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.
|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