Materials and Methods
To achieve the aims of this study, existing literature on South Africa’s
urban traditional medicine markets was reviewed for records of trading
in anuran amphibians and reptiles, traditional health practitioners were
interviewed to increase understanding of their practices, and herptiles
species were identified through visual confirmation during visits to the
traditional medicine markets and with DNA barcoding targeting cytochromec oxidase marker 1 (COI) of herptile specimens from those
markets.
Literature review
A search of the keywords: animal + traditional medicine + “South
Africa” on Google Scholar (https://scholar.google.com/) returned
results of literature whose titles and abstracts were pre-screened for
mentions of animal use in South African traditional medicine. Following
this initial screening, the suitable articles were studied to find
records of herptiles sold specifically in South Africa’s urban
traditional medicine markets or shops. Subsequently, availability of
reference DNA sequences for the herptiles species matching the inclusion
criteria of the literature review was verified with searches on the
National Center for Biotechnology Information (NCBI) GenBank database
(using the search query ((Species name ) AND (CO1[Gene Name]
OR COI[Gene Name])) and the Barcode of Life Data Systems (BOLD)
database (using the search query “Species name ”).
Fieldwork: interviews, tissue sampling and visual
observation
Visual confirmation surveys of herptile specimens available for sale at
traditional medicine markets/shops by the first author involved visiting
a total of six markets/shops in Polokwane, Pretoria, Johannesburg,
Pietermaritzburg and Durban from August to December 2020 (Figure 1).
Morphology-based identification of species using visual confirmation was
based on wildlife guides for reptiles (Alexander & Marais, 2007;
Marais, 2008). In accordance with North-West University Health Research
Ethics Committee’s guidelines, the participation of traditional health
practitioners at the markets/shops was sought after the first author
explained the purpose of this study in SePedi (language spoken by people
of Pedi culture) to practitioners from Limpopo and IsiZulu (language
spoken by people of Zulu culture) for Gauteng and KwaZulu-Natal
practitioners. SePedi was the preferred language for the Limpopo
participants, IsiZulu was the most spoken language at the markets/shops
in Gauteng and KwaZulu-Natal. Following explanation of the study, 11
traditional health practitioners consented to participation in this
study (two in Limpopo, two in Gauteng and seven in KwaZulu-Natal). An
informal conversational interview approach was used to collect data
about herptiles of traditional medicine value; their Indigenous names,
and the collection and preservation methods used for those herptiles.
This interview approach relies on continuous participant observation
without predetermined questions (Gall et al., 2003). The approach was
chosen due to traditional health practitioners expressing apprehension
towards researchers based on what they explained as past unpleasant
experiences with researchers and conservation practitioners. This
interview was guided by the first author’s conversation with
participants and questions were introduced to the conversation when
participants were forthcoming with details about their practices.
Answers to these questions were written in a field book once the
practitioners gave permission for their answers to be recorded in that
manner. The reasons for the practitioner’s apprehension towards
researchers were also noted.
Tissue samples were collected from specimens sold by nine participants
at four of the six localities visually surveyed in Gauteng and
KwaZulu-Natal markets/shops (Figure 1) as they gave consent for this
collection while the two participants in Limpopo said they could not
give consent as they did not own the traditional medicine shops. A total
of 111 samples were collectively obtained from Gauteng and KwaZulu-Natal
(Dataset S1). Practitioners were asked the IsiZulu names for each
sampled specimen and notes were made of any morphological features that
were still visible on those specimens. Notes about morphology were
written as the traditional health practitioners did not allow use of
cameras at the markets/shops.
Distinctive morphological traits were not visible on all sampled
specimens as sometimes all that remained were ventral scutum, or bones
with flesh but no skin. Opting for collection of tissue samples instead
of taking entire specimens minimises this study’s environmental impact
as removal of entire specimens may prompt traditional health
practitioners to acquire replacement specimens to satisfy demand from
customers or patients.
DNA extraction and absorbance
measurements
From the acquired samples, outside layers of tissue that were most
likely exposed to contamination were shaven/scraped off and discarded
before taking ~25mg of tissue for DNA extraction. This
tissue’s genomic DNA was extracted using the standard extraction
protocols for animal tissue provided by the manufacturer in the
NucleoSpin®Tissue Genomic DNA Tissue Kit (Macherey-Nagel, Duren,
Germany).
To assess the suitability of the extracted DNA samples for downstream
applications, (amplification and sequencing) their purity was determined
through measures of absorbance using ultraviolet-visible spectroscopy
(UV–visible spectrophotometry) where the peak absorbance of pure
nucleic acid is 260 nm (Desjardins & Conklin, 2010; Koetsier & Cantor,
2019). These absorbance measurements were carried out on the NanoDrop
One Spectrophotometer (Thermo Scientific) according to manufacturer’s
instructions. Blank measurements were first performed with 2 μl of the
reference solution (elution buffer used during DNA extractions) to
minimise this solution’s contribution to the absorbance of the extracted
DNA. To be able to make inferences about purity of the extracted DNA,
spectrophotometry results from this study’s sample are compared to
typical absorbance of pure nucleic acid for DNA; a 260/280 nm (A260/280)
absorbance ratio of ~1.8 (1.85 – 1.88) and a 260/230 nm
(A260/230) absorbance ratio in the range of 1.8 – 2.3 (Desjardins &
Conklin, 2010; Koetsier & Cantor, 2019). Samples with 260/280 nm and
260/230 nm absorbance ratios greater or equal to 1.8 are generally
considered suitable for downstream applications (Koetsier & Cantor,
2019), but there are likely to be exceptions to these general guidelines
for interpreting absorbance ratios. Negative absorbance ratios could be
an indication of contamination that is emitting light instead of
absorbing it, absorbance ratios that are minor outliers generally give
an indication that DNA extraction procedures need to be improved, while
major outlier absorbance ratios suggest presence of impurities in the
sample (Desjardins and Conklin 2010). Outliers were determined using the
interquartile rule where the minor outliers in the absorbance ratios are
lower than the first quartile value minus 1.5 times the interquartile
range (i.e., Q1 – 1.5(IQR)), the major outliers are higher than the
third quartile plus 1.5 times the interquartile range (i.e., Q3 +
1.5(IQR)), and interquartile range is calculated by subtracting the
first quartile value from the third quartile value (i.e., IQR = Q3 –
Q1).
Following the absorbance measurements, all extracted DNA samples were
used to amplify DNA barcode fragments with a polymerase chain reaction
(PCR). In addition to PCR being a step towards obtaining DNA barcodes,
it also provides an indication of whether the success of this barcoding
conforms to absorbance ratio guidelines. This amplification targeted a
region of a length of maximum 664 bp of the COI gene with a primer set
from a previous study by Nagy et al., (2012); RepCOI-F (5’-TNT TMT CAA
CNA ACC ACA AAG A-3’) and RepCOI-R (5’-ACT TCT GGR TGK CCA AAR AAT
CA-3’). For DNA barcoding animals, the mitochondrial 5’ end of the
cytochrome c oxidase subunit 1 marker (COI) is proposed as a
universal barcode marker (Hebert et al., 2003b). The PCR reactions were
performed in total volumes of 25 μl: 12.5 μl Thermo Scientific DreamTaq
Green PCR Master Mix (X2) (with DreamTaq DNA Polymerase, 2X DreamTaq
Green buffer, dNTPs, at 0.4 mM each and 4 mM MgCl2),
1.25 μl (10 μM) of each of the two RepCOI primers mentioned above, 3 μl
of the template DNA elution and 7 μl Thermo Scientific Nuclease-free
water (PCR-grade). The reactions were carried out in the Applied
Biosystems SimpliAmp Thermal Cycler (Thermo Fisher Scientific Inc) using
the following PCR protocol: initial denaturation at 95°C for 3 minutes,
40 cycles of denaturation at 95°C for 30s, annealing at 48.5°C for 30s,
and extension at 72°C for 1 minute, followed by a final extension at
72°C for 10 minutes, and subsequent storage of PCR products at 4°C. The
PCR products were visualised on a 1% agarose gel under ultraviolet
light on the E-BOX CX5 stand-alone gel imaging system (Vilber Lourmat
Deutschland GmbH).
Sequencing protocol
Purification and sequencing of PCR products was outsourced to a
commercial sequencing company (Inqaba Biotechnical Industries (Pty) Ltd,
Pretoria, South Africa). They cleaned PCR products using the ExoSap
Protocol: 10 µl amplified PCR product and 2.5 µl ExoSAP master mix
(Exonuclease I 20 U/ul and Shrimp Alkaline Phosphatase 1 U/ul) mixed
well and incubated at 37°C for 15 minutes then held at 80°C for 15
minutes. The Nimagen, BrilliantDye™ Terminator Cycle Sequencing Kit
V3.1, BRD3-100/1000 was used to sequence fragments according to
manufacturer’s instructions. The cycle sequencing protocol provided by
the sequencing company was as follows: 10 μl NEB OneTaq 2X MasterMix
with standard buffer, 1 μl genomic DNA (10-30ng/μl), 1 μl of forward and
reverse primer each (10μM) (using the same primers, RepCOI-F and –R, as
in initial amplification), and 7 μl Nuclease free water. The sequencing
PCR profile was 94°C for 5 min, 35 cycles of 94°C for 30 seconds, 50°C
for 30 seconds, and 68°C for 60 seconds, followed by 10 minutes at 68°C
and subsequently held at 4°C. Subsequently, products were cleaned with
the ZR-96 DNA Sequencing Clean-up Kit and the cleaned products were
injected on an Applied Biosystems ABI with a 50cm array (using POP7).
Sequence chromatograms were analysed using the FinchTV analysis
software.
Sequences obtained from the commercial sequencing company were trimmed
with the Decontamination Using Kmers (BBDuk) trimmer, paired, then
assembled using De Novo assembly on the Geneious Prime® 2022.0.2
(https://www.geneious.com/prime/) sequence analysis software
(Biomatters New Zealand Ltd). The BOLD Identification System (IDS) was
used to compare this study’s sequences to reference samples on the BOLD
database
(https://v3.boldsystems.org/index.php/IDS_IdentificationRequest)
for verification of the sequence and species identity using
neighbour-joining placement (Ratnasingham & Hebert, 2007).
Subsequently, The Basic Local Alignment Search Tool (BLAST) (Altschul et
al., 1997) was also used for a second comparison of all of this study’s
sequences with published sequences on the NCBI Nucleotide collection
(nr/nt) database (http://blast.ncbi.nlm.nih.gov/Blast.cgi) to
determine sequence and species identity using the MegaBlast (Zhang et
al., 2000) algorithm for identifying highly similar sequences. A
difference of 2% or less between DNA sequences was used as a limit for
discriminating between species (Hebert et al., 2003a; Pereira et al.,
2013). Morphology of specimens was used to supplement molecular
identification. The sequences obtained from study were deposited in the
NCBI GenBank database under the accession numbers [GenBank: XXX-XXX]
(accession numbers to be added later).