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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 (Universidade Federal do Rio de Janeiro)
U.S. Partner: Mark Hahn (Woods Hole Oceanographic Institution)
Project Dates: September 2013 to July 2015

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

As the project got under way in the fall of 2013, Dr. Parente recruited Paula Andrade and Maithê Gaspar, two female undergraduate students from Universidade Federal do Rio de Janeiro, to join his research team. In addition, Dr. Paulo Buckup and his group (two technicians, one female PhD and one master’s student) were engaged to help with logistical arrangements and fish identification. During the first month, the two undergraduate students were trained in RNA isolation from fish liver, complementary DNA (cDNA) preparation, and polymerase chain reactions (PCR). From October 2-7 2013, a group of eight researchers traveled to north Rio de Janeiro State and south-central Espírito Santo State. The team later made a one-day trip to Visconde de Mauá and a six-day excursion (November 20-25) to central Rio de Janeiro State and southeast Minas Gerais State. In total, the research team collected 135 individual samples from 31 different genera at 52 different sites.

Brazil Partnership Photo 1
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).

The RNA from all of the samples has been isolated and used for cDNA preparation. PCR with primers for amplification of the entire CYP1A coding sequence has been performed for at least one individual from each genus. PCR products at the expected molecular weight were obtained for fish from six genera. Those PCR products are to be cloned into bacterial vectors for the first DNA sequencing. The research team plans to obtain the full CYP1A sequence for the six genera that had positive PCR. Having these sequences, it will be possible to design more specific primers, which will be used in PCR tol amplify the CYP1A gene from several other genera. The team is also analyzing Next Generation Sequencing possibilities.
 
A trip to the Amazon is scheduled for the second semester of 2014 to sample selected genera. Exchange visits are planned for Dr. Parente and Dr. Hahn, as well as for Ms. Andrade and Ms. Gaspar. The latter are applying for the Summer Student Fellowship of the Woods Hole Oceanographic Institution and are expected to work at the lab of U.S. partners Drs. Hahn and Stegeman from early June to early August 2014. Dr. Parente is applying for the Workshop on Molecular Evolution and Phylogenetics at the Marine Biological Laboratory (MBL) in Woods Hole from July 27 to August 6. After that period, Dr. Parente and co-PI Dr. Mauro Rebelo are expected to stay a couple of weeks working in Dr. Hahn’s lab.

Link to Project Blog

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