Baseline Susceptibility of Spodoptera frugiperda Populations Collected in India towards Different Chemical Classes of Insecticides

Simple Summary Fall armyworm (FAW) is a highly destructive moth pest and its larvae feed on many different host plants, including major crops such as maize. This pest is native to the Americas but invaded the Eastern hemisphere in 2016. It is highly migratory and was first detected in India in 2018, where it rapidly became a major threat to maize production across India. FAW control mostly relies on the application of chemical insecticides and transgenic crop plants expressing insecticidal proteins of bacterial origin. FAW has developed resistance against insecticides, and for management purposes, it is important to assess its sensitivity status against different chemical classes of insecticides, particularly in invaded regions. In this study, we conducted more than 400 bioassays with nine different insecticides from seven mode-of-action groups on 47 FAW populations collected in 2019 and 2020 across different geographical areas in India. The baseline susceptibility determined for all these insecticides will help to set-up appropriate resistance management strategies to keep FAW infestations in India below economic damage thresholds. Abstract Fall armyworm (FAW), Spodoptera frugiperda, is a major pest of maize in the Americas and recently invaded the Eastern hemisphere. It was first detected in India in 2018 and is considered a major threat to maize production. FAW control largely relies on the application of chemical insecticides and transgenic crops expressing Bacillus thuringiensis insecticidal proteins. Assessing FAW resistance and insecticide susceptibility is a cornerstone to develop sustainable resistance management strategies. In this study, we conducted more than 400 bioassays to assess the efficacy of nine insecticides from seven mode-of-action classes against 47 FAW populations collected in 2019 and 2020 across various geographical areas in India. The resistance status of the field-collected populations was compared to an Indian population sampled in 2018, and an insecticide susceptible reference population collected in 2005 in Brazil. Low to moderate resistance levels were observed for thiodicarb, chlorpyriphos, deltamethrin, chlorantraniliprole and flubendiamide in several populations (including the reference population collected in 2018). The highest resistance ratios were observed for deltamethrin which likely compromises recommended label rates for pyrethroid insecticides in general. Our data provide a useful baseline for future FAW resistance monitoring initiatives and highlight the need to implement insecticide resistance management strategies.


Introduction
Fall armyworm (FAW), Spodoptera frugiperda (Lepidoptera: Noctuidae), native to tropical and subtropical regions of the American continents has recently invaded the Moreover, we determined insecticide resistance ratios for FAW populations collected across India in comparison to an Indian population established in 2018 and a susceptible laboratory population collected in Brazil in 2005. The obtained data provide an important reference for future resistance monitoring initiatives and will help to detect early changes in insecticide efficacy to guide regional insecticide use and the implementation of IRM strategies.

Insect Origin and Rearing
Larvae of S. frugiperda were collected in the years 2018-2020 from maize fields at different sites in India (Supplementary Figure S1, Table S1) and reared on commercial artificial diet under controlled climate conditions (25 • C, 65% relative humidity and L16/D8 photoperiod) [36]. Larvae of the first generation (F1) were used in bioassays. Population I-2018 was collected in Bangalore, Karnataka in 2018, the year when S. frugiperda was first detected in India [4]; 24 FAW populations were collected in 2019 and 23 populations in 2020 (Supplementary Table S1). This population was maintained without insecticide selection pressure under the same conditions as described above and served as the Indian baseline population. A S. frugiperda population, collected in 2005 in Brazil named SUS-2005 was herein used as an insecticide susceptible reference population as described elsewhere [29,36].

Bioassays
Baseline susceptibility data were collected using a diet incorporation assay according to IRAC test method number 20 [37] with minor modifications. In brief, third instar larvae were individually placed onto 1 mL of artificial diet in each well of insect bioassay trays (Frontier Agriculture Sciences, Newark, DE, USA) mixed with serial dilutions of insecticides covering 0-100% mortality. Seven concentrations of each insecticide within the respective dose range stated in Table 1 were tested with distilled water serving as a negative control. Each replicate consisted of 16 larvae and the bioassay was repeated three times. The bioassays were incubated at 25 • C, 65% relative humidity and L16h/D8 photoperiod. Larval mortality was scored after five days. Data were not corrected for control mortality since control mortalities did not exceed 10%. Growth retarded larvae of approximately 1/3 in size of control larvae were considered strongly affected and therefore scored as dead.

Data Analysis
Mortality data were analyzed using Excel 365 (Microsoft Corporation, Redmond, WA, USA) and GraphPad Prism v9.0.2 (GraphPad Software, San Diego, CA, USA). Four parameter non-linear regression models (constraints: bottom = 0, top = 100) were applied to calculate EC 50 -and EC 95 -values and 95% confidence intervals. For the calculation of composite EC 50/95 -values, mortality data of all populations collected in 2019 or 2020 were combined and subjected to four parameter non-linear regression analyses. Resistance ratios (RR) were calculated by dividing the EC 50 -value of the respective field populations by the EC 50 -value of populations I-2018 and SUS-2005, respectively. For a direct comparison to EC 95 -values, recommended field doses of insecticides (Table 1) were converted to spray solution concentrations with an estimated average water usage of 500 L/ha.

Insecticide Susceptibility of Indian FAW Populations
Third instar larvae of 47 different FAW populations collected across India in 2019 and 2020 were exposed to insecticides from different chemical classes for five days in diet incorporation bioassays to evaluate their efficacy. Some of the field-collected populations, particularly those sampled in 2019, could not be tested against all insecticides due to low numbers of F1 larvae. EC 50 values for thiodicarb ranged from 2.91 to 16 Table S10).
The EC 50 values obtained for the field-collected populations were compared with the EC 50 value of I-2018, a FAW population considered as the Indian reference, as it was collected at the time of the FAW invasion in 2018. The resulting resistance ratios (RR) for the populations collected in 2019 and 2020 were typically close to 1 in most populations, indicating similar in- secticide sensitivity as population I-2018 across chemical classes, except for a few populations exhibiting higher RRs ( Figure 1A, Supplementary Tables S2-S7 and S10). The EC 50 values for flubendiamide (2019, 2020) and chlorantraniliprole (2020) were slightly, but significantly increased in several field-collected populations compared to I-2018, with maximum RRs of 7.36 and 6.84, respectively ( Figure 1A, Supplementary Tables S8 and S9). Table S8), chlorantraniliprole 0.005-0.035 µ g/mL (Supplementary Table  S9), tetraniliprole 0.008-0.036 µ g/mL (Supplementary Table S10).

(Supplementary
The EC50 values obtained for the field-collected populations were compared with the EC50 value of I-2018, a FAW population considered as the Indian reference, as it was collected at the time of the FAW invasion in 2018. The resulting resistance ratios (RR) for the populations collected in 2019 and 2020 were typically close to 1 in most populations, indicating similar insecticide sensitivity as population I-2018 across chemical classes, except for a few populations exhibiting higher RRs ( Figure 1A, Supplementary Tables S2-S7 and S10). The EC50 values for flubendiamide (2019, 2020) and chlorantraniliprole (2020) were slightly, but significantly increased in several field-collected populations compared to I-2018, with maximum RRs of 7.36 and 6.84, respectively ( Figure 1A, Supplementary Tables S8,sS9).  Table S1).
However, the resistance status of invading FAW populations prior to settlement in new geographies-such as I-2018 in India-is usually unknown. Consequently, while comparison of field-collected FAW populations to I-2018 reveals general changes in insecticide resistance levels since FAW detection in India, it might still lead to misinterpretations of the actual insecticide resistance level. Therefore, we compared the EC50 values obtained for I-2018 with those of SUS-2005, an insecticide susceptible FAW reference population originally collected in Brazil. The differences in EC50 values between I-2018 and SUS-2005 were ≤2-fold for thiodicarb, fipronil, spinetoram, emamectin benzoate, chlorantraniliprole and tetraniliprole, suggesting similar susceptibility and lack of resistance. Whereas chlorpyrifos, flubendiamide and deltamethrin were significantly less effective against population I-2018 compared to SUS-2005 (Supplementary Tables S3, S5 and S8). The highest RR (21.6-fold) was observed for deltamethrin.
By utilizing SUS-2005 as a reference, high levels of deltamethrin resistance in all fieldcollected FAW populations collected in 2020 were found ( Figure 1B Table S1).
However, the resistance status of invading FAW populations prior to settlement in new geographies-such as I-2018 in India-is usually unknown. Consequently, while comparison of field-collected FAW populations to I-2018 reveals general changes in insecticide resistance levels since FAW detection in India, it might still lead to misinterpretations of the actual insecticide resistance level. Therefore, we compared the EC 50 values obtained for I-2018 with those of SUS-2005, an insecticide susceptible FAW reference population originally collected in Brazil. The differences in EC 50 values between I-2018 and SUS-2005 were ≤2-fold for thiodicarb, fipronil, spinetoram, emamectin benzoate, chlorantraniliprole and tetraniliprole, suggesting similar susceptibility and lack of resistance. Whereas chlorpyrifos, flubendiamide and deltamethrin were significantly less effective against population I-2018 compared to SUS-2005 (Supplementary Tables S3, S5 and S8). The highest RR (21.6-fold) was observed for deltamethrin.
By utilizing SUS-2005 as a reference, high levels of deltamethrin resistance in all fieldcollected FAW populations collected in 2020 were found ( Figure 1B Table S9). The data did not reveal a distinct pattern of insecticide resistance based on geography for those populations collected in 2020 ( Figure 1B,C).

Temporal Development of Insecticide Resistance
The quantification of resistance levels over the years helps to detect early changes in insecticide efficacy allowing adjustment of IRM strategies in affected regions. Here, we focused especially on chlorpyrifos, flubendiamide and deltamethrin, because particularly for these insecticides significant levels of resistance were observed (Figure 1) tamethrin resistance level of 71.3-fold, followed by HR-KS from Haryana state with 45.9fold resistance ( Figure 1B, Supplementary Table S5). A significant increase in resistance levels by 3-and 4-fold could also be observed for flubendiamide and chlorpyrifos, respectively, resulting in RR as high as 24.5 for flubendiamide and 27.5 for chlorpyrifos ( Figure  1B, Supplementary Tables S3 and S8). For thiodicarb, spinetoram, emamectin benzoate, and tetraniliprole, RR did not change when EC50 values of field populations were compared to population SUS-2005 (Figure 1B, Supplementary Tables S2, S6, S7 and S10), whereas slight changes were seen for chlorantraniliprole ( Figure 1B, Supplementary Table  S9). The data did not reveal a distinct pattern of insecticide resistance based on geography for those populations collected in 2020 ( Figure 1B,C).

Temporal Development of Insecticide Resistance
The quantification of resistance levels over the years helps to detect early changes in insecticide efficacy allowing adjustment of IRM strategies in affected regions. Here, we focused especially on chlorpyrifos, flubendiamide and deltamethrin, because particularly for these insecticides significant levels of resistance were observed (Figure 1)

Comparison of Recommended Field Rates to EC95-Values for all Insecticides
To estimate the potential field efficacy of all tested chemical insecticides against FAW, we calculated and plotted the respective EC95-values for all field-collected populations along with the recommended label rates in India (Figure 3). For almost all compounds, EC95 values were considerably lower than the recommended field application rates irrespective of the collection year of the populations. The only exception is deltamethrin, where the EC95-value was close to or in the case of population AP-PB above the recommended label rate ( Figure 3D). For fipronil, no label rate was provided because this compound is not recommended for use against FAW in India ( Figure 3C). The lowest

Comparison of Recommended Field Rates to EC 95 -Values for all Insecticides
To estimate the potential field efficacy of all tested chemical insecticides against FAW, we calculated and plotted the respective EC 95 -values for all field-collected populations along with the recommended label rates in India (Figure 3). For almost all compounds, EC 95 values were considerably lower than the recommended field application rates irrespective of the collection year of the populations. The only exception is deltamethrin, where the EC 95 -value was close to or in the case of population AP-PB above the recommended label rate ( Figure 3D). For fipronil, no label rate was provided because this compound is not recommended for use against FAW in India ( Figure 3C). The lowest variation in EC 95 -values across sampling sites was observed for tetraniliprole and spinetoram ( Figure 3E,I).
Insects 2021, 12, x FOR PEER REVIEW 7 of 11 variation in EC95-values across sampling sites was observed for tetraniliprole and spinetoram ( Figure 3E,I).  Table 1 with an average water usage of 500 L/ha, except for fipronil which is not registered for FAW control in India. Number of populations (n) tested in the respective sampling years for each insecticide are indicated in each graph. The full data set including all calculated values can be accessed in the supplementary information (Supplementary Tables S2-S10).

Discussion
After its first detection in Karnataka state in May 2018 [4,17,38], FAW spread to almost all states in India by August 2019 [39,40]. To provide the basis for sound recommendations to control this new invasive lepidopteran pest species, we conducted a baseline susceptibility study for the Indian sub-continent covering nine different insecticides out of six MoA classes using 47 FAW populations collected in 2019 and 2020.
Low resistance ratios were observed for thiodicarb, spinetoram, emamectin benzoate, chlorantraniliprole and tetraniliprole across field-samples of both collection years when compared to a laboratory susceptible reference population SUS-2005. Our results correspond with Indian FAW laboratory (leaf-dip) and field experiments conducted in 2018, and which previously demonstrated high efficacy of spinetoram, emamectin benzoate and chlorantraniliprole [35]. In contrast, chlorpyrifos, deltamethrin and flubendiamide were less effective against field populations collected in 2019 and 2020 in comparison to the reference population SUS-2005 (Figure 1, Supplementary Tables S3, S5 and S8). Likewise, population I-2018 (collected in the year of FAW invasion) did also show low-tomoderate levels of resistance to these insecticides when compared to SUS-2005. A finding underpinning the importance of utilizing established susceptible reference populations to assess the extent of the resistance levels present at the time of invasion.
Genomic analyses of a FAW population collected in 2018 from Karnataka state in India detected the presence of the F290V target-site mutation in the acetylcholinesterase (AChE), along with the less prevalent A201S mutation [34]. The F290V substitution is known to confer resistance to organophosphates in FAW [20], but also cross-resistance to carbamates has been described, e.g., against carbaryl in Cydia pomonella [41]. The high frequency of target-site mutations in AChE in the invasive FAW population in India might  Table 1 with an average water usage of 500 L/ha, except for fipronil which is not registered for FAW control in India. Number of populations (n) tested in the respective sampling years for each insecticide are indicated in each graph. The full data set including all calculated values can be accessed in the supplementary information (Supplementary Tables S2-S10).

Discussion
After its first detection in Karnataka state in May 2018 [4,17,38], FAW spread to almost all states in India by August 2019 [39,40]. To provide the basis for sound recommendations to control this new invasive lepidopteran pest species, we conducted a baseline susceptibility study for the Indian sub-continent covering nine different insecticides out of six MoA classes using 47 FAW populations collected in 2019 and 2020.
Low resistance ratios were observed for thiodicarb, spinetoram, emamectin benzoate, chlorantraniliprole and tetraniliprole across field-samples of both collection years when compared to a laboratory susceptible reference population SUS-2005. Our results correspond with Indian FAW laboratory (leaf-dip) and field experiments conducted in 2018, and which previously demonstrated high efficacy of spinetoram, emamectin benzoate and chlorantraniliprole [35]. In contrast, chlorpyrifos, deltamethrin and flubendiamide were less effective against field populations collected in 2019 and 2020 in comparison to the reference population SUS-2005 (Figure 1, Supplementary Tables S3, S5 and S8). Likewise, population I-2018 (collected in the year of FAW invasion) did also show low-to-moderate levels of resistance to these insecticides when compared to SUS-2005. A finding underpinning the importance of utilizing established susceptible reference populations to assess the extent of the resistance levels present at the time of invasion.
Genomic analyses of a FAW population collected in 2018 from Karnataka state in India detected the presence of the F290V target-site mutation in the acetylcholinesterase (AChE), along with the less prevalent A201S mutation [34]. The F290V substitution is known to confer resistance to organophosphates in FAW [20], but also cross-resistance to carbamates has been described, e.g., against carbaryl in Cydia pomonella [41]. The high frequency of target-site mutations in AChE in the invasive FAW population in India might support the lower relative RRs when EC 50 values of the field populations were compared to I-2018 rather than SUS-2005, because SUS-2005 has been characterized earlier and is known to lack the F290V mutation in AChE [42]. However, we did not find a correlation between the RR's calculated for chlorpyrifos and the carbamate thiodicarb (Pearson's r 0.24; Tables S2 and S3). More investigations at the biochemical level are required to understand and explain potential cross-resistance issues between organophosphates and carbamates in Indian FAW populations.
Based on our findings we can conclude that moderate levels of deltamethrin resistance were already present in FAW populations invading India in 2018. However, this fact only became obvious after a comparison of the efficacy data obtained for the Indian field-collected populations to the susceptible reference SUS-2005 ( Figure 2). A recent mechanistic investigation revealed that the resistance to pyrethroids in FAW might be metabolic and based on the increased expression and activity of cytochrome P450 enzymes, particularly those belonging to the CYP9A subfamily [42]. Interestingly, CYP9A enzymes were recently shown to detoxify pyrethroid insecticides-including deltamethrin-in the cotton bollworm Helicoverpa armigera [43]. An increased metabolic turnover could explain the relatively high RR values obtained for deltamethrin in our study. Nevertheless, it is still unknown for FAW whether this mechanism would confer cross-resistance to all pyrethroids at similar or different levels. However, a previous study on a field-collected Brazilian population selected for pyrethroid resistance, three voltage-gated sodium channel mutations, T929I, L932F and L1014F, were detected-albeit at very low frequency despite continuous selection pressure, thus reinforcing the importance of metabolic pyrethroid resistance in FAW [20,42]. Another study on FAW samples collected outside Brazil revealed that only a single population from Indonesia carried a target-site mutation (L1014F) in the voltage-gated sodium channel among populations from different invaded countries analyzed [30,32,34].
For flubendiamide, population I-2018 exhibited low levels of resistance compared to the Brazilian susceptible population SUS-2005. Significant differences between the EC 50/95value of population I-2018 and calculated composite EC 50 -and EC 95 -values of 2019 and 2020 FAW field populations indicated additional selection pressure that further enhanced flubendiamide resistance levels. High levels of cross-resistance among diamide insecticides (including flubendiamide) in FAW have been recently described in a population from Brazil after laboratory selection with chlorantraniliprole [22] and was shown to be mediated by a mutation (I4790M) in the ryanodine receptor, the diamide target-site [44]. The same mutation has been shown to confer diamide resistance in a few other lepidopteran pests such as diamondback moth, beet armyworm and tomato leafminer [45]. However, the frequency of this resistance allele in FAW field populations seems very low, as it was not yet detected in populations collected outside Brazil [32,34,44], except China where it was detected at very low frequency in a FAW population showing low levels of diamide resistance [46]. However, it remains elusive and needs to be analyzed if the moderate RR obtained for flubendiamide in our study is due to an increased detoxification capacity or the presence of a ryanodine receptor target-site mutation, or both. The lack of any cross-resistance to tetraniliprole and almost no cross-resistance to chlorantraniliprole might indicate the absence of a ryanodine receptor target-site mutation affecting diamide binding in Indian FAW populations. As it is generally known that the presence of such a mutation confers cross-resistance among diamide chemotypes [45].
The absence of tetraniliprole cross-resistance renders it a valuable tool in future resistance management strategies. Nevertheless, an adequate IRM strategy should consider the rotation of diamide applications with insecticides belonging to other modes of action in order to increase the life span of diamides in the market. Future resistance monitoring efforts should include all chemical classes of insecticides available for FAW control in India. This is a laborious task that could be simplified by establishing discriminating dose bioassays for the individual insecticides, e.g., based on the EC 95 -values (or slightly higher concentrations) against a susceptible reference population such as SUS-2005 used in this study. This would allow the detection of early signs of resistance development.
Our study demonstrated resistance to some chemical classes of insecticides such as organophosphates and pyrethroids in Indian FAW, most likely already present in populations that invaded Karnataka in 2018. The initial populations from Karnataka state were shown to be closely related to South African populations of corn and rice strain haplotype hybrids according to original host preference classification in the Americas [9,47]. Since then, FAW has rapidly adapted to the Indian landscape despite the different agroclimatic zones and experienced an expansion mediated by its population dynamics [9]. This is in line with our findings of a generally homogenous distribution of resistance with only a few geographically unlinked hotspots.

Conclusions
Our study described a robust and reliable bioassay format and provided a useful baseline for future FAW resistance monitoring initiatives in India. The presence of resistance alleles in Indian FAW populations conferring low-to-moderate levels of resistance to some chemical classes of insecticides highlights the need for continued monitoring efforts to limit the dispersal of resistant haplotypes. Furthermore, this will facilitate the implementation of appropriate IRM strategies at regional and local levels.

Data Availability Statement:
We will provide all data generated in this study upon request.