MethodsX 8 (2021) 101255 
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MethodsX 
j o u r n a l h o m e p a g e: w w w . e l s e v i e r . c o m / l o c a t e / m e x 
Method Article 
An optimized high-quality DNA isolation protocol 
for spodoptera frugiperda J. E. smith (Lepidoptera: 
Noctuidae) 
Diana Victoria Marína , b, Diana Katherine Castillob,         
Luis Augusto Becerra López-Lavalleb    ,  Jairo Rodríguez Chalarcab,  ∗ , 
Cristo Rafael Pérezc  
a Universidad Nacional de Colombia, Sede Palmira, Palmira, Valle del Cauca, Colombia 
b The Alliance of Bioversity International and the International Center for Tropical Agriculture (CIAT), Recta Cali-Palmira km 
17, Valle del Cauca, Colombia 
c Fedearroz-Fondo Nacional del Arroz, Monteria, Córdoba, Colombia 
a b s t r a c t 
An optimized high-quality DNA isolation protocol was developed using body segment tissue from the Fall 
Armyworm (Spodoptera frugiperda), that will allow documenting genetic variability based on biotypes, facilitating 
studies on the appearance, distribution and population dynamics of the fall armyworm at the molecular level. The 
resulting protocol is an easy-to-use, timesaving method that can rapidly achieve high quality, high-yielding total 
genomic DNA, using chemicals and everyday consumables available in a molecular laboratory. This new method 
of DNA extraction avoids the contamination of polysaccharides, salts, phenols, proteins and other cellular by- 
products that can interfere with subsequent reactions. DNA purity estimates reveal A260: A280 ratios greater 
than 1.9, which were evidenced by quality test on agarose gel, observing complete integrity and high purity of 
the resulting samples, and yielded 30–99 μg/g of total DNA. Therefore, the quality of the DNA produced from 
this extraction is suitable for subsequent molecular applications: (i) next generation whole genome sequencing, 
(ii) conventional polymerase chain reaction for genotyping, (iii) barcodes and (iv) gene cloning. In addition, to 
become an anticipating diagnostic tool for invasive lepidopteran larval stages: 
• The resulting protocol is an easy-to-use time-saving method. 
• This new extraction method prevents contamination from polysaccharides, salts, phenols, proteins, and other 
cellular sub-products. 
• DNA purity estimations reveal A260:A280 ratios above 1.9. 
© 2021 The Author(s). Published by Elsevier B.V. 
This is an open access article under the CC BY license ( http://creativecommons.org/licenses/by/4.0/ ) 
∗ Corresponding author. 
E-mail address: j.chalarca@cgiar.org (J.R. Chalarca). 
https://doi.org/10.1016/j.mex.2021.101255 
2215-0161/© 2021 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY license 
( http://creativecommons.org/licenses/by/4.0/ ) 
2 D.V. Marín, D.K. Castillo and L.A.B. López-Lavalle et al. / MethodsX 8 (2021) 101255 
a r t i c l e i n f o 
Method name: DNA isolation standardization for fall armyworm 
Keywords: Fall armyworm, DNA, COI, Barcoding, DNA isolation, CTAB method 
Article history: Received 31 May 2020; Accepted 28 January 2021; Available online 3 February 2021 
 
 
 
 
 
 
 
 
 
 
Specifications table 
Subject Area: Agricultural and Biological Sciences 
More specific subject area: Molecular entomology 
Method name: DNA isolation standardization for fall armyworm 
Name and reference of original Doyle, J. (1991). DNA protocols for plants. In Molecular techniques in taxonomy (pp. 
method: 283–293). Springer, Berlin, Heidelberg. 
Resource availability: Reagents: 
Ribonuclease A from bovine pancreas (Merck; Cat. No. R4875) 
UltraPureTM  1 M Tris–HCl ( pH 8.0; Thermo Fisher Scientific; Cat. No. 15,568,025) 
Sodium chloride solution 5 M (Merck; Cat. No. S5150) 
UltraPureTM  0.5 M ethylenediamine tetraacetic acid (EDTA; pH 8.0; Promega; Cat. No. 
V4233) 
Hexadecyltrimethylammonium bromide (CTAB; Merck; Cat. No. H5882) 
2-mercaptoethanol (Sigma-Aldrich; Cat. No. M6250) 
UltraPureTM  DNase/RNase-free distilled water (Thermo Fisher Scientific; Cat. No. 
10,977–015) 
Chloroform-isoamyl-alcohol mixture 24:1 (Merck; Cat. No. C0549) 
Absolute ethanol for analysis (Merck; Cat. No. 1.00983) 
Chloroform for analysis (Merck; Cat. No. 366,919) 
2-propanol (Merck; Cat. No. 109,634) 
Materials : 
Nanodrop©R  ND-10 0 0 spectrophotometer (Thermo Fisher Scientific) 
ELIMINase (Decon Laboratories Inc.; Cat. No. 1101) 
VWR©R  Mini Shaker 
Eppendorf Centrifuge 5427 R, Germany 
Method details 
Introduction 
The fall armyworm, Spodoptera frugiperda (Lepidoptera: Noctuidae), is the most important 
polyphagous pest from an economic point of view in South America, and it is associated with more
than 27 plant families [1] . The distribution of S. frugiperda has changed dramatically from its first
report in Africa (Senegal) in January 2016 on maize [2] . To date, S. frugiperda has been reported in
more than 30 countries in Africa [3] . It was reported during May and June 2018 in Asia [4] . In China
alone, in January 2019, S. frugiperda was reported in Yunnan Province and by May it had been reported
in more than 13 provinces [5] . For S . frugiperda , two biotypes (rice and maize) are reported [6 , 7] ,
which exhibit identical morphological characteristics although they differ in genetic, physiological, 
and reproductive isolation characteristics. In these two biotypes, cytochrome oxidase I and II and ITS1
genes have been sequenced in different populations of the United States, Mexico, Colombia, Brazil, 
Argentina, and Paraguay [6 , 8–10] . 
Therefore, the early detection and monitoring of economically important pests is critical for 
preventing their dissemination and establishment in valuable commodity crops [11–14] . To accomplish 
this, tools for rapid and accurate species identification are needed. DNA-based species identification 
using short DNA-barcode sequences (50 0–70 0 bp) such as mitochondrial cytochrome c oxidase 
subunit I–CO I in animals has proven effective for accurate species identification [15 , 16] . Furthermore,
DNA barcoding has shown its potential as a tool for rapid and accurate detection of economically
important insects [17–19] . 
Similarly, the quality of the DNA sequencing is an important criterion to achieve an accurate
identification of the targeted biological entity; hence, access to a high-quality DNA sample is 
D.V. Marín, D.K. Castillo and L.A.B. López-Lavalle et al. / MethodsX 8 (2021) 101255 3 
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mperative. Thus, a reliable, easy-to-use, fast, and inexpensive DNA extraction method is critical as
 first step in generating accurate high-quality DNA barcodes. Otherwise, this can represent a limiting
actor in the implementation of barcoding studies. Hence, it is crucial to have access to a time-
aving/cost-effective high-quality DNA isolation method that effectively removes contaminants such
s polysaccharides, salts, phenols, proteins, and other cellular sub-products [20] . 
For most Hemiptera species, high-yield/-quality DNA isolation is severely affected by a variety
f inhibitory compounds found in the DNA extraction matrix, mainly coming from the processed
iological sample (e.g., polysaccharides or phenols) or the chemical used for making the DNA
ccessible (e.g., CTAB) [21–23] . However, few studies deal with a comparison of extraction methods
ntended to find the most appropriate method to use in each species or family [21 , 24 , 25] . These
nsects contain plant phenolics and tannins in their digestive tracts, especially when the insects
re not adults, and they are not easily dissected for DNA isolation. Phenolics and other secondary
ompounds cause direct damage to DNA or inhibit enzymatic activity in downstream molecular
nalysis, particularly restriction endonucleases or Taq polymerases that decrease efficiency for
arcoding, genomic library construction, or Southern blot analysis [26 , 27] . Although the CTAB-based
NA extraction method is widely used, it occasionally fails to remove all phenolics from DNA
reparations, so antioxidants, such as β-mercaptoethanol and PVP, are commonly used to overcome
roblems related to phenolics [26] . 
Nevertheless, several modified CTAB methods [28] are available for DNA isolation of species-
pecific insects; however, in some instances, they have been erroneously referenced [29] . Moreover,
he implementation of other published DNA extraction methods does not ensure high-quality DNA
solation from the different body structures of fall armyworm [30] ; among these, CTAB total DNA
xtraction [31–33] . There is not even a consensus about the specific body section to be dissected
o conduct a successful DNA extraction [28 , 34–36] . In addition, several commercially available DNA
xtraction kits are recommended for insect DNA isolation, such as DNeasy Blood & Tissue Kit from
iagen [37] , DNAzol©R  Reagent [38] , and Puregene©R  Kit [39] . These kits, however, are not suitable for
NA isolation of some insect species, particularly those with high polysaccharide, polyphenol, protein,
nd other cellular sub-products. More importantly they are costly per sample and produce low DNA
ield in comparison with homemade protocols [40] such as SDS [34] or CTAB [26] . Previous research
n the identification of S. frugiperda biotypes used a CTAB method to isolate DNA from the FAW and
chieved success in only 10% of the analyzed samples [41] because of the low quality or degradation of
he extracted samples. To improve quality, researchers used DNeasy Blood & Tissue Kit from Qiagen,
entioned a range of concentrations from 25 ng/μL to 100 ng/μL, and did not report A 260:A  280 or 
 260:A  230 ratios.  
Given the evident low success of the proposed CTAB method in other studies [26 , 29 , 35 , 40] , the
eed to develop an optimized high-quality DNA isolation method for FAW, using all body sections
nd tissue from different storage conditions, is acknowledged. Thus, we developed a modified CTAB-
ased DNA isolation protocol that can produce high-yield/-quality total genomic DNA with minimal
ontamination using low-cost chemicals. Furthermore, our high-quality DNA isolation protocol is fast
nd simple and produces sufficient quantities of high-quality DNA. 
aterials and methods 
iological materials 
Different larval stages of Spodoptera frugiperda were sampled from four agricultural sub-regions
f Colombia: the Magdalena River Valley, the Humid Caribbean, the Cauca River Valley, and the
rinoquía. A sub-sample of 64 was stored in 70% ethanol at 4 ᵒ C. Additionally, adults from
arvae collected in maize, which were stored dry for a year at 4 °C, were used for DNA
xtraction. 
4 D.V. Marín, D.K. Castillo and L.A.B. López-Lavalle et al. / MethodsX 8 (2021) 101255 
Table 1 
Summary of the main characteristics of the DNA extraction protocol implemented for fall armyworm in other studies. 
Sample input Protocol 1 [35 , 41] Protocol 2 [29] Protocol 3[36] Protocol 4 [43] 
Head- abdominal section Thorax Abdomen Post-abdomen 
Volume of buffer per sample (μL) 400 400 500 400 
Extraction buffer 
0.1 M Tris-HCL pH 8.0 x x X X 
1.4 M NaCl x x X X 
0.02 M EDTA x x X X 
2x CTAB x x X X 
1% β-mercaptoethanol x x X X 
PVP 1% X 
Proteinase K x X X 
Incubation time (h) 0.5 1.0 3.0 1.0 
Washing steps 
Chloroform IAA x 2 x x X X 
Chloroform x x X X 
Isopropanol x x X X 
100% EtOH 1 1 2 2 
70% EtOH 1 
Purification step 
10 mg/mL RNase A X x x 
 
 
 
 
 
 
 
 
 
 
 
 
 
Solutions, reagents, and supplies 
All reagents and solutions were purchased at the required concentration from commercial 
suppliers, except for ribonuclease A from bovine pancreas (Merck; Cat. No. R4875) that was purchased
as powder and prepared in a working solution of 10 mg/mL. The stock solutions of UltraPureTM    
1 M Tris–HCl (pH 8.0; Thermo Fisher Scientific; Cat. No. 15568025), sodium chloride solution 
5 M (Merck; Cat. No. S5150), UltraPureTM  0.5 M ethylenediamine tetraacetic acid (EDTA; pH 8.0;
Promega; Cat. No. V4233), hexadecyltrimethylammonium bromide (CTAB; Merck; Cat. No. H5882), 
2-mercaptoethanol (Sigma-Aldrich; Cat. No. M6250), UltraPureTM  DNase/RNase-free distilled water 
(Thermo Fisher Scientific; Cat. No. 10977-015), chloroform-isoamyl-alcohol mixture 24:1 (Merck; Cat. 
No. C0549), absolute ethanol for analysis (Merck; Cat. No. 1.00983), chloroform for analysis (Merck; 
Cat. No. 366919), and 2-propanol (Merck; Cat. No. 109634) were of molecular biology grade and were
free of RNAses, DNAses, and pyrogens. All disposable plasticware for the preparation of extraction 
buffer and the tubes used for extraction were free of RNAses, DNAses, and pyrogens. 
DNA extraction procedure 
To obtain high-quality DNA from S. frugiperda , a total of four DNA isolation protocols were assessed
( Table 1 ) [11 , 35] . The initial sample input for DNA extraction depended on the protocol: head, thorax,
abdomen, and post-abdomen, in a weight range of 0.1 g to 1.0 g. Prior to DNA extraction, benchtop
areas were cleaned using 70% ethanol and ELIMINase (Decon Laboratories Inc.; Cat. No. 1101).
Pipettes were also cleaned using the same procedure and exposed to ultraviolet light for 30 min.
Mortars and pestles were washed with soap, rinsed with 70% ethanol, and autoclaved at 118 ᵒ C for
15 min. 
All tissue samples (head, thorax, abdomen, and post-abdomen) were macerated and homogenized 
with extraction buffer pre-heated at 65 ᵒ C. Proteinase K was added in the buffer according
to other protocols ( Table 1 ). Subsequently, all other steps were followed as described by each
protocol ( Table 1 ). Quantities and qualities of isolated DNA were evaluated spectrophotometrically
by determining absorbance ratios of A ©R  260:A  280 and concentration using a Nanodrop ND-10 0 0 
spectrophotometer (Thermo Fisher Scientific). DNA quality was further assessed electrophoretically 
D.V. Marín, D.K. Castillo and L.A.B. López-Lavalle et al. / MethodsX 8 (2021) 101255 5 
Fig. 1. DNA-quality gel product of the implementation of the CTAB protocol for identification of S. frugiperda biotypes. 
Table 2 
Yields and A 260:A 280 ratio of isolated total DNA from previously reported protocols and the protocol modified in this study.   
Component Protocol 1 Protocol 2 Protocol 3 Protocol 4 CIAT modified 
protocol 
DNA yield rate 46 ± 15.15 63 ± 24.75 45 ± 25.66 47 ± 23.89 72 ± 16.66 
(mean ± SE) (μg) 
Absorbance ratio 1.84 ± 0.08 1.45 ± 0.43 1.70 ± 0.12 1.77 ± 0.08 1.84 ± 0.07 
(mean ± SE) and range 
Color of DNA pellet light to dark light to dark light to dark light brown to light brown to 
brown brown brown brown brown 
Estimated time 5.5 6.0 16.0 5.5 6.0 
(h)/sample 
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s  n 1% agarose gels, which were prepared by adding 1.5 g of agar powder to 100 mL of 1X BS buffer
2% NaOH; 10% boric acid) and boiling until melted, followed by the addition of 4 μL of SYBR©R     Safe.
els were run at 90 V for about 35 min. 
esults and discussion 
Initially, we extracted DNA from the head, thorax, abdomen, and post-abdomen separately
ccording to previously described methods [29 , 35 , 36 , 42] ( Table 1 ). However, the resulting DNA was
f low yield and purity, and degraded in all cases when protocol 2 [28] or 3 [40] was used ( Fig. 1 ,
able 2 ). These preliminary assays indicated that these protocols were not suitable for Spodoptera
issues, likely reflecting the prevalence of inhibitory compounds during the extraction process that
re not efficiently removed during the washing steps proposed in the protocols and the lack of an
fficient enzymatic lysis. 
Therefore, according to the quality visualized in Fig. 1 , to improve the quality of the DNA, we
odified the methods reported [11 , 35] by adding mixing steps in a shaker to fully incorporate the
rganic compounds and increase the effectiveness of the washing steps, and also by increasing the
recipitation time and improving the astringency of the RNase A treatment. The larval body section for
NA extraction must be dissected and liquid-nitrogen ground on the day of extraction, when samples
ave been stored with 70% ethanol. These stored FAW samples, once processed, cannot be placed back
nto ethanol and stored for later DNA extraction since they showed clear evidence of degradation.
owever, if the liquid-nitrogen ground samples are stored at −80 °C, they can be stored for several
ays before DNA extraction. 
Our modified DNA isolation procedure was used for polymerase chain reaction (PCR), cloning, and
equencing to assess the quality and reliability of the DNA obtained. The protocol was used for all
6 D.V. Marín, D.K. Castillo and L.A.B. López-Lavalle et al. / MethodsX 8 (2021) 101255 
Fig. 2. Quality gel of the DNA isolated using the modifications to the CTAB-based protocol proposed here. (A) DNA from FAW 
samples collected in field and stored in 70% ethanol. (B) DNA from hind legs of FAW adults subjected to long-time storage at 
4 o  C. 
 
 
 
 
 
 
 
 
 
body segments, including samples collected from the field and reared in the laboratory. Here, we
describe the modified protocol as follows: 
Extraction buffer containing 100 mM Tris-HCL pH 8.0, 1.4 M NaCl, 0.02 M EDTA, 2x CTAB, and 1%
2-mercaptoethanol was prepared in ultra-pure water that was free of RNase and was then preheated
to 65 °C prior to its use. 
Tissues were (1) ground to a fine powder using a mortar and pestle in liquid nitrogen and stored
at −80 °C until DNA isolation or (2) dissected and immediately hand-macerated using a pestle in
a 1.5 mL tube with 200 μL of pre-heated extraction buffer for the homogenized sample. In this case,
when the samples were macerated with liquid nitrogen, it was provided continuously to avoid melting
of frozen tissues and to prevent DNA degradation until the addition of pre-heated buffer. 
Pre-heated extraction buffer (400 μL) was added to fresh body part samples ground in liquid
nitrogen; however, when the samples were either fresh tissue or tissue stored in 70% ethanol,
D.V. Marín, D.K. Castillo and L.A.B. López-Lavalle et al. / MethodsX 8 (2021) 101255 7 
Fig. 3. Amplification of the COI∗ region of FAW DNA isolated by the application of the DNA extraction protocol modified and 
proposed here. (∗   ) Using as a template 10 ng/μL isolated with the modified protocol implemented in this study. The negative 
controls correspond to the control without template to rule out the presence of cross contamination. 
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a  hey were macerated in 200 μL of pre-heated extraction buffer (in a 1.5 mL tube) to complete
omogenization. The remaining 200 μL of pre-heated extraction buffer was added to the final
xtraction volume of 400 μL. After adding the extraction buffer, the samples were incubated for
0 min at 65 ᵒ C and mixed by inversion every 5 min. 
After incubation, one volume of chloroform-isoamyl-alcohol (24:1) was added to the samples and
ixed by inversion at 90 rpm for 1 min in a shaker (VWR©R  Mini Shaker). Later, the samples were
entrifuged at 18,700 rcf for 6 min at room temperature (Eppendorf Centrifuge 5427 R, Germany).
hen, the supernatant was transferred to a new sterile 1.5 mL tube, to which one volume of
hloroform was added and mixed thoroughly by inversion for 1 min, followed by a centrifugation
t room temperature (18,700 rcf). This step was repeated one more time. 
The resulting supernatant was then transferred to a new sterile 1.5 mL tube with care not to
isturb the lower chloroform phases. Then, one volume of cold ( −20 °C) 2-propanol was added. The
amples were incubated for ≥2 h at −20 °C and, after that, centrifuged at 18,700 rcf for 12 min at
 °C. After discarding the supernatants, precipitated pellets were washed immediately with 200 μL
f cold ( −20 °C) 100% ethanol and centrifuged at maximum speed for 6 min. The pellets were then
ashed with 200 μL of cold ( −20 °C) 70% ethanol. Finally, the supernatants were discarded, the pellets
ried at room temperature for 1 h, resuspended in 50 μL of T 10E 1 and 5 μL of RNase A (10 mg/mL),  
ncubated for 1 h at 37 °C, and stored at −20 °C. 
The protocol described above was used on (1) the whole FAW larval body ( Fig. 2. A, wells 36, 50, 55,
9, 60), (2) head tissue ( Fig. 2. A, wells 64, 67, 68, 71, 72) stored with 70% ethanol, and (3) in posterior
egs of FAW adults stored for 1 year at 4 °C ( Fig. 2. B). Quantitative spectrometric assessments of DNA
eached A 260:A 280 ratios above 1.8, indicating very low protein contamination ( Table 2 ). Qualitative  
ssessment of the DNA in an agarose gel did not depict evidence of degradation ( Fig. 2 ). In addition,
he extraction protocol described here efficiently yielded 30–99 μg/g of high-quality total DNA from
ll samples, under several storage conditions. These yields were higher than with all other methods
ested here ( Table 2 ). 
Although very few commercially available DNA isolation and purification methods reported
cceptable concentrations and absorbance ratios, very few kits have been designed exclusively for
8 D.V. Marín, D.K. Castillo and L.A.B. López-Lavalle et al. / MethodsX 8 (2021) 101255 
Fig. 4. Resulting chromatogram of the COI region sequencing. The yellow arrows show the relative position of the primers 
flanking the COI fragment of interest. 
 
 
 
 
 
use in insects [43] . Our modified CTAB method resulted in higher DNA yield (ng DNA vs. mg tissue)
and quality when assessed on FAW body tissue. This protocol becomes a suitable option, especially
when a large number of samples need to be extracted or if DNA of high concentration and quality is
required for downstream molecular applications. 
On the basis of our proposed protocol, EDTA starts cell wall rupture to release the nucleic acids
[44] . Tris-HCl equilibrates the pH (close to 8.0), ensuring lysis. β-mercaptoethanol delays sample
D.V. Marín, D.K. Castillo and L.A.B. López-Lavalle et al. / MethodsX 8 (2021) 101255 9 
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xidation, facilitating DNA recovery. On the other hand, the addition of β-mercaptoethanol helps to
educe browning in DNA preparations produced by the oxidation of phenolics. Sodium chloride is
fficient in extracting nucleic acids from polysaccharides [45 , 46] . Cetyl trimethylammonium bromide
CTAB) in buffer has been used to obtain good quality, given that it acts on the membrane lipid
ayer to remove proteins. Chloroform-isoamyl-alcohol washing promoted protein precipitation and
ecreased polysaccharide contents. We also performed two chloroform washes to remove debris and
roteins in the initial stages of DNA isolation. The mixing step with chloroform-isoamyl-alcohol in
he shaker enables the disruption of the cell wall, which improves enzymatic lysis, decreases the
hances of DNA fragmentation, and ensures a high DNA yield. The longer incubation period with
Nase A decreases the possibility to obtain absorbance ratios lower than 1.9, which can be caused by
NA contamination. These observations suggest that CTAB and its modifications increase DNA yields
nd eliminate possible contamination with lipids, proteins, and other cellular compounds that may
nterfere with the downstream DNA purposes. 
Using the DNA obtained from our modified protocol, we amplified the cytochrome oxidase I region
COI) [45] to evaluate FAW biotype composition and diversity ( Fig. 3 ) in 83 field populations. The COI
ene is an ideal candidate to be used as a DNA barcode system. Its amplification and sequencing are
seful when rapid detection and identification of a pest are required for regulatory purposes [47 , 48] .
he targeted DNA fragment was cloned and sent to Macrogen (Korea) for sequencing. The resulting
hromatogram depicted evenly-spaced peaks, each with only one color ( Fig. 4 ). Little baseline noise,
n a few cases, was present, but it was minimal. Even so, the real peaks were still easy to call.
he fragments sequenced were blasted against the NCBI database and the sequences had a 99%
imilarity with previously reported sequences of S. frugiperda in other experiments [49] . There was
o contamination of the sequences that allowed associating the sequences with other species. 
In conclusion, we optimized a protocol to isolate high-quality DNA from FAW tissues, achieving
igh yields and DNA integrity. The extractions carried out confirmed that the implementation of this
rotocol for the isolation of DNA allows the selection of DNA samples free of inhibitors and generating
 suitable product for PCR amplification, for its subsequent cloning and sequencing. It was determined
hat this protocol is successful in all body sections of FAW larvae when the specimens are collected
n 70% ethanol and in adult legs when they are stored at 4 °C. A total duration of 6 h was established
or the implementation of this protocol, of which 3 h correspond to the incubation period. The total
umber of samples that can be processed per day varies between 24 when the tissue is dissected
ithin the same day just before DNA isolation from samples stored in 70% and 96% ethanol, and 96
hen the tissue was previously ground in liquid nitrogen and stored at −80 °C until DNA isolation.
inally, the quality of the DNA obtained suggests that this protocol can be implemented as a tool for
he detection of Spodoptera species and biotypes. 
eclaration of Competing Interest 
The Authors confirm that there are no conflicts of interest. 
cknowledgments 
We are grateful to the staff who provided support for the field collections and to Carlos Blanco for
omments on the manuscript. 
upplementary materials 
Supplementary material associated with this article can be found, in the online version, at doi: 10.
016/j.mex.2021.101255 . 
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Identificación de biotipos de Spodoptera frugiperda de diferentes áreas costeras, Biotecnol. Sust. 1 (2018) . 
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