will unbalance the evolved equilibrium of CYP1 dual roles to the beneficial or to the detrimental side. This knowledge will be crucial to better evaluate the risks of oil drilling activities for Amazonian biodiversity.

Summary of Recent Activities

During the most recent quarter (October-December 2014), Dr. Parente and his team assembled the transcriptome of Pterigoplichthys anitisiti, made progress on its analysis, collected several species in Manaus, and finished preparing all libraries for transcriptome sequencing. A total of 57,644,658 nucleotides were assembled to construct the transcriptome of Pterigoplichthys anitisiti. In this transcriptome, 51,530 components were detected and BLAST searches against the UniProt database for Human and Zebrafish found that around 33,000 of those had positive hits with known gene sequences. The components whose BLAST top hit was a “defensome" gene were selected for further analysis. So far, the team has analyzed the Cytochrome P450 (CYP) genes of P. anitisiti. Results from the studies indicate that certain genes are evolving under selective pressure. A scientific paper is in preparation to publish the results on the defensome genes in P. anitisiti. This part of the work was performed with the invaluable assistance of the recently hired bioinformatician on the project.

On November 10, 2014, Dr. Parente conducted a weeklong excursion to Manaus, where the team sampled 36 fish from the 15 species of the Loricariidae subfamilies found in the Amazon. The team also managed to sample two species that were not planned but proved to be very important. Of particular note is the Hypancistrus zebra, an endangered species due to its overexploitation by international aquarists and the construction of the Belo Monte Dam, the largest dam in the world. For H. zebra the team sequenced the transcriptomes from the liver and six other organs.

Brazil Partnership Photo 1Brazil Partnership Photo B
The research team at the Andorinhas waterfall on the Roncador river in the district of Santo Alexio. Pictured are Paolo Buckup (top), Emmanuel Neuhaus (center), Thiago Parente (left foreground), and Jose Gomes (right foreground). (Photo: Carla Quijada).Labwork to obtain sequencing of genes (Photo: Dr. Parente).

Meanwhile, the two undergraduate students continued to construct cDNA libraries for sequencing by Illumina Hi-Seq2500 with RNA samples from previously collected fish. The fish collected in the Amazon had RNA extracted from their livers as soon as the samples arrived in the laboratory in Rio de Janeiro. The team finished preparing all libraries for sequencing on the second week of December 2014, and each was subjected to quality control by checking the size range of cDNA fragments and their quantity using a bioanalyzer. Libraries will be precisely quantified by quantitative PCR during the first week of January 2015 and the sequencing is schedule to begin immediately thereafter.

In January 2015, the project team also plans to complete analysis of the defensome genes from P. anitisiti, publish a scientific paper describing the results, and obtain the sequences of 37 other transcriptomes by Illumina Hi-Seq 2500. The team will then assemble these transcriptomes and perform preliminary analyses in the following two months. Finally, in April and May, select species will be chosen for toxicological assays. Thanks to a recently approved supplement to his PEER grant, Dr. Parente and his team will also be expanding sampling efforts on the project to include one additional rare species that was not initially included in his proposal. With the addition of this species he will have representatives of all six subfamilies of the Loricariidae, including the previously missing subfamily, Lithogeneinae. In collaboration with Prof. Paulo Buckup from the Brazilian National Museum of Natural History of the Rio de Janeiro Federal University, Dr. Parente is currently planning a field excursion to the Potaro river, upstream Kaieteur Falls, Guyana, in order to collect a representative from Lithogeneinae subfamily and from the Corymbophanes genus.

Link to Project Blog

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