Illegal hunting cases detected with molecular forensics in Brazil
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Illegal hunting is one of the major threats to vertebrate populations in tropical regions. This unsustainable practice has serious consequences not only for the target populations, but also for the dynamics and structure of tropical ecosystems. Generally, in cases of suspected illegal hunting, the only evidence available is pieces of meat, skin or bone. In these cases, species identification can only be reliably determined using molecular technologies. Here, we reported an investigative study of three cases of suspected wildlife poaching in which molecular biology techniques were employed to identify the hunted species from remains of meat.
By applying cytochrome b (cyt-b) and cytochrome oxidase subunit I (COI) molecular markers, the suspected illegal poaching was confirmed by the identification of three wild species, capybara (Hydrochoerus hydrochaeris), Chaco Chachalaca (Ortalis canicollis) and Pampas deer (Ozotoceros bezoarticus). In Brazil, hunting is a criminal offense, and based on this evidence, the defendants were found guilty and punished with fines; they may still be sentenced to prison for a period of 6 to 12 months.
The genetic analysis used in this investigative study was suitable to diagnose the species killed and solve these criminal investigations. Molecular forensic techniques can therefore provide an important tool that enables local law enforcement agencies to apprehend illegal poachers.
KeywordsSpecies identification mtDNA Wildlife forensics Bush meat Poaching Neotropical region
cytochrome c oxidase subunit I
Barcode of Life Database
Instituto Brasileiro do Meio Ambiente e dos Recursos Naturais Renováveis
sample meat of the mammal seized in case 1
sample meat of the bird species seized in case 2
sample meat of the mammal seized in case 2
polymerase chain reaction.
It is estimated that illegal hunting kills millions of vertebrates per year in tropical rainforests . A number of reports have shown that the volume of wild game harvested is unsustainable and has led to the local extinction of several populations [1, 2]. The use of wild foods or the bush meat crisis is one of the major challenges for the conservation of large-bodied vertebrates. Poor local law enforcement and corruption allow the hunting of large vertebrates to continue to be widespread even in protected areas .
Even when hunters are captured, the precise identification of bush meat is often questionable. Moreover, the remains of meat, fur, skin and bone are the only evidence recovered from the crime scene. In such cases, species identification can only be resolved with molecular tools [3, 4, 5].
The sequences were manually edited using the software BioEdit Sequence Alignment Editor 126.96.36.199 . This software was also used to align both strands and obtain consensus sequences for each sample. The sequences obtained were submitted as independent entries in a BLAST search for the most similar sequences using the default Megablast algorithm parameters . For the species diagnosis, we considered the percentage homology between query and reference sequence pairs. In order to minimize the chance of incorrect species assignment, we adopted a threshold identity value of ≥98 % between the sequences . Since this study aimed at the taxonomic identification of the seized meat samples, without any prior evidence or indication of the species, we regarded uploading the sequences to GenBank inadequate. The query sequences can be obtained directly from the authors.
Cyt-breference sequences of species that would likely be hunted or consumed at the seizure region
GenBank accession numbers (ID reference sequence)
AY126697 (Bind1), EF693799.1 (Bind2), EF061238.1 (Bind3), EF061239.1 (Bind3), EF061242.1 (Bind4)
FJ971088 (Btau1), GU249572.1 (Btau2), DQ186288.1 (Btau3), DQ186284.1 (Btau4), AY952963.1 (Btau5)
GQ351599 (Sscr1), GU135819.1 (Sscr2), AM492594.1 (Sscr3), AY237529.1 (Sscr4), GU135716.1 (Sscr5)
AY206574.1 (Cpac1), AY206563.1 (Cpac2), AY206572.1 (Cpac3), AY206561.1 (Cpac4), AY206560.1 (Cpac5)
DQ789176.2 (Bdic1), DQ789173.2 (Bdic2), DQ789175.2(Bdic3), DQ789174.2 (Bdic4)
Red brocket deer
DQ789230.2 (Mame1), DQ789201.2 (Mame2), DQ789225.2 (Mame3), DQ789209.2(Mame4), DQ789224.2 (Mame5)
Gray brocket deer
DQ789200.2 (Mgou1), DQ789189.2 (Mgou2), DQ789182.2 (Mgou3), DQ789203.2 (Mgou4), DQ789184.2 (Mgou5)
L48404.1 (Obez1), DQ789199.2 (Obez2), DQ789198.2 (Obez3), DQ789191.2 (Obez4), DQ789192.2 (Obez5)
DQ179085.1 (Ptaj1), DQ179079.1 (Ptaj2), DQ179082.1 (Ptaj3), DQ179074.1 (Ptaj4), DQ179065.1 (Ptaj5)
AY534303.1 (Tpec1), U66290.1 (Tpec2), AY726775.1 (Tpec3)
AF056030.1 (Tter1), GQ259949.1 (Tter2), GQ259923.1 (Tter3), GQ259954.1 (Tter4), GQ259936.1 (Tter5)
Gallus gallus domesticus
HQ122606 (Ggal1), AF195628.1 (Ggal2), AY029583.1 (Ggal3), AF354171.1 (Ggal4), AF028795.1 (Ggal5)
FJ899152 (Csou1), FJ899151.1(Csou2), FJ899149.1 (Csou3), FJ899150.1 (Csou4), FJ899147.1 (Csou5)
AY659790.1 (Cfas1), AY141923.1 (Cfas2)
Ortalis canicollis/Ortallis pantanalensis
AF165472.1 (Ocan)/AY659783.1 (Opan)
AY354484.1 (Ptub1), AF165469.1 (Ptub2)
The cyt-b sequencing of BIRD and mammal species (MAM1 and MAM2) produced readable sequences of approximately 550 and 1100 bp, respectively, which did not present insertions, deletions or stop codons. BLAST analysis of cyt-b indicated that the MAM1 meat sample was highly similar (99%) to the capybara (Hydrochoerus hydrochaeris) reference sequence, the MAM2 was similar (98%) to Pampas deer (Ozotoceros bezoarticus), and the BIRD sample was similar (98%) to Chaco chachalaca (Ortalis canicollis).
Contrary to results with cyt-b, species identification was not possible using COI. The amplification of COI in BIRD, which could be solved using primers designed for bird species, was not successful. For the mammal species, the BLAST analysis did not match a reference sequence based in our threshold (≥98%). At the time of the manuscript writing, Genbank and BOLD did not contain COI reference sequences for capybara or for Pampas deer. Although the COI gene is considered the DNA barcoding gene , reference sequences for several species are still unavailable in these genetic databases. Researchers have used the cytochrome b gene for species identifications [5, 19] since it is one of the better represented genes in GenBank  and has superior ability for separating species when compared with COI [20, 21].
The three species killed are widely reported in hunting studies [1, 22, 23]. The capybara is widely distributed in South America and is the largest living rodent, weighing around 50 kg . Pampas deer, the most endangered Neotropical cervid, formerly occupied a range of open habitats such as grassland, pampas and savanna (cerrado) in Brazil . Its populations are decreasing because of habitat conversion for agriculture and cattle farming, hunting and attacks by feral dogs . Its former range has been reduced to less than 1% . The Chaco Chachalaca is a galliform commonly found in the Pantanal of Brazil. Cracid species are becoming rare because of hunting, and the loss and fragmentation of suitable habitats .
In this study the cyt-b molecular marker was suitable to diagnose the species killed and solve these criminal investigations. The suspected poaching in all three cases was confirmed with the identification of three wild species, capybara, Pampas deer and Chaco Chachalaca. In Brazil, hunting is a criminal offense, and based on the results of our molecular analysis, the defendants were found guilty and punished with fines; they may still be sentenced to prison for a period of 6 to 12 months.
The authors are very grateful to IBAMA, FAPESP (2007/03392-6), FUNDUNESP and Idea Wild. AS receives a fellowship from FAPESP (2007/04073-1). MG receives a fellowship from CNPq.
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.