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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 June 2016

Link to Project Blog

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 first four months of 2015, Dr. Parente and his team continued to work on the transcriptome of Pterigoplichthys anitisiti, drafted a section of a paper on these results, assembled the transcriptome of Ancistrus spp. and Corydoras nattereri, began their analysis, assembled the mitogenomes of these three species, and sequenced the transcriptome of 12 other species. As noted in their previous quarterly report, more than 57 million nucleotides were assembled to construct the transcriptome of P. anitisiti. In this transcriptome, 51,530 components were detected. BLAST searches against the UniProt database for Human and Zebrafish found that around 33,000 of those had positive hits with known gene sequences. Similar results were found for Ancistrus spp. and C. nattereri. The defensome genes of P. anitisiti were studied in detail, and Dr. Parente and his team have begun analyzing defensome genes in the two other species, but the results are still very preliminary.

The team encountered some difficulties in mid-January 2015 when the next-generation sequencing equipment they are using at the Brazilian National Institute of Cancer (INCA) broke down while running their samples. No samples were lost, but the breakdown cost them a significant quantity of expensive NGS reagents. Fortunately, INCA is covering the cost of sequencing of their samples, but this unexpected problem delayed the project schedule by four to six weeks. Part of their samples (12 of 36) had already been sequenced, and another sequencing run was scheduled for early May.

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).

The PI attended the international meeting PRiMO 18 (Pollutant Responses in Marine Organisms) in Trondheim, Norway, May 24-27, 2015, where he met with U.S. collaborators on the project. Dr. Parente, accompanied by the two undergraduate students and the computational biology specialist on the project, also attended the international meeting Evolution 2015, held in Guarujá, Brazil, June 26-30, 2015. Dr. Parente and his colleagues are now working to finish sequencing the transcriptomes from the previously sampled species and analyze the evolution of at least CYP1A and a receptor gene. Two more field trips are planned, one to Manaus and one to Guyana. The excursion to Manaus is aimed at exposing two selected species (most probably Pterygoplichthys pardalis and Dekeyseria amazonica) to water enriched with humic acids (from the Rio Negro) and to crude oil. The excursion to Guyana will provide an opportunity to sample two particular species, Lithogenes sp. and Corymbophanes sp. For this excursion, the team is applying for a licence to collect biological samples in Guyana. In order to increase the chances to collect those species, and also for safety reasons, the excursion must happen in one of the few dry months in Guyana, which are September-October or February-March. A no-cost extension on this project has been issued through June 2016 to allow for more time to complete the field work and subsequent analyses.

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