Rapid identification of the invasive fall armyworm Spodoptera frugiperda (Lepidoptera, Noctuidae) using species-specific primers in multiplex PCR

The fall armyworm (FAW), Spodoptera frugiperda (Smith), is a major pest native to the Americas. A recent invasion of FAWs from Africa eastward to South Asia, the Indochina Peninsula, and mainland China has received much attention due to the considerable economic losses in agriculture. FAWs can rapidly colonise a new area, likely due to the wide range of host plants, good flying capability, and high egg production. Therefore, a convenient, quick, and accurate tool for FAW identification is urgently required to establish a FAW invasion management strategy. In this study, FAW-specific primers were designed to recognise FAWs on the basis of internal transcribed spacer 1 (ITS1). The results revealed the accurate FAW recognition of the three congeneric species and eight common corn lepidopteran pests, especially at their larval stage. Furthermore, species-specific primers have confirmed their efficacy by using 69 FAW specimens from Taiwan, Thailand, and the United States, with a 96% success rate, excluding 3 decayed specimens. By using the simple, reliable, and convenient FAW-specific primers, a pest management programme can be developed not only to reduce sequencing costs and experimental time from 2 days to 4 h, but eradicate the FAW as soon as it enters a new area.


Stability of species-specific primers for FAW. The stability test of FAW-specific primers on the three
Spodoptera species revealed that the expected amplified products were only amplified in target species, with no cross-amplification in the other two congeneric pests (Fig. 1). In each reaction, the successful amplification of universal primers of 28S rDNA with 520 bp confirmed the qualitative aspect of DNA and the experimental www.nature.com/scientificreports/ procedures. Additional weak bands were found with fragment sizes smaller than the universal primer products of Sfru1F/Sfru1R and Sfru1F/Sfru2R PCR amplifications, indicating no influence on the recognition of the specific products (Fig. 1a,b). For the eight common corn pests of noctuid moths, the stability of these species-specific primers showed that multiplex PCR can distinguish the FAW from other lepidopteran corn pests (Fig. 2). Although a weak band found in a nontarget species (Fig. 2c, lane 13: O. furnacalis) would somewhat lower the stability of the species identification, the relative DNA concentration without cross-amplification of other samples can eliminate this suspicion.

Efficacy of specific primers for FAW identification. Considering intraspecific variations of ITS1 in
FAWs, the efficacy of four species-specific primers on 69 FAW specimens from Taiwan, Thailand, and the United States showed that specimens were amplified with a 96% success rate, except for three suspected decayed samples, which also led to failure or weak amplification in universal primers (Fig. 3, see Supplementary Fig. S4). However, their DNA concentrations ranged from 19.8 to 129.9 ng/μL did not show great difference from the other samples (see Supplementary Table S1). Furthermore, the dilution test of DNA concentration also revealed that the decreasing efficacy of FAW-specific primers occurred when DNA concentration was diluted to 100X and 1000X (see Supplementary Fig. S6).      www.nature.com/scientificreports/

Discussion
Among the PCR-based approaches for species identification, multiplex PCR using universal primers, namely 28S rDNA, has ensured the DNA quality of sampled individuals 35,36 , which was seldom used. In this study, a few specimens died and rotted rapidly during their delivery process to our laboratory. Universal primers were useful to confirm the DNA quality for these decayed specimens in PCR experiments. Although the DNA concentration of the decayed samples showed no great difference to other samples, DNA fragmentation may be the possible explanation for their present status of failed amplification (see Supplementary Table S1 and Supplementary  Fig. S6). Similarly, the DNA concentration dilution tests, which mimicked the DNA amounts of different field specimens, also affirmed that the failure of PCR amplification of 28S rDNA in these specimens was due to their spoiled status, particularly the DNA concentration diluted > 100X. In addition, the advantages of multiplex PCR can reduce the sequencing costs and time required for purification of DNA products from at least 2 days to 4 h, provided the experimental equipment is available. The identified results can provide the FAW management faculties with information for controlling this pest immediately as they collect the specimen in the field, especially in FAW-free countries.
Although the species-specific primers using mitochondrial genes are common in insects [37][38][39] , the more efficient nuclear ITS region with hypervariable sites, particularly the distinct inter-and intraspecific genetic variations of ITS sequences, is more suitable for developing species-specific primers for species identification 32,[34][35][36][40][41][42] . However, the application of species-specific primers based on ITS sequences in insect identification remains rare. A few studies of mosquitos, thrips, and stored-product psocids have been proposed using ITS species-specific primers 35,36,41,[43][44][45][46] . The results of the present study suggest that the ITS1 sequence variations of Spodoptera with high interspecific variations of > 16% and low intraspecific divergence of < 1.2% (Table 1) can be favourable markers with which to develop species-specific primers for FAW identification. The FAW-specific primers have accurately identified the FAW specimens from congeneric species and the other common lepidopteran pests in the field.
Doubts concerning the stability and efficacy of specific primers for FAW identification are raised as many noctuid moths or FAW populations are involved. For example, previous studies on thrips have shown that the detection ability of specific primers might fail if the number of mismatched positions of primers is more than four, but only one or two sporadically variable positions have little influence 41 . In the case of FAWs, Lewter et al. 23 showed that no variation in ITS1 could be detected in samples belonging to both corn and rice strains from several types of crops in its native countries, namely the United States. Additional information from the nuclear ITS1 gene also revealed that gene flow may frequently occur in these two genetically differentiated strains, namely only 0.66% to 0.99% of the mitochondrial genes 23 . The slight genetic variation showed that these two host-specific strains may represent a recently diverged evolutionary units. Because no variation of the two strains was found in the ITS1 gene from its native habitat, the ITS1 of FAWs may have accumulated few or no mutations in the invasive populations within the past 5 years. Thus, the consistency of ITS1 among populations helped confirm the efficacy of the developed specific primer for FAWs. If the multiple invasion events occurred in the following years, the present species-specific primers should still work efficiently for FAW identification because nucleotide drift cannot occur in a short interval-several 1000 years. In addition, although a low concentration of the nontarget amplification products was observed in a specimen, which may have resulted from supercoiled DNA or a secondary structure of PCR product, the relatively high product yield of the target species could be applied concordantly for FAW identification (Fig. 2). The other three species-specific primer sets without PCR products amplification in the target position has eliminated this query. In this situation, the DNA barcodes are only required to confirm the identity of these two species, thus significantly elevating the efficiency of FAW identification.
An effective species recognition approach for FAW identification would be beneficial for establishing management strategies. On the basis of the biological habits of FAWs, FAW development from larva to adult requires approximately 30 days to establish its second generation in a new invasive area 1,31 . Because the damage related to FAW invasion predominantly occur in their larval stage, a rapid identification method is urgently required, especially considering that identifying the young larvae is more difficult than other larval instars and adults. Although pheromone traps are efficient for collecting adults for pest control, the FAW-invaded regions must take action at the initial invasion period to decrease its population establishment. Thus, it would be possible to eradicate FAWs as soon as it invades a new area if a quick identification method could be applied easily and conveniently. This study has displayed the efficiency of multiplex PCR using four pairs of specific primers in a systematic manner, which has also proved that these FAW-specific primers are simple, reliable, and convenient diagnostic tools for distinguishing FAW from other noctuid moths.

Methods
Sample collection. Fifty field larvae of FAW were collected from corn farms in Taiwan and Thailand (Taiwan: 31 specimens, including individuals of 2, 2, and 26 for 4th-6th instar larvae and 1 adult; Thailand: 19 individuals of 1, 11, 3, and 4 for 2nd, 4th-6th instar larvae; see Supplementary Table S2). Nineteen larvae specimens of a laboratory strain of FAW, including 2 eggs, individuals of 2, 4, 1, 1, 3, 2 for 1st-6th instar larvae, 3 pupae, and 1 destroyed specimen, were purchased from Benzon Research (Carlisle, PA, USA) (see Supplementary Table S2). The congeneric pests of Spodoptera, including Spodoptera litura (Fabricius) and Spodoptera exigua (Hübner), from Taiwan and Thailand as well as common corn pests in maize fields, namely Euproctis taiwana (Shiraki), Helicoverpa armigera (Hübner), Mythimna separata (Walker), Mythimna loreyi (Duponchel), Ostrinia furnacalis (Guenée), and Spoladea recurvalis (Fabricius), were used to test the stability of species-specific primers (for detailed information of instar stage and individuals, see Supplementary Table S2). The detailed information collected regarding the examined specimens is listed in Supplementary DNA extraction, amplification, and sequencing. Genomic DNA was extracted from adult/larval legs or eggs. In both adults and larvae, one leg was exploited for DNA extraction. The entire egg was used for DNA extraction. The tissue was ground in a 50-μL solution of the QuickExtract DNA extraction kit (Epicentre Biotechnologies, Madison, WI, USA) and was incubated at 65 °C for 10 min; it was then vortexed for 15 s followed by incubation at 98 °C for 2 min. After incubation, the sample solution was stored at − 20 °C for PCR. For each sample, the DNA concentration was measured using the Nanodrop 2000 Spectrophotometer (Thermo Scientific, Wilmington, DE, USA) before PCR amplification.
To confirm the identity of field larvae, the universal primer set of LCO1490 and HCO2198 47 was used to amplify the mitochondrial COI gene, namely DNA barcodes, for species recognition. The PCR assay was conducted in 25 μL, including 19.8 μL of ddH 2 O, 2.5 μL of 10X GenTaq Buffer, 0.2 μL of 25 mM dNTP, 0.5 μL of both forward and reverse primers, 0.5 μL of GenTaq DNA polymerase (GenMark Technology, Taipei, Taiwan), and 1 μL of template DNA. The PCR conditions were as follows: incubation at 94 °C for 2 min as initial denaturation, 35 cycles of 94 °C for 40 s, 45 °C for 1 min, and 72 °C for 40 s, and then 72 °C for 10 min as a final extension. DNA products were checked by using 4.5 μL mixed with 1 μL of 6X EZ-Vision DNA Dye (Amresco Inc., Solon, OH, USA). Next, PCR products were run on a 1% agarose gel at 100 V. After electrophoresis, the gel was imaged using a Canon EOS M50 digital camera (Canon, LA, USA). A comparison based on DNA concentration of products and referable image (provided by Protech Technology Enterprise Co., Ltd., Taipei, Taiwan) was made to decide the DNA quality for sequencing. If the concentration of DNA products reached 109 ng, 20.5-μL samples of the remaining products were sent for sequencing. Samples were submitted to the two laboratories for sequencing, namely the Genome Research Center, Sequencing Center, National Yang Ming University and the Biotechnology Center, National Chung Hsing University. In the Genome Research Center, purification of PCR products was conducted using the QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany) with 1% agarose gel. DNA products were sequenced using the BigDye Terminator 3.1 Sequencing Kit (Applied Biosystems, Foster City, CA, USA) and an ABI 3730XL sequencer. In the Biotechnology Center, DNA products were sequenced using an ABI 3730 DNA Analyzer.

Sequence analyses of the noctuid ITS1 variation. ITS1 fragments of FAW (23 sequences), S. litura
(2), S. exigua (3), and other noctuids (15) were retrieved from the GenBank (see Supplementary Table S2) and then aligned using Muscle Alignment option in SeaView4 48 . Genetic divergences among the noctuid moths were analysed on the basis of the ITS1 gene using MEGA 7.0 by assessing the p-distance 49 . Substitution saturation analysis was conducted through making a comparison of transitions and transversions versus the K80 distance using DAMBE v. 5.2.13 50 .

Design of species-specific primers and multiplex PCR. Four pairs of species-specific primers of
FAWs were designed on the basis of the variable regions of the ITS1 sequences of the three Spodoptera species, namely FAW, S. litura, and S. exigua, and noctuid moth sequences retrieved from GenBank. The stability of these species-specific primers was examined and determined among (1) the three Spodoptera pests and (2) the eight common corn pests, namely FAW, S. exigua, E. taiwana, H. armigera, M. separata, M. loreyi, O. furnacalis, and Spoladea recurvalis. Furthermore, the capability of species-specific primers was confirmed using 69 FAW specimens from Taiwan, Thailand, and the United States.

Data availability
All sequences have been deposited in GenBank. Accession numbers and information of voucher specimens are listed in Supplementary Information.