Acute and Persistent Toxicity of Newer Insecticide Molecules Against Invasive Pest of Maize, Fall Armyworm Spodoptera

Laboratory and pot culture experiments were conducted to assess the acute and persistent toxicity of new molecular insecticides against the notorious invasive pest of maize, Spodoptera frugiperda during 2020-2021 at Department of Agricultural Entomology, Tamil Nadu Agricultural University, Coimbatore. Results revealed that, the LC 50 value of emamectin benzoate, chlorantraniliprole, spinetoram, flubendiamide and novaluron were 0.05, 4.08, 0.1, 85.89 and 0.91 ppm, respectively for second instar larvae and 0.03, 5.63, 0.02, 162.99 and 0.99 ppm for third instar larvae of fall armyworm. Among insecticides tested emamectin benzoate and spinetoram showed high toxicity to S. frugiperda than other insecticides by registering a minimum LC 50 value. But in the persistent toxicity studies, the same insecticides showed less persistence on maize crop. Hence, insecticides like flubendiamide and chlorantraniliprole need to be recommended in the early stage of the crop period (within 20 days after sowing) and less persistent insecticides viz., emamectin benzoate and spinetoram should be recommended in the middle stage (25 to 40 days) of the maize crop for the management of S. frugiperda so that the residues may not accumulate in the harvested produce both in the stalk and grain.


INTRODUCTION
Maize (Zea mays L.) is one of the extensively cultivated crops occupying around 180.63 m ha in 165 countries worldwide (APEDA, 2019).It is the third most important cereal crop after rice and wheat in India is extensively used for food, feed, fodder and raw material for industrial applications (Rakshit, et al., 2017).Maize is mostly grown as rainfed crop in tropical and sub-tropical conditions and the productivity (3.1 tonnes/ha) is much lower than the world average (5.62 tonnes/ha) (Rakshit and Chikkappa, 2018).Among various factors responsible for the low productivity of maize, insect pests contribute to the maximum loss.This crop is encountered by more than 141 insect pest species causing a different level of damage, of which only two pests viz., spotted stem borer (Chilo partellus Swinhoe), and shoot fly (Atherigona spp.) were causing strains infesting crops belongs to Poaceae, primarily on maize, rice and fodder grasses (Dumas et al., 2015).There are about 353 host plants recorded as alternate host plants for S. frugiperda (Montezano et al., 2018).This pest invaded Africa during 2016 (Goergen et al., 2016) and Asia during 2018 and is threatening food security of the millions of people.In India FAW has been reported first in the state of Karnataka (Shylesha et al., 2018;Sharanabasappa et al., 2018a) followed by Andhra Pradesh, Madhya Pradesh, Maharashtra, Tamil Nadu, Telangana, Gujarat and Chhattisgarh (Mahadevaswamy et al. 2018;Sisodiya et al. 2018;Sonali Deole and Nandita Paul, 2018).In the absence of natural biological control, fall armyworms can cause significant yield loss in maize and other crops (Kebede and Shimalis, 2019).
Insecticides are used as a prime tool in fall armyworm management in western countries (Tomquelski and Martins 2007;Sisay et al. 2019).Farmers in the invaded area were not prepared for this devastating pest, resulting in heavy losses on one hand and a drastic increase of insecticide use on the other (Kansiime et al., 2019).Use of insecticide cocktails and over dose of insecticide in the maize ecosystem to manage the notorious invasive pest is common among the maize growing farmers in the FAW infested areas.After intensive discussion with the State Agricultural Universities, the Central Insecticide Board and Registration Committee (CIBRC) of Government of India had given the ad-hoc recommendation for the management of FAW during 2019.There is no baseline data available for any of the insecticides used for the management of FAW in India.Hence, this study was conducted to develop the baseline toxicity and persistence toxicity data for the important and effective insecticides used for the FAW management to optimize the dose and accommodate the right insecticide in the right stage of the crop.

Target Insect:
Second and third instar larvae of S. frugiperda were used for the bioassay study.Larvae of uniform age group were taken from the fall armyworm mass culture laboratory, Department of Agricultural Entomology, Tamil Nadu Agricultural University, Coimbatore where the insects were reared on the specially prepared lab lab based semi synthetic diet (composition pending patent).Three hundred uniform -sized larvae were obtained per bioassay.Totally 32 bioassays were conducted for the range finding test, baseline susceptibility study and persistent toxicity study in which more than ten thousand larvae (both second and third instar larvae) were utilized for the study.

Insecticides
Five newer insecticides viz., emamectin benzoate 5 SG, chlorantraniliprole 18.5 SC, spinetoram 11.7 SC, flubendiamide 20 WG and novaluron 10 EC were used for this study.These insecticides were selected based on the ad-hoc recommendation of the CIBRC, Government of India for the management of S. frugiperda on maize in India.All the formulations were purchased from the pesticide dealers in Coimbatore.The insecticide dilution required for various bioassays were freshly prepared by dissolving the required quantity of insecticide formulations in water.

Baseline susceptibility of the FAW to different insecticides
Six well bio assay tray was used for the bioassay study.Tender maize leaves were taken from the field, washed under tap water, and sandwiched gently in blotting paper to remove the water.Leaf discs of 5 cm diameter were taken from the leaves using a metal lid.To sustain the turgidity of the leaf in the bioassay plate, one per cent agar medium was added in to the bioassay plates up to 0.5 cm thick layer.This was prepared by adding 1 gm agar to 99 mL water and dispersing the agar properly by constant stirring.The solution was heated until boiling, allowed to cool until it reaches 65ºC after which 0.3 mL of anti-mould solution (4.5 mL phosphoric acid and 42 mL propionic acid to 53.5 mL water) was added.
A preliminary range finding test was conducted with laboratory-cultured populations of S. frugiperda with wide range of test insecticide to fix the test dose range causing 20 to 80 per cent mortality approximately.Based on this, 6 doses were fixed in geometric progression and working standards were prepared.Triton X 100 @ 0.01 % was added as surfactant.Leaf discs were dipped individually in the test liquid for 5 s with gentle agitation and placed on a paper towel for surface drying.
Each bioassay study was performed with seven treatments (six insecticide doses and one control) replicated thrice with ten insects in each replication.Thirty leaf discs were treated in each dilution.After treatment, the leaf discs were placed gently inside the well over the agar medium and one larva was released per well.After release of larva the plate was covered with white muslin cloth to avoid the escape of the larva and kept undisturbed in the ambient environment (28ºC ± 5ºC and 75 ± 5 % RH).Untreated control was maintained by dipping the leaf disc in the distilled water over which larvae were released.After 24 hrs of exposure, live larvae were fed with untreated leaves.Larval mortality was recorded at 8 h interval up to 72 h after exposure period.A larva was considered dead if it failed to move in co-ordinated manner, when probed with camel hair brush.Based on the mortality in different insecticidal treatments, the LC50 value of insecticide was calculated (Finney, 1971) after applying the correction for the control mortality (Abbott, 1925).

Persistent toxicity of insecticides against S. frugiperda on maize
Pot culture experiment was conducted to assess the persistent toxicity of insecticides against FAW.One week old maize seedlings (grown under hydroponics) were transplanted on the 30 cm wide earthen pots @ 4 seedlings per pot.Fifteen days after transplanting (DAT) following insecticides were sprayed in X (recommended dose) and 2X dose (double the recommended dose) on the 20 potted plants each, respectively.Leaf samples were collected on 0 (2 hrs after spray), 1,2,3,4,5,7,10,15,20,25 and 30 days after spray and bioassay was conducted.Six well bioassay tray was used for bioassay study.Two 3 cm leaf bits from each treatment were placed gently inside the well over the agar medium and one larva was released per well.After 24 hrs exposure live larvae were fed with untreated leaf.For each treatment thirty larvae were used and percent mortality of larva was observed on 24, 48 and 72 h after treatment.

RESULTS AND DISCUSSION
The results of the bioassay study performed for emamectin benzoate against S. frugiperda revealed that, the LC50 value is 0.05 ppm for second instar larva and 0.03 ppm for third instar larva.The LC99 and χ 2 value recorded were 7.51 & 6.40 and 2.23 & 0.37 respectively for second and third instar larva (Table 1).The slope of the baseline curve was almost equal (6.38 and 6.53) for two stages of the larva tested.The LC50 values of chlorantraniliprole against second and third instar larva of S. frugiperda were 4.08 and 5.63 ppm, respectively.The slope, LC99 and χ 2 value recorded was 4.38, 853.64 ppm and 0.12 for second instar larva and 4.35, 12.37.79ppm and 1.55 for third instar larva.The values of LC50, LC95 and LC99 values for flubendiamide against second instar larva of S. frugiperda were 85.89, 1648.38 and 5606.04 ppm and third instar larva were 162.99, 4821.66 and 19618.43ppm, respectively.Spinetoram recorded LC50 values of 0.1 and 0.02 ppm for for second and third instar larva, respectively.The χ 2 value and slope of the baseline curve were 3.74 and 0.82, 5.69 and 7.05, respectively for second and third instar larva of S. frugiperda.Insect growth regulator novaluron recorded LC50 value of 0.91 and 0.63 ppm, LC95 value of 64.16 and 12.79 and LC99 value of 374.78 and 44.60 ppm, respectively.
The results of the persistent toxicity study revealed that, toxicity of emamectin benzoate persisted up to 10 days in the maize leaves and produced mortality of S. frugiperda larva (Table 2).More than 90 per cent mortality was observed during first four days after treatment in X dose, whereas in 2X dose cent percent mortality was observed during the same period.On 10 DAT, 6.67 and 10.0 per cent mortality was observed on 72 HAT in emamectin benzoate 5 SG @ X and 2X dose, respectively (Table 2).Chlorantraniliprole 18.5 SC persisted in the maize leaves up to 20 days in X dose and 25 days in 2X dose (Table 3).

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It was observed that the toxicity of chlorantraniliprole 18.5 SC on the day of spraying was less than one day after spraying.Up to 5 DAT, more than 80 per cent mortality of the test population was observed which declined later.
The toxicity of flubendiamide 20 WG against the third instar larva of S. frugiperda remained up to 25 days after treatment (DAT) in both the doses but only 3.33 percent mortality in X dose and 16.67 percent mortality in 2x dose was observed during 72 hours after treatment (HAT) at 25 DAT (Table 4).Cent per cent mortality was observed up to 2 DAT at X dose and 3 DAT at 2X dose but in 2 X dose 90 per cent mortality was observed till 5 DAT.Toxicity of novaluron 10 EC was very less at 24 hours after exposure but it attained peak during 72 HAT (Table 5).Cent percent mortality of test population was observed up to 1 DAT in the plants treated with novaluron 10 EC @ X dose where as it was up to 3 DAT in the plants treated with novaluron 10 EC @ 2X dose (Table 5).More than 80 per cent mortality was observed up to 5 DAT at both the test doses of novaluron 10 EC.Toxicity of novaluron against S. frugiperda persisted upto 10 and 15 DAT respectively on novaluron 10 EC @ X and 2X dose.Toxicity of spinetoram 11.7 SC against S. frugiperda persisted up 15 and 20 DAT on maize plant treated with spinetoram 11.7 SC @ X dose and 2X dose respectively (Table 6).Cent per cent mortality was recoded up to 3 DAT in spinetoram 11.7 SC @ X dose and 4 DAT in spinetoram 11.7 SC @ 2X dose.
The studies were conducted to identify the median lethal concentration of different insecticides which the farmers in India predominantly use to manage the recently introduced polyphagous pest S. frugiperda on maize.The results revealed that the LC50 value of emamectin benzoate, chlorantraniliprole, spinetoram, flubendiamide and novaluron were 0.05, 4.08, 0.1, 85.89 and 0.91 ppm, respectively for second instar larva and 0.03, 5.63, 0.02, 162.99 and 0.99 for third instar larvaof fall army worm.Present results are in accordance with the findings of Zhang et al., (2021) who reported that the LC50 values of susceptible strain of S. frugiperda against chlorantraniliprole, spinosad and emamectin benzoate were 0.07 -2.00, 8.00 -10.00 and 0.40 to 0.90 µg mL -1 , respectively and different populations S. frugiperda to indoxacarb, chlorantraniliprole, cyantraniliprole, spinosad, emamectin benzoate and chlorfenapyr were 16.35 -99.67 µg mL -1 , 1.28 -2.34 µg mL -1 , 1.49 -4.64 µg mL -1 , 598.57-3878.74µg mL -1 , 1.94 -4.59 µg mL -1 and 87.03 -128.43 µg mL -1 .The population of S. frugiperda tested for this study has less LC50 than the susceptible strain of the above findings.It shows the strain present in India is highly susceptible than the strains present in China.During 1990, S. frugiperda collected from southern and central Florida showed 3 to 264 fold resistance to pyrethroid, 11 to 517 fold resistance to organophosphorus and 10 to 507 fold resistance to oxadiazine (Yu, 1992).Yu et al., (2003) reported that population of S. frugiperda collected from Citra, Florida recorded 562 fold resistance to carbaryl and 354 fold resistance to parathion -methyl.Field strains of S. frugiperda collected from northern Florida during 2007 registered 30 to 39 fold resistance to parathion-methyl and 626 to 1159 fold resistance to carbaryl (Yu and McCord 2007).It infers that this polyphagous pest can develop resistance to insecticides faster than other lepidopteran pests.Even though the LC50 is very less in the populations collected at Coimbatore, Tamil Nadu the probability of development of resistance is more due to increased frequencies of insecticide spray to supress this pest in this region.Song and Wu et al. (2020) reported that the spraying frequency of emamectin benzoate, which had a high efficiency on FAW, increased to 6.83 times in summer 2019 in west Yunnan since FAW invaded China.Hence continued monitoring of resistance development especially for the recommended insecticides is important to overcome massive destruction expected from this pest on the various food crops.
The results of persistent toxicity study revealed that, all the recommended insecticides cause more than 80 percent mortality up to 5 DAT except emamectin benzoate and spinetoram (Fig. 1 and 2).Among insecticides tested emamectin benzoate and novaluron have the shortest persistency of 10 DAT followed by spinetoram, chlorantraniliprole and flubendiamide in the recommended dose.Toxicity of emamectin benzoate, chlorantraniliprole, flubendiamide, novaluron and spinetoram at double the recommended dose persisted upto 10, 25, 25, 15 and 20 days, respectively.Vinothkumar et al., (2018), reported that the residues of emamectin benzoate persisted up to 3 and 5 days after treatment and further dissipated to Below Detectable Limit (BDL < 0.05 μg g -1 ) on 5 and 7 days after treatment at @ 10 g a.i ha -1 and 20 g a.i ha -1 , respectively.Wang et al. (2012), reported that 110|1-3|95 the dissipation half-life of emamectin benzoate in cabbage, apple and soil were 1.34 -1.72 day, 2.75 -3.09 day and 1.89 -4.89 day, respectively.Minghui Li et al. (2011), reported that the half-life of emamectin benzoate in paddy plants, water and soil were 2.04-8.66days, 2.89 -4.95 days and 3.65 -5.78 days with a dissipation rate of 90% over 7 days after application, respectively.Emamectin benzoate (Proclaim 5 SG) at 68.1 and 136.2 g a.i.ha −1 , dissipated below the limit of quantification (LOQ) of 0.05 mg kg −1 after 5 days at both the dosages on okra fruits (Gagan Jyot et al., 2014).Preethi et al., (2019) reported that the persistence of flubendiamide in cabbage at tropical region sprayed at recommended dose 18.24 g a.i ha -1 and double the recommended dose 36.48 g a.i ha -1 reached Below Detectable Limit (BDL) of less than 0.01 µg g -1 on 15 days after treatment.Sharma and Parihar (2013), recorded the initial deposit of flubendiamide on tomato fruits were 0.295 and 0.641 µg g -1 and the residues reached below detectable level of 0.01 mg kg -1 after 5 and 7 days of application of single dose (48 g a.i.ha -1 ) and double the dose (96 g a.i.ha -1 ).Deepak et al. (2017) reported the initial deposit of flubendiamide on okra fruits was 1.49 µg g -1 and it dissipated below detectable level of 0.01 µg g -1 on 10 th day when sprayed at 60 g a. i. ha -1 .
Persistence of chlorantraniliprole on cabbage sprayed at 10 g a.i ha -1 and 20 g a.i ha -1 reached Below Detectable Limit (BDL) of less than 0.05 µg g -1 on 7 and 10 days after treatment, respectively (Preethi et al., 2019).Chlorantraniliprole residues on cauliflower reached BDL on 3 and 5 days after treatment (Ioriatti, et al., 2009).The spinetoram residues in tomatoes were below the codex maximum residue level (0.06 mg kg -1 ) after 10 days of application when sprayed at recommended concentration (Malhat, 2013).Whereas Hafez et al., (2016) reported that spinetoram residues reach below detectable limit of (below the quantification limit 0.03 mg / kg) in tomato fruit on 21 days after spraying.The efficacy studies revealed that spinoteram, emamectin benzoate and spinosad recorded significantly higher mortality ranging from 90.40 to 96.22, 92.47 to 98.73 and 98.28 to 100 per cent under in vitro and in vivo condition, respectively (Mallapur et al., 2019).Piw das et al., (2007) and Anita et al., (2018) reported that novaluron @37.5 g. a.i ha -1 and 75 g.a.i ha -1 persisted for seven and ten days, respectively in chilli, brinjal and tomato ecosystem whereas it was 10 and 15 days on fresh and dry chilli peppers (Visalkumar et al., 2018).
A mostly persistence study was carried out through quantification of insecticide residues (physical method) on the plant using chromatographic techniques and the impact of trace amount of insecticide on the target organism was not evaluated.The fates of insecticides reported below quantification level on target organism were also not assessed.In this study, the biological evaluation of the persistence of insecticide using the susceptible stage of target organism was assessed.Presence of trace quantity of insecticide residue will create mortality on susceptible insects.This study clearly indicated that the persistence of test insecticides were more than the earlier reported period which indicated that the presence of minor quantity recorded below the limit with sophisticated instruments also greatly impacted the target organism in the field.Hence, the more persistence insecticides like flubendiamide and chlorantraniliprole need to be recommended in the early stage of the maize crop (within 20 days after sowing) and less persistence insecticides viz., emamectin benzoate and spinetoram should be recommended in the middle stage (25 to 40 days) of the maize crop for the management of FAW so that the residues may not accumulate in the harvested produce both in the stalk and grain.

Conclusion
The LC50 value of emamectin benzoate, chlorantraniliprole, spinetoram, flubendiamide and novaluron were 0.05, 4.08, 0.1, 85.89 and 0.91 ppm, respectively for second instar larva and 0.03, 5.63, 0.02, 162.99 and 0.99 for third instar larva of fall armyworm.Among insecticides tested emamectin benzoate and spinetoram showed high toxicity to S. frugiperda than other insecticides by registering minimum LC50 value.But in persistent toxicity study, same insecticides show less persistence on maize crop.Hence, insecticides like flubendiamide and chlorantraniliprole need to be recommended in the early stage of the maize crop (within 20 days after sowing) and less persistence insecticides viz., emamectin benzoate and spinetoram should be recommended in the middle stage (25 to 40 days) of the maize crop for the management of S. frugiperda so that the residues may not accumulate in the harvested produce both in the stalk and grain.

Table 1 .
Baseline susceptibility of the maize fall armyworm to different insecticides