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Standardization of molecular analysis techniques for DNA identification of bird species of the Psittacidae family

Published:September 29, 2022DOI:https://doi.org/10.1016/j.fsigss.2022.09.037

      Abstract

      Birds of the Psittacidae family belong to one of the groups with the most negative impact from wildlife trafficking, which has consequences beyond removing these species from the wild. This work aimed to standardize DNA extraction techniques from blood, feathers, and eggshells of Psittacidae to molecular identification and help track the place of origin of the seized bird. Blood and feather samples from adult of the Turquoise-fronted Parrot, Amazona aestiva, individuals (n = 5) were collected, and additionally, eggshells from nests (n = 3). We tested five nucleic acid extraction techniques.DNA concentrations and purity were evaluated by fluorimetry and spectrophotometry. As a result, the extraction by phenol provided a higher concentration of DNA from blood (20 µg/ml) and feathers (0.4 µg/ml), while for the eggshells, the acetate technique was the most efficient (33 µg/ml). Polymerase chain reaction amplification of mitochondrial cytochrome B (CytB) and 16 S genes were positive for all DNA samples. We obtained favorable conditions for DNA extraction from blood, feathers, and eggshells of the Turquoise-fronted Parrot using the five DNA extraction methods tested. These protocols can be used as a reference for other species of Psittacidae, and can be applied for biodiversity and forensic studies.

      Keywords

      1. Introduction

      The trafficking of wild animals, including birds of the Psittacidae family, is one of the most practiced environmental crimes in Brazil and its negative effects extend to those caused by release in inappropriate places due to the absence of data from its origin. Since lineage markers are free of recombination, we assume their geographic specificity from well-defined geographically bird populations should be carefully evaluated. Besides, the specificity of the vectors [
      • Ricklefs R.E.
      Embryonic development period and the prevalence of avian blood parasites.
      ], the isolation of populations [
      • Hoberg E.P.
      • Brooks D.R.
      A macroevolutionary mosaic: episodic host ‐ switching, geographical colonization and diversification in complex host – parasite systems.
      ,
      • Agosta S.J.
      • Janz N.
      • Brooks D.R.
      How specialists can be generalists: resolving the ' parasite paradox ' and implications for emerging infectious disease.
      ], and the infection by parasites during the life of vertebrate hosts [
      • Valkiūnas G.
      Avian Malaria Parasites and Other Haemosporidia.
      ], favor the emergence of new lineages of blood parasites [
      • Hoberg E.P.
      • Brooks D.R.
      A macroevolutionary mosaic: episodic host ‐ switching, geographical colonization and diversification in complex host – parasite systems.
      ,
      • Agosta S.J.
      • Janz N.
      • Brooks D.R.
      How specialists can be generalists: resolving the ' parasite paradox ' and implications for emerging infectious disease.
      ]. Based on these assumptions we also consider possible that species of Psittacidae present different strains of blood parasites according to their regions of origin. Standardizing DNA extraction techniques of blood and other biological samples is an important initial step to approach that. This work aimed to standardize DNA extraction techniques from blood, feathers, and eggshells of Psitacidae to molecular identification as a way to help track the place of origin of the seized bird.

      2. Material and methods

      We collected blood and feather samples from adult of the Turquoise-fronted Parrot, Amazona aestiva, individuals (n = 5) at the Wild Animal Screening Center of Rio de Janeiro, and additionally, eggshells from nests in the Várzeas do Rio Ivinhema State Park of Mato Grosso do Sul (n = 15). Blood smears were prepared for each sampled bird and examined for detection and morphological identification of hemosporidian parasites under optical microscopy (Fig. 1). Molecular analysis through PCR screening and DNA sequencing are being carried out for detection and identification of parasites lineages. We tested five nucleic acid extraction techniques (Organic: Phenol:chlorophorm), magnetic bead (Promega), column (Qiagen®), Chelex100®(Bio-Rad), and ammonium acetate salt. In addition, DNA concentrations and purity were evaluated by fluorimetry and spectrophotometry. Samples were amplified using CytB gene and 16 S molecular markers.
      Fig. 1
      Fig. 1Sample collection and DNA analysis of Amazona aestiva. Blood sample (A) Feather samples (B) Eggshell sample (C), Hemosporidian parasite (indicated by the arrow) (1000x) visualized in blood sample (D) and Agarose gel electrophoresis analysis (E): Agarose gel electrophoresis for amplified CytB gene of Psitacidae eggshell DNA samples extracted by using five techniques. L: 50–700 bp ladder; Lanes 1–3 (Column) Lanes 4–6 (Chelex); Lanes 7–9 (Magnetic beads); Lanes 10–12 (organic); Lane 13: Ammonium acetate; 14: PCR positive control; 15: PCR negative control.
      Source: the authors (2021).

      3. Results

      As a result, the extraction by phenol provided a higher concentration of DNA from blood (20 µg/ml) and feathers (0.4 µg/ml), while for the eggshells, the acetate technique was the most efficient (33 µg/ml). Regarding the purity of DNA from blood samples, all protocols provided DNA with a 260/280 ratio between 1.7 and 1.9, except Chelex. For feather samples, the best results were those from phenol and column extractions (1.8 and 2.0). For eggshells, on the other hand, extractions with magnetic particle, column, and acetate were most satisfactory (1.8–2.0). Polymerase chain reaction amplification of mitochondrial CytB and 16 S genes were positive for all DNA samples, even for those from feathers that presented the lowest concentrations: column and acetate (0.1 µg/ml) (Table 1). We obtained the most favorable conditions for DNA extraction from blood, feathers and eggshells of the Turquoise-fronted Parrot using the five DNA extraction methods tested.
      Table 1Nucleic acid extraction techniques using blood, feathers and eggshells of the Turquoise-fronted Parrot, Amazona aestiva. DNA concentrations and purity were evaluated by fluorimetry and spectrophotometry. Samples were amplified using CytB gene and 16S molecular markers.
      SamplesOrganic Phenol: ChloroformMagnetic bead DNA IQ™Column QIAamp DNA InvestigatorChelex ResinAmmonium acetate
      Conc. µg/mlRation 260/280Conc. µg/mlRation 260/280Conc. µg/mlRation 260/280Conc. µg/mlRation 260/280Conc. µg/mlRation 260/280
      Blood20.01.714.61.80.041.34.10.711.01.7
      Feathers0.41.70.22.50.12.00.31.10.11.4
      Eggshells3.11.62.71.913.402.01.90.613.51.9

      4. Discussion

      In an analysis performed with DNA extracted from canines for the typing of markers with autosomal single nucleotide polymorphism (SNP), Jiang et cols [
      • Jiang H.H.
      • Ma Y.
      • Bai S.Y.
      • et al.
      Forensic validation of a panel of 12 SNPs for identification of Mongolian wolf and dog.
      ] obtained positive typing results with a minimum DNA concentration of 6.25 pg/uL (0.00625 ug/ml). Using fecal material and elephant ivory, Ngatia et cols [
      • Ngatia J.N.
      • Lan T.M.
      • Ma Y.
      • et al.
      Distinguishing extant elephants ivory from mammoth ivory using a short sequence of cytochrome b gene.
      ] were able to detect short fragments of the mitochondrial gene of the cytochrome b enzyme with an amount of 1 pg/uL (0.001 ug/ml). In a study carried out with captive-bred parrots, Coetzer et cols [
      • Coetzer W.G.
      • Downs C.T.
      • Perrin M.R.
      • et al.
      Testing of microsatellite multiplexes for individual identification of Cape Parrots (Poicephalus robustus): paternity testing and monitoring trade.
      ], managed to develop a multiplex system for the analysis of 16 microsatellite markers in which 2–5 ug/ml of DNA was used as a minimum concentration. These data suggest that all protocols tested in the present dissertation produced enough DNA to at least detect autosomal SNP markers, mitochondrial DNA regions or even microsatellite markers of autosomal chromosomes.

      5. Conclusion

      These protocols can be used as a reference for other species of Psittacidae family, and can be applied for biodiversity and forensic studies.

      Funding

      This Project was supported by the Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).This study was also financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brazil (CAPES) - Finance Code 001, that awarded a master's scholarship to one of the authors (P. Succo)

      Acknowledgments

      We thank biologist Caio Vinícius de Oliveira Prates, Assistant of the Papagaio-verdadeiro Project, who helped to collect samples and the Mato Grosso do Sul Environment Institute (IMASUL) for the authorization (AA 0004/2020) of research in Várzeas do Rio Ivinhema State Park (PEVRI).

      Conflict of interest

      None.

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