Laboratory efficacy of selected synthetic insecticides against second instar invasive fall armyworm, Spodoptera frugiperda (Lepidoptera: Noctuidae) larvae

Maize is the most essential crop of China and its productivity has been recently endangered by the fall armyworm (FAW), Spodoptera frugiperda. Chemical pesticides are one of the most important strategies for managing FAW on a short-term basis. The seven synthetic insecticides including novel and conventional belong to four chemical group, spinetoram and spinosad (spinosyns), lambda-cyhalothrin, cypermethrin and bifenthrin (pyrethroids), abamectin (avermectins), broflinilide (diamides), were assessed for their efficiency in causing mortality to second instar S. frugiperda larvae at 24, 48 and 72 h post-treatment at five different serial concentrations (10 to 0.625 mg liter-1). The second instar S. frugiperda larvae were susceptible to the tested synthetic insecticides, however, the toxicity index of synthetic insecticides was estimated based on lethal concentration 50 (LC50), while, LC50 was calculated from the data of larval mortality. The broflanilide and abamectin proved to be the most toxic having the highest toxicity index of 100 and 78.29%, respectively, followed by cypermethrin and bifenthrin were showed toxicity index of 75.47 and 66.89%, respectively. The LC50 values were 0.606 and 0.774 mg liter-1 for broflanilide and abamectin, respectively, followed by cypermethrin and bifenthrin were showed LC50 values of 0.803 and 0.906 mg liter-1 at 72 h post-treatment. Rest of the other synthetic insecticides were showed moderate toxicity index of 42.11 to 62.09%, based on LC50 values were 1.439 to 0.976 mg liter-1 at 72 h post-treatment. The efficiency of synthetic insecticides was increased by increasing concentration level and exposure time. The screened synthetic insecticides among seven insecticides perhaps, provide basis for the development of novel insecticides for controlling S. frugiperda population after further research to evaluate and validate the laboratory results in the field.


Introduction
The application of novel synthetic insecticides is an effective emergency-based control method that might be an essential component of integrated pest management strategies to tackle invasive S. frugiperda in China [45]. Because of this, evaluating the efficiency of synthetic insecticides against S. frugiperda laboratory populations is a top priority [46]. Farmers and agricultural managers have no experience dealing with S. frugiperda, which is necessary for the development of effective management strategies [47]. To meet the food demands of a growing population, the maize production systems deployed by smallholders in China have tended towards the highly excessive use of chemicals, which have caused severe environmental impacts [48]. However, there is little evidence on the efficiency of locally available synthetic insecticides against S. frugiperda.
Hence, the objective of the present work is to evaluate the laboratory efficacy of selected synthetic insecticides against new invasive pests in China to develop an emergency-based approach by novel insecticides for minimizing yield losses by suppressing this notorious pest in China and other affected countries.

Insects
Individuals of fall armyworms were reared on an artificial diet containing, 26.0g of agar, 1.0g of choline chloride, 0.2g of myo-inositol, 2.0g of sorbic acid and propyl 4-hydroxy benzoate, 0.1g of streptomycin sulphate and penicillin GNa salt, 8.0g of ascorbic acid, 100g of soyabean, 80g of wheat bran, 26g of yeast extract, 8g of casein and 100 ml of double distilled water. Fall armyworms were kept at 25 ± 2˚C and were subject to a 12:12 (light: dark) h photoperiod and relative humidity (RH) between 50 and 70% in an environmental growth chamber at the Institute of Zoology, Guangdong Academy of Sciences, Guangzhou, China.

Synthetic insecticides
This experimental study tested the efficacy of seven synthetic insecticides from four different chemical groups to second instar S. frugiperda larvae after being purchased from different manufacturers. Chemical groups, active component percentages, formulation type, and supplier's information are captured in Table 1. The five serial concentrations of each synthetic insecticide were generated prior to the bioassay using repeated dilutions with distilled water.

Bioassay
The artificial diet mix method assessed the efficiency of seven synthetic insecticides against second instar S. fruigiperda larvae. The insecticide stock solutions were prepared by dissolving each in 1 ml acetone; and then diluted with distilled water to prepare five serial concentrations (Conc.1 to 5) for each insecticide i.e., 10.0, 5.0, 2.5, 1.25, 0.625 mg liter -1 , respectively, for spinetoram, spinosad, lambda-cyhalothrin, cypermethrin, bifenthrin, abamectin and broflinilide. The artificial diet mixed insecticides for each concentration were placed in clean rectangular plastic boxes (28 × 17 × 18 cm), with perforated lid. Second instar S. frugiperda larvae collected from culture were released onto artificial diet. Larval mortality was assessed at 24, 48, and 72 h after exposure to the treated artificial diet using a camel hairbrush. The larvae that responded to the gentle touch of a camel hair brush were considered alive, while those who failed to move were considered dead. Thirty larvae were considered one replicate; five replicates were performed for each insecticide concentration, while an artificial diet mixed with water was used as the control.

Statistical analysis
The mean percentage larval mortality and means numbers of S. frugiperda larvae obtained from the laboratory were subjected to one-way analysis of variance (ANOVA) using a generalized linear model. The percent larval mortality data of synthetic insecticides was transformed using an arcsine transformation to normalize the variance [49]. The significant level was set at (p < 0.05), and the means were separated using Tukey's honest significant different test. Lethal concentrations (LC 50 ), 95% confidence limits (CLs), slope, and chi-square (χ2) were calculated using the POLO Plus software (version 2.0, LeOra Software, Berkeley, CA, USA), while degree of freedom (df) and p value were calculated using the IBM SPSS statistics software package version 23.0 (Armonk, New York, USA). The letters were calculated using software Statistix, version 8.1. Relative potency ratios to estimate the potency of the active ingredients were calculated as the LC 50 of the least toxic compound divided by the LC 50 of the most toxic compound [40].

Susceptibility of the second instar S. frugiperda larvae to synthetic insecticides
The toxicity regression equations, lethal concentration 50 (LC 50 ), and toxicity index were estimated based on larva mortality at 24 h post-treatment in Table 5. The results presented the  Table 5.
The results presented the toxicity of seven synthetic insecticides, including broflanilide and abamectin were found to be lower than those of the other five synthetic insecticides, spinetoram, spinosad, lambda-cyhalothrin, cypermethrin and bifenthrin. The broflanilide and abamectin were showed lowest LC 50 values of 2.664 and 2.796 mg liter -1 , respectively, followed by lambda-cyhalothrin (3.123 mg liter -1 ), while, bifenthrin and cypermethrin were showed highest LC 50 value of 5.942 and 5.505 mg liter -1 , respectively. The obtained results demonstrated that broflanilide and abamectin outperformed among all other insecticides causing toxicity to the second instar S. frugiperda larvae. Based on the toxicity index at the LC 50 level, broflanilide and abamectin were the most effective insecticide by indicating the toxicity index of 100 and 95.28% followed by lambda-cyhalothrin and spinetoram were showed toxicity index of 85.30 and 75.30%, respectively, while, the least toxicity index of 44.83% was observed in bifenthrin. The toxicity index of (> 48.0%) for all other insecticides to second instar S. frugiperda larvae at 42 h post-treatment in Table 6.
The results presented the toxicity of seven synthetic insecticides, including broflanilide and cypermethrin were found to be lower than those of the other five synthetic insecticides, spinetoram, spinosad, lambda-cyhalothrin, abamectin and bifenthrin. Broflanilide and cypermethrin were showed lowest LC 50 values of 0.606 and 0.783 mg liter -1 , respectively, followed by abamectin (0.803 mg liter -1 ), while, spinosad and lambda-cyhalothrin were showed highest LC 50 value of 1.439 and 1.001 mg liter -1 , respectively. The obtained results demonstrated that broflanilide, cypermethrin and abamectin outperformed among all other insecticides causing toxicity to the second instar S. frugiperda larvae. Based on the toxicity index at the LC 50 level, broflanilide and cypermethrin were the most effective insecticide by indicating the toxicity index of 100 and 77.39%, respectively, followed by abamectin (75.47%), while, the least toxicity index of 42.11% was observed in spinosad. The toxicity index of (> 60.0%) for all other insecticides to second instar S. frugiperda larvae at 72 h post-treatment in Table 7.

Discussion
Pesticide residue levels in produce have become stricter due to a growing demand for highquality and safe food. Synthetic insecticides although have residual effects but could be used after evaluating the optimum dose level with least residual effects as an emergency control of many arthropods especially lepidopteran pests. The present study was conducted to assess the susceptibility of second instar larvae of Fall armyworm Spodoptera frugiperda (Lepidoptera: Noctuidae) at five different concentrations after different time exposures to seven synthetic insecticides belong to different chemical groups including two spinosyns, three pyrethroids, one avermectin and one diamides in China. The present study results revealed that all of the tested synthetic insecticides showed significant efficacy against second instar S. frugiperda larvae. Recently, many researchers around the world have been investigating the different management strategies against FAW in field and laboratory to develop registered pesticides as an emergency approach including India, Africa, Brazil and Indonesia [50][51][52][53], where this invasive pest caused severe damage to maize crop. However, this is the first study to our knowledge reporting the laboratory toxicity of seven synthetic insecticides to S. frugiperda larvae.
The results of present study revealed that second instar S. frugiperda larvae was susceptible to seven synthetic insecticides; however, spinetoram, broflanilide and spinosad were caused highest larval mortality 72 h post-treatment. The present study results align with Kulye et al. [51], who observed S. frugiperda susceptible to spinetoram across field samples. The highest toxicity of spinetoram in causing larval mortality to second instar S. frugiperda larvae [50]. Spinosad and abamectin displayed lower resistance and still effectively manage fall armyworm resistance in Puerto Rico [40]. Another study reported that cypermethrin caused 76.7% mortality to the larvae of corn earworm, Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae) [53]. According to a recent study, the beet armyworm, Spodoptera exigua (Hübner) (Lepidoptera: Noctuidae), was highly susceptible to broflanilide. Hence, broflanilide could be an important new tool alternative to synthetic insecticides to control lepidopteran pests [54].
Another study demonstrated that broflanilide is expected to become a prominent insecticide because it is effective against S. litura with resistance to cyclodienes and fipronil [55]. The resistance was higher in bifenthrin to American bollworm, Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) [56]. The results of present study are in lined with [41] reported that more than 80% mortality to third instar S. frugiperda larvae at 96 h after application of spinosad. Another study demonstrated that spinosad showed highest toxicity against S. frugiperda larvae [57]. The higher concentrations and time exposure of synthetic insecticides increase larval mortality of S. frugiperda both in the field as well as in laboratory assay [57,58]. S. frugiperda was resistant to lambda-cyhalothrin due to continuous application of conventional insecticides in Colombia and China [59,60]. Some of the pests such as the diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae) [61] and two-spotted spider mites, Tetranychus urticae (Trombidiformes: Tetranychidae) [62], have become resistant to abamectin while spinetoram still proved to be efficient in suppressing FAW in field trials. Even though chemical pesticides are critical in FAW control, the insect has become resistant to many of them as a result of their widespread usage.
The results of present study are in lined with [50] reported that the LC 50 values of spinetoram were lower than lambda-cyhalothrin. The findings of the present study in lined with Hardke et al. [63] who reported that LC 50 value of spinetoram was significantly lower as compared to the indoxacarb. Although synthetic insecticides are effective against S. frugiperda, but also have harmful effect on human and ecosystem due to a lack of appropriate information about insecticides dose recommendation and safety precautions which is an important issue for the application of synthetic insecticide in the field [64]. This situation suggests that there is a dire need to develop novel insecticides with optimum dose as a component of IPM.
The recent invasion of an invasive pest has alarmed many affected countries such as Africa, Ethiopia, India [18]. Therefore, there is an urgent need of insecticides spraying program as an emergency response in S. frugiperda invaded countries, especially in the maize fields, to protect crop damage and prevent the further expansion of the invasive pest. Farmers are applying unregistered synthetic insecticides in many countries, including Ethiopian and Kenya [65]. This work adds to our understanding of the efficacy of novel insecticides in the management of S. frugiperda. These pesticides recommend only as a last option in treating lepidopteran pests as part of an integrated pest management strategy.

Conclusions
Since very low resistance levels have been determined with broflanilide, cypermethrin and abamectin, hence, these chemicals should therefore be used cautiously and appropriately in management plans to retain their effectiveness for as long as possible. The highest discriminating concentrations of novel synthetic insecticides dramatically increase the cumulative larval mortality of early instar S. frugiperda. The findings of present study suggested that larval mortality of early instar significantly increase with increasing concentrations. Additionally, these insecticides could be used on an emergency basis at recommended dose against S. frugiperda larvae after further investigation on their efficacy are obtained in the field. The results of this study provided valuable information for choosing alternative insecticides substitute of S. frugiperda resistant insecticides.