Simulating the Bird Eggs Illegal Trade and Improve the DNA Barcode Amplification to Combat Bird Trafficking Through Species Identification
DOI:
https://doi.org/10.17063/bjfs12(4)y2025340-354Keywords:
Case-type samples, DNA amplification, DNA mini-barcode, forensic analysis, illegal bird trade, wildlife crimeAbstract
The illegal bird trade is changing from capturing live animals to smuggling eggs, making trafficking even more dangerous. Eggs can exhibit intraspecific variations or show very similar traits between different species, making the morphological species identification inaccurate. Thus, genetic species identification has become an essential tool in solving wildlife crimes. However, the conditions to which eggs are subjected during trafficking, from capture in the wild to seizure, make egg samples increasingly difficult even for DNA analysis, limiting the availability of biological material for species identification. We simulated different scenarios where eggs could be found and determined the best DNA barcode size to amplify in each situation. Chicken muscle tissue (Gallus gallus) and japanese quail eggs (Coturnix japonica) were submitted to adverse conditions resulting from trafficking: dried eggs, decomposing eggs, broken eggshells, and egg fluid deposited on various substrates. We assessed three salting-out DNA extraction protocols to find a cheaper and less toxic method. Different molecular markers from cytochrome oxidase I (COI) gene, fragments of 747, 405 and 164 bp, were tested. We successfully amplified the DNA mini-barcode (164 bp) in all simulations. However, the amplification of larger fragments was more challenging in complex samples such as decomposed eggs, egg fluid, and broken eggshells. These results demonstrate that DNA barcoding and mini-barcoding are powerful tools for identifying bird egg samples in various states of preservation, including decomposed samples. To apply this methodology as evidence in combating environmental crimes, we present a standardized flowchart for identifying bird species using molecular techniques.
References
Sodhi NS, Sekercioglu CH, Barlow J, Robinson SK. Conservation of Tropical Birds. Wiley Blackwell; 2011. https://doi.org/10.1002/9781444342611
Utermohlen M, Baine P. In plane sight: wildlife trafficking in the air transport sector. 2017:214.
Halle BO. Bird’s-eye view: Lessons from 50 years of bird trade regulation & conservation in Amazon countries. 2018:198.
Sollund R, Maher J. The Illegal Wildlife Trade: A Case Study Report on the Illegal Wildlife Trade in the United Kingdom, Norway, Colombia and Brazil. 2015.
Illes A. Wildlife Crime in the United Kingdom. Inst Eur Environ Policy. 2016:31.
UNODC. World Wildlife Crime Report: Trafficking in Protected Species. New York: United Nations; 2016.
Hughes LJ, Morton O, Scheffers BR, Edwards DP. The ecological drivers and consequences of wildlife trade. Biol Rev. 2023;98(3):775-91. https://doi.org/10.1111/brv.12929
Johnson RN. The use of DNA identification in prosecuting wildlife-traffickers in Australia: Do the penalties fit the crimes? Forensic Sci Med Pathol. 2010;6(3):211-6. https://doi.org/10.1007/s12024-010-9174-9
Gonçalves PFM, Oliveira-Marques AR, Matsumoto TE, Miyaki CY. DNA Barcoding Identifies Illegal Parrot Trade. J Hered. 2015;106(S1):560-64. https://doi.org/10.1093/jhered/esv035
Formentão L, Saraiva AS, Marrero AR. DNA barcoding exposes the need to control the illegal trade of eggs of non-threatened parrots in Brazil. Conserv Genet Resour. 2021;13(3):275-81. https://doi.org/10.1007/s12686-021-01209-4
Koenig WD. Ecological and social factors affecting hatchability of eggs. Auk. 1982;99:526-536.
Hemmings N, West M, Birkhead TR. Causes of hatching failure in endangered birds. Proc Biol Sci. 2012;279(1737):1446-52. https://doi.org/10.1098/rsbl.2012.0655
Stoddard MC, Yong EH, Akkaynak D, et al. Avian egg shape: Form, function, and evolution. Science. 2017;356:1249-54. https://doi.org/10.1016/j.semcancer.2013.09.001
Iyengar A. Forensic DNA analysis for animal protection and biodiversity conservation: A review. J Nat Conserv. 2014;22(3):195-205. https://doi.org/10.1016/j.jnc.2013.12.001
Barreira AS, Lijtmaer DA, Tubaro PL. The multiple applications of DNA barcodes in avian evolutionary studies. Genome. 2016;59(11):899-911. https://doi.org/10.1139/gen-2016-0086
Dawnay N, Ogden R, McEwing R, Carvalho GR, Thorpe RS. Validation of the barcoding gene COI for use in forensic genetic species identification. Forensic Sci Int. 2007;173(1):1-6. https://doi.org/10.1016/j.forsciint.2006.09.013
Hajibabaei M, Smith MA, Janzen DH, Rodriguez JJ, Whitfield JB, Hebert PDN. A minimalist barcode can identify a specimen whose DNA is degraded. Mol Ecol Notes. 2006;6(4):959-64. https://doi.org/10.1111/j.1471-8286.2006.01470.x
Carvalho CBV. Identificação genética de aves vítimas do tráfico de animais silvestres. Atualidades Ornitológicas On-Line. 2012;165:40-4.
Coghlan ML, White NE, Parkinson L, et al. Egg forensics: An appraisal of DNA sequencing to assist in species identification of illegally smuggled eggs. Forensic Sci Int Genet. 2012;6(2):268-73. https://doi.org/10.1016/j.fsigen.2011.06.006
Meiklejohn KA, Burnham-Curtis MK, Straughan DJ, Giles J, Moore MK. Current methods, future directions and considerations of DNA-based taxonomic identification in wildlife forensics. Forensic Sci Int Anim Environ. 2021;1(September):1-11. https://doi.org/10.1016/j.fsiae.2021.100030
Minakshi P, Shafiq M, Alisha A, et al. Animal Forensics and Applications. In: Salar RK, Gahlawat SK, Siwach P, Duhan JS, eds. Biotechnology: Prospects and Applications. New Delhi: Springer; 2013:265-86. https://doi.org/10.1007/978-81-322-1683-4_20
FWG FWG. Wildlife Crime: A Guide to the Use of Forensic and Specialist Techniques in the Investigation of Wildlife Crime. 2014:106.
Gaur A, Reddy A. DNA Techniques in Wildlife Forensics (Animals): Standard Operating Procedures (SOP). 2017:37.
SWGDAM. Validation Guidelines for DNA Analysis Methods. Sci Work Gr DNA Anal Methods. 2016:1-15.
Aljanabi SM, Martinez I. Universal and rapid salt-extraction of high-quality genomic DNA for PCR-based techniques. Nucleic Acids Res. 1997;25(22):4692-3. https://doi.org/10.1093/nar/25.22.4692
Pilotto M, Goncalves A, Vieira F, et al. Exploring the impact of the biofloc rearing system and an oral WSSV challenge on the intestinal bacteriome of Litopenaeus vannamei. Microorganisms. 2018;6(3):83. https://doi.org/10.3390/microorganisms6030083
Rivero ERC, Neves AC, Silva-Valenzuela MG, Sousa SOM, Nunes FD. Simple salting-out method for DNA extraction from formalin-fixed, paraffin-embedded tissues. Pathol Res Pract. 2006;202(7):523-9. https://doi.org/10.1016/j.prp.2006.02.007
Kerr KCR, Birks SM, Kalyakin MV, Red’Kin YA, Koblik EA, Hebert PD. Filling the gap - COI barcode resolution in eastern Palearctic birds. Front Zool. 2009;6(29):1-13. https://doi.org/10.1186/1742-9994-6-29
Lijtmaer DA, Kerr KCR, Stoeckle MY, Tubaro PL. DNA Barcoding Birds: From Field Collection to Data Analysis. In: Kress WJ, Erickson DL, eds. DNA Barcodes: Methods and Protocols. Washington: Humana Press; 2012:127-12. https://doi.org/10.1007/978-1-61779-591-6_7
Birkhead T. The Most Perfect Thing: Inside (and Outside) a Bird’s Egg. London: Bloomsbury; 2016.
Downloads
Additional Files
Published
How to Cite
Issue
Section
License
Copyright (c) 2025 Brazilian Journal of Forensic Sciences, Medical Law and Bioethics
This work is licensed under a Creative Commons Attribution 4.0 International License.
