Molecular characterization of the Fall armyworm, Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae) on maize in southern Rajasthan, India


 The fall armyworm (FAW), Spodoptera frugiperda (J.E. Smith) is a polyphagous Lepidopteran pest, a native to tropical and sub-tropical America and recently it has invaded the African and Asian countries. Presently, the mitochondrial Cytochrome Oxidase Subunit I (COI) - gene based molecular characterization of FAW samples from the maize fields of southern Rajasthan has revealed the occurrence of corn and rice strains there. The occurrence of such S. frugiperda population of Rajasthan region could be traced its origin from the Florida-Caribbean region or African region. Further, the Tpi gene region analysis showed that the S.frugiperda forms found in the maize fields are only the corn strains. In the Indian Rajasthan populations of FAW, the Tpi-variant2 category is the highest one and is then followed by the Tpi-variant1 and Tpi-variant3 was unique with C and T at Tpie4192 and Tpie4198, respectively. Further research is needed towards the confirmation of these tentatively identified strains of S. frugiperda that would in turn helpful for the proper monitoring, host-plant identification and the effective management of such pests.


Introduction
An invasive pest, the fall armyworm (FAW), Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae) is a tropical American insect that is responsible for huge economic loss (Prasanna et al. 2018). This pest had invaded Central and Western Africa during early 2016 and due to its voracious feeding nature and rapid migratory behavior it could able to rapidly spread across the Sub Saharan countries that have caused severe damages there (Goergen et al. 2016). As it is a serious pest, it could cause severe damage to almost 100 plant species representing the 27 families including the maize-crop and other graminaceous crops like millet, sorghum, rice, wheat and sugarcane (Goergen et al. 2016) and thus it would pose threat to the food security of the millions of people (De Groote et al. 2020). Montezano et al. (2018) have also reported a host range of 353 plant species representing 76 plant families like; Poaceae, Asteraceae and Fabaceae from Brazil. Over 30 countries have reported its occurrence within their borders including the island countries (Prasanna et al. 2018). The yield losses in maize ranging from 8.3 M to 20.6 M tons per year have been reported from 12 maize producing countries (CABI 2016). Fall armyworm attack occurs in maize crop right from emergence to tasseling, silking and cob formation stage. This pest can reduce corn grain yield up to 34% and is estimated at U$400 million loss annually in Brazil (Sena et al. 2003;Lima et al. 2010).
In 2018, FAW has been reported in many Asian countries like India, Yemen, Thailand, Myanmar and Sri Lanka , Bangladesh (Chhetri and Acharya 2019) and subsequently its spread has been reported in China, Korea, Japan and Australia (Zhang et al. 2019;Guo et al. 2018;Lee et al. 2020;Acharya et al., 2021). In India, the FAW was rst time reported in Karnataka state, on maize, in May, 2018 (Sharanabasappa et al. 2018) and subsequently, within a year, the pest was found to spread in the other states viz., Andhra Pradesh, Bihar, Chhattisgarh, Gujarat, Madhya Pradesh, Maharashtra, Odisha, Rajasthan, Tamil Nadu, Telangana, West Bengal (Ramesh Babu et al. 2019), Arunachal Pradesh, Meghalaya, Manipur, Sikkim, Mizoram, Nagaland and Tripura (Firake et al. 2019). In Rajasthan, we have reported this pest for the rst time in early 2019 from the Southern region of the state on winter maize which was con rmed by the morphological and molecular characterization (Ramesh Babu et al. 2019).
However, its infestation was already been reported in some of the popular hybrids, inbreds of maize and sweet corn in the region.
The FAW species consists of morphologically identical but genetically distinct subpopulations namely the corn strain (CS) and the rice strain (RS) (Pashley et al. 1985;Prowell et al. 2004). While the corn strain's preferred feeds are; maize, sorghum and other grasses, the rice strain prefers rice and other large grasses (Pashley et al. 1986). Both these strains are region-speci c and they differ in their response to bioagents like Bt and various groups of insecticides and they also exhibit different dispersal patterns (Pashley et al. 1992). Pest management strategies are formulated based on such differences between the two strains (Adamczyk et al. 1997).
Molecular genetic markers are one of the most reliable methods to differentiate the diverse strains of FAW although their morphometric features can be analyzed based on their wing shapes (Cañas-Hoyos et al. 2014). The allozymes, esterases, PCR-RFLP (DNA ampli cation and digestion), FR (For Rice) fragment repeats and AFLPs have been generally used to differentiate the corn strain (CS) with that of rice strain (RS) in FAW (Pashley 1986;Levy et al. 2002;Nagoshi and Meagher 2003;Prowell et al. 2004;Groot et al. 2010). Mitochondrial cytochrome oxidase subunit I (COI)-gene sequence was shown polymorphic variation between C and R strains whereas, the populations collected from the maize elds having an Rstrain in the mtDNA COI gene region (Acharya et al. 2021). Mahadeva Swamy et al. (2018) have recently studied the molecular aspect of FAW and reported the prevalence of RS FAW in maize (using mtDNA gene regions). In the present study, we have used molecular markers based on the partial regions of Cytochrome Oxidase Subunit I (COI) and also another genetic marker, the nuclear triosephosphate isomerase (Tpi) gene which is linked with Z-chromosome to con rm the species identi cation, differentiate the corn and rice strains and the origin of haplotypes. The single nucleotide polymorphisms (SNPs) in the Tpi gene region perhaps could form one of the reliable methods to detect the strain identity than the mtDNA COI gene regions ) and which could be also helpful to nd out the host plant preference by the two strains.

Materials And Methods
During the present experiments, the standardized procedures of Levy et al. 2002;Ve'lez-Arango et al. 2012;Laura Juarez et al. 2012; and 2018 were followed to detect FAW strains in Rajasthan populations and compared the COI haplotype pattern with the other region (Western and Eastern) populations.

DNA Extraction
Fall armyworm larvae were collected from the maize elds at various places of Rajasthan (India) during Rabi and Kharif seasons, 2019 (January to September 2019) ( Table 1). The collected specimens were preserved at -20 o C. Total genomic DNA was extracted using DNA Sure Tissue mini kit (Nucleo-pore, Genetix Brand, India) by following the manufacturer's instructions. Presently, all the PCR reactions were carried out by using C1000Touch™ Thermal cycler of Bio-Rad, USA.
The PCR ampli cation was performed for 50µL containing 25 µL DreamTaq PCR Master Mix (2X) (Thermo Fisher Scienti c Inc.), 2µL of template DNA, 10 pmol of each forward and reverse primers ( Table 2) and nal volume was made by using nuclease free water. PCR was performed according to the different primers used with initial denaturation for 4 min at 94°C, followed by 35 cycles of 30 sec denaturation at 94°C, 45 sec primers annealing at 47°C, 45 sec initial extension at 72°C and a nal extension of 20 min at 72°C ). The ampli ed PCR products were separated by electrophoresis in a 1.2% agarose gel containing ethidium bromide (0.5µg/µL) for 60 min at 80 V (BIO-RAD, USA) and visualized in gel documentation system (Gel Doc™ EZ Imager, BIO-RAD, USA). PCR puri cation, sequencing and analysis The PCR products were puri ed by using GeneJET PCR puri cation Kit (Thermo Fisher, Scienti c Inc.) and sequenced by using ABI PRISM 3730xl Genetic Analyzer by Applied Biosystems, USA (Agile Life science Technologies India Pvt. Ltd, Pune). The obtained sequences were aligned through BioEdit sequence alignment editor (version 7.0.5.3) and homology was con rmed by using National Center for Biotechnology Information (NCBI)-Basic Local Alignment Search Tool (BLAST) (BLASTn, http:// www.ncbln1m.nih.gov). A phylogenic tree was graphically constructed in MEGA x program (Kumar et al. 2016).

FAW Genotyping (FAW strain analysis): Restriction Site Length Polymorphism (RFLP)
The PCR reaction was carried out for the ampli cation of Cytochrome Oxidase Subunit I (COI) gene of ~ 600 bp, using the forward primer JM76 (5'-GAGCTGAATTAGG(G/A)ACTCCAGG-3') and the reverse primer JM77 ( Characterization of the other CO1 regions and Tpi gene region Presently, we have also used another COI primer (apart from the universal COI primers) to con rm and determine the strains (Corn and Rice strain) of S. frugiperda. To determine the regions speci c haplotypes and the host strain identity, another COI region was ampli ed with the primers 891 F or 893 F and 1472 R or 1303 R (Table 3). To identify the host strain and region-speci c haplotypes, the COI-II primers 891F and 1472R were used (Nagoshi et al. 2012 and. The SNPs analysis at COI-II 1164 (T) and COI-II 1287 (A) identi es as rice-strain whereas, four corn-strain haplotypes (h1-h4) were found for the corn-strain group. Polymorphisms in the genetic marker, the Triosephosphate isomerase gene (Tpi) also used to identify and con rm the two strains of FAW and comparable with the CO1 gene markers. Tpi region was ampli ed by using primers 412 F and 850 R (Table 3) Phylogenetic analysis revealed that out of eight samples collected from maize in different places of the region, six were clustered to the S. frugiperda rice-strain haplotype RS9 whereas the two were clustered to the corn-strain haplotype CS1 of the United states and also with African strains, Indian strains and also with China region corn-strains (Fig. 1).
The PCR-RFLP analysis revealed that the two fragments of about 510 and 90 bp size were obtained due to cut by MspI of PCR fragments for the corn strains but not by SacI whereas PCR fragments of the rice strain cut by SacI showed a reciprocal pattern by producing 450 and 150 bp fragments, but not by MspI (Fig. 2). The corn and rice strain nucleotides differences are given in Fig. 5. In another analysis, the EcoRV enzyme digested PCR products derived from the primers COI-II: COI-893F/COI-1303R showed a single band for the corn strains (DPRM and RJD) whereas, strains of other regions showed typical diagnostic two bands which is a characteristic of rice strain (Fig. 3). The single band PCR products were puri ed and sequenced to con rm the absence of EcoRV site. Based on the COI-III: COI primers 891F/1472R, the FAW samples from RJD and DPRM regions were having similar polymorphic sites (G 1164 ; G 1287 haplotype) which are designated as corn strains. Similarly, the six regions of Rajasthan displayed the polymorphic sites at T 1164 ; A 1287 and identi ed as Rice strains (Table 4). The analysis of the Tpi strain markers revealed that all the FAW populations collected from maize elds at Rajasthan were C-strain (Tpi-C haplotype) as de ned by Tpi e4 183 (

Discussion
In the present study, the molecular markers of both Tpi and COI genes were used to characterize the populations of FAW collected from the maize elds of Rajasthan region. The COI gene sequence analysis by using universal primers revealed that all the specimens collected from the maize were of S. frugiperda and a similar COI gene fragment was earlier reported by Shylesha et al. 2018 andMahadeva Swamy et al. 2018. The specimens collected from different regions of Rajasthan revealed that the two types of haplotypes are present in the FAW populations. This has been represented in the phylogenetic tree analysis where out of eight populations, six were clustered with the fall armyworm rice-strain speci c haplotypes and two with corn-strain speci c haplotypes. Earlier,  have also observed similar types of haplotypes i.e., corn and rice strains from Togo, Africa based on the COI region analysis. Their sequences were identical with US corn and rice-strain haplotypes. As per the present study, both the rice and corn strains are prevalent in the Indian region and they are identical to the Western hemisphere as well as with the Eastern hemisphere. In another earlier study by Nagoshi et al. (2019) found one Ecuador haplotype clustered with the rice-strain and others were similar to the corn-strain haplotype (when they analyzed the COI gene sequences). Jing et al. (2019) have studied the phylogenetic tree which was constructed by using the Maximum likelihood method and which revealed that the majority of FAW populations belong to the corn-strain haplotype in China. Similarly, in Korea, Lee et al. (2020) reported the incidence of S.frugiperda in maize and they analyzed the haplotypes by using COI gene which comprised of two haplotypes based on the phylogenetic analysis.
Molecular markers have been used to differentiate the corn and rice strains of S. frugiperda. In the present study, PCR-RFLP method was adopted by using JM76 and JM77 primers and the ampli ed COI region PCR products were digested with the restriction enzymes, MspI and SacI. Out of the eight populations from different regions of Southern Rajasthan, six (BSW;UDR;CTG;PTG;DPRP;SLB) and two populations (DPRM;RJD) were identi ed as rice strains and corn strains, respectively. Earlier also, Machado et al. (2008) have used several molecular markers to differentiate the strains of S. frugiperda and they used the PCR-RFLP of the mitochondrial gene Cytochrome Oxidase Subunit I (COI) and the PCR of the tandem repeated fragment FR. Based on a PCR-RFLP product of 569 bp of the COI gene that is digested with restriction enzymes, MspI and SacI. Two cleavage sites of 497 and 72 bp were observed in the corn strain when PCR products were digested with MspI (Levy et al. 2002;Nagoshi and Meagher 2003), whereas when the enzyme was SacI, two cleavage sites of 500 and 69 bp have been observed in the rice strain (Lu et al. 1994).
The analysis of another COI region by using the primers COI-893F/COI-1303R generated a PCR product having a single EcoRV site which is a characteristic of the rice strain. PCR products of the two regions Dungarpur (DPRM) and Rajsamand (RJD) didn't have the EcoRV site which was con rmed by the sequencing of the PCR products and con rmed that the FAW populations belong to the corn strain. Nagoshi (2019) revealed that the EcoRV recognition site in the COI-RS haplotype and this strain-speci c polymorphism site were not present in COI-CS haplotype in African populations. Nagoshi et al. (2007Nagoshi et al. ( & 2015 reported the geographically divided sub-groups for the corn strains of S. frugiperda based on differences in the mitochondrial haplotypes derived from polymorphisms at sites COI 1164 and COI 1287 in the Western hemisphere. The western hemisphere contains most of the TX-type pro le whereas FL-type belongs to Florida and Caribbean regions. Populations of corn strains collected from Togo (African country) revealed the differences in haplotype pattern which was most consistent with the pattern of FL-type. In the present study, similar COI region primers 891F/1472R were used to study the haplotypes of S. frugiperda in maize. The analyzed COI-CS FAW populations were from Rajsamand (RJD) and Dungarpur (DPRM) regions that had G 1164 G 1287 SNP con guration which is most consistent with the Florida-Caribbean region and Africa and Togo region corn haplotyes . The FAW populations present in the Southern Rajasthan may be the source or originated from Sub-Saharan African region or Florida-Caribbean region.
Single Nucleotide Polymorphisms (SNPs) in the genetic marker and sex-linked Triosephosphate isomerase (Tpi) gene could also help in identifying the host-speci c strains which could provide more accurate identi cation information of the host speci c strains of S. frugiperda. The results of this gene can be comparable with the COI gene markers for con rmation (Nagoshi et al. & 2018. Earlier, based on the results of such studies Nagoshi reported the presence of two strains of S. frugiperda in the Western as well as Eastern Hemisphere. Tpi coding region was PCR ampli ed using the Tpi primers 412 F and 850 R. The e4183 SNP varies as C or T for C-strain allele (Tpi-C) is indicated by a C183 or the R-strain (Tpi-R) is by T183. Based on this the African FAW populations showed 60% and 65% corn strains based on COI and Tpi markers. This indicates that the corn strains have been predominant in the FAW populations of African countries which were similar to that of Western hemisphere populations based on the Tpi gene markers. Based on the Tpi marker, all FAW populations collected from maize at Southern Rajasthan revealed that all of them were of corn-strain based on the polymorphism in the Tpi e4 183 site.
In Indian Rajasthan populations, apart from two Tpi variants one unique variant3 was found in the FAW populations.
The present analysis of mtDNA COI gene regions con rmed that both the rice and corn strains are present in different regions of maize elds of Southern Rajasthan. The same set of FAW populations were tested with Tpi marker revealed that the corn-strain haplotypes are predominant in the collected populations of S. frugiperda from maize elds of Rajasthan. This indicates Tpi marker-based corn-strain populations showed expected associations with the host plants ie. maize than the mitochondrial markers. Therefore, this study gives a preliminary idea of the occurrence/presence of two strains of S. frugiperda and it is rare/unusual that both the strains have been present in the samples of maize plants of Rajasthan. A more detailed survey has to be undertaken over some time by covering different regions of Rajasthan and also from different host plants. So that the exact origin and strains of S. frugiperda will be identi ed and that would have a positive impact on the timely management strategies of this pest in the future.