Genetic Variation in the Invaded Population of the Fall Armyworm, Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae), in India

Abstract

The fall armyworm, Spodoptera frugiperda, an invasive, polyphagous pest, causes significant damage to corn. The majority of insects rely on a broad range of digestive enzymes and an intricate detoxifying mechanism to consume chemically diverse host plants. The genetic variation in S. frugiperda was analyzed using cytochrome oxidase subunit I (COI) and triose phosphate isomerase (Tpi). In addition, a new attempt was made to determine the variation with respect to two detoxifying genes, viz., carboxylesterase and glutathione S-transferase. The highest genetic variation (3.03%) was found between the S. frugiperda populations of Tirupati and Delhi with respect to the Tpi gene and between the populations of Punjab and Hosur regarding COI (3.30%). The results for various genes revealed that populations of the fall armyworm were homogeneous, showing low genetic distance using COI, ranging from 0.40 to 3.30%, and, using Tpi, ranging from 0.43 to 3.03%. The variation in carboxylesterase and glutathione S-transferase ranged from 0.04 to 0.15% and from 0.01 to 0.02, respectively. Amino acid sequences were also produced using DNA sequences from several fall armyworm populations. Populations in Tirupati, Solapur, and Hyderabad shared 98.7% of their sequence with that in Delhi. Fall armyworm amino acid sequences showed 79.7 to 82.0% identity with S. exigua and 69.6 to 73.0% identity with S. litura. Our study provides vital information for understanding the genetic variation in the fall armyworm following its invasion of India.

1. Introduction

The fall armyworm (FAW), Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae), is native to the Americas and is known to attack a large number of crops. More than 350 plant species have been reported as host plants, meaning that the fall armyworm causes significant damage to economically valuable crops [1]. The FAW is divided into two strains, the rice strain and the corn strain, based on host plant preference. These strains differ genetically, with 2.09% genome divergence, although they have similar morphologies [2]. While the “corn strain” is more common in corn and sorghum, the “rice strain” is more frequently detected in pasture grass and millet. In 2016, the fall armyworm invaded the African continent and then spread rapidly to more than 28 countries in southern and eastern Africa [3,4], reaching India in May 2018 [5]. Recent findings suggest that migratory fall armyworm populations from Puerto Rico have undergone significant genetic exchange with populations in Florida [6,7]. Such genetic exchange between Indian populations and migratory populations of the fall armyworm across continents might have resulted in the introduction of resistance alleles into India. Thus, these occurrences provide clues that the migratory patterns of the fall armyworm involve significant genetic exchange between populations. Resistance build-up in S. frugiperda to various insecticides from different groups has been reported in different parts of the world [8,9,10]. Resistance in insects is usually a complex phenomenon with more than one mechanism operating simultaneously within the same insect strain. Insects employ a number of biochemical and genetic mechanisms to resist insecticides. Multiple applications of insecticides in one cropping season can lead to the rapid development of resistance [9]. In Spodoptera, three major detoxifying enzymes have been implicated in the metabolic detoxification of insecticides: carboxylesterase, mixed-function oxidases [11,12], and glutathione S-transferases [11]. The development of insecticide resistance in S. frugiperda is one of the major obstacles to the profitable cultivation of corn crops in countries to which it is native and in those it has invaded. The molecular basis of similarity in S. frugiperda populations has been studied using both universal primers and detoxifying genes. Efforts have been made to collect information on genetic diversity in S. frugiperda, in addition to other closely related species, which have been considered as outgroups in phylogenetic analyses, as knowledge of the similarities is also useful in tracking the subsequent evolution of the FAW in India [13]. In this study, we evaluated the association between fall armyworm populations from a number of remote locations. DNA sequencing was utilized to examine four gene segments, including the nuclear (triosephosphate isomerase gene, or Tpi) and mitochondrial (cytochrome oxidase I, or COI) segments that are commonly used for DNA barcoding. The genes responsible for detoxification, carboxylesterase, and glutathione S-transferase are represented in the third and fourth genomic segments. This is the first study to pinpoint variations brought about by detoxifying genes, allowing researchers to understand the chemical pressure on the introduced FAW population in order to control the pest. Knowledge of geographical and host plant variation in the susceptibility of S. frugiperda to insecticides, the mechanism of insecticide detoxification, and information on susceptibility to new molecules would be useful in formulating location-specific resistance management strategies.

2. Materials and Methods

2.1. Fall Armyworm Collection

Egg masses and different stages of FAW larvae were collected from unsprayed corn fields from different states of India between 2019 and 2022 (Figure S1, Table S1). The susceptible population (F16) was the field-collected population from corn fields in Shivamogga, Karnataka, India, in July 2018 and reared under laboratory conditions without exposure to the insecticides at the Department of Entomology, College of Agriculture, Shivamogga. To avoid cannibalism, later instar larvae were collected and kept individually in breeding dishes and provided an artificial diet until they were brought safely to the laboratory for further analysis. The laboratory culture was maintained at the Department of Entomology, College of Agriculture, Shivamogga, Karnataka, at 26.9 to 29.2 °C, 80% RH, and a 16:08 h (L:D) photoperiod.

2.2. DNA Preparation

Populations of FAW were collected from different locations and were stored in 90% ethanol at −20 °C until DNA extraction. The stored samples were gathered in bulk and examined simultaneously. Genomic DNA was extracted from the fourth instar larvae of fall armyworms from the different populations using the Cetyl trimethyl ammonium bromide (CTAB) method [14]. The Tpi gene of S. frugiperda was amplified via reference to the method [15,16]. The larvae were crushed in 1.5 mL microcentrifuge tubes using a micro pestle with 0.5 mL of CTAB extraction buffer. The samples were incubated at 60 °C for 1 h with intermittent mixing. The samples were cooled for 5 min at room temperature and an equal volume of chloroform–isoamyl alcohol 24:1 was added. The samples were centrifuged at 13,000 rpm for 15 min. The top aqueous phase was transferred into a new Eppendorf tube, and 2 µL of 10 mM of RNase was added to each tube and incubated at 37 °C for 30 min. Thereafter, 500 µL of ice-cold isopropanol was added to each tube, and they were then incubated overnight at −20 °C. Centrifugation at 13,000 rpm for 15 min at 15 °C was carried out following overnight incubation. The supernatant was then discarded to avoid loss of the pellet formed at the bottom of the tube. The pellet was washed in 0.5 mL of 70% ethanol via centrifugation at 13,000 rpm for 5 min. The ethanol was discarded, and the pellet was air-dried at room temperature for 15 min. Lastly, the dried pellet was suspended in 50 μL 1xX TE buffer and stored at −20 °C. A mitochondrial gene (COI), a nuclear gene (Tpi), and detoxifying genes (carboxylesterase and glutathione S-transferase) were used to study the genetic diversity of the S. frugiperda population.

2.3. Agarose Gel Electrophoresis and PCR Amplification

A total of 200 mL of 0.5% (w/v) of agarose gel was prepared by accurately weighing 1 g of agarose powder, made up to 200 mL with 1 × TAE (196 mL of water + 4 mL of 50 × TAE) as a solvent, heated in a microwave oven until the agarose dissolved entirely, and then allowed to cool. Ethidium bromide (DNA intercalating agent) was added when the solution was slightly cooled as the staining agent, which helps to visualize the DNA bands under UV light.

PCR was performed in a total reaction volume of 35 µL, containing 17.5 µL of PCR master mix, 2.1 µL of both forward and reverse primers, 3.5 µL of the DNA solution, and 11.9 µL distilled water. The reaction mixtures were amplified under the following conditions: a COI gene of 658 bp (initial denaturation at 92 °C for 5 min, followed by 35 cycles at 92 °C for 1 min, 55 °C for 1 min, and 72 °C for 1 min and a final extension at 72 °C for 5 min) [17], a Tpi gene of 444 bp (initial denaturation at 94 °C for 5 min, followed by 35 cycles at 92 °C for 30 s, 56 °C for 45 s, and 72 °C for 45 s, and a final extension at 72 °C for 3 min) [18], and a carboxylesterase gene of 568 bp (initial denaturation at 94 °C for 5 min, followed by 35 cycles at 92 °C for 30 s, 56 °C for 45 s, and 72 °C for 45 s, and a final extension at 72 °C for 3 min). Since cDNA sequences were not available for glutathione S-transferase, primers were designed from the whole-genome sequence of S. frugiperda, with accession number NC049722.1. Then, 661 bp glutathione S-transferase (initial denaturation at 94 °C for 5 min, followed by 35 cycles at 94 °C for 30 s, 60 °C for 1 min, and 72 °C for 1 min, and a final extension at 72 °C for 5 min) was amplified in a 96-well thermal cycler. The details of the primers are provided in Table S2. The primers were custom synthesized at Sigma Aldrich in Bengaluru, India, supplied as lyophilized products of desalted oligos, and used to amplify the S. frugiperda. Amplified DNA samples were securely packed in ice packs, labeled separately, and sent to Barcode Biosciences, Bengaluru, for gene sequencing after being validated via gel electrophoresis.

2.4. Comparison of Fall Armyworm Population Using Amino Acid Sequences

DNA sequences of various populations were used to generate amino acid sequences to compare the variations in amino acids in different populations of S. frugiperda. Amino acid sequences of carboxylesterase were generated using the Translate online tool Expasy (https://web.expasy.org/translate/ (accessed on 5th February 2022)). Single and largest open reading frames were retrieved among the other frames. Lastly, the sequences were used to compare the variation in amino acids. Percent identity was also calculated to determine differences between the populations using Clustalo Emb software 1.2.2 (https://www.ebi.ac.uk/Tools/msa/clustalo/ (accessed on 10th April 2024)). Carboxylesterase genes from S. litura (KF835888), S. exigua (EF580101), and S. littoralis (HQ12260) were aligned with the inferred amino acid sequence of carboxylesterase from different populations of S. frugiperda.

2.5. Sequence Analysis

The sequences for the four genes were individually edited and aligned using Bio Edit version 7.2.5; poor-quality regions and gaps from the aligned sequences of various genes were removed using an automated option and then analyzed using MEGA X software (https://www.megasoftware.net/ ((accessed on 24 June 2022)). Following identification, the sequences were deposited in the NCBI GenBank database with different accession numbers. Phylogenetic analysis was carried out using the UPGMA method in MEGA-X software (https://www.megasoftware.net/ ((accessed on 24 June 2022)). The evolutionary divergence between sequences was estimated. All ambiguous positions were removed for each sequence pair (pairwise deletion option). Outgroups of closely related species were used, and a phylogenetic tree was constructed. The reliability of the phylogram was tested using bootstrap analysis with 1000 replicates [19,20,21].

3. Results

Genetic relatedness was studied among the populations of S. frugiperda collected from different locations using different genes. The genetic relatedness of S. frugiperda populations was studied using a mitochondrial gene, i.e., cytochrome oxidase subunit I (COI), a nuclear gene, i.e., triose phosphate isomerase (Tpi), and detoxifying genes, i.e., carboxylesterase and glutathione S-transferase. The sequences were aligned and analyzed. All of the sequences were submitted to the NCBI GenBank database and the accession numbers were obtained (

Table 1. Accession details of MtCOI, Tpi, carboxylesterase, and glutathione S-transferase genes of Spodoptera frugiperda deposited in the NCBI GenBank database.

S.N.	Populations	MtCOI	Tpi	Carboxylesterase	Glutathione S-Transferase
1	Susceptible	OL638179	OL555556	OL555549	OL555565
2	Davangere	OL638153	OL555557	OL555550	OL555563
3	Tirupati	OL624642	OL555558	OL555551	OL555564
4	Solapur	OL638191	OL555559	OL55555	OL555566
5	Hyderabad	OL638185	OL555560	OL555553	OL555567
6	Hosur	OL797984	OL555561	OL555554	OL555568
7	Delhi	*	OL555562	OL555555	*
8	Punjab	OL638970	*	*	*

* Samples with poor amplification.

).

3.1. Mitochondrial Marker: Cytochrome Oxidase Subunit I (MtCoI) Gene

UPGMA (Unweighted Pair Group Method with Arithmetic Mean) cluster analysis showed that the genes were identical in all populations and that the sequences were closely matched with the nucleotide sequences of the fall armyworm sister species S. litura.

Pairwise distance analysis for the COI sequence indicated that the fall armyworm populations are genetically distinct from their sister species, S. litura, which was used in isolation as an outgroup. The highest genetic variation (3.30%) was found between the populations of Punjab and Hosur. Fall armyworm populations showed a consistent genetic variation of 6.60% with S. litura (

Table 2. Dissimilarity in the MtCOI gene of the Spodoptera frugiperda population (in percent).

Populations	Susceptible	Davangere	Tirupati	Solapur	Hyderabad	Hosur	Punjab	S. litura
Susceptible	-
Davangere	0.4	-
Tirupati	0.4	0.6	-
Solapur	0.6	0.4	0.30	-
Hyderabad	0.4	0.7	0.60	0.60	-
Hosur	2.10	1.8	2.20	2.40	2.20	-
Punjab	1.60	1.30	1.80	1.60	1.50	3.30	-
S. litura	6.60	6.60	6.60	6.60	6.60	6.60	6.60	-

).

For phylogenetic analysis of the COI gene, S. frugiperda populations collected from different locations were used along with a single out-group, S. litura. The phylogenetic analysis showed two main clusters that differentiated the S. frugiperda populations from S. litura (Figure 1a).

3.2. Triose Phosphate Isomerase (Tpi) Gene

The results of our pairwise distance analysis indicated that the fall armyworm populations were genetically distinct with respect to the Tpi gene. The highest genetic variation (3.03%) was found between the S. frugiperda populations of Tirupati and Delhi. When compared with the sequence of South Korean fall armyworm populations submitted to the NCBI GenBank database, the genetic variation in fall armyworm populations ranged from 0.74 to 2.55% (

Table 3. Dissimilarity in the Tpi gene of the Spodoptera frugiperda population (in percent).

Populations	Susceptible	Davangere	Tirupati	Solapur	Hyderabad	Hosur	Delhi	South Korea
Susceptible	-
Davangere	0.44	-
Tirupati	1.75	1.76	-
Solapur	0.87	1.10	2.65	-
Hyderabad	1.09	1.32	2.87	0.87	-
Hosur	1.09	1.32	2.87	0.88	0.43	-
Delhi	1.75	2.01	3.03	2.02	2.01	2.01	-
South Korea	0.92	1.15	2.55	1.16	1.15	1.15	0.74	-

).

For phylogenetic analysis of the Tpi gene, S. frugiperda populations collected from different locations were used together with the NCBI sequence of South Korean (Accession no.—MT894240.1), Chinese (Accession no.—OR724712.1), Congolese (Accession no.—OQ857569.1), and Ugandan (Accession no.—MT894233.1) fall armyworm populations. The phylogenetic analysis showed two main clusters, 1 and 2. Group 1 consisted of fall armyworm populations, viz., Hyderabad, Hosur, Solapur, Susceptible, Davangere, and Tirupati. Group 2 consisted of Delhi and South Korean, Chinese, Congolese, and Ugandan populations, closely related to each other (Figure 1b).

3.3. Detoxifying Gene: Glutathione-S-Transferase

The glutathione S-transferase gene was amplified and sequenced from the DNA of the Spodoptera frugiperda population. The results of our UPGMA cluster analysis showed very few variations among the populations studied, and the sequences were closely linked with the nucleotide sequences of S. litura, S. exiua, and S. littoralis (Figure 1).

The results of our pairwise distance analysis indicated that the fall armyworm populations were genetically distinct from their sister species, such as S. litura, S. exigua, and S. littoralis, with the genetic variation in the fall armyworm with S. litura ranging from 2.02 to 2.13%, and that with S. littoralis ranging from 2.29 to 2.43%. In comparison, all of the populations of fall armyworm were consistent with the genetic variation of 1.46% with S. exigua (

Table 4. Dissimilarity in the glutathione S-transferase gene of the Spodoptera frugiperda populations (in percent).

Populations	Susceptible	Davangere	Tirupathi	Solapur	Hyderabad	Hosur	S. litura	S. exigua	S. littoralis
Susceptible	-
Davangere	0.02	-
Tirupati	0.02	0.02	-
Solapur	0.01	0.02	0.02	-
Hyderabad	0.01	0.02	0.02	0.01	-
Hosur	0.01	0.02	0.02	0.01	0.01	-
S. litura	2.12	2.05	2.02	2.12	2.05	2.13	-
S. littoralis	2.40	2.29	2.39	2.43	2.37	2.39	2.14	-
S. exigua	1.46	1.46	1.46	1.46	1.46	1.46	1.25	1.40	-

).

With respect to the glutathione S-transferase gene, there was minimal genetic variation between the populations of S. frugiperda. All of the populations of fall armyworm showed genetic variation in the range of 0.01 to 0.02 (Table 4).

For phylogeny analysis of the glutathione S-transferase gene, S. frugiperda populations collected from different locations were used together with out-groups (sister species of the fall armyworm). The phylogenetic analysis results showed three main clusters that differentiated the S. frugiperda populations from the S. litura, S. exigua, and S. littoralis populations. However, the sub-clusters of the glutathione S-transferase gene of the fall armyworm showed no distinct genetic variation among the populations of S. frugiperda (Figure 1c).

3.4. Detoxifying Gene: Carboxylesterase

The results of our UPGMA cluster analysis showed that the gene sequence was identical in all populations and that the sequences were closely matched with the nucleotide sequences of S. litura, S. exigua, and S. littoralis (Figure 1d).

The results of our pairwise distance analysis indicated that the fall armyworm populations were genetically distinct from their sister species, such as S. litura, S. exigua, and S. littoralis, with genetic variation with S. litura ranging from 0.96 to 1.06%, that with S. exigua ranging from 1.29 to 1.43%, and that with S. littoralis ranging from 1.13 to 1.35%. The highest genetic variation (0.15%) was found between the populations of Tirupati, Hosur, and Hyderabad and susceptible populations (

Table 5. Dissimilarity in the carboxylesterase gene of the Spodoptera frugiperda population (in percent).

Populations	Susceptible	Davangere	Tirupathi	Solapur	Hyderabad	Hosur	Delhi	S. litura	S. exigua	S. littoralis
Susceptible	-
Davangere	0.13	-
Tirupati	0.15	0.06	-
Solapur	0.08	0.05	0.06	-
Hyderabad	0.15	0.04	0.07	0.05	-
Hosur	0.15	0.04	0.08	0.08	0.04	-
Delhi	0.09	0.08	0.06	0.07	0.07	0.05	-
S.litura	1.00	0.98	1.00	1.01	0.96	1.01	1.06	-
S.exigua	1.38	1.38	1.43	1.29	1.38	1.34	1.30	0.14	-
S.littoralis	1.25	1.32	1.35	1.18	1.31	1.24	1.13	2.53	2.03	-

).

For phylogenetic analysis of the caroboxylesterase gene, S. frugiperda populations collected from different locations were used together with out-groups (sister species of the fall armyworm). The phylogenetic analysis results showed three main clusters that differentiated the S. frugiperda populations from S. litura, S. exigua, and S. littoralis populations. However, the sub-clusters of the carboxylesterase gene of the fall armyworm showed no distinct genetic variation among the various populations of S. frugiperda (Figure 1d).

3.5. Comparison of Different Populations of Spodoptera Frugiperda Using Amino Acid Sequences

DNA sequences of various populations of the fall armyworm were used to generate amino acid sequences to compare the variations in the amino acid sequences of Spodoptera frugiperda. Utilizing amino acid sequences, the percent identity was verified. The sequence of the Tirupati, Solapur, and Hyderabad populations showed 98.7% identity with the Delhi population. All of the amino acid sequences of the fall armyworm exhibited a range of 79.7 to 82.0% identity with S. exigua and a range of 69.6 to 73.0% identity with S. litura (

Table 6. Percent identity matrix using the amino acid sequence of carboxylesterase.

Populations	S. litura	S. exigua	Susceptible	Hosur	Delhi	Davangere	Tirupati	Solapur	Hyderabad
S. litura	-
S. exigua	80.8	-
Susceptible	71.0	80.0	-
Hosur	71.0	80.0	100.0	-
Delhi	68.9	79.7	96.0	96.0	-
Davangere	73.0	82.0	98.0	98.0	98.0	-
Tirupati	69.6	81.1	98.0	98.0	98.7	100.0	-
Solapur	69.6	81.1	98.0	98.0	98.7	100.0	100.0	-
Hyderabad	69.6	81.1	98.0	98.0	98.7	100.0	100.0	100.0	-

).

The average amino acid composition of fall armyworm populations with respect to the carboxylesterase gene was 4.3% Ala (alanine), 2.4% Cys (cysteine), 3.2% Asp (aspartic acid), 4.4% Glu (glutamic acid), 6.9% Phe (phenylalanine), 2.8% Gly (glycine), 1.7% His (histidine), 2.8% lle (isoleucine), 10.3% Lys (lysine), 10.0% Leu (leucine), 7.8% Met (methionine), 5.1% Asn (asparagine), 3.6% Pro (proline), 4.1% Gln (glutamine), 4.7% Arg (arginine), 5.7% Ser (serine), 6.9% Thr (threonine), 5.0% Val (valine), 2.0% Trp (tryptophan), and 6.5% Tyr (tyrosine) (Figure 2)

4. Discussion

The Tpi and COI genes are genetically characterized in African and Asian specimens, and the Tpi gene was found to be a better molecular marker of host plant preference than the COI gene [22]. There has been a significant genetic change in S. frugiperda species [23,24]. The geographical range of S. frugiperda is thought to cause genetic heterogeneity within this pest [23,24,25]. Variation and genetic diversity are critical for determining management strategies and monitoring resistance development [24,26]. The present results are consistent with those of other studies [23,24,25]. We found genetic variation among the populations of S. frugiperda; however, these results are contradictory to other studies showing no genetic variation in S. frugiperda populations [27,28]. Comparing our dataset to earlier research from India and Africa, less genetic variation could be the potential reason owing to the rapid migration within two years [13,18,22]. A single introduction followed by quick dispersion through natural and trade-related processes could account for the low number of haplotypes found in other studies conducted in the Republic of Congo and the Philippines [29,30]. These findings may be explained by its recent migration or more likely by the weak selection forces that accompanied its establishment [29,30]. In this regard, a clearer understanding of the genetic differences among polyphagous pests such as S. frugiperda will help scientists in better understanding the structure and population dynamics of these pests, their behavior, and their responsiveness to diverse selection pressures. More in-depth knowledge of the gene flow of sympatric and allopatric S. frugiperda populations linked with distinct locales and host plants is needed to provide insights into the evolutionary history of S. frugiperda in India.

In this study, phylogenetic trees were constructed using mitochondrial (COI), nuclear (Tpi), and detoxifying genes (carboxylesterase and glutathione S-transferase); the topologies of the trees revealed that all S. frugiperda populations belong to a single main clade. As a result, no distinct genetic diversity in COI, Tpi, or detoxifying genes was found in S. frugiperda from different geographical regions in India. In the future, the above findings can be validated by including large fall armyworm populations and a variety of host plants. The results of genetic diversity and phylogenetic analyses of S. frugiperda using COI, Tpi, carboxylesterase, and glutathione S-transferase revealed that S. frugiperda from different geographical regions are homogeneous, with populations showing overall low genetic distance for COI at 0.40 to 3.30%, Tpi at 0.43 to 3.03%, carboxylesterase at 0.04 to 2.53%, and glutathione S-transferase at 0.01 to 0.02, with low genetic distance across S. inferans populations found in rice in the Punjab state of India.

The Indian population of S. frugiperda differed significantly from other populations, including that of South Korea. The Tpi gene is specific to S. frugiperda; hence, populations in the present study were compared with the Tpi gene of the NCBI sequence of South Korean fall armyworm populations. However, the genetic distance in the Indian population was less between different geographical locations in India. Overall, it must be noted that there is a distinction between laboratory and fieldwork results. Differentiation in S. frugiperda most likely occurs during adaptation to mass rearing, leading to the selection of specific alleles that are not generally indicative of field populations. While our findings indicate that laboratory strains exhibit genetic diversity compared to field populations, the former may have unique allele frequencies that are not representative of field populations [31].

Based on the amino acid substitutions at various residues in S. litura populations from Delhi, Sonepat, and Varanasi, in addition to selected members of carboxylesterase from other Spodoptera species, we concluded that metabolic detoxification appears to be primarily due to the overproduction of the CarE enzyme in respective populations rather than amino acid residue changes [13]. The results of this study demonstrate that S. frugiperda is genetically less variable with the present collected populations, implying that future research should include more populations from a wider geographic area to acquire more genetic information on the host strain, geographic isolation, and reproductive incompatibility.

5. Conclusions

In conclusion, the analysis of all four genes, COI, Tpi, glutathione S-transferase, and carboxylesterase, suggested that invading FAW populations have low genetic variation. Glutathione S-transferase and carboxylesterase exhibited very little or no variation compared to COI and Tpi. Although the two detoxifying genes utilized in this study cannot be verified at the molecular level to determine diversity, we cannot rule out the possibility that invading FAW populations have a high capacity for genetic adaptability to new environments. Variation in detoxifying genes may occur in the future, depending on the extent of pest pressure and evolution, which is critical for determining management strategies. This study forms the basis for future studies on variation including detoxifying genes.